sample preparation techniques in trace element … · element analysis by x-ray emission...

lAEA-TECDOC- 300

SAMPLE PREPARATION TECHNIQUESIN TRACE ELEMENT ANALYSIS

BY X-RAY EMISSION SPECTROSCOPYV. VALKOVIC

INSTITUTE RUDER BOSKOVICZAGREB, CROATIA

YUGOSLAVIA

A TECHNICAL DOCUMENT ISSUED BY THEINTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1983

SAMPLE PREPARATION TECHNIQUES IN TRACE ELEMENT ANALYSISBY X-RAY EMISSION SPECTROSCOPY

IAEA, VIENNA, 1983IAEA-TECDOC-300

Printed by the IAEA in AustriaNovember 1983

FOREWORD

This text on sample preparation technique in traceelement analysis by x-ray emission spectroscopy has beenwritten for I.A.E.A. under provisions of research contract2947/RB and 3^0/TC.

The text has been written with the aim of assistinglaboratories introducing x-ray emission spectroscopy as ananalytical tool with the most difficult problem in ac-complishing this: mastering sample preparation techniques.

Many of the preparation steps have been used in theauthor's laboratory for over a decade. Many more have beenused by researchers all over the world, and some of themhave been described in detail, some only mentioned in thereference list. Many have been omitted, not because theauthor wanted that to do but because of being pressed intoproducing this manuscript (version 1.0). The author will beglad to accept any suggestions for addition, revisions orimprovements of this text.

Let us help those laboratories who want to introducex-ray emission spectroscopy and work with it.

Note: Mention of commerical products or company names does notconstitute endorsement by the author or publisher.

Please be aware that all the Missing Pages

in this document were originally blank pages

CONTENTS

Preface

1. SAMPLING ............................................. ~........... 71.1 Introduction .............................................. 7

1.1.1 Aerosol sampling ................................. 111.1.2 Water sampling ................................... 191.1.3 Soil sampling ..................................... 211.1.4 Sampling of biological materials .............. 211.1.5 Sampling of petroleum and its products ........ 26

1.2 Sample storage ............................................. 291.2.1 Loses from water by sorption on surfaces .... 33

1.3 Sample fragmentation, powdering and homogenization . 381.4 Contamination of sample ................................. 39

2. SAMPLE PRETREATMENTS .......................................... 432.1 Preconcentration ......................................... 432.2 Dry ashing ................................................. "452.3 Wet ashing ................................................. 462.4 Low - temperature ashing ................................ 492.5 Loss of elements during sample pretreatments ........ 522.6 Chelation and Solvent extraction ....................... 562.7 Ion exchange ............................................... 612.8 Electrodeposition ......................................... 63

3. SAMPLE PREPARATIONS FOR PIXE ................................ 663.1 Backing'materials ......................................... 673.2 Target uniformity and homogenity ....................... 743.3 Reproducibility ........................................... 743.4 Effects of irradiation ................................... 763.5 Internal standards ....................................... 803.6 Examples of sample preparation for PIXE .............. 85

3.6.1 General .,...................................'....... 853.6.2 Aqueous samples ................................... 873.6.3 Biological samples ............................... 913.6.4 Blood serum samples .............................. 933.6.5 PIXE targets preparation for solid samples 96

4. SAMPLE PREPARATIONS FOR EXCITATION WITH RADIOACTIVE .... 99SOURCES OR TUBE EXCITATION ................................... 994.1 Water ....................................................... 994.2 Liquid samples ............................................ 1074.3 Solid samples ............................................. 1094.4 Soil ........................................................ 1124.5 Geological samples ....................................... 1124.6 Atmospheric particulate .................................. 1174.7 Plants ..................................................... 1194.8 Tissues .................................................... 119

5. STANDARDS ....................................................... 1245.1 Standard solutions ........................................ 1255.2 Reference materials ...................................... 1335.3 Other standards ........................................... 1465.4 Intercomparisons .......................................... 156

6. LITERATURE ON SAMPLE PREPARATION METHODS ................. 164

1. SAMPLING1.1 Introduction

Sampling is often discussed because the greatest sourcesof error in many studies are usually in the sampling steps. Asa general rule the analyst himself should be directly involvedin the sampling procedure. In such a way it is easier to ensurethat the samples are representative and that no significantchanges in composition occur during sampling.

The major concern in sampling must be that the sampleaccurately reflects the variations in the material being sampledSamples for laboratory assays may be selected on the bases ofthe capabilities of the analytical methods used: precision andaccuracy, sensitivity, time considerations, costs, single versusmultielement analyses - but special consideration must be givento specific media sampled. For example, greater homogeneity isoften encountered in natural waters or in body fluids than insoils or tissues of organisms. Such characteristics always de-termine the sample sizes necessary to measure the variation ac-curately. The specific technique employed must be one capableof handling the analytical sensitivity required.

Great diversity in techniques and sampling proceduresexist . For some cores general sampling designs have been de-veloped. For example general sampling designs have been devel-oped for environmental surveys and can serve as models forfuture programs (U.S. Geological Survey, 1972). Sampling plansinvolving large regions begin with a decision concerning thematerials to be investigated from the lithosphère, hydrosphere,biosphere, or atmosphere. Increased detail can be ascertainedfrom successive stages of sampling to a point determined bythe demands of the problem or by the economics of the situation.

General and theoretical considerations of sampling arediscussed in details by Sansoni and lyengar (1980). In theprocess of chemical analysis only a small part of the totalmaterial is generally used to provide the analytical signalfrom which the concentrations of the components of interestare calculated. In general, a large sample is taken from thebulk material and transported to the laboratory (laboratorysample). Subsequently an aliquot is taken to provide a muchsmaller analytical sample. Of this, very often only a smallfraction is actually used to produce the analytical signal. Thesample and its subsamples must satisfy several requirementswhich can be listed as (Sansoni and lyengar, 1980):

1. The mean composition of the laboratory and analyticalsamples should, in principle, be exactly the same as that ofthe bulk material to be evaluated (representative mean compo-sition). However, this is an ideal condition which cannotusually be met in practice. 'Compromises are to increase thenumber of random aliquots to be analysed and to homogenize thebulk material before sampling.

2. The variance of the concentration levels within thelaboratory and analytical samples should be the same as thatof the bulk of material (representative variance).

3. The total error introduced during the entire samplingoperations should be less than, or only of the same order ofmagnitude as, the error of the subsequent analytical procedure.

There are several sampling methods used. In random repre-sentative samples cannot be obtained by random sampling ofinhomogeneous material unless the number of random samples takenis quite large. The overall error in this case largely depends•on the degree of inhomogeneity.

For large samples that cannot be homogenized, it isnecessary to use a sampling approach. This involves systemati-cally sampling several constituent parts of a given material,e.g. by collecting samples from a population covering differentage groups, sexes, geographical regions and nutritional habits.

In some cases the bulk material is made up of well-definedparts which are individually fairly homogeneous, e.g. differentorgans of the human body. The composition of the whole humanbody may be computed by analysing the individual homogeneoussections. This method is known as differential sampling. However,it is normally applied only to bulk materials that have clearlyidentifiable subdivisions and that cannot easily be homogenizedin a single batch, as in the case of total body analysis (San-soni and lyengar, 1980).

There are many papers written on this subject some ofthem are listed in the reference list. One of the importanteffects in sampling is caused by random particle distribution.This effect is based on the number of particles extracted persample, i.e., the greater the number, the smaller the effect.Therefore, it can be generally stated that random particledistribution is of greatest influence when extracting smalllaboratory size samples•having few particles. On the other hand,particle segregation exerts its greatest influence for bulksamples which cannot be conveniently mixed or processed througha device that eliminates particle segregation.

General sampling equation can be written as:S2 = A/W + B/N (1.1)

where S2 = the total variance for the systemA = sampling constant (random variance)B = sampling constant (segregation variance)W = size of the gross sampleN = number of samples collectedThe random variance constant A is estimated by taking

a series of small samples where the sampling variance isprimarily due to random effects, and it is assumed that thesegregation variance is negligibly small. Similarly, thesegregation variance constant B is estimated by taking aseries of large samples where the sampling variance is dueprimarily to segregation effects; and it is assumed thatrandom effects are negligibly small.

Further, W and N in Equation (1.1) are in the natureof operating variables that can be manipulated within certainlimits by the sampler. For instance after A and B have beendetermined and for a given S2 and N, the sample weight, w,can be determined by substituting the equality Nw = W into

Equation (1.1). This substitution gives--w = A/(NS2 - B) (1.2)

Conversely, by specifying w, N can be determined.Therefore, the number and size of samples can be calculatedfor a-specified sampling variance.

This approach is most applicable for the continuousanalysis of similar lots of bulk material. This method isnot, however, as helpful in estimating the expected samplingerror for laboratory size samples.

Since segregation of particles in laboratory samplingcan be largely eliminated, one needs to emphasize errorsresulting from random particle distribution. Benedetti-Pichlerconsiders this situation; and for a binary population BernoulliTheorem yields the equation—

G; - !â!s (. . p., i/RO^Ei"Prwhere Ç' = the absolute standard deviation of the percentp of component x in a mixture of particles A and B

dA = the density of the A particlesdg = the density of the B particlesd = the weighted average density assuming all particles

have the same volumePA = the percent of the component x in the A particlesPg = the percent of the component x in the B particlesp = the fractional number of the total particles which

are type A1-p = the fractional number of type B particlesn = the total number of particles

This equation is idealized by assuming only two speciesof particles. However, this restriction can be overcome byconsidering that the components are either rich in x or poorin x. Equation (1.3) is also idealized by assuming that allparticles are the same size.

Equation (1.3) can be modified to employ terms ofparticle size, sample weight, concentration, and density. Forthe simple case of two distinct particulate species of thesame particle size, one obtains

(.) (1.4)

where SE = the absolute standard deviation of the concentra-tion of element E

t- = the weight fraction concentration of element Ein species 1

tp = the weight fraction concentration of element E inspecies 2

W.j = the weight proportion of species 1W2 = the weight proportion of species 2d. = the density of species 1dp = the density of species 2d = the weighted average densityV = the volume of the individual particlesw = the weight of sample takenLet us consider the determination of trace elements in

powder samples. If the element under consideration is a minoringredient of one or more of the major constituents, its re-lative sampling error will be comparatively small because(t., - t«) will be very small. If on the other hand the traceingredient is present as a major constituent of the speciesof minor abundance, its relative sampling error will becomparatively large. The latter situation would pertain toelements in certain mineral mixtures such as zirconium inbeach sands where the zirconium is almost exclusively as-sociated with individual zircon particles, gold in mineralsands, and metals in synthetic powder mixtures used tocalibrate analytical systems for trace analysis.

For the case of more than two mineral species and withthe simplifying assumption that all species have the samedensity, the following equation can be derived:

W WW "<*i - v * (ta - v * fts - v - (1-5)Usually the interest is primarily to determine trace elementswhich are major components of species in minor abundance . Inthis case one can assume a binary system in which everythingother than the species of interest is the second component.

For samples of wide particle size distribution theaverage volume, V, be replaced by a weighted average volume,V, calculated from —

kh=1

where k = the number of groups of different particle size*V, = the average volume of the individual particlesof each groupg, = the fraction by weight of the h group

One major drawback to the use of any estimate of particlevolume is that, after grinding, different species are • not likelyto show the same particle size distribution due to dif-ferences in hardness and brittleness. For rock samples, mineralssuch as zircon, chromite, and magnetite which are harder thanthe bulk materials, may be concentrated in the larger fractions.

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Since these coarse particles have the greatest influence onthe standard deviation of sampling, such segregation couldcause a serious discrepancy between expected and observed sampleerrors. " ' '

Another sampling equation sometimes used is:

s = Lb/p J "& (1 .7)where s = relative sampling standard deviation

b = weight of largest individual particle (assumed cubic)x100weight of sample

c = weight percent of the analyte

1.1.1 Aerosol samplingAerosol sampling and analysis by the method of charac-

teristic x-ray detection has received great attention inrecent years. Sampling is ussualy done by collecting airparticulates using a low volume total suspended particulatesampler.

Samples are usually collected on filters or by cascadeimpactors. As filter material Milipore, Nucleapore and otherfilters have been used. Some filter materials tend to becontaminated with elements of int erest but are sometimesused when the sample is transferred to a solution for furtheranalyses. Aerosol size tfractionation is obtained by cascadeimpactors, where the aerosol to be sampled is drawn throughjets of decreasing diameters.

An example of a commercially available impaction deviceis shown in Figure 1.1. This single impactor consists of fiveimpaction stages in series plus an after filter. Air is takenin at the top and exhausted via a pump connected to the orificeat the bottom. The speed of the air flow is greater at eachsuccessive stage so that the largest particle (~H jim) impacton the surface below stage 1 and the smallest particlesC'A» 0.25 .urn) impact on the surface below stage 5. Very thin(50 to 100 ug/cm2) polystyrene supported by glass impactionsurfaces may be used to minimize the mass of the backingmaterial of the sample. This device however does not producea deposit of uniform areal density. Size fractionated samplescontain information on aerosol generation mechanisms and arethus of interest in atmospheric chemistry and air pollutionwork.

Particulate matter can be filtered from the air andthe filter examined directly by x-ray spectrometry.Detection limits of a few ng/cm2 are now routinely achievedbut require that background intensity be reduced to aminimum. To reduce the background care must be taken toremove sample supports or other material from the areawhich can be seen by the x-ray spectrometer; vacuum operationis necessary in order to eliminate air scatter. Usuallythe total sample is less than 1 mg/cm2 and the filter is1 to 10 mg/cm2 which makes it the largest contributor tobackground intensity.

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AIR OUT AIR OUT

*• DUST OUT WATER IN

Fig. 1.1 The principle of a commercially availableimpaction device for aerosol sampling.

SI

a)

TIME

kiK.S

TIME

Fig. 1.2 Flow rate through a filter; (a) linearrelationship caused by the loading ofparticulate matter on the filter device(b) more realistic dependence of flowrate on time (after Morrison, 1967).

Each of components used in collection of airparticulates needs to be considered individually sinceit may be a potential source of error. According to Morrison(1967) the components to be considered are: a filter holder,a filter media, a flow-measuring device or a volumetotalizer such as a dry-gas meter, a timing device torecord length of sampling time, and a pump.

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A flow-rate device must be used in conjunction with amechanism for recording the length of time of sample col-lection to allow for calculation of total quantity of airpassed. This is a major source of error in the samplingprocess because a plot of flow rate versus time is not alinear relationship caused by the loading of particulatematter on the filter device. Figure 1.2 (a) (after Morrison,1967) illustrates the assumed nature of flow versus timeby this procedure. The determination of volume of airsampled is calculated by averaging the initial (F1 ) andfinal (F2) flow rates :

X length of time = volume of air sampled. (1.8)

Actually, the flow rate through a filter subjected to par-ticulate buildup follows Figure 1.2 (b). The shape of thecurve depends .on .the actual loading characteristics, whichare the result of the total particulate burden of the air atany time t. It thus becomes clear that. this is not a prefer-red method of estimating total volume of air sampled (Morrison,1967).

A volumetric totalizer, such as a dry-gas meter, isnot subject to errors of the extent described for the previousmethod and use of it probably represents the best method ofmeasurement of air flow. However, this method will lead toincorrect estimates of the total volume unless certain pre-cautions are taken. The major error lies in the change intemperature of the air being sampled and the resultant vacuumhead produced between the filter head and the pump. If avacuum recorder is placed in the airline to record continuouslythe pressure drop on the upwind side of the meter, appropriatemathematical corrections can be made. Another source of dif-ficulty lies in the selection of a filter medium: because theparticle-size distribution of suspended particulate matter inair ranges from submicron to very large, the experimentaldesign must take this factor into account.

Any study designed to investigate the possible relation-ships between heavy metals in air and health or disease mustemploy the use of filter media that will have a high collectionefficiency for submicron particles, because these sizes arerespirable by man and animals . The foliar deposition and intakeinto the plants are also significant for these smaller-sizeparticles (Morrison, 1967). The analysis of exposed filterpaper poses another problem; it has been shown that metal par-ticles are not uniformly distributed over the surface of thefilter itself. This is presumably true of all airborne parti-cles, making it necessary to ash the entire filter if reliabledata are to be obtained. Kometani et al., (1972) have shownthat particulate air pollutants collected on paper filterscan be dry ashed at 500 C without serious loss of trace metalsby volatilization. Conversion of metal salts to sulfates bythe addition of H-SO^ prior to dry ashing ensures virtuallycomplete recovery of the metals tested. Losses reported duringdry ashing of particulate matter collected on glass fiberfilters are not necessarily ascribable to volatilization ashas commonly been supposed. Metals such as Pb, Zn , Cu , and

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Cd react to varying extents with glass at high temperature toform insoluble metal silicates. Comparative studies of par-ticulate matter collected on paper filters indicate that theresults obtained by dry ashing compare favorably with thoseof accepted methods such as wet ashing and low-temperatureashing. Good recoveries of Pb, Cu, Zn, and Cd from sampleswere obtained by dry ashing at 500 C, for 1 hr even withoutthe prior use of HpSO^.

Aerosol sampling is also discussed by Kemp and Miller(1981).

When determining the composition of an aerosol, twoparameters are often of special interest: the time variationsand the particle size distributions. Through the possibilityof analysing very small amounts of material using x-ray emis-sion spectroscopy, time sequential measurements can be achievedin a simple and handy way using a so-called "streaker", i.e.a sampler collecting a series of samples on a filter by movingthe filter over a suction orifice (Nelson, 1977).

Valuable information about the normally "bimodal" sizedistribution of urban aerosols may be obtained by measuringonly two size fractions, and discriminating the valley of thedistribution, i.e., at a particle diameter of approximately2 urn. Adding one more stage to the streaker may then increasethe usefulness of the measurements considerably.

Compatibility with other samplers, e.g., standarizedhigh volume samplers, is essential for intercomparing resultsfrom different projects. Due to the very different physicaldimensions of the streaker from most other samplers, it isnot obvious that this compatibility exists.

The aim in the construction of the streaker describedin the following is to separate the two humps mentioned aboveand as far as possible to get compatibility with normally usedhigh volume samplers. To get precise time resolution, streakingis performed in steps, thus creating discrete spots on thefilters. The performance has been checked through simultaneousmeasurements in the field with a two-filter-high-volume sampler,fitted with a 12 /am pore size Nuclepore filter for collectionof particles >2 /am (Heidam, 1979).

The main advantages of the streaker are the small sizeand the low power consumption. The air flow through the sampler,which is an important parameter determining its collectionproperties, is limited to approximately 1 1/min. In work byKemp and Miller (1981) the air flow was chosen to be 0.58 1/min,which is well below the maximum capability of the pump used.The suction tube is open and points downwards (see Fig. 1.3).The diameter is 0.6 cm, giving3" air velocity of 35 cm/s. TheReynolds number for the flow is approximately 150, whichindicates that the flow is laminar. Compared to standard highvolume samplers tlie velocity is approximately the same, butthe very much smaller tube diameter may give rise to considerabledifferences in the collection efficiency for large particles.

An impaction stage is used for the size separation. Thejet is formed in a linear slit having a length of 0.5 cm anda width of 0.025 cm. Using a channel length (i.e., the lengthof the tube having the slit cross section), which is long

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AIROUTLET!

PUMP HOUSE AND WIND KETTLECLOCK ANDCONTR.ELECTRONICS

AIRINTAKE

Fig. 1.3 Schematic of the device used by Kempand Miller (1981) for aerosols sampling.

compared to the width, the air velocity in the centre of_ theslit is increased. In this way a cut at diameters of 2-2.5 urnis obtained, which would otherwise be difficult to reach withthe low flow rate. However, since the boundary layers in thechannel play an important role, the steepness of the cut-offmight by somewhat smaller. Another factor which tends to dif-fuse the cut-off is that the distance between the slit andthe impaction plate has to be rather high (^0.1 cm) so as toreduce the effect of wobble in the plate position. The parti-cles are impacted on a 12 m thick mylar film coated withApiezon L grease in order to avoid "bounce off".

Small particles are collected on a 0.4 jurc pore sizeNuclepore filter, covering, a 0.25 cm x 0.75 cm oval suctionorifice. The orifice has a smooth end, so the filter can passover it without being damaged.

The filter and the impaction film are mounted on discswhich can rotate around a common axis_. The distance from theaxis to the middle of the impaction jet and to the middle ofthe filter orifice is 3-75 cm. Ey rotating the discs it ispossible to obtain 64 samples separated 5° from each other,on each film/filter. Exact positioning of the discs isobtained by pressing them towards a fixed stop duringexposure. The exposure times are controlled by means of aquartz clock and the shifts are programmable in intervalsbetween _L and 24 h in a 24 h cycle.

The pressure drop over the filter depends on itsload, which makes flow stabilization necessary. Stabilizationis obtained by regulating the supply voltage for the pumpby means of a flow meter equipped with two sets of opto-couplers "looking" at the flow meter ball. In this way, theflow is kept constant to within 5-10%.

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LIMITS ON PARTICULATE SAMPLES(APPROXIMATE)

REGION OFQUANTITATIVE

ANALYSIS

No Mg At Si S C* A

ELEMENT« K•aK Ca Sc Ti

LINE)

Fig. 1.4 Limits on total sample mass in aerosolsampling (Cahill, 1981).

The total power consumption of the streaker is ap-proximately 3 W, with a 12 V supply voltage. More than 75%of the energy is used in the pump motor (Kemp and Miller,1981).

Let us mention work by Adams and Grieken (1975)who have described a method for the evaluation of fluores-cence radiation absorption in the analysis of air particulatematerial collected on depth filters and imperfect screenfilters. The total absorption effect is divided into twocomponents, one due to the particulate itself, and one dueto the filter material. The first effect is calculatedafter evaluating the mass absorption coefficient by transmis-sion measurements. The second correction is obtained bydetermining experimentally an hypothetical equivalent depth •defined as the one hypothetical depth from which the par-ticulate material would give rise to the. same filterabsorption effect as that obtained from the actual distri-bution. Measurements of the ratio of the fluorescentintensities obtained from the front side of the filter andfrom its back side allow this hypothetical depth to becalculated.

Cahill (1981) has discussed aerosol sampling foranalysis by PIXE. Because of limiting pentrating power ofcharged particle beams used, only thin deposits of material,regardless of the penetrating power of the resultantcharacteristic x-rays, can be tolerated. Quantitativedetection of these x-rays, however, causes even more severerestraints on total thickness. This is especially true forthe elements sodium trough calcium, elements extremelyimportant for atmospheric physics and chemistry, comprisingabout 25% of the particulate mass. Fig. 1.4 illustratesthe limits on total sample mass set by degradation of ionbeam energy and concomitant changes in x-ray productioncross-sections, and by x-ray attenuation corrections inmass layers and particles. One must also keep in mind the65% or so of the mass in elements hydrogen through fluorinewhich PIXE cannot do quantitatively.

The only method certified in the United States forsampling of particulate matter is the high volume filter

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sampler (Hi-Vol). Typical filters are about 500 cm2 inarea, so that ion beams sample no more than 0.5% of thecollected matter and occasionally as little as two partsin 105.

Large number of analyses are usually requiredtogether with both high time resolution and detailed sizeresolution. The characteristic temporal variations occuron several scales, from wind gusts (seconds) to diurnalpatterns (hours) through synoptic behavior (days) toseasonal behavior scaled in months. The latter three areparticularly informative, but to generate data at 2-hourresolution at one site with no size cuts for a yearinvolves 4,380 analyses, entailing costs between about$ 40,000 and $ 100,00 (Cahill, 1981). However, diurnalpatterns are often highly repetitive, and a limited numberof such profiles, probably each season, might be adequatefor many sites. Synoptic patterns are themselves character-istic of seasons, and seasons are among the most stablepatterns since they average over many synoptic periods andhundreds of diurnal cycles.

These considerations also demand that one be carefulto average over the shorter time cycles when interested inlonger behavior. Thus, measuring for only 2/3 of a day maygive misleading results, while sampling for two days mightcoincide with just one phase of a strong synoptic shift.Obviously, sampling in one season allows one to say littleor nothing about annual cycles. One major time scale usedfor samplers involves the diurnal cycle, and such samplersgenerally use 2,3, or 4 hour increments, with the FloridaState streaker being a prime example of such a de-vicematched to PIXE. Many devices use a 24 hour integrationtime to smooth out diurnal patterns, and in some circumstancesoccasionally allow 2 or 3 day periods in remote and/orstable conditions. The one-day-in-six 24 hour criterion usedfor many Hi-Vol networks defies statistical understandingeven for seasonal averages. Ideally, one would like to takesamples that would contain detailed time information thatcould be later used if desired. The rotating drum Multidayimpactor used by the University of California, Davis, hassuch a capability for the >0.5 /urn stages, as the ion beamis magnetically swept so as to average over 24 hours in a2.5 cm strip. Resolution of 2 hours is easy for such samples,but since the filter switches every 24 hours, the sizesample is incomplete.

Such considerations also hold for the streaker when asingle time-averaged coarse stage filter is used to imposea 2.5 urn size cut on the device. Both the Multiday impactorand the streaker are being modified to give more completetime-size information, and these will allow detailed analysesof a single day if desired, while minimizing total numbersof analyses during routine monitoring. One might call suchinvestigation "ex-post-facto experimentation", with thedetailed analyses triggered by any unusual event that wouldbenefit from detailed study in 2 hour increments, whilenormal analyses cover 24 hour durations (Cahill, 1981).

Compromises in size resolution are also aided by thesystematic behavior illustrated by the bimodal distribution,

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Fig. 1.5

0,50,25

0,120.06

<0 06 AFTER< ' FILTER

TO PUMPBATTELLE-TYPE IMPACTOR

( WITH LOW PRESSURE STAGES)

Battelle type impactor with lowpressure stages.

and by rejection of very large particles by the humannose. Thus, a natural first step would be to limit collectionof very large particles that have minimal health effect, andsuch a limitation at 15 pm is proposed by the U.S. Environ-mental Protection Agency. This cut greatly aids PIXE (andXRF) as particle size corrections become very uncertain above15 to 20 jum diameter. The next logical cut is at the minimumof the bimodal distribution, around 2 to 3 juin. Such a cutserves several purposes: it separates modes of differentsources, making analysis both easier and more meaningful;it corresponds roughly to the partition between fine particlesdeeply penetrating the lung and the coarser particles lodgedin nose, throat, and upper bronchial passages; it alsoputs minimal mass at the point of the cut, making resultsrelatively insensitive to the shape of the separation functionbetween coarse and fine. Four major devices operate at thiscut, including a cyclone preseparator that eliminates thecoarse particles, selective filtration through a nucleporefilter, physical impactors such as the Multiday, and virtualimpaction. The latter depends on separation of air streams sothat fine and coarse fractions largely end up on two filters.

The next cut that has proved useful lies at about thewavelength of light,« 0.5 /jm. This cut bisects the accumulationmode into fractions that may scatter light differently, anda physical impactor is usually employed although other methods(spiral centrifuge), electrical methods, diffusion batteries)can be so utilized. There is a distinct difference between

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particles above and below this cut in terms of both composi-tion and impact upon visibility. The latter effect is predictedby Mie theory, as particles significantly smaller than thewavelength of light have minimal ability to scatter light.

Further size separations occur for the Lundgrenimpactor (5 fractions), the Batteile or Delrcn impactor (5fractions), and the Anderson impactor (8 fractions), amongothers. Recently, Battelle impactors with low pressurestages have been designed and built, allowing 10 or so cutsdown to 0.06 urn diameter, a diameter so small that massvalues are falling rapidly with size (see Fig. 1.5). Evenso, use of a filter is recommended, as in unusual circum-stances, a significant mass mode at 0.03 JJm, the condensationmode, can occur for short times in the presence of hightemperature sources . The width of impactor separation functionsset limits on the total number of cuts possible withoutserious overlap, and this number lies between 20 and 30for the 0.05 to 15 urn range. This constraint of a fixedtotal number of analyses normally demands that bestinformation is obtained when some'size information ismixed with some time information, either through differentdevices (streaker and Battelle) or the same device(3 stage Multiday impactors), although exceptions can occurwhen size information is not required, or previouslydetermined (lead from automobiles rarely varies much insize) (Cahill, 1981).

Table 1.1 lists the characteristics of a number ofsampling devices heavily involved in PIXE analyses.

1.1.2 Water SamplingThe principal problems in water sampling consist of

obtaining a representative sample, avoiding contaminationand separating dissolved and particulate phases (Morrisonet al., 197^). Many research groups have described samplingmethods used. For example, often used sampling deviceconsists of a Van Dorn bottle (sampling volume 3-51) madefrom PVC, that does not release any element in detectableamount to the water sample. Immediately after collecting,water should be transferred to polyethylene bottlespreviously cleaned with 10% nitric acid and rinsed withdistilled water.

According to Morrison et al.,(1974) very shallowstreams, only a few meters wide, that are well mixedlaterally as well as vertically may be sampled by dippingat mid-depth. Larger streams may require the compositionof numerous samples according to some meaningful system.Each cross-section sample needs to be taken in such a waythat it is velocity-integrated over the distance from thewater surface to the water-bed interface. In larger streamsthe sampling is -done from a bridge, cable, or boat.

The sample-bottle holders currently in use for samplingstreams should have the brass intake nozzle replaced withone made of Teflon and the rubber gasket replaced with onemade of silicone. It is important that the sampler be im-mersed in a flowing stream to wash off dissolved metals

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Table 1.1Particulate Samplers (after Cahill, 1981)

Unit( flow )

High volume sampler(1500 1/min)

Streaker(1 1/min)

Virtual impactor(15 1/min)

Stacked filter unit(10 1/min)

Multiday impactor(30 1/min)

Battelle impactor( 1 1/min)

Size Volume/areafractions (m3/cm2)

1 «t

1 2.2

2.72 (1.3)

1 .02 (0.5)

7.5/2.5F3 (2.5/0.8F)

D f1 11 / 0 Q F*6 (2.1/0.15F)

Advantages

common, simple, cheap,. legally acceptable

simple, cheap, goodtime resolution,automatically, for1 to 2 weekssizing; two membranefilters

sizing; two membranefilters; simple andcheap

extra cut at wave-length of light;automatic for 1 weekmany size cuts«

Problems

manual unit;no sizing;glass fiberfiltersno sizing;clogging (?)inlet (?)

cost; manual;maintenance;particle lossin handling;clogging (?)particlebounce /losson coarsefilter; manualcost;maintenance

manual; heavydeposits may

Improvements

inlet controlto 15 p™;teflon filters

two stagesizing streaker

coated filters;automation

coated filters

time variationfor filter stage

low pressurestages

a)Volume of air (m3) per area of substrate (cm2) per 2^ hour day, (values in parentheses are divided bythe number of size fractions). F = filter stage for impactors.assumes 0.1 cm2 beam.

before inserting the sample bottle. With all plastic filters,filtration is best done in the field or as soon as practical.The membranes used for filtration should have a nominalpore size of no more than 0.1 urn. The actual particle sizecutoff is unknown because, as sediment collects on themembrane, it generally filters more effectively than doesthe membrane itself (Morrison et al., 1974).

1.1.3 Soil SamplingIn soil sampling it is essential to recognize that

soils are complex systems resulting from weathering proces-ses. A soil profile reflects the magnitude of local weatheringeffects. Changes occur with depth in the distribution oforganic matter in texture and structure, and clay contents(Morrison et al., 1974).

Some theoretical consideration of trace elementsources, their redistribution by transport and deposition,and their behavior during weathering processes is essentialin the evaluation of trace elements in soils. Models ofsoils need to be translated to real soils on landscapesthat have properties important to a particular study. Soilsurvey maps are a means of locating potential sampling sitesof specific soils. Natural soils rarely have sharply definedboundaries on a landscape and it is therefore necessary tocheck the actual soil to determine if it is suitable forstudy. Surface samples of soils are usually taken whereinformation is needed about the role of soils on the traceelement composition of shallow-rooted plants. Studies oftrace element mobility and redistribution, whether fromnatural weathering or pollution, often -require the collectionof samples to greater depth. Only a few grams are usuallyneeded for laboratory determinations. Samples are best col-lected from one face of a small hole. Because sampling toolsare possible sources of trace metals, the use of a cleanspade is preferable to the use of a soil auger, and-unlessmost samples are needed-the samples collected can beplaced in a clean cloth bag.

For studies of most trace elements, it is necessaryto break down soil aggregates. Riffle samplers may be usedto reduce the amount of soil that might be fine-ground fortrace element determinations. Sieves made of silk boltingcloth or nylon are free of trace metals and are useful forsieving ground-soil materials (Morrison et al., 1974).

When sampling rocks the methods must be designed toreduce the sampling error to a level commensurate with orless than the effects being studied. The sample must beground to a sufficiently fine powder to yield an acceptablenumber of particles of each component of the heterogeneousmaterial. This process introduces contamination, which mustbe minimized during the sample preparation.

1.1.4 Sampling of biological materialsSampling of biological materials is discussed by many

authors involved in trace element analysis . Processing and

21

storage of foods often result in drastic changes in traceelement concentrations naturally present in plants . Somepertinent factors concerned with plant sampling are discus-sed below. It is stressed that in the plant sampling thenumber of individual plants needed to obtain a samplerepresentative of the soil on which plants were grown. Thespecies of plants collected and studied often differ aswidely as do the kinds of soils on which they are grown.This problem was studied by Lazar and Beeson (1956). Sixsoil areas nearly chemically uniform and representingfour soil series were used. Within each soil area, theindividual plants were tagged and samples were collectedfrom them over a 2-year period. At least five individualgrass plants were needed to characterize the cobalt andcopper status of soils when a grass was used, but the sameinformation could be obtained by using leaves of two black-gumplants. Later, tests using leaves of black-gum (Kubota andLazar, 1958; Kubota et al., 1960) showed that use of thisplant can distinguish the cobalt concentrations of total, aswell as extractable, forms of soil cobalt. Studies byBeeson and MacDonald (1951) of the effect of sampling dateson trace element concentration of alfalfa in New York showedthat manganese, cobalt, and iron increase with the growingseason. Marked initial decreases in trace element concentra-tions, however, were later observed by Loper and Simith(1961) in Wisconsin, also using alfalfa. When effects ofhigh rates of fertilization used in the Wisconsin study(Loper and Smith, 1961) were reduced with cropping, theseasonal changes were essentially the same those of theNew York study.

In terms of trace element concentration, plants ap-pear to be species-distinct. Browse plants, herbaceousplants, sedges, and grasses grown in Alaska were all studiedwhere they were growing in the surficial peaty mantle(Kubota et al., 1970). Marked species effects on the concen-tration of molybdenum were observed among species of com-mon forage plants in Nevada, USA (Kubota et al., 1961).

The necessity for sampling specific parts of plantsto identify trace element deficiencies or toxicities af-fecting growth of agricultural crops is well recognized.A comprehensive review was prepared, for example, by Tanakaand Yoshida (1970) to diagnose trace element deficienciesand toxicities using specific parts of the rice plant.Increases in trace element concentration with plant growthoften result from increases in leaves of forage plantsbut not of stems or petioles (Beeson and MacDonald, 1951).Patterns of trace element changes evident in browse plantsimportant for game- animals (Kubota et al., 1970). Studiesby Arkley et al., (1960) showed that trace elements carriedin peat dust and deposited on plants are easily removed bywashing, but those applied by sprays are not. Absorptionof trace elements through leaf surfaces was noted as afactor. Surface absorption of trace elements by plants wasalso observed by Lagerwerff (1971) to be an important factorin increases caused by absorption of trace elements onleaf surfaces may also be enhanced with drying and changesin leaf-surface characteristics with plant maturity.

22

Sampling techniques and sample processing were examinedsome years ago in the study of vitamin levels in turnipgreens (Southern Cooperative Group, 1951), but no systematicstudy has been made of sampling of fruits and vegetablesfor trace elements.

More attention has been paid to food processing andits effect on changes in trace element composition of fruitsand vegetables. Losses of zinc and manganese from spinach,beans, and tomatoes in canning, as well as gains in zincand manganese in canned 'beets, have been observed bySchroeder (1971). Peeling of some common fruits and vegeta-bles has been found to result in losses of chromium, copper,and zinc, as well as lead (Cannon et al., 1972).

Sampling of animal and human tissues have been discus-sed in many papers .

Two types of sampling are used in experiments withanimals and humans. In the first type the investigator usesexperimental animals which can be sacrificed as necessaryand dissected to produce whatever specific tissues are desiredHere replication of animals is common, total weight of samplesis not limiting, and the analyst is generally present duringthe sampling. The major decisions involve selection of thespecific tissue that will, on analysis, yield the mostimportant data.

In the second type of experiment the animal or personbeing investigated must remain alive and experience minimumdiscomfort. Here the number of individuals is usually limited,and the analyst receives the samples from a nurse, technician,or other person who is generally not a part of the researchteam. In these instances, blood or serum, urine, hair, andperhaps needle biopsy materials are the tissues that aregenerally available. In this type of sampling, decisionsinclude the kind of tissue or tissues most useful in charac-terizing the status of the subject with respect to theelement in question and whether the data on environment, diet,health or disease, water supply, etc., are adequate for thepurposes of the experiment. Very frequently the investigatoris forced to use less than adequate samples rather than donothing (Morrison et al., 197*O.

Another decision in sampling under these conditionsconcerns contamination of samples during sampling. Bloodsamples drawn with stainless-steel needles may be unsatis-factory for chromium, and those drawn through rubber tubingmay be unsatisfactory for zinc. Plastic urine containersmay irreversibly absorb heavy metals . It would be useful forsome central organization to sponsor the production anddistribution of syringes, blood needles , and containers forblood and urine that would be suited to trace elementstudies on samples obtained from humans and domestic animals.A second desirable, but perhaps unattainable, feature wouldbe the establishment of a uniform terminology suited tocoding, for describing the environment, health and disease,diet, etc., of the subject sampled (Morrison et al., 1974).

Several authors have used following sampling methodfor fish and shellfish. Immediately after collection, samplesof fish and shellfish should be frozen at -20 C. The dis-

23

section of the fish should be carried out with the organismsonly partially thawed thereby obtaining muscle uncontaminatedby drip and allowing easier cutting. As to shellfish shellsshould be allowed to open by gentle heating, taking care toavoid contamination of the soft tissues as much as possible.

In both cases dissection is to be carried out usingplastic or quartz knives. The species should be chosen ac-cording to their taxonomic position and typical environmentencountered in the region of interest.

Many procedures for tissue sampling have been describedin the literature. Here we shall describe in some detailsprocedure for human milk sampling as described by InternationalAtomic Energy Agency (Byrne et al., 1979).

PUMP PUMP

nFig. 1'. 6 IAEA/WHO sampling device for human milk.

Human milk should be sampled by direct expressien intoa 500 ml pyrex freeze drying flask (i), or via the IAEA/WHOdevice with a pump (iii) (see Fig. 1.6). Device (ii) is com-monly used for withdrawing excess milk in maternity homes,but is not be recommended, as the extra tubing and difficultiesassociated with cleaning the reservoir and its stopperincrease the risk of contamination. This was confirmed inpractise (Byrne et al., 1979).

All vessels and tubes were cleaned with a concentratedHNO-j-HpSO^. mixture and rinsed with doubly distilled water.After removal of excess water, drying is not necessary andonly introduces further contamination risks. The nipple areaof the breast was washed before sampling with soap andwater, rinsed-with distilled or de-ionized water and driedwith a tissue. Ideally, both breasts should be emptied, andthe sample mixed, or failing that, one breast. Protocolfor this aspect have been established earlier (Byrne et al.,1979). However, some of the samples analysed in the studyby Byrne et al. (1979), particularly of maturer milk, werenot the whole -contents of the breast, but representedexcess milk from volunteers. In any case, for those elementsof primary interest there, i.e. the "difficult" elements,magnitudes and methodological differences are of primeinteres

Very often trace element analysis is performed duringautopsy studies. The following recommendations are usefulto keep to a minimum the mineral contamination of, and theloss of, mineral elements from the samples.

24

1 . Instruments used for the collection of specimens andtheir handling prior to the analysis must be acid-washed and cleaned with water of the highest purity.These instruments should be preferably of quartz,polyethylene, titanium and/or teflon; however,stainless steel instruments can also be used. (Incase, the specimens cannot be collected followingthe recommended procedure, it should be the responsibilityof the analytical laboratory to remove surfacecontamination before sampling the specimens foranalysis) .

2. Specimens should be collected as soon as possibleafter death (not more than 48h), suitably identified,and placed in sealed individual plastic bags. Theymust be kept frozen until analysis. (Hair samplesneed not to be kept frozen).

3. Clean metal-free plastic gloves should be wornduring the handling of the samples which must bekept to a minimum.

4. No chemical fixatives' should be used and samplesshould not be rinsed with water or any other medium,nor should they be pierced with a metal instrument.

Here are procedures for pre-analysis procedures forsoft tissues, hand tissues and blood.

Soft tissues:1. Allow the sample to thaw slowly in refrigerator for

at least 24 hours. Remove plastic storage bags andallow the blood or any other fluid to drain. Obtainthe wet weight. Carry out the entire procedure ina clean dust-free enclosure.

2. Using clean instruments and plastic gloves cut thesamples into 2-cro cubes. The working surface shouldbe a clean plastic sheet in a glove-box or laminarflow hood.

3 . In cases where doubts exist as to the contaminationof the specimen at collection the outer surfacesof the sample should be removed with the recommendedinstruments.

4. Homogenise sample cubes under liquid nitrogen usingfor instance the brittle fracture technique.Thoroughly mix the homogenate in clean tightly closedplastic containers. Store the homogenate at atemperature below -10 C.

5. Freeze-dry aliquots of homogenate prior to analysisand determine their dry weight; record the weightless.

Hard tissues:A. BONE1 . Allow the sample to thaw as outlined above for soft

tissues. Obtain the wet weight.2. Freeze the sample in liquid nitrogen and break up

into small suitable pieces using instruments of

25

recommended material. Mix pieces and homogeniseinto powder using the brittle fracture technique.Mix the powder thoroughly and store at low temperature.

3. Aliquots are freeze-dried prior to analysis; careshould be taken to record the weight loss.

B. HAIR1. Wash hair (in a tied lock) successively once in

acetone, thrice in water and once more in acetone.(Acetone should be of reagent grade and water ofthe highest purity.) Add sufficient amounts ofthe'above solvents to cover the sample entirely.At each wash, allow the sample to stand at roomtemperature for 10 min in contact with the solventwith constant stirring. After each wash, decantthe liquid and add fresh solvent. Carry out thewashing in a dust-free enclosure (e.g. glove-box,laminar flow hood).

2. Allow the sample to air-dry over-night at -roomtemperature between clean chromatography gradefilter paper in a dust-free enclosure. Usingplastic scissors or a titanium knife obtain thefirst centimeter (from the proximal end) sectionsof the lock.

3. Homogenise the proximal end sections obtained asdescribed above into powder. One effective procedureis the brittle fracture technique. About 2g ofhair are placed in a teflon container along witha teflon ball, and the lid is closed tightly. Thecontainer is cooled in liquid nitrogen for 3 minand then vibrated at 3,000 cycles per min for 2 minusing a "micro-dismembrator" (B. Braun MelsungenAG, Melsungen, FRG). fine hair powder is obtainedby repeating the procedure thrice. Mix the powderthoroughly and store at room temperature in aclean plastic container closed tightly .BLOOD:Elements of interest should be analysed in whole

blood, plasma and blood cells. Blood should be collectedby venipuneture using a heparinised evacuated blood col-lection tube (e.g. Venoject, Terumo Corp., Tokyo, Japan)to minimise metal contamination. Collected blood samplesshould be stored at a cold temperature to avoid microbialaction. Blood cells and plasma should be separated bycentrifugation.

1.1.5 Sampling of petroleum and its productsSampling, which precedes analysis, should be carefully

planned and executed because its quality determines the valueof everything that follows .Generally, in the first step, alaboratory sample is removed from the bulk product to beanalysed. The second step includes storage, transportation tothe laboratory, with or without addition of some preservatives.In the third step this sample is then divided into theanalytical subsamples .

26

The quantitative results generated from an analyticalsubsample are limited by how well the subsamples representsthe bulk. Trace elements are generally not uniformly distrib-uted in nonhomogeneous materials as most fuel oils. A traceelement of interest in fuel oil may be associated with theparticulate matter of sediment, with the solution, or both.Highly sensitive instrumental techniques often require a verysmall specimen, thereby increasing the danger that thespecimen being analysed may not constitute a representativeportion of the original sample.

Some of the recommended methods for the bulk samplingof petroleum and its products have been described in thefollowing documents:

1. American Society for Testing Materials, Manual onMeasurement and Sampling of Petroleum and PetroleumProducts, 1957 Methods D270-57T, D923-56, D1145-53.

2. Institute of Petroleum (London), Standard Methodsfor Testing Petroleum and Its Products, Method1P 60/61 .

3. American Petroleum Institute, API Standard 2500,Measuring Sampling and Testing Crude Oil.

Although these provide sound guidelines, in practicethe sampling of petroleum and related materials, as withother products, is largely a matter of sound judgment. Inpetroleum trace analysis, the problem of sampling oftenresolves itself to one of avoiding contamination (Milner,1963). Let'us describe the sampling method used by Witherspoonand Nagashima (1975):

Crude oil samples were collected in two-quart Masonjars by attaching a short length of small rubber hosethrough appropriate connections to a 2.5 cm (1 inch) gatevalve normally present at the wellhead of any producing well.After the sample line was connected, the gate valve wasopened so that the amount of oil and water being producedcould be observed and the line purged. If the water-oil ratio

.was above approximately, 1.0, the produced fluids were firstcollected in a glass separator, the water drained off, andthe oil poured directly into the sample bottle.

If the water-oil ratio was less than 1.0, the producedfluids were collected directly in the sample bottle. Eachsample bottle was covered with aluminum sheet foil and themetal cap screwed on. Occasionally it was necessary, dependingon atmospheric temperatures, to loosen the cap and to ventany natural gas that had been released. The crude oils werestored in these containers until investigated.

In the studies and in the monitoring of water pollutionby oil, sample collection is also very important. A relativelylarge sample makes possible more accurate analysis and islikely to be more representative than a small sample. Aboutone liter of the orginal petroleum sample should be collectedalong with similar amounts of oil from the water's surfaceand, if the oil reaches, from beaches and shores. Water col-lected with the oil should be kept at a minimum, to reducebacterial activity.

27

Commonly available devices for collection of oil aredescribed by Kawahara (1969). When the amount of spilled oilis appreciable, skimming oil into a container is recommended.Thin oil films can be collected using treated glass cloth.In all cases care must be taken to avoid contamination, anddata on the collection location, conditions, date, time,and persons should be recorded.

Preservation procedure involves the containment oflow boiling components and protection against oxidation andmicrobial attack. Procedure depends on the amount of watermixed with the oil. Crude oils and products containing lessthan 3% water can be preserved by sealing in glass- bottlesand storing at ambient temperature, upright, and in thedark (Kawahara, 1909). This will usually be the case withcrude oils and products from tankers and pipelines .Petroleum from slicks and producing wells may contain asignificant amount of water.

The bulk of the water should be removed and the samplecontainer frozen and maintained at -20 C until the time ofanalysis to minimize bacterial alteration of the oil. Analternative procedure is to add about 100 ppm of mercuricchloride to the water phase to inhibit bacterial activity.The sample can then be stored at ambient temperatures. Thisalternative procedure may be preferred for oils that maybecome heterogeneous when held at -20 C.

Low-molecular-weight hydrocarbons can readily escapeto the atmosphere. Rubber seals should be used on thesampling devices when the waters are to be analyzed forlow-molecular-weight hydrocarbons. The addition of mercuricchloride or sodium azide is recommended before capping thebottle. The problems connected with the determination ofhigh-molecular-weight hydrocarbons are more difficult tosolve. Extreme care must be taken to use proper samplingequipment and carefully cleaned collection and storage vesselsSampling devices should be constructed of glass, stainlesssteel, and/or Teflon. U.S. National Academy text on Petroleumin the Marine Environment (1975) prescribes the followingprocedure :

The retrieved samples should be transferred throughclean Teflon tubing into sample bottles that have been cleanedpreviously with carbon tetrachloride. The bottles should befrom 0.5 to 20 liters, depending on the sensitivity required,and an appropriate volume of hydrocarbon-free CClj, should beadded to each sample. For certain analyses, it will also beappropriate to add hydrocarbon-free sodium chloride andhydrochloric acid.

The collection bottles should be capped immediately,using a clean Teflon liner, and the sample should be storedupright. Immediately after collection, the sample should beshaken in order to initially extract the hydrocarbons intothe CC14.

28

1.2 Sample storageStorage of the sample obtained after the sampling

procedure may have several purposes. First, the sample mayhave to be kept awaiting sample preparation and the fol-lowing analytical steps if these cannot be carried outimmediately after sampling. This is important in long-terminvestigations when it is desired to analyse all the samplesin one series. Second, it may be necessary to prepare andstore duplicate samples (identically prepared aliquots),one of which remains with the user of the analytical datafor independent cross-checking at a later time, if necessary.Storage of duplicate samples for a definite interval isalso necessary sometimes in forensic and clinical analysisfor legal reasons. Analytical reference materials are alsooften produced in very large quantities and have to be storedfor years. A last argument for long-terns storage is thepreservation of characteristic ecological, environmental orbiological- samples as specimen banks for use in futuremonitoring investigations (Sansoni and lyengar, 1980).

For some samples the problems are not difficult.However, for biological samples there are number of dif-ficulties. In-many cases there is even a maximum acceptablestorage time. It depends on the organ or tissue in question.

The storage temperature may be 2-4°C for short-termstorage but should be -15°C for longer preservation.

The total, surface area of the container and the freespace in it should be kept to a minimum. Containers withnon-porous, smooth and non-wettable surfaces are generallypreferable. As far as possible, whole organs or tissuesshould be stored without dividing them into smaller parts .The larger the amount of sample stored in the container,the less significant will be the influence of container surface .

Containers with non-wettable walls (teflon, polyethylene,etc.) are commonly used. Surface preconditioning, especiallyof quartz and glass containers, can be carried out by mineralacids such as HNO-. HC1 and diluted HF, by chelating reagents(EDTA) and oxidants (H-Op), followed by thorough rinsingwith demineralized distilled water (Sansoni and lyengar, 1980).

These authors also list some common materials usedfor containers in decreasing order of importance in each group .

Polymers: pluyfluorohydrocarbons (teflon, kel-F, tetzel,halar, etc.), polyethylene (high-pressure PE generallypreferred to low-pressure PE), polypropylene (hostalen,hostaflon, etc.), silicone rubber (one of the purest rubbers,but contamination risk by Zn is reported), polymethylmethacrylate (plexiglas, perspex; relatively low in traceelement imuriti'es). Glasses: ultrasilica (synthetic quartz),borosilicate glass. Metals: High-purity aluminium foil,platinum, high-purity titanium, etc.

The goal of any storage technique is the maintenanceof sample integrity. Considerations of the container materialare necessary regarding absorption from solution on thewalls, leaching from the walls, loss through volatilization,degradation through photochemical or biological acitvity,and other factor. At present, such relatively inert materials

29

TABLE 1.2

T R A C E ELEMENT IMPURITIES IN L A B O R A T O R Y - W A R E MATERIALS

Element Glass Polyethylene(high pressure)

AlAsBrCaCdClCoCrCsCuFeHgKMgMnMoNaNiPbRbSbSeSiSrThUZn

g/gioooooa

10003

0.082

0.12

30003, 280b

30003600aioooa3oooooa

2.92

looooo3

0.73

ng/g80-3100

200-20000

16005

15-3001

600-2100

80-150010

170-10000

200

5

2000800

90

Process unknown

ng/g230-3000

186200

800-30000.07-0.31

19-760.056.60-1710500

510lil-25000

200

0.1813

2000

381028

Tygon Plexiglas

g/g ng/g55

5

0.056 10

0.0610 9.5050 110

1202 32

200200

0.010.002

100

5 10

Synthetic quartz Teflon

ng/g

0.17700.38

37000.331.600.122.00

1600.03

500

0.10

50

2.130.10-3-80

0.70

0.16

31

ng/g

0.33-1.7030

0.052235

2500-5000

8

Pyrex.

Borosilicate.

TABLE 1.3CONTAMINATION OF SOME MINERAL ACIDS Bï LEACHING OF

CONTAINER WALLS DURING EVAPORATION

Elementleached

AlBrCaCdCoCrCuFeMgMnNaNlPbSiSnTiVZn

Glass(ng/ml)

10

37902.3030

HC1Poly-ethylene(ng/ml)

3

0.0240.70.00520.030.80.2

7

Poly-propylene(ng/ml)0.1

0.03

0.030.010.60.020.001

0.010.060.4

0.07

0.02

Teflon(ng/ml)

0-350.020.9430.50.3

44010.040.04

Quartz(ng/ml)

1060

0.61110100.430

0.50.42

HNOPoly-ethylene(ng/ml)

38

2.6160

507-5

Teflon(ng/g)

240.270.011470.21.08

0.760.04

Quartz(ng/ml)

2060

20200.6

1

HF

Poly-propylene(ng/ml)0.54

0.0007

0.20.30.12

0.020.03

0.1

Teflon(ng/g)

31

0.40.4330.10.40,1

2

as quartz, teflon, polyethylene, hard glass, and so forth,have both attractive and undesirable attributes. An over-riding consideration may be the duration of storage; short-term requirements clearly differ from those of longer periods

Table 1 .2 shows usual trace element impurities insome of often used laboratory-ware materials. Sometimes acidsare used for sample preparation for storage. Contaminationof some mineral acids by leaching of container walls duringevaporation is shown in Table 1.3-

The systematics of storage have not as yet been ade-quately explored. The increasing need for baseline studiesdemands an evaluation of currently used storage methods .There are difficulties in predicting future disposition ofstored samples, whether it be analysis of yet unconsideredspecies or the extension of present assays through enhancedcapabilities of analyses.

During sample storage some changes in mean traceelement composition of a sample may result from physicalor chemical changes. The most often encountered changeis the change in sample weight due to loss of water, ifno special precautions are taken. This is a difficultyoften experienced with biopsy samples of soft tissues. Thenecessary precautions are storage in closed systems, freezingat the sampling site, or repeated weighing of the sampleas a function of time followed by extrapolation of theweight to the sampling time. In contrast, the residue fromashing or evaporation may absorb water from the surroundings.

Segregation of a heterogeneous mixture of solidparticles of different size, shape and density can cause aconsiderable change in composition if aliquots are takenwithout prior re-mixing (Sansoni and lyengar, 1980).

During reconstitution of frozen fluids by thawing,the protein part tends to form small lumps resulting inconcentration gradients if the solution is not mixed thor-oughly. Since many metals are bound to proteins or arecomponents of specific enzymes, inhomogeneity in the fluidwill affect the accuracy of the determination. Chemicalprocesses that may result in changes in mean compositionare hydrolysis, oxidation (e.g. decomposition), haemolysisof whole blood, denaturation of proteins by excess heatingor chemical reagents, fermentation, photochemical reactionsand microbial attack, e.g. fungus growth (Sansoni andlyengar, 1980).

During sample storage loss of some elements may °ccurdue to adsorption on container walls and tools particularly •for low-level trace elements in body fluids.

With occasional exceptions quartz, teflon and high-purity polyethylene containers are generally suitable forstoring dilute solutions, e.g. those used in the preparationof standards, Interactions between trace elements in dilutesolution and various container materials have been reportedfor various elements (see list of references).

32

1.2.1 Loses from water by sorption on surfaceVarious studies have been published dealing with

sorption phenomena in relation to matrix composition,concentration and chemical form of elements being determined,nature of container material, contact time, and additionof complexing agents and acids. Here, we shall describethe work by Masse et al (1981) on sorption behaviour ofselected trace elements in distilled water and artificialseawater using polyethylene, poly tetrafluorethylene (PTFE;teflon) and borosilicate glass as typical container materials.The reagents and materials used in their work were discribedas: distilled water was prepared by distillation in quartzof demineralized water. For the preparation of artificialsea-water the following reagents were dissolved in 10 1of distilled water: 69,9 g of MgSO, x 7H?0, 50.5 g ofMgCl2 x 6H-0, 14.8 g of CaClp x 2 0, 1.00 g of KC1, 1.77 gof NSpCO,, and 268 g of NaClT Batcnes of distilled waterand artificial sea-water were adjusted to pH 1, 2, 4 or8.5 by suitable additions of nitric acid or sodium hydroxide.7U The following radiotracers were used: 7C.Ag'(1.2 mCi mg'-),' As6i0.9 mCi mg-1), ]09cd (0.27 mCi mg-1 ), 'DSe (3.8 mCi mg ),and Zn (1.3 mCi mg~ ). From these radionuclides stocksolutions were prepared at radioactive concentrations of5-10 uCi ml'"' and a mass concentration of 10~^ M by addingamounts of the corresponding stable elements. The acidity ofthe stock solutions was adjusted to pH 2 by adding nitric acid.

The containers tested were 200-ml borosilicate glassbottles 100-ml high-pressure polyethylene bottles and 100-mlpolytetrafluoroethylene bottles. New bottles were usedexclusively. The differences in specific surface values wereachieved by adding pieces of the material considered. Toavoid the possibility of highly active sites for sorptionarising from fresh fractures, the edges of the added piecesof borosilicate glass were sealed in a flame. Prior to theuse of all materials, the surfaces were cleaned by shakingwith 8 M nitric acid for at least 3 days and by washing fivetimes with distilled water.

Specific surface is defined as the ratio of the innercontainer surface in contact with solution to the volume ofthe solution, denoted by R(cm-1).

The following procedure was used by Masse et al., (198l):working solutions (1 1) which were 10"' M in one of theelements to be studied were prepared by appropriate additionof the radioactive stock solutions to pH-adjusted distilledwater and artificial sea-water. After the pH had been checked,100-ml portions were transferred to the bottles to be tested.The filled bottles were shaken continuously and gently in anupright position, at room temperature and in the dark. Atcertain time intervals, ranging from 1 min to 28 days, 0.1-mlaliquots were taken. These aliquots were counted in a 3 x 3-in.Nal(Tl) well-type scintillation detector, coupled to a single-channel analyser with a window setting corresponding to the-rays to be measured. The counting times were chosen in such away that at least 15 000 pulses were counted. The sorptionlosses were calculated from the activities of the aliquots andthe activity of the aliquot taken at time zero.

33

Results obtained by Massée et al., (1901) are shown inTables 1.4, 1.5 and 1.6. In these tables the perdent loss ofAg, Cd and Zn from distilled water and artificial seawaterstored in different containers is shown. Losses smaller than3% are not indicated.

Here are the results for different elements as describedby Massée et al., (1981).

Silver; At pH 1 and 2 sorption from either distilledwater or artificial sea-water was not observed for any of thetypes of container materials. However, from distilled waterat pH 4 silver was substantially sorbed on polyethylene,borosilicate glass, and PTFE. In polyethylene, silver wasalmost completely lost; in the case of larger R values therate of loss increased. In borosilicate glass, inconsistentbehaviour was observed which could not be explained, e.g. thepercent sorption at pH 4 for R = 1.0 cm-"1 and R = 4.2 cm-1after 24 h. After 28 days the loss of silver appeared to beindependent of the R value considered. In PTFE, silver wasstable in solution up to 24 h in distilled water. At theend of a 28-day storage period the loss of silver in thePTFE vessel with R = 5.5 cm-1 was almost 4 times higher thanin the vessel with R = 1 .0 cm~^.

From artificial sea-water at pH 4, losses of silverwere observed only in borosilicate glass containers. AtpH 8.5, silver was sorbed from both distilled water andartificial sea-water regardless of the container material.The same anomaly as in distilled water was observed, thoughto a lesser extent.

Cadmium: At pH 1 and 2, there was no significantsorption for any of the three container materials . At pH 4sorption of cadmium was observed only from artificial sea-water stored in borosilicate glass. In distilled water atpH 8.5, cadmium was lost onto all three materials, whereassorption of cadmium was not observed from artificial sea-water. In all cases, the amount of cadmium sorbed increasedwith increasing R value.

In general, the results of this study indicatelosses of cadmium lower than those reported in the literature.However, unambiguous conclusions cannot be drawn because ofthe lack of information on various parameters, especiallyabout the specific surface. This parameter seems to becritical in considering the sorption phenomena of cadmium,as may be seen from Table 1.5. Although the pH seems to be thedominant factor in preventing sorption of cadmium, it shouldbe noted that at pH 8.5 and in the case of the highest Rvalue cadmium was substantially sorbed from distilled water,whereas sorption was not observed from artificial sea-water.The absence of sorption of cadmium from sea-water may beexplained either by the formation of chloride complexes ,analogously to silver, or by competition between cadmiumand other bivalent ions (Mg, Ca) in occupying active sorptionsites.

In general, it may be concluded that for the determinationof trace concentrations of cadmium, the sample has to beacidified to pH 4 and it is usually advantageous to use poly-ethylene or teflon containers.

34

Table 1.1

Sorption behaviour of silver (af ter Massée et al., 1981)

Matrix

Material

PHRtcnf1)

Contacttime1 min

30 min1 h2 h4 h8 h24 h2 d3 d7 d14 d21 d28 d

Distilled waterPolyethylene

41.4 3.4Sorption

87 1210 1512 2014 2817 4225 6630 8337 9151 8883 9595 10096 100

8.51.4(%:

1925364551727675666l5959

3-4)

1233364449494960747898100100

Borosilicateglass

41.0

-

-34932728384858482

4.2

34581218315679707580

8.51.0

6915182326286570707272

4.2

192127354248585660576463

PTFE

4 8.51.0 5-5 1.0

_

_- - 3- - 3

44 6 53 5 75 18 96 32 117 55 1910 54 2015 55 22

5.5

101013141825242627282728

Artificial sea-waterPolyethylene

4 8.51.4 3-4 1.4Sorption (%)

76101416

- - 24- - 35

446466

- - 5846

Borosilicateglass

3-4

78591318283645647277.78

4 8.51.0 4.2 1.0

_ _ _- - 3- - 3- - 33 4 54 4 66 7 106 11 3174 60 2781 76 3980 73 3982 71 40

4.2

_354109

318073846467

PTFE

4 8.51.0 5-5 1.0 5.5

- - - 3- - - 4

678

6 12- - 13 17

13 2314 2920 30

- - 26 37- - 27 37

Denotesta loss smaller than 3%-to

Table 1.5Sorption behaviour of cadmium (after Massée et al., 1981)

MatrixMaterial

pHR(cnf1)Contacttime1 mln30 min1 h

l h8 «_h

n la t.

2 d3 d7 d1l d21 d28 d

Distilled water

. Polyethylene

1 8.51.1 3.1 1.1 3.1Sorption (%)

- - 5 32- - 7 69- - - 70

"• 1 v

- 59- - - 17

- - 30- - - 29- - - 31- - - 30- - - 30- - - 31

Borosllicateglass1 8.51.0 1.2 1.0

- - 56

1010119

1.2

3226« f26f\f

2932323031

PTFE

1 8.51.0 5.5 1.0

- - 755

- - 610

12- - 13- - 1l

1515

Artificial sea-waterPolyethylene Borosilicate PTFE

glass

1 8.5 1 8.5 1 8.55.5 1.1 3-1 1.1 3.1 1-0 1.2 1.0 1.2 1.0 5.5 1.0 5-5

Sorption (%)

3 6 - - - - _ - _ _ _ , _ _oo _ _ _ _ _5 3 _ _ - _ _ _ _ _ _ . _ _SA _ _ _ _ _ _R7 — — —Jl — — — — — _ — _ — —II Q

ll^ - - -

H 3 _ _ - _ 5 _ _ - _ _ _ -1 6 - - - - 1 1 1 0 - - _ _ _ _1 1 - - - - 1 3 1 3 - - - - - -1 5 - - - - 1 5 1 i _ - _ _ _ _1 6 - - - - i l 3 6 - - _ _ - -

aDenotes a loss smaller than 3%.

Table 1.6Sorption behaviour of zinc (after Massée et al., 1981)

Matrix

Material

pHR(cm~1)

Contacttime1 min

30 min1 h2 h1 h8 h21 h2 d3 d7 d11 d21 d28 d

Distilled waterPolyethylene

1 8.51.1 3.1 1.1Sorption (%)

-

-- - 3- - 3

58

- - 9- - 6

11111012

3.1

666561605856525357575556

Borosilicateglass

1 8.51.0 1.2 1 .0 1.2

21 2021 2123 2221 2125 21

- - 25 2326 2225 2020 22

- - - -_- - - -_

PTFE

1 8.51.0 5-5 1.0

- - 5- - 3_ _ c

1- - 3

5- - 3- - , 3

1- - 5

16

Artificial sea-water

Polyethylene

1 8.55-5 1.1 3-1 1 .1 3.1

Sorption (%)

12 . -16222833 - - - -2725 - - - -19 - - - -20 - - - -202020

Borosilicateglass

1 8.51.0 1.2 1.0

12- - 9

109

- - 95

- - 11

10 1 327 19 -25 17 320 19 1

5.5

313129302826211891099

PTFE

1 8.51.0 5.5 1.0 5.5

__5 - - -n _5 - - -n _ _ _145 - - -1 -5 _ _ —5 - - -5 - - -

co

Zinc: At pH 4 losses of about 20% of zinc were observedfrom artificial sea-water stored in borosilicate glass. Therewas no apparent relation between the R value and the size ofthe losses. At pH 8.5 zinc was lost from distilled water inall container materials. The rate of loss in polyethylene andPTFE increased with increasing R value-. In the case ofborosilicate glass, an immediate loss of 20% was observed,whereas after 7 days all the initially sorbed &5zn activitywas found to be in solution again. At pH 8.5 artificial sea-water showed some loss of zinc in polyethylene after 28 days .In borosilicate glass similar effects were observed for sea-water and distilled water, i.e., a decrease of loss withincreasing storage time.

Arsenic and selenium: For arsenic (added as sodiumarsenate) and selenium (added as sodium selenite), losseswere insignificant in all the container materials considered,irrespective of matrix composition. For arsenic, literaturedata on sorption from aqueous matrices could not be found.For selenium, a some authors have mentioned small losses.

It was concluded that the sorption behaviour of traceelements depends on a variety of factors which, takentogether, make sorption losses rather difficult to predict.However, the data from this study and from the literatureindicate for which elements sorption losses may be expectedas a function of a number of factors, such as trace elementconcentration, container material involved, pH, and salinity.

1.3 Sample fragmentation, powdering and homogenizationThe preparation of samples for trace element analysis

generally involves fragmentation of the bulk material, grindingto the desired particle size, and homogenization. lyengar andSansoni, (1980) have discussed the procedures in the case ofbiological samples.

Fragmentation and powdering of soft tissues can beaccomplished by conventional means, provided that sufficientprecautions are taken to avoid contamination. A particularlyuseful apparatus is a "microdisraembrator" comprising a teflonvessel in which the sample is vibrated rapidly, together witha teflon-covered metal ball, at liquid-nitrogen temperature.Other suitable materials for grinders and homogenizers includeultrapure quartz, polymethylmethacrylate, high pressure poly-ethylene and high-purity titanium.

Fragmentation and powdering of hard tissues such as boneand teeth present formidable problems for the trace elementanalyst.

Homogenization involves not only fragmentation andpowdering, but also mixing of different batches, and testingto confirm that the distribution of elements is adequatelyuniform. It is particularly necessary (a) for preparingbiological standard reference materials, (b) before subdividingsamples when comparing different analytical methods, and (c)to prevent the segregation of particles in samples containinga wide range of particle sizes.

38

Homogeneity tests may be carried out by determiningcertain elements in randomly selected subsamples, e.g. K, Zn,Se, Ag, which represent major, minor, trace and ultra tracelevels, respectively. For examining different locations onthe surface of pellets prepared from the powder, electron andion microprobes can be used. It is also possible to measurethe bulk density at different locations in the sample byexamining the degree and rate of weight loss during drying,or by determining the constancy of the ash content at dif-ferent locations. The uniformity of the particle sizes canbe conveniently checked by sieving and observing the dif-ferent sieved fractions.

1.4 Contamination of sampleContamination of the sample by elements is often dif-

ficult to avoid. Contamination during sampling may occurfrom the environment of the sample, the sampling operationitself and the operating personnel. During the samplingoperation, contamination may arise from dust and volatilecontaminants in the air. In addition, the sampling toolsmay contribute contamination to a marked degree. Numerouspossibilities of contamination of the samples by the operatingpersonnel also exist. Exhaled air, spit, phlegm, sweat,cosmetics, tobacco ash, tobacco smoke or even clothing debrismay contribute.

Sansoni and lyengar (1980) have summarized types ofcontamination which may be derived from th.e laboratoryatmosphere. This is shown in Table 1.7.

The basic requirement is to keep potential contaminationhazards small in relation to the concentration levels in thesample. lyengar and Sansoni (1980) have discussed the case ofbiological materials. For example, the elements Cu, Fe and Znare present at mg/kg levels in most biological materials anda reasonably clean laboratory is usually adequate for thepreparation of such samples. At the other extreme is thepreparation of blood serum samples for the determination of Mn.The concentration of this element in blood serum is only"»0.6 jjg/1 and unusual precautions are prerequisites for theavoidance of external contamination since Mn is widelydistributed in the environment, e.g. as air-borne dust. Fordeterminations at the jag/kg level, clean air benches orglove boxes are usually indispensable for sample preparation.

Problems of contamination and loss of elements arepresent at every stage of analysis. They demand a minimumof sample manipulation and the use of appropriate equipment.Dust-free containment is a basic necessity at all stages ofpreparation. However, the magnitude of the contaminationproblem and its prevention are dependent upon the elementinvolved. For example, Cr, Mn and Pb create more problemsthan Bi, Te, Th, Tl and U. Air-borne contamination is especiallydangerous for Pb analysis at low concentrations (lyengar andSansoni, 1980).

The major source of contamination are usually chemicalreagents used for the treatment of sample. Table 1.8 shows

39

Table 1.7Trace element levels and laboratory atmosphere

(after Sansoni and lyengar, 1980)

Element

AlAsBrCaCdClCoCrCsCuFFeHgIKMgMnMo'NaNiPPbRbSSbSeSiSnSrThTiTlUVZn

Non-filteredair

ug/g dust30005523

26902.81.59391.3

2131.0

32X

2.97920

2.4116

295070

1150 ••215023

2000014.80.6

9.613.5

258

0.12591640

Filteredair

ug/g dust

6<0.01<0.02<0.0040.1<0.0050.1<0.006<0.01<0.020.10<0.006

<0.01<0.004<2390<0.006

1340.501.50<0.04<0.01<0.003<0.03<0.02

<0.05<0.01

3

<0.010.005<0.02

Tobacco „Cosmetics Sweatsmoke

ug/g ' ug/g ug/ml

2.8571.50 4000 0.2-0.5

6x10 4-10*

630 1054-20410.034 1.12 0.020.350.01

1.0-1.50.2-2.0

7.X 1100 0.5-1.5

0.01250 176-350

1-480.06

1017-33700.05-0.2

1400 0.2-1.50.05-2.7

4000.150.229x10 "

6300

10 3.5x10 1

Skin

ug/g wet

1-20.06-0.10

4-102500.032

26540.050.100.0310-20

100.100.10

3067580.10.05

2000.0.053270.102

15000.040.25

100-200

0.10

0.500.03

0.156-20

Hair

ug/g

4-290.2-3.7<l-53200-31900.24-2.7950-4805

0.1-3.60.4-1.111-32

5-681.3-7.6<1-15150-86319-1630.3-5.70.06-0.2118-17200.6-6.583-165

* 3-700.2-O.50

477000.1-30.6-2.5320-1950

?0.05-0.92?0.05-14

0.0120.00013

0.005-0.5399-450

trace element impurities in some reagents used for samplepreparation as summarized by lyengar and Sansoni (1980).

Tap water shown in Table 1.8 is from UK, Belmont area,Surrey. It will be different composition in other localitiesDistilled water in Table 1.8 was obtained by thermal distil-lation in quartz and then passed through a double stagemixed-bed ion exchanger followed by filtration through acomplex teflon filter.

40

Table 1.8Trace element impurities in some reagents used for sample preparation (ug/1)

(after lyengar and Sansoni, 1980)

Element

Al

As

Br

Ca

Cd

Cl

Co

Cr

Cs

Cu

F

Fe

Hg

I

K

Mg

Mn

H20 HC1

Tap

57

-

9555000

0.70

11100

-

-

0.02

-

1.10

--

9.^0

28000

10100

2.20

Deminera-lized

0.10

-

0.10

1

4. 0.10

1

<0.10

40.10

-0.20

-

0.20

<1

-

0.01

0.30

0.05

Singledistillât.

^0.002

-

-

< 0.0003< 0.007

<0:0001

0.02

0.0002

< 0.00001

< 0.002

< 0.0002

< 0.0005

-

< 0.001

< 0.0001

< 0.0002

< 0.0005

p.a.

8

-

-

72

0.03

-

0.09

1.10

0.002

0.20

-

1

-

-

200

7

4.2

Ultrapure

0.80

-

2.60

0.30

0.003

-

0.01

0.008

< 0.002

0.03

-

-

- "

-

0.10

0.30

0.001

HF

p.a.

1

-

-

0.1

8

-

<1

5

-

0.50

-

60

OO

-

0.10

2

0.60

Ultrapure

0.5

-

-

52

0.005

-

1

0.6

-

0.30

-

0.60

00

-

1

0.1

0.03

HNO

p.a.

7

-

-

0.2

0.1

-

0.018

72

<0.01

1.30

-

1300

-

-

•00

3

9

3Ultrapure

1

0.005

7

0.1

0.03

-

0.01

0.10

<0.1

0.2

-

0.80

-

-

9

0.10

2

H2S°1

Ultrap.a. pure

8

_

10 2

< 1 <1

-

<1 < 1

25 2

_

3 3-

8

<10 -

-

<10 1

3-30 2

8 0.8

HC10.

Ultrap.a. pure

-

-

760 0.2

0.1 0.05

-_

10 9

-

11 0.10

-330 2

-

-

200 0.6

500 0.2

-

K)

Table 1.8 ( con t . )

Element

Mo

Na

Ni

P

Pb

Rb

S

Sb

Se

Si

Sn

Sr

Th

Ti

Tl

U

V

Zn

t — ——— —— —

Tap

-

8100

30

138.50

10

11100

0.60

3-30

1900

0.60

11000

-

-

-

-

18.50

5.60

H20 HC1

Deminera-lized

0.02

0.03

<0.1

0.001

0.10

-

1

<0.50

-

0.50

0.10

0.06

-

<0.1

--

<0.1

«KI

Singledistillât. P"a '

0.02

< 0.0002 500

< 0.0002 0.20

< 0.0003

<0.003 0.20

< 0.001

< 0.0003< 0.002 0.20

-

20

< 0.001 0.07

< 0.007 2

40.0002

-

< 0.0001 0.10

-0.10

< 0.002 1

Ultrapure

- "

0.20

0.005

0.20

0.0015

-

30.38

-1

0.002

0.06

-

0.006

0.10

-

0.08

0.03

HF

Ultrap.a. pure

-

100 0.60

0.50 0.05

7

2.20 0.002

-

-

3-0

-

1

11 0.05

0.50 0.10

-

0.50

0.20 0.10

-

-

6 0.10

HNO H2S01 HClOjj

p.a.

-

80

0.07

0.80

0.20

-

0.60

0.03

0.20

30

0.10

0.20

-

0.50

0.20

-

0.05

1

Ultra Ultrap.a. p .a.pure r pure r

_

0.01 20 9 600

0.03 4 1 0.20 8

0.50 - -

0.01 1.2 1 2_

15 -0.01 -

0.09 - 200

8 18

0.002 0.60 0.20 0.30

0.01 0.10 0.30 1l

_

0.80 -

0.10 0.10 0.10

0.003 - , -<2.10

0.08 <1 <1 7

Ultrapure

-

2

0.50

-

0.20

-

-

-

-

-

0.30

0.02

-

-

0.10

--

0.10

For the handling of biological samples tools shouldbe made of materials that contain very low concentrationsof the trace elements such as Cr, Mn and Ni are to be deter-mined, it may even be acceptable to use steel knives forsample preparation, provided they are not freshly sharpened.Disposable plastic gloves, teflon tweezers, polyethyleneand teflon foils, and wax paper (parafilm) are other handyaids .

If it is necessary to use tools made from materialsthat could be a potential source of contamination, appropriât6monitoring should be carried out under realistic conditions.Problems of this kind arise particularly in the collectionof biopsy samples (lyengar and Sansoni, 1980).

2. SAMPLE PRETREATMENT2.1 Preconcentration

Depending on the as-received state of the samples, thetarget preparation technique to be applied and the analyticalresults required, pretreatment of the samples may be necessaryin order to obtain suitable targets. Pretreatment should ingeneral be avoided as far as possible, as it is time consumingand may lead to contamination or loss of analytes. Pretreatmentmay be necessary for:

- homogenisation of the sample material;- concentration of trace elements by removing matrix

constituents ;- selective concentration of the elements of interest;- selective removal of non-relevant interfering elements;- selection of a certain chemical form of an element;- selection of a specific constituent of .the sample.Whether required or not solutions have several general

advantages over solid samples. In particular, it is much easierto prepare, mix, aliquot, and dilute samples and standardsif they are in the form of solutions. Therefore we shalldescribe the ways in which various types of samples may beput into solution and discuss the errors that may be introducedby the solution procedure.

Most metals are easy to dissolve in mineral acids. HNO~and HC1, either singly or in combination, are commonly used .Iflarge quantities of tungsten are present, the use of H^POj,helps keep the tungsten in solution. HF may be added to destroyany residue of silica. Zirconium and its alloys can be dis-solved in HF and HNO-,. Some alloys may contain carbide nitride,oxide, or intermetalxic inclusions which are resistant tosimple acid treatment. The use of fusion or Teflon bombtechniques may be required in such cases (0'Haver, 1976).

Fusion, often in Na-Op or NapCO,, is the classic way ofsolubilizing geochemical materials; especially if the analysisof silicon is desired. A more recent development is the lithiummetaborate (LiBOp) fusion. In this procedure, 0.2 g samplesare fused with 1 g LiBOp and poured while still molten into100 ml 3% HNO,.

43

Fusion techniques retain silicon in solution and arethus useful if silicon is to be determined. The majordisadvantages are the time and special equipment required(platinum ware, muffle furnace), and the large excess offlux added to the sample. Obviously, the major cation in theflux cannot be determined and, furthermore, trace impuritiesin the flux material may lead to excessively high blanks.

HF, in combination with other mineral acids, is oftenused to dissolve"silicateous material. The use of HF drivesoff silicon as the tetrafluoride and thus is suitable only ifsilicon is not to be measured. Teflon containers are recom-mended. If large amounts of calcium are present, CaF~ mayprecipitate. Also, HF does not dissolve zircon or carbonresidue.

A dissolution technique based on a pressure decompositionvessel, commonly called a Teflon bomb is often used. The sampleis placed in a specially designed Teflon container, treatedwith aqua regia and HF, tightly sealed in a stainless steelbomb, and heated at 110 C for 30-40 min. This technique has beenfound useful with glasses, nitrides, and other refractorymaterial that is difficult to dissolve by other methods. Theadvantages are that excess alkali salts are not added and thatsilicon is retained.

Cement samples can be treated with 4 N HC1. Silica isnot dissolved. Glass can be dissolved in HF in a Teflon beaker,evaporated to dryness, and the residue taken up in HC1. H^BCumay be added to aid in the elimination of F~. Coal ash isbasically a silicate material; dissolution in HF, sometimeswith HClOj. has been found useful if silica is not determined(O'Haver, 1976).

Samples containing large amounts of organic matterrepresent a large and important class of materials for whichthe sample preparation steps are often long and involved andsometimes a major source of analytical error. This includessolid biological samples such as plant and animal tissue,man-made organic materials such as plastics, and liquid samplessuch as blood, urine, oils, and liquid fuels, when analyzedfor elements at concentration levels too low to allow the useof dilution methods.

The two most widely used methods for the destruction oforganic matter are dry ashing and wet digestion. This isdescribed in paragraph 2.2 and 2.3.

A very often used step in sample preparation is evaporation.This is a simple, but slow, proconcentration method for'solutions. Its main advantage is that it does not involve theuse of large amounts of reagents or of complicated glasswarethus contamination is minimized. The main disadvantage is thatthe total dissolved solids content of the solution is increased.Also, the procedure is very slow, requiring many hours evenfor modest concentration factors. It is necessary to use a dustcover over the evaporation dish to prevent contamination. Clean,dry, filtered air should be passed over the dish to carry awaythe vapor. Polyethylene or Teflon evaporation dishes are best.The dust cover can also be fabricated from a large plasticbeaker fitted with a side arm ion the flushing air. The sampleis usually acidified (e.g. with 1 or 2% concentrated HC1 by

44

volume) to prevent hydrolysis. Even so precipitation losses fromCaSOn, BaSOj. , silica, and so on, may be expected in some cases.Volatility losses also occur, especially for mercury and to alesser extent for arsenic and antimony. The lowest possibletemperature should be used consistent with reasonable evapora-tion times. Boiling should be avoided. Either a heat lamp ora hot plate may be used, or both.

2.2 Dry ashingThe purpose of the ashing is to destruct the organic

matrix, there by concentrating the trace elements in the sample.In dry ashing, the organic matter is decomposed at high

temperature in the presence of atmospheric oxygen. In a typicalprocedure, the sample is weighed into a clean silica or platinumdish, covered with a crystallizing dish, and dried under a heatlamp until the water has evaporated and the sample has a brit-tle, charred appearance. The sample dish is then placed in amuffle furnace, usually at 500°C, until ashing is complete.After cooling the ash is taken up in dilute mineral acid (O'Haver,1976).

Dry ashing is relatively simple, can accommodate largesamples, and does not require the addition of large amounts ofpotentially contaminating reagents. However, its principaldisadvantage is the serious losses of trace elements that canoccur because of (a) volatilization, (b) retention on the wallsof the ashing dish, and (c) retention in the acid-insolublefraction of the ash. Volatility losses are especially seriousfor mercury and selenium, for which dry ashing procedures arenot recommended. Under certain conditions, arsenic, boron,cadmium, chromium, iron, lead, phosphorus, vanadium, and zinchave also been reported to be lost. Elements that occur asvolatile organic complexes, such as copper, iron, nickel, andvanadium porphyrins in petroleum, can be lost through volatili-zation even at comparatively low temperatures. Nonmetals formmany volatile compounds which are easily lost.

Wall retention has been found to be a problem, especiallywith cobalt, copper, iron, silver, aluminium, and manganese,when using silica dished. Some elements for example, silicon,aluminium, calcium, copper, tin, beryllium, iron, niobium, andtantalum, may react to form acid-insoluble compounds in theash, especially atashing temperatures above 500 C.

Kometani et al., (1972) have described procedure for dryashing of samples in this way. Samples and metal salts in openPt crucibles were heated in air in a carefully regulated mufflefurnace. Filter paper samples were placed in a vented furnaceat 300 C for 1/2 hour. At this temperature the paper was charredslowly and did not ignite to a flame. The preliminary heatingensures that sample loss will not occur by turbulence andhigh temperatures associated with flaming. The temperaturewas raised to and held at 500 C for 1 hour. The residue inthe crucible was dissolved with HF + HNO- (1+3), evaporatedto dryness and diluted for analysis by AÄS. Unused filterswere similarly treated for determination of metal blankcorrections.

45

Dry ashing is often used for the preparation of crudeoils for trace element analysis.

Wet samples to be analyzed usually contain materialsother than oil, such as brine and sand, which have to beremoved before the oil is analyzed. The raw crude oil shouldfirst be washed several times with distilled water in orderto remove the brine, each time draining off and discardingthe water layer. To remove the last traces of waterand any suspended sand or clay particles , the oil should befiltered through a dry medium-fine filter paper.

A dry ashing method is described by Horr et al., (1961);first a convenient volume, usually a liter, should be weighed.Small volumes ( 25 ml) of the weighed sample are thentransferred to a 250-ml tarred platinum dish, and each incrementignited and allowed to burn freely in the open dish untilreduced to dry char before the next increment is added. Thesample is added in small portions to minimize possible lossescaused by too rapid combustion, spattering, or mechanicalentrainment. This is a slightly modified ASTM D482-46 procedure.The charred mass is then heated in an electric muffle furnaceat 500 C until all carbon is removed.

Morgan and Turner (1951) have shown by radioactive tracertechnique that no significant losses in inorganic ash occurif the ashing is carried out below 550°C. The losses of metalsin the dry ash method have been found not appreciable forcrude oils and residual stocks; however, in charge stocks andoverhead fractions obtained by vaccum distillation, losses ofmetals may be considerable (Gamble and Jones, 1955); Karchmerand Gunn, 1952). Loss of some metals during the ignition hasbeen recognized (Milner et al., 1952; Davis and Hbcck, 1955).This affects nickel and vanadium primarily because they arepresent in petroleum in the form of volatile porphyrins .

2.3 Wet ashingIn wet digestion, the sample is treated with concentrated

mineral acids and/or strong oxidizing agents in solution.Oxidizing conditions are maintained throughout tne procedure.Most often, the mixture is heated to 100-200 C to aid thedigestion process. Wet digestion is fast and is much lesstroubled with volatilization losses, because of the lowertemperature. The major disadvantage is the possibility ofcontamination from the large excess of reagents employed.

The most widely used acids for wet digestion are HNO,,O^, and HC10. . A 3:1:1 mixture, respectively, dissolves'5

its weight of most organic materials. HpSOj. is eliminatedif the sample contains large amounts of calcium, because ofthe danger of the coprecipitation of trace elements on CaSCk .The insolubility of certain other sulfates (e.g., of Pb2*,Ag+, Ba2 + ) and chlorides (e.g., of Ag+, Pb2+) limits thechoice of suitable digestion acids in certain cases. Otheroxidizing agents, such as H-O- and permanganate are occasionallyemployed in wet digestions. Salts of molybdenum (VI) aresometimes used as catalysts to speed up the oxadation reactions.

Although volatility losses are less severe in wetdigestion than in dry ashing, they are not entirely absent.

46

Marcury is lost when wet digestion are performed in openbeakers: an enclosed reflux system is recommended. Elementsthat form volatile oxides, such as ruthenium" and osmium, arelost. If the organic material is allowed to that during thedigestion, reducing conditions are temporarily induced andlosses of selenium, arsenic, and antimony, which formvolatile hydrides, may occur. The presence of organicallybound chlorine has been shown to lead to losses of germaniumand arsenic which form volatile chlorides.

The use of concentrated HClOj. in wet digestion proceduresrequires several precautions. Its use is desirable, becausethe hot, concentrated acid is an exceedingly strong-oxidizingagent, capable of destroying the most resistant organic materialsHowever, this high reactivity can lead to violent explosionsif the acid is mishandled. First, it must be realized that theacid is an oxidizing agent only if both hot and concentrated.The cold, concentrated (70%) acid, although a strong acid, isnot an oxidizi-ng agent and will not even oxidize iodide to .iodine or iron (II) to iron (III), Basically, the rules ofsafe handling are (O'Haver, 1976):

1 . Never bring undigested organic matter directly intocontact with hot, concentrated HCIO^; a fire orexplosion may result.

2. Always predigest organic matter with HNO_, first todestroy the easily oxidized compounds.

3. If hot, concentrated HCIO^ is spilled, dilateimmediately with quantities of water. This dilutesand cools the acid and effectively "turns off11 itsoxidizing power.

4. Never boil HCIO^, to dryness. If the digestion cannotbe watched.

Wet ashing is also applied to crude oil sample prepara-tion. Horeczy et al., (1951) have shown that wet ashing(ashing with sulfuric acid) recovers more metal than dry ashing.They added synthesized porphyrin complexes of nickel, copper,,iron and vanadium to crude oil, and after ashing by the twoprocesses, analyzed for the metals. Their results indicatedessentially complete recovery for samples that were wet ashed,but significant losses from the dry ashed samples.

To eliminate such losses a number of investigators haveutilized methods of fixing the metals by pre-sulfating the oilbefore igniting it.

Wet oxidation of oil is similar to the wet ashingprocedure of biological and other organic material. A techniquefrequently used is to char the sample by heating it withsulfuric acid and then adding concentrated nitric acid in 1 or2 ml increments; alternatively or additionally, strong hydrogenperoxide is added dropwise directly into the charred digestionmixture.

In general, the characteristics of the wet-oxidationtechniques are that relatively large acid-to-sample ratios areneeded and limited amounts of sample can be decomposed in areasonable length of time. The two methods of ashing showgenerally close agreement for the metals contained in oil.

47

Table 2.1

Comparison of Results for Samples Preparedby Dry Ashing and Wet Oxidation

(After Horr et al., 1961)

Method of Ashashing (%)

dry

wet

8.6

9.7

Concentration inCu

75

61

Ni

690

720

Co

115

115

V

17

17

Cr

18

22

Pb

150

160

Mo

230

220

crude oilY

100

116

As

7.70

8.30

(ppm)U

37.00

33.00

Fe

2.60

2.40

Zr

70

55

Sr

10

9.2

Table 2.1 shows the comparison of data for metals in crude-oilsamples as obtained by dry ashing and wet oxidation of the oil(Horr et al., 1961 ) .In another report Agrawal and Fish (1972) compared three

methods of ashing for the determination of V, Fe, Ni, Cu, Mg,Na, K, and Ca. Wet ashing'gave the most reliable results.

Barney (1955) and Barney and Haight (1955) comparedresults when a variety of concentration procedures were used.These included (a) dry ashing, (b) total sulfated ashing, (c)partial sulfated ashing, (d) extraction with iodine solution,(e) extraction with a mixture of acetic acid and hydrobromicacid, (f) extraction with a mixture of acetic acid and hydriodicacid, and (g) extraction with sulfuric and hydrochloric acid.Their results indicated quantitative recovery of copper, iron,lead, nickel, and vanadium by procedures (b), (e), and (g).

Gleim et al., (1975) describe the sample preparationtechnique for crude oils. In their measurement a sulfuric andnitric acid wet digestion process was selected for samplepreparation. This process differs from the wet ashing procedurein that the former is carried out in a liquid acid medium fromstart to finish at a relatively low temperature (280 C),whereas in the wet ashing procedure after acid charring theoil, the car is subjected to a 600°C muffling step. Theprocedure used by Gleim et al., (1975) was as follows:

Two portions of oil, 55 g - 0.1 g, were wet-digestedseparately with sulfuric and nitric acids in a glass laboratorydigestion apparatus. Prior to wet digestion, 1,000/of aConostan molybdenum sulfonate was added to one of the portionsof the oil. After digestion, these two portions of the digestedoil would now represent a modification of the classical AASmethod of standard additions with one added desirable feature:the added molybdenum standard would be subjected to the sameharsh acidic treatment as any of the molybdenum present in theoriginal oil. The authors refer to this procedure as the"cooked" method of standard additions, and find it to be theultimate in matching atomic absorption response between thesample, and the sample plus the added standard. Smith et al.,(1975) have summarized the sample preparation techniques fortrace element analysis of crude oils by X-ray fluorescence.

48

2.4 Low-temperature ashingFeasibility of low temperature ashing as a sample

preparation technique for X-ray fluorescence analysisof biological samples has been investigated in detailsby Pallon and Malmqvist (1981).

The purpose of the ashing is-to destruct the organicmatrix, therebly concentrating the trace elements in thesample. The traditional method of dry ashing organic materialis to use an oven in which the sample can be heated totemperatures of several hundred degrees centrigrade. Dueto the high temperature, volatile compounds or elements canbe lost (Gorsuch, 1959). In the low temperature ashingtechnique (Gleit, 1963, Mangelson et al., 1979), oxygenmolecules in a gas stream are transformed into highly activeoxygen atoms by using a high frequency electromagnetic field.The excited atoms react with the sample, converting primarilyhydrogen, carbon and nitrogen into volatile oxides. Thusthe matrix can be ashed without the use of a high temperature.

The probability of losing volatile elements duringlow temperature ashing is expected to be much smaller than intraditional ashing in an oven, but it cannot be neglected.The behavior of elements such as Hg, Br and Se is particularlydifficult to predict. Another problem is the risk of samplecontamination, which increases with the number of samplepreparation steps. To check if such effects occur, well-knownstandard materials can be analysed. NBS reference materialshave well-known compositions, which have been certified bythe U.S. National Bureau of Standards (NBS Special Publication260), and determined by many independent analytical techniques.In the study reported by Pallon and Malmqvist (1981), twocommon NBS standard materials, bovine liver and orchard leaves,were chosen. The NBS reference materials, originally freeze-dried and homogenized, were put into a glass crucible forashing. The sample masses were determined before and afterashing by weighing. The blood plasma was first freeze-driedat liquid nitrogen temperature thus reducing its mass by afactor of 11 . Next, 450 mg of dried blood plasma was lowtemperature ashed for 12 h yielding a mass reduction factor of 7

Targetswere prepared from both the freeze-dried matterand the ashed residue. Thick pellets were pressed from thefreeze-dried parts of all three materials. From the ashedresidues, thick targets were made from blood plasma and bovineliver but not from orchard leaves, due to lack of material.The thick pellets had diameters of 6 mm.

Targets were also prepared by dissolving the rest ofthe ashed residue in 0.5 ml of 7-molar supra-pure nitric acidand pipetting between 5 and 20 /il on to Nuclepore fitlers(N 040, pore size 0.4 pm, diameter 25 mm). In the study byPallon and Malmqvist (1981) targets were irradiated by 2.55 MeVprotons from the 3 MV Pelletron accelerator in Lund. In orderto decrease the count rate from dominant elements such as S,K and Ca, an external pin-hole absorber was used. It consistedof a 340 urn thick Mylar film with a. small hole for the lowenergy X-rays to pass through. This absorber was chosen fromour standard set of different absorbers as being the mostsuitable one. It would, however, have been better to use onewith a ten times smaller hole.

49

Table 2.2

Ratios of element abundances in NBS bovine liver(SRM 1577) as measured by -PIXE (Pallon and

Malmqvist, 1981) to the stated values (in percent)

Element

PClKÇaMnFeCuZnSeRb

Dried, thick(pellets)

92.712210511511593.398.196.210098.1

- 2.7- 1- 6t 8- 1- 5.6- 5.2- 1.6- 27- 5.5

Ashed, dissolved(plletted on Nuclepore)

63.6 -0.888.7 -115 -99-0 -89.6 -88.1 t92.3 -8295.6 -

3-6

1.1122.93-72.67-7

5.5

Ashed, thick(pellet)

116 -0.9

127 -169 -90.3 -101 -95.9 -100 -2790

9

9167.887.88

NBS

(100)100100 - 6.2100100 - 9.7100 - 7.5100 - 5.2100 - 7-7100 - 9.1100 - 5.5

Table 2.3

Ratios of element abundances in NBS orchard leaves(SRM 1571) as measured by PIXE (Pallon and Malmqvist,

1981) to the stated values (in percent)

Element Dried, thicka' Ashed, dissolved

SClKÇaMnFeCuZnAsBrRbSrPb

a)b)

84.0 -82. 6 î109 -100 -114 î110 ±117 î96.0 -150 î100 -100 î83.8 ±133 ±

cellulose matrix*» ** »w t^ T ^* r* i«\^TSA^^Ay"]

101433517428401085

22

has„., 4

65.8 -58.0 î100 -95.7 -100 î73.3 -100 ±324 î160 i50 ±100 -97.3 -122 î

been assumed .

2.64.334338

162010171118

4-- A ** n

b) NBS

(100(100100100100100100100100(100100

(100100

"

))•«•+

+

+

•f++

)-I-

)-»•

21468

1220

8

6

.0

.4

.4

.7

.3

.3

.7

Normalized to Mn.

The thi'ck pellets were analysed for 300-500 s using abeam diameter of 4 mm giving a total accumulated charge of3 pC. The pipetted targets were analysed for 300-700 s usinga beam diameter of 8 mm.

The total accumulated charge was 10 juC. All irradiationswere performed in a vacuum chamber and for some hot carbonfilament was used to avoid charge build-up on the samples.

The obtained results are summarized in Table 2.2 and 2.3which show the contents of the NBS standards bovine liverand orchard leaves, as found in the PIXE-analysis, as percent-ages of the values stated by NBS. The left-hand column inTable 2.2, named dried thick, gives the values for the originalfreeze-dried matter. Except for Ca these agree quite wellwith the certified values. In the case of orchard leaves themeasured values, shown in Table 2.3, show good agreement withthe certified values.

During ashing of bovine liver, more than 90% of thechlorine and part of the selenium present are lost. Whencalculating the contents of the pellet, thick target correctionsdue to proton slowing-down and X-ray attenuation must be used.The low-Z element values are especially sensitive to the matrixcomposition, which is not completely known in the ashed specimen

51

From the results in Tables 2.2 and 2.3, one can concludethat volatile elements are lost to some degree, but that mostof elements are well preserved after low temperature ashing. Ifenough ashed material is available and it is possible to makea good estimate of the major constituents of the ash matrix,thick pellets are to be preferred. This approach has severaladvantages :

(a) Easier target handling, including fewer preparationsteps and minimum requirements of chemical treatment,decreasing the risk of contamination.

(b) The sensitivity (pulses/(ppm xjuC)) is often higherfor thick targets, but the uncertainties"whencalculating the corrections are larger especiallyfor lighter elements.

(c) Corrections made to thick target data can be basedon the fact that the matrix is infinitely thick forprotons and that absorption occurs for all X-rayenergies. Pipetted samples can in general not beregarded as thin targets with negligible X-rayabsorption. When the pipetted ash solution dries onthe thin filter, crystalline structures may result.These crystals can be too thick, thus making theself-absorption of low energy X-rays and slowing--down of protons severe problems. To avoid this,pipetted solutions have to be diluted, or a tediousabsorption control of each target made. The firstalternative decreases the sensitivity, the secondis time consuming: neither is very attractive (Pallonand Malmqvist, 1981 ).

2.5 Loss of elements during sample pretreatmentDuring the process of sample pretreatment element

losses can occur as well as possible contamination . Themost usual steps in sample pretreatment are drying andashing. These two steps are almost always done when analysingbiological material.

lyengar and Sansoni (1980) have discussed the lossesof elements in biological material which was subject to dryashing at temperatures around 500 C. Their results are shownin Table 2.4. Losses are observed for elements such as Ag,As, Co, Cr, Hg, I. K, Na, Pb, Se, Sn and Te.

Some losses are observed even during oven drying ofdifferent biological matrices. Data presented by lyengar andSansoni (1980) on the loss of elements during oven dryingare shown in Table 2.5. The losses range from not detectableup to more than 50%.

Table 2.6 shows the recovery of various elements indifferent matrices following freeze drying as reported by thesame authors.. Basically freeze drying is well suited fordrying biological samples, yet care should be taken to preventcontamination from the metallic housing of the freeze drierfor elements such as Cr, which may volatilize and be trappedby the sample. Use of non-metallic components such asperspex, or preferably quartz, is desirable for the construc-

52

Table 2.4Loss of elements during dry ashing of biological samples

(After lyengar and Sansoni, 1980)

Element

Ag

Al

As

Ba

Ca

Cd

Co

Cr

Cu

Fe

Matrix

Animal, liverkidney

Animal, liverkideny

Ox, blood (dry)

Rat, bonebloodkidney

Animal, liverkidney

Human , rib

Animal , liverkidney

Rat, liverliverkidney

Animal, liverMollusc

Sugar, refinedbrownunrefined

MollassesSugar , refined

brownunrefined

MollassesAnimal, kidney

liverRat, liver

Rat, blood

Animal, kidneyliver

Animal, kidneyliver

Rat, liverblood

Procedure ormode ofincorporation

Chemical analysis

Chemical analysisChemical analysisRadioisotope,spiking

Radioisotope,intravenous

Chemical analysis

Radioisotope,spiking

Chemical analysis

Chemical analysisplatinum dish

Chemical analysisRadioisotope,metabolizedGraphite furnaceChemical analysis

Chemical analysismuffle furnace

Chemical analysis

Radioisotope,platinum dish

Chemical analysis

Chemical analysis

Chemical analysisplatinum dish

Temperature(°C)

450450450450850550450450450450450450420600710450450600500500450450800450450450450450450450450450450700500700500450450450450500500

Time(h)

????161616161616??161616??16161*6???????????7?16161616????1616

Lossobserved(%)

5201612352928448682

41110.761,624.41426220134752636286892572.26.1

' 51.340.40.20.10.3

No loss0.4

53

Table 2.4 (cont . )

Element Matrix

HgK

Mn

Mo

Na

Ni

Pb

Sn

Sr

Zn

Fish (whole)Human, rib

MolluscmetabolizedAnimal , kidney

liverAnimal , kidney

liverHuman, rib

Ox, bloodAnimal , kidney

liverAnimal , kidney

liverHuman , rib

Animal, kidneyliver

Animal , kidneyliver

Ox, blood

Rat, bonebloodkidney

Mollusc

Seaweed

Mussels

Ox, blood

Rat, blood

Rat, bloodAnimal , kidney

liverRat, liver

Procedure ormode of Temperatureincorporation ( C)

Radioisotope

Chemical analysis

Chemical analysis

Radioisotope,spiking

Chemical analysis

Chemical analysis

Chemical analysis

Chemical analysis

Radioisotope ,spikingRadioisotope,intravenous

Radioisotope,metabolizedChemical analysis

Chemical analysis

Radioisotope ,spiking

Chemical analysis ,new porcelainChemical analysisChemical analysis

Chemical analysis,etched porcelain

no420600710450800150450450450420600710450450450450450600710450450450450450

45045045045080050010005001000450550850700

500450450700500

Time(h)

24161616?.7????161616167???1616????16

161616?71616161616161616

16??1616

Lossobserved(%)

81 .41

559015210.40.31.50.43

1020Slight153122.45400.3

110.52.59

Slight1653344

No lossNo lossNo lossNo lossNo lossNo lossNo loss

1

No loss111 .11.3

54

Table 2.5Loss of elements during oven drying of biological

samples (After lyengar and Sansoni, 1980)

Element

Cd

Co

Cr

Fe

Hg

r

Mn

Pb

Sb

Matrix

Oyster

Rat liverkidney

Oyster

Mollusc

Rat , manytissues

Rat liverblood

OysterRat bloodHuman urine

PlanktonRat liver

Rat brainmuscle

Human urine

Rat musclebloodserumerythrocytesbrainkidneylung

OysterMolluscOyster

Rat blood

Rat brainkidneylungspleen

Procedure ormode ofincorporation

Radioisotope,metabolized

Radioisotope,intravenousRadioisotope,metabolizedRadioisotope,metabolizedRadioisotope,intravenous

Radioisotope,intravenousRadioisotope,intravenous?n "2Hg-organic,intravenous

Chemical analysisRadioisotope ,metabolized

Radioisotope,metabolizedRadioisotope,metabolized


Radioisotope,metabolizedRadioisotope,metabolized


Temperature(°0

1209050110110120

no80110120120110no1058010512060801051201201208010512012012012012012012012050-12011060100120105120120120120120

Time(h)

484848161648

?

72242448161648722424

' 5072242424247224242424242424242448?484848242424242424

Lossobserved(%)

No lossNo lossNo loss

11

No loss

14

No lossNo lossNo lossNo loss

35

No loss3

1525

51-605

3-107-155-165-21247577a10157

No loss14101720558967

55

Table 2.5 (con t .}

Element MatrixProcedure ormode ofincorporation

TemperatureC°C)

Time(h)

Lossobserved(%)

Se

Zn

Herbage

Rat bloodbrainlungmuscle

Human urine

Oyster

Rat bloodliver

Rat, manytissues

Mollusc

Chemical analysis


75Se organic,intravenous


Radioisotope,intravenousRadio-isotope,metabolized


3060100120120120120801051206010012011011080110120110

121212242424247224244848481616722424

No lossNo lossNo loss

5555

12-3030-5050-65

5520

No lossNo lossNo lossNo lossNo loss

tion of the sample housing compartment of the freeze drier.Generally freeze drying of bilogical samples has beenreported to be satisfactory for most elements in differentmatrices.

2.6 Chelation and Solvent ExtractionSolvent extraction can be conveniently used for

preconcentration and for separation of transition metalsfrom large amounts of alkali metals. Most commonly, metalsin an aqueous solution are extracted into an inimiscibleorganic solvent, usually with the use of a chelating agent,and the organic phase is analyzed directly without back--extracting into the aqueous phase.

The most common used chelating agents for the solventextraction of metals are listed in Table 2.7. The recom-mended pH ranges for the extraction of metals by dithizone,cupferron, oxine, sodium diethyidithiocarbamate (Na DDC),and ammonium pyrrolidine dithiocarbamate (APDC) are listedin Table 2.8. Note the APDC is the most comprehensive, thatis the least selective reagent. It can conveniently usedbecause several trace metals can be extracted simultaneouslyfrom an aqueous solution and determined in a single extract(O'Haver, 1976).

The most commonly used extracting solvents are CHC1~,, methyl isobutyl ketone (MIBK), esters such as ethyl"3

56

Table 2.6

Loss of elements during freeze drying of biologicalsamples (After lyengar and Sansoni, 1980)

Element Matrix

Co

CrFeHg

I

Mn

PbSe

Oyster

OysterOysterFishFish homogenate

ButterfishHuman brain (pons)PlanktonGuinea-pig, rat:

muscleliverkidneyheartbloodfaecesmuscleliverkidneybloodfaeces

Sea cucumberWaterHuman urine

WaterHuman urine

Oyster

Human urine

Procedure ormode of Pressureincorporation (Torr)


Chemical analysisChemical analysisRadioisotope, spikingChemical analysis

Methylraercury ( Hg)

Phenylmercury ( Hg)

Chemical analysis

Hg-organic,intravenousChemical analysisRadioisotope,metabolizedRadioisotope,metabolized

75Se-organic,intravenous

?

???7?????0.05

0.05

?0.01-0.050.05

0.01-0.050.05

?

70.05

Time(h)

24

2424????7?724

24

?24-7248

48-7248

24

2448

Lossobserved

No loss

No lossNo loss

2016-39No loss

7018-5750-64

33.31.7

No loss1.52.8

No lossNo loss

2No lossNo loss

9.359392

322

No less

No less3

acetate or propionate, and ethers such as ..ethyl ether. Thesolubility of MIBK in water (20 ml liter" at 25 C) is togreat to allow very large concentration factors. Methyamylketone (MAK) is much less soluble in water and is a suitablealternative. However, MIBK is less expensive and more widelyavailable .

O'Haver (1976) has described an example of a typicalextraction procedure suitable for many metals:

57

Table 2.7Chelating agents commonly used in preconcentration

(After Ü'Haver, 1976)

Common Name Chemical Name Commonly Used for

APDC Ammonium pyrrolidinedithiocarbamate

Cupferron Ammonium salt of N-introsophenylhydroxylamine

Oxine 8-Hydroxyquinoline

Dithizone DiphenylthiocarbazoneACAC Acetylacetone;

2,4-pentanedioneNADDC Sodium

diethy1dithiocarbamate

Cu, Pb, Cd, Co, Mn,Fe, Ni, Bi, Zn, As,Ir, Pd, Pt, Se, Te,Tl, Mo, V, CrV, Ti, Cu, Mn, Fe, Ni

Al, alkaline earths,othersAg, Pb, Cd, Zn, CrTransition metals

Pb, Cu, Fe, Mn, Te

Adjust 100 ml of the aqueous solution to the appropriatepH range (usually between 3 and 4) by the addition HC1 or NH,.Transfer to a separatory funnel. Add 5 ml of a freshly prepared1% solution of APDC in water and shake to mix. Add 10 ml MIBK,shake 2 minutes, and allow the phases to separate. The organic(upper) phase may be used for the analysis.

It is sometimes convenient to use a narrow-neckedvolumetric flask or similar vessel instead of a separatoryfunnel. After the phases have separated, additional water canbe poured in to raise the level of the organic phase intothe neck, if needed.

In solvent extraction work, it is always necessary tocarry the blank and the standards through the same extractionprocedure (O'Haver, 1976). The purpose of this is to (1)allow for less than 100% extraction, (2) correct for theconcentration of reagent impurities, (3) eliminate calculationof the exact concentration ratio, and (4) provide standardsolutions in the appropriate chemical form and matrix.

Cronin and Leyden (1979) have described a method ofuranium concentration on to a solid substrate in a single step .The method utilized a silica-cellulose filter chemically--modified by bonding a silanedithiocarbamate to the silicasurface. The aqueous sample is continuously cycled throughthe filter in a closed-loop flow system. In their work theextraction and subsequent enrichment of uranium onto the filteris accomplished by ion-pairing of the tris-carbonato complexof uranium (UO-CCO-, )^~ ) with an immobilized derivative ofethylenediamine. Tne^silica-cellulose filter is chemicallymodified by silylation with a silane-ethylenediamine. Thismethod combines the advantages of a one-step filter pretreatment,

58

Table 2.8

Recomended pH ranges for solvent extractionof metal chelates (After O'Haver, 1976)

Element

AgAlAsBeBiCdCoCrCuGaInFeHgPbMnMoNiSe •

SbSnTlVWZn

Dithizone

0-7--

26-146-80.52-5---0-47-10--6-8------6-9

Cupferron Oxine

3-63.5-9.0 5-6

-6

-65-6

-7; 1 2-6--7 6-

67

1.8-2.67 6--

2.5-6-1-

6

NADDC

8-11-6-

8-118-113.6; 8-113-63.6; 8-11--3.68-113.6; 8-113.6-3.6; 8-116--

8-116-

3.6; 8-11

APDC

1-10-2-6-1-101-6;2-4;3-90.1-83-71-102-5;2-4;0.1-62-4;3-4;2-4;3-6i; 3,3-63-10;1-2;1-32-6;

1-101-10

; 1-10

5-0.31-10; 1-105 0.33-61-10

7

3-104; 3-6

1-10

a single step U0?+ extraction and enrichment onto a solidsubstrate and the simplicity of an x-ray fluorescence deter-mination. The details of experimental procedure are as follows:

N-fi-ethyl-^-aminopropyl trimethoxysilane (Dow-CorningZ-6020) was used as a 10% v/v solution in toluene. The filters(22 or 25 ram diameter) were punched from sheets of SG-81(Whatman, Inc.), 20% w/w silica gel in cellulose after silylation.Stock solutions were prepared from reagen^ grade chemicals, cop-per from the chloride salt as a 1000 mgl~ .aqueous solution anduranium from uranyl acetate as a 5000 mgl" solution. Workingstandards were subsequently prepared from these solutions asneeded. Samples from various stages in the uranium solution miningprocess were obtained from Wyoming Minerals, Inc. These samplesrepresented ar range of uranium concentrations in a varying matrix,although all samples were aqueous solutions.

59

10 11

^

Fig. 2.1 The relative amount of Cu extracted bythe silyloted filters at various pHvalues (Cronin and Leyden, 1979).

Thefilters at2 ppm Cu2+with NaCIO0.1 N NC1meter withof Cu2+ onas in the

Orelative amount of Cu * extracted by the silylatedvarious pH values was determined using 50 ml ofsolution, adjusted to constant ionic strength (/J = 0.5)

M. The pH of the solutions was adjusted using eitheror 0.1 N NaOH. The final pH was determined using a pHa combination pH electrode. The relative concentrationthe filters at the various pH values was determined

chemical capacity study.Uranium enrichment on the silylated filter involves the

ion-pairing of U02(CO,)^~ with the immobilized ethylenediaminebetween pH 6 and o. Thus the uranium working standards and theprocess samples were prepared as follows: the required aliquotof either the stock solution or process sample was pipettedinto a volumetric flask and brought to volume so that the sampleused for enrichment would be 0.1 M in (NH^-CO., and at apH = 6.5 - 0.2. In the case of the process samples, the aliquotwas first pretreated by acidifying to pH2 and heating to justboiling followed by cooling and neutralization. The pretreatmentis required to remove any carbonate that might be present.

The procedure for uranium extraction involves a 25 mlsample and a cycling time of 60 min. This is followed bywashing the filter with three 5 ml portions of 0.1 M (NHa)?CO,solution (pH = 6.5). The filter is then prepared for XRF *analysis by air drying and placing it in a 25 mm diametersample holder (Chemplex) between two sheets of 6.3 urn thickMylar film (Cronin and Leyden, 1979).

The results showing the extraction of Cu "*" on SG-81 arepresented in Fig. 2.1 and it has the same pH dependence as onthe bulk silica gel. it was concluded that the immobilizedethylenediamine on the SG-81 filters was similar to theethylenediamine immobilized on bulk silica gel. In addition,even though the capacity of each filter is low, their convenientphysical form makes the filters attractive as preconcentrationtools in XRF analysis .

60

Two experiments were done to determine the suitabilityof the filters for XRF analysis. The first study dealt withdetermining the necessity of counting both sides of the filterin the XRF spectrometer. A series of solutions of variousuranium concentrations was preconcentrated by this method fol-lowed by x-ray counting of the UL^ line on both sides of thefilters. A plot of cps of the contact side of the filters (theside facing the sample solution) versus cps of the average ofthe front and back counts of the filter was constructed and astraight line was obta-ined with a slope of 0.96 - 0.03 and acorrelation coefficient of 0.9991- This indicates that countingof only one side of the filter is sufficient for the uraniumdetermination. In the second experiment pressing of the filtersto improve the precision of the counting statistics was inves-tigated. A series of filters containing varying amount ofuranium was first measured by XRF followed by pressing at10.000 psi for 20s in a one inch die and remeasured. Usingtwo-group statistics, no significant difference at the 95%confidence level was observed between the XRF signals from theunpressed and pressed filters.

Two methods were used to determine the percent recoveryof uranium of the filters. In the first method, two filterswere used in succession; a sample was preconcentrated onto onefilter, the filter was removed and a second filter was usedto concentrate the uranium remaining in the sample. The secondmethod involved a comparison of the x-ray counts of a samplefilter with another filter spiked with a known amount ofuranium. Mean recovery ranges from 70 to 95% for 10 ppm (Croninand Leyden, -1979).

2.7 Ion exchangeIon exchange is becoming an increasingly popular method

of separation and preconcentration in trace element analysis ,especially for water analysis at ultratrace levels. Basicallythree different types of ion exchange resins are used for thesepurposes: cation exchange, anion exchange, and chelating-typeresins. Cation (acid) exchange resins are those that exchangecations with the solution, replacing all the cations in solutionwith H"1" or Na~. Anion exchange resins replace the anions insolution with OH" or Cl~. Chelating resins contain functionalgroups similar to those in conventional chelating agents. Theseresins remove from solution any ions with which the functionalgroups can form a chelate bond. They are somewhat more selectivethan cation or anion exchange resins. All three types arewidely used for trace element concentration. The large ionexchange capacity of many resins means that large volume ofdilute solutions can be passed through the exchange column withnearly complete retention of the ionic species. The retainedions are then eluted with a relatively small volume of a strongacid (e.g. 2 M HNÛ2) or for anion exchange resins, an alkali(e.g. 4 M NHijOH). Nearly 100% recovery can be obtained for manymetals. If the sample contains large amounts of major cation(e.g. Na"1", K"1", Mg2*, Ca2~), a cation exchange column may notbe practical because it may be saturated by the major cations,thus preventing complete retention of trace cations . In such

61

cases, a chelating resin that does not retain alkali or alkalineearth elements is preferred (O'Haver, 1976).

Beamish (1967) has reported methods of separating bothmicrogram and milligram amounts of the six platinum metals fromlarge proportions of iron, copper and nickel by cation-exchangecolumns. The isolation of rhodium, iridium, platinum and palladiumby anion-exchange columns has also been subject of number ofpapers. In the work by Taylor and Beamish (1968) quantitativeseparations of microgram quantities of osmium and ruthenium fromlarge proportions of copper, iron and nickel were accomplishedby the use of anion-exchange paper. Accurate determinations ofosnijum and ruthenium were made by adaptation of X-rayfluorescence.

Use of chelating ion exchange resin in the determinationof uranium in ground water by x-ray fluorescence is describedin details by Hathaway and James (1975).'In their work all testsolutions were prepared from a common ground-water sample .Spiked solutions were prepared using a solution of uranyl nitrate.Solutions used in the studies on extraction time, pH dependence,and recovery in multiple extractions were spiked to 50 ppb(ug/1) above the natural uranium level of the ground- water.Preacidification of the water samples consisted of treatmentwith 3 ml concentrated HC1 per liter of water 12 hours prior toaddition of resin.

Samples used for the standard additions-calibration curvewere triplicate sets of unspiked ground water, and ground waterspiked to 20, 60, and 100 ppb above the natural uranium level.

The procedures used were as follows: a 0.3-ml glass scoopwas used to add two level scoopfuils (0.6 ml equa-ls 60 mg dryweight) of prepared Chelex-100 resin to 1-liter samples ofwater contained in 1-liter Erlenmyer flasks. The pH of thesolutions was then adjusted to the desired value, with the aidof a pH meter, using a dilute NaOH solution.

All samples, except those in the extraction time study,were stirred for 3 hours on a magnetic stirring table. All flaskswere sealed during the extraction period in order to minimizecontamination and CÛ2 uptake in the higher pH solutions. Afterstirring the solutions for the proper length of time, the resinsamples were collected on 0.45-u filter and then dried for1 hour at 45 C.

Filtrates from solutions used in the multiple extractionstudies were treated as new samples and run through the aboveprocedure two more times .

The dried resin samples were each mixed about 500 mg ofSomar Mix (a binder), and then spread uniformly over the surfaceof 1.25-inch planchets which were half-filled with boric acidas a backing agent. The samples were then pressed to 10.000 psi.The resulting pellets were quite stable when stored in aChamber with silica gel.

Pellets were counted for 100-second intervals at boththe analyte and background lines.

The use of ion-exchange resin-loaded filter paper forautomatic analysis of dissolved metal pollutants in water isdescribed in an article by Ho and Lin (1982). The systemconsists of a sample tank, a roll of 3.75 cm wide ion-exchange

62

resin-loaded filter paper on a supply reel, a filter-papertransport mechanism, a collection tank, and a spent-water tank.A water sample as large as 500 ml can be processed from thesample tank through the ion-exchange filter paper and intothe collection tank. The water in the collection tank willeither recycle back to the sample tank or go to the spent-watertank. The water transport lines and tanks are made of plexiglassand Teflon in order to reduce the possibility of samplecontamination. The filter-paper transport mechanism includesa precision motor that moves the filter paper forward andbackward in precise increments, a photoelectric sensor thatdetects seams in the paper, a cutter, and a transporter thatmoves the cut filter paper to the x-ray chamber for analysis.

The use of ion-exchange resin-loaded filter paper forwater sample preparation is also described by Campbell et al.,(1966) and Law and Campbell et al., (197*4). In their workschemical and x-ray characteristics of Reeve-Angel cation andanion exchange resin-loaded paper disks were investigated.Chemical characteristics include exchange capacity, effectsof pH, salt concentration, and competing ions, and distributionof collected ions. X-ray characteristics include x-rayransmission coefferent as a function of wavelength, relationshipof x-ray intensity to quantity atomic number, and distributionof collected ions, reproducibility of intensity measurements,and limits of detection.

2 .8 ElectrodepositionThe use of constant current electrodeposition of reducible

metal ions upon a pyrolytic graphite roll to prepare samplesfor wavelength dispersive x-ray fluorescence analysis has beendescribed by Vassos et al., (1973). In the of Boslett et al.,(1977) a technique by which trace amounts of" the aqueous metalions nickel (II), copper (II) and zinc (II) are preconcentratedon the end face of an ordinary spectrographic graphite roll bypotentiostatic electrodeposition is described. The thin metalfilm that results from the electrodeposition is analyzed byXRF. Controlled potential electrodeposition has the capabilityto selectively separate trace concentration metal ions froma solution that may contain interfering metal ions. In thework by Boslett el al., (1977) stock solutions of zinc (II)acetate, copper (II) perchlorate, and nickel (II) chloridewere prepared by dissolving reagent grade salts in distilled-deionized water. The stock solutions were standardized againstdried primary standard disodium dihydrogen ethylenediaminetetra-acetate dihydrate (Na2H2EDTA-2H20) and determined to be0.0996 F Zn(C2H302)2, 0.1039 F Cu(C10i|)2 and 0.0991 F N1C12 .Solutions containing trace-levels of the metal ions were pre-pared by diluting microliter amounts of the stock solution indistilled-deionized water.

Supporting electrolyte was prepared in concentratedform by dissolving reagent grade sodium acetate in distilled-deionized water and adjusting to pH 6.0 with glacial acetic acid.The total analytical concentration of acetate was 2.60 F. Thissolution was cleaned of metal contaminants by electrolysisfor 18h orar a cathodic mercury pool at -1.700 V US. saturated

63

calomel electrode (SCE). The details of the procedure used areas follows:

A solution of distilled-deionized water and sufficientamounts of stock supporting electrolyte to make the solution0.10 F in total acetate at pH 6.0 was prepared and degassed bypurging with nitrogen for 10 min. The appropriate microliteramount of previously standarized stock metal solution was added.The final volume in all cases was 120.0 ml. Electrodepositionproceeded at -1.300 V vs. SCE, with vigorous stirring. Allelectrode positions were run at ambient temperature. On completionof the deposition, the graphite rod was removed from the cell,with the voltage applied, and rinsed quickly in distilled-deionized water. After air drying, the working surface wassprayed lightly with the acrylic lacquer to fix the deposit,and a 1/4- to 1/2-inch length of the carbon rod was cut off foranalysis.

Counting times were generally 400 s (live time), exceptfor the analysis of 'solutions of very low concentrations (<20 ppb ) ,for which counting times as long as 1200 s were used and nor-malized to an equivalent 400-s count. Date for the calibrationcurves were obtained from digitized spectra by a computer-assistedintegration of the analytical peaks.

The concentration of a metal ion in solution that is beingpotentiostatically electrodeposited on an electrode surfacedecreases with time according to equation:

C = C0e~kt (2.1)

where CQ is the initial concentration, and k is a constantcharacteristic of the electrodeposition system, the metal ion,and its environment. The amount of material Y. deposited onthe electrode at time t also depends upon the initial concen-tration, as shown in equation:

Y = CQVM(1-e~kt) '(2.2)

in which V is the solution volume and M is the gram-atomicweight of the metal ion.

The electrode upon which the thin metal film was depositedmay then be inserted directly into the modified x-ray spec-trometer sample holder for quantitation. For a given depositiontime, the intensity of characteristic fluorescence emitted fromthe sample is proportional to the amount of metal deposited, upto the critical thickness, beyond which additional metaldeposited results in no incremental fluorescence emitted. Thus,Equation (2.2) may be rewritten as:

It = I d-e" ) (2.3)

in which It is the intensity of characteristic fluorescenceemitted from the electrode surface after electrodeposition forsome time t, and 1^is that intensity which would be emittedafter all of the metal ion has been deposited on the electrode(t =*-).

64

Bosiett et al., (1977) have concluded that MDL for Cuusing a 6-h. deposition is 0.9 ppb. Similar results were alsoobtained for Zn.

In spite of encouraging results, a more widespreadanalytical application of the technique is limited to theelements which can be recovered from aqueous solution in suf-ficient yield.

An improvement of this method has been reported byWundt et al., (1975). They have developed an analyticalprocedure for transition elements based on the electrode-position of their anionic cyano complexes from mixed organicaqueous media in a high potential electric field wasdeveloped .

For optimizing the electrodeposition procedure, solutionsof the individual elements (10~^M) containing cyanide of dif-ferent concentrations (5xlO~3M - 7.5 x 10~2M, pH 11) werestudied. For most mono- and bivalent transition metals, theywere obtained from weighed amounts of the respective chlorides(Co, Ni, Cu, Zn, Cd, Pt, Au, Hg) or nitrates (Ag). Because oftheir redow and hydrolytic behavior, Fe(II), Fe(III), and Co(III)were used in the form of their cyano complexes. Those of V(III),Cr(III), and Mn(II) were prepared by standard methods. Becauseof the limited stability of these cyano metalates in aqueoussolution, the oxo compounds NH^VO^, F^CrOi}, and KMnO^ were also -examined. All reagents were Suprapur or of analytical grade, thewater was deionized and bidistilled. For deposition, aliquotsof these solutions were diluted with methanol, ethanol, and2-propanol to give alcohol mole fractions between 0.4 and 0.9.

To favor the elution of the cyano complexes, which ispossible only from basic medium, the cation-exchange resin(Dowex 50W-X8, 200 400 mesh) was preconditioned with a mixedsolution of KOH (1 M) containing KCN (1 M) for eliminatingcontaminants, repeatedly washed with bidistilled water, anddried at 70°C.

The effectiveness of deposition is most sensitive to thenature of the organic component and its ratio to water. Becauseof their solvating properties and their miscibility with water,polar organic solvents like alcohol proved to be most suitablefor the eldctrodeposition technique. Preliminary experimentsshowed, however, that deposition was incomplete in aqueousmixtures of methanol and ethanol, irrespective of their compo-sition and the variation of the other parameters. Also, thecyano metalate films tended to become less adhesive. This seemsto be due to the relative high dielectric constants of themedia causing high current densities (10 mA cm~2 at 400 V) andconsequently the corrosion of the aluminum anode.

Quite different results were found with 2-propanol asthe organic component. With all other parameters optimizedquantitative deposition as well as thin, uniform, and firmlyadhering films were obtained, when aqueous cyanide solutions(pH 11) with Fe(III), Co(III), Ni(II), Cu(I), Zn(II), andCd(II) were mixed with appropriate amounts of 2-propanol to givemole fractions of the deposition solution between 0.50 and0.78. For Fe(II) quantitative deposition is possible only ina small region of 2-propanol mole fractions, whereas Co(II) isdeposited with an incomplete but constant yield (70%). These

65

conditions were realized after 30-min deposition time at high-voltages above 1000 V and current densities of about 10 mA cm ,Extending the range of mole fractions beyond 0.50 and 0.75leads to a steep decrease of the deposition yield and to badlyadhering deposits.

The deposition yield is sensitive also to the ioniccomposition of the aqueous solution, namely the pH and thedegree of complex formation (Wundt et al., 1979).

3. SAMPLE PREPARATION FOR PIXEA major problem in quantitative PIXE analysis is the

target preparation; most of the positive characteristics ofPIXE may be spoiled by inadequate target preparation. Inthis context it should be noted that PIXE is a surfaceanalysis technique. For instance, the range of 3-MeV protonsin organic material is less than 20 mg/cm2 .

If targets thicker than the proton range ("infinitely"thick) are analysed for their "bulk" composition, the surfaceunder irradiation should be representative for the wholetarget. This means that such targets should be homogeneous,implying constant target composition throughout the targetvolume to be irradiated. Because of the homogeneity, matrixcorrections can then be applied when a quantitative analysisis required.

For thin targets (£1 mg/cma ) PIXE may be consideredas a quantitative trace element analysis method, since matrixcorrections are negligible. Thick targets (}£1 mg/cm2) shouldmeet the requirement of uniformity, implying a constant areadensity over the target area to be analysed. For thin targetsuniformity is not a strict requirement, when the beam spotis bigger then the area of sample.

Thick targets have the following disadvantages (Kivits,1980) :

- data analysis is complicated and uncertainties arerelatively large due to the varying cross sectionsfor the x-ray production by protons which are sloweddown in the target as' well as due to absorption andenhancement effects;

- charge build-up in the target may cause in increasedBremsstrahlungs continuum, especially if the protonsare stopped in the target;

- target damage may occur by heating, even at low beamintensities (e.g. 10 nA/cm2).

For homogeneous thick targets, the disadvantage ofinaccuracy can be overcome, to a certain extent, by calculationof the matrix corrections. The effect of charge build-up maybe reduced by using targets with thicknesses less than theproton range. Special additions may allow targets to withstandheat generation, at the expense of loss of sensitivity andthe risk of contamination, however.

On the other hand the use of thick targets also hasadvantages. Thick targets are generally less complicated and

66

faster to prepare than thin targets, and involve less riskof contamination and loss of elements to be analysed. Withrespect to a bulk analysis, the preparation of thick targetsis less dependent on sample inhomogeneity.

3 .1 Backing materialsWith charged particles the simplest is the direct

bombardment of a specimen. This method is often used forsuch materials as certain biological tissues, e.g., teeth,or metallurgical samples, where it is difficult or.impos-sible to obtain thin targets. The drawbacks are mainly thelower sensitivity and the need for various corrections incalculating the results.

When thin targets are prepared the material to beanalyzed should be deposited on some suitable backing.There exists a great number of methods for bringing a sampleinto a form suitable for analysis and for depositing it onthe backing. The method chosen depends .on the type ofmaterial to be analyzed.

When charged particles are used for the excitation ofcharacteristic x-rays then a sample to be analyzed has tobe maintained under vacuum during ion bombardment. Thismeans that volatile compounds or gas contained in the samplewill be removed to a large extent during pumpdown. For examplewater will disappear from tissue samples so that one isanalyzing mainly dry matter, and the sensitivity to Br andHg (for instance) will depend on the chemical form they havein the original sample.

A backing material is necessary for many types ofsamples. Mechanical strength and good electric and thermalconductivity is desired and it has to be of high puritymaterial, able to withstand high beam intensities. Thecontinuous background radiation produced by the backing oughtto be as small as possible, and this favors thin backingsconsisting of low Z elements.

Many different kinds of backing materials are used indifferent laboratories. Some investigators favor thin carbon _foils, some others are using different plastic foils (Formvar ,Kapton, MylarR, polysterene and Hostaphane). Often MilliporeR,Whatman^, Nucleapore^ and other filters are used.

Here we shall present procedures for the floating offormvar: A solution of 50 ug/ml formvar in spectroanalyzed1,2-dichloroethane should be prepared and stored in a glasscontainer, (the container must be glass since 1 ,2-dichloro-ethane, a strong organic solvent, will dissolve polyethylene).Target frames"should be cleaned by soaking overnight inacetone. The frames should be stored in acetone, since theyoxidize in .water and air, and should be rinsed with doublydistilled water (DDW) just prior to use. All work should bedone in a clean box to reduce dust contamination.

Details are described inapaper by Valkovic (1974). Aplexiglass trough 2.5 cm wide by 17.7 cm long and 5 cm deepwas cleaned with ethanol and rinsed with DDW. The troughwas then filled with DDW and a drop of the formvar solution

67

was placed at one end of the trough. The formvar was drawnacross the water as a thin film with the edge of the frameheld by an alligator clip. The formvar film was then drapedover the hole in the target frame by dipping the frame inand out of the coated water and folding the formvar back onitself. Four layers of formvar film proved to give the sup-port necessary for the backing. The wet targets were thenstored in a special dust free holder until dry. Preparationof Formvar films is also discussed by Bearse et al., (1973).

X-ray spectrum from Formvar obtained by Valkovic et al.,(1974) exhibits no characteristic x-ray peaks. The thinnessand low effective atomic number of Formvar^ foils confer onthem the advantage of extremely low bremsstrahlung background:if trace metal impurities can be eliminated, FormvarR willafford better detectibility limits than its competitors.. Thedisadvantage is that FormvarR is very fragile and is a badheat conductor; breakage rates are likely to be high and beamcurrents must be kept low, increasing running time. Valkovicet al., (197^) have circumvented the heat dissipation problemby evaporating a 100 ug/cma aluminum layer onto 100 to 150 ug/cm2FormvarR. This allowed them to use 300 nA beams with normalfilms. Another disadvantage of Formvar^ relative to carbon isthat it is destroyed by acidic solutions. Figure 3.1 shows thespectrum of x-ray radiation resulting from 3 MeV bombardmentof Al-FormvarR backing. It is essentially free of any linesand it can be easily approximated by a polynomial for backgroundsubstraction.

When higher concentrations have to be measured in powderedsamples different adhesive plastic tapes can be used. Figure 3.2shows the spectrum from the Scotch tape (3M Company, Minneapolis)with the significant contaminant being bromine only.

The use of thin films to support liquid, powdered andslurry samples in x-ray spectroscopic sample cups is a state-of-the-art. Polyester film supports are the most commonly usedand preferred because of their unique properties. The chemicalcomposition of polyester attenuates absorption of the primaryx-rays and characteristic radiation emitted by the sample. Thedegree of attenuation is further controlled by the gauge ofthe film used; the thinner the gauge, the less the absorptionof x-rays. The inherent high strength of polyester film alsopermits safe sample handling and retention concurrent withmaintaining taut film surfaces to define statistically repro-ducible target-to-saraple distance. For example, Chemplex"X-Ray Mylar Films" are polyesters (polyethyleneterephthalate )and have all of the unique combination of properties requiredfor x-ray spectrbscopy.

Chemplex "X-Ray Mylar Film" is available in threegauges covering the entire x-ray spectral range:

One has a gauge thickness of 2.5 /im. It is used forapplications requiring reduced absorption of the primaryx-rays and characteristic long wavelengths, including the"L" spectral line series.

Another has a gauge thickness of 3.6 _um. This is ageneral purpose film gauge for both short and long wavelengthinvestigations. It is particularly well suited for analyzingsamples containing mixtures of both heavy and light elements.

68

Al-FORMVAR BACKING

Ep-3MeV

200 400 600 800 1000

CHANNEL NUMBER

Fig. 3-1 X-ray spectrum resulting from 3 MeVbombardment of Al-Formvar backing.

K Br Sr

600 800KANAL ANALIZATORA

1000

Fig. 3.2 X-ray spectrum resulting from 3 MeVproton bombardment of Scotch tape.

Third is a 6.3>»m gauge film used primarily for shortwavelength (heavy element) determinations. Its applicationsmay be extended to include moderately high concentrations ofelements having long wavelengths.

Chemplex "X-Ray Mylar Films are supplied in 7.6 cm x 91.*l mrolls in dispensers with cutting edges. Each roll adequatelyprepares over 1200 samples at two inches (5 cm) per sample, isconvenient to use, clean and eliminates waste.

Fig. 3-3 can serve as a guide in helping to select theappropriate gauge of Mylar film for any given x-ray spectralwavelength investigation.

69

100 0.4 0,6 0,8 1,0 1,2

WAVELENGTH/ nm

Fig. 3.3X-ray transmission through Mylar filmsof different thickness.

t.TRANSMISSION VS WWELENGHTFOR CHEMPLEX 6.3 ji POLYPROPYLENEX-RAC FILM

Fig. 3-4X-ray transmission through 6.3polypropylene x-ray film.

0,2 0,4 0,6 0,8WAVELENGTH/nm

Percent transmission for each of the three differentgauges is related to wavelength. Simply determine the wavelengthof the element to be investigated and select the gauge ofMylar film displaying the greatest transmission. For multi-element determinations the transmission values for the shortestand longest wavelengths should be considered.

Elements having wavelengths of 0.2 nm or less exhibitvirtually 100 percent transmission through each gauge.

The possibility of pinholes, pores and variations ingauge thickness existing in all thin film sample supportsregardless of form and packaging may present leakage of asample with subsequent potential contamination and damage tothe analytical instrumentation and its components, variationsin quantitative data and impose bodily injury to the user. Itis strongly recommended that each product and section to beused be subjected to judicious testing, use, applications andevaluation prior to actual use by the user.

Polypropylene x-ray films are unique by combining a lowmass absorption coefficient value with a gauge thinness of6.3 ^UID and a density of 0.9 gm/cm3 . These two propertiescontribute to the transmission of primary x-rays and character-istic radiation emitted by a specimen as shown in Fig. 3.4.To further enhance low level elemental determinations, Chemplexpolypropylene x-ray film is processed without additives,stabilizers or lubricants, which frequently cause limitationson detectability.

The high sample retention strength of polypropylenex-ray film permits safe sample handling in XRF Sample Cups.The sample planes are maintained taut for uniform target-to-sample distances and statistically replicate intensity

70

measurements. Chemical resistance to acids, alkalies andoils is excellent, and good for organic solvents.

In analyzing liquid and powdered samples in vacuumcontainment of the sample and maintaining a flat reproduciblesample plane, as defined by the thin-film sample support areimportant considerations. Samples encases in closed samplecups without provision for venting present the possibilityof internal pressure build-up and distension or rupture ofthe thin-film sample support. Samples introduced in open samplecups may abruptly and vigorously outgas and spatter.

Microporous film permits permeation of entrapped airand sample vapors in a closed sample cup through tortuousmicrometer-size channels. An equalization of pressure withinthe closed sample cup and the vacuum environment is estab-lished and avoids distension of the thin-film sample support.Intensity variations are minimized by maintaining a flatuniform sample plane and a reproducible target-to-sampledistance. The 0.1 jam micro pore channels prohibit the pene-tration and escape of sample material.

The opacity of microporous film is an indicator forgaseous permeation. The disappearance of opacity indicates"wetting" by low surface tension liquids which may causediminished gas permeability with continued use. High surfacetension liquids "bead" and resist penetration. Generally,gentle heating restores microporous film to original porosityfor reuse .

Microporous film may be used with Chemplex "XRF SampleCups". An "XRF Sample Cup" is equipped with a Chemplex samplesupport the sample is introduced, and microporous film isaffixed to the-top opening with a snap-on ring. Care shouldbe exercised in avoiding contact of the microporous filmwith a liquid sample to prevent unnecessary permeation of themicro pore channels.

Thin carbon foils (20 to 60 ig/cm2 ) are very convenientbackings. They are mechanically strong, and the breakage rateis usually less than 5%. Their high heat conductivity enablesthem to withstand A 1 /iA proton currents for over 60 min, butthis advantage over Formvar^ becomes less significant in viewof the need to restrict currents to é. 0.5 pA to prevent lossof volatiles from the specimen. For these thicknesses brems-strahlung background is negligible in comparison with back-ground .generated in the specimen material.

The effects of self-absorption are very important andshould be considered when the sample thickness exceeds somevalue. An expression can be obtained for the magnitude ofthe emitted radiation as a function of sample thickness ifone idealizes the geometry and makes several simplifyingassumptions. It is felt that although the problem is greatlyidealized, the answers so obtained are useful in estimatingthe errors one can expect due to thickness or absorptioneffects in the sample (Zeitz, 1969).

The necessary condition for a quantitative analyticalmethod is that the magnitude of the detected line must beindependent of sample thickness, or stated in another way,that the magnitude of the detected line must be a function

71

10- —————Zn

go.5-oj,o-§Q5-

Fig. 3-5MO-Cy 0.5-IC-0,5-0

Self-absorption for K^ lines of differentelements in biological tissue (Zeitz, 1969)

0 0,040,160,64 35610,24THICKNESS}'mgcm2

of the amount of element present in the sample and not itsthickness. One can develop an expression which will indicatethe error in accuracy (precision) to be expected due to theabsorption or thickness effect. The results of the calculationby Zeitz (1969) are shown in Figure 3.5. The ordinate isessentially the ratio of the power of K radiation of elementA actually emitted from the sample into a relatively small,well-defined solid angle to the power which would be emittedinto this same solid angle if there were no absorption withinthe sample.

The actual numbers will depend on the tissue compositionand for different tissue composition will be slightly differentIt can be shown that similar curves can be constructed for thecase of "fluorescence in reflection", or the detection offluorescent radiation emitted from the same side of the sampleas that of the entering exciting radiation. Curves for thiscase vary little from those given in Figure 3-5.

Careful choice of backing materials is very importantin trace element analysis since the background highly affectsthe accuracy of the measurement and the detection limit. Theideal backing is a thin film composed of elements of lowatomic number and possessing the properties of high mechanicaland chemical strongth and of high electric and thermal con-ductivities. Further-more, it must be of very high purity sothat the characteristic x-rays from impurity elements can beignored. In the study by Kaji et al., (1977) the advantagesand disadvantages of carbon, Formvar and Mylar as backingmaterials have been examined from the practical viewpoint.

Several authors have compared background spectra fromthin plastic films such as Mylar, Kapton and Teflon. Accordingto some reports carbon films of 40 ;jg/cm2 thickness canwithstand up to 1 h irradiation by 2.5 ;uA of -1.5 MeV protons.The use of thin films as backing will reduce the brems-strahlung background significantly compared to a thick backing.Heat dissipation by radiation is no doubt important at hightemperature for carbon films, but for thin aluminium filmsof good conductivity thé temperature rises only a few degreesbecause of heat dissipation. According to some reportsMylar can withstand proton irradiation by 150 A at 3-3 MeV,with some evidences of deformation, while 0.3 mil. of Kapton

72

can withstand a 30 min irradiation by 200 uA protons at3.5 MeV, but will burn away after a few minutes irradiationwith 350 uA.

In the study by Kaji et al., (1977), carbon filmswere prepared by evaporating onto a clean glass platecoated previously with a thin layer of glucose. The highsolubility of glucose facilitated the removal of thecarbon film from the glass plate on the surface of waterby dipping it in water and then the carbon film was mountedon a brass-plate target holder. The x-ray spectrum fromthe carbon foil thus prepared contained characteristicpeaks of impurities such as Fe, Cu and Zn superimposed onthe rather low continuous background. These suggest thatcarbon films are inadequate as backings unless ultra-purecarbon is employed.

Background spectra of commercial Mylar films bombardedby 3-5 MeV protons show small amounts of Ca, Fe and Zn super-imposed on the fairly high background were found in 10 jamMylar film. However, the background level of 4 jjm Mylar issufficiently low for the analysis of trace elements. Thus,Kaji et al., (1977) have adopted Mylar as the backing, takinginto account its mechanical and chemical strength and therelatively low background.

In their work Johansson et al., (1970) have used thinfoils of polystyrene as a backing materials.

Samples to be analysed are applied on thin foils( «s 40 ;ug/cm2 ) or polystyrene, either by being directly col-lected on these foils, as in the aerosol analysis program,or transferred to them. Solutions, e.g. can be spotted witha pipet and, after evaporation, the residue can be analyseddirectly provided care is taken to avoid absorption problems.Polystyrene is easy to handle and has considerable strengthIt also withstands sufficient.beam intensities to allowcount-rate limited analysis .

The carrier foils are prepared from a 4% solution ofpolystyrene in benzene. Microscope slides previously coatedwith NaOH are inserted and then withdrawn at a constant rateof several cm/s. After evaporation of the benzene, the poly-styrene coating can be cut to suitable pieces (e.g. 1 cm2),then floated off by obliquely lowering the glass slide intodistilled water. The foils are then easily picked up on analuminium frame with an ellipsoidal hole. The whole targetpreparation procedure is performed in a dust free laminarflow work station to reduce contamination. Eight frames withsamples are mounted together on a rod, held in a barrel that,when closed, shields the targets from airborne contaminantsduring the transportation to the irradiation site. The foilsgenerally have low blank values. In 33 blank foils, Ca wasdetected in 28 foils with an average of 3.4 ng Ca per foil inthe beam, Cl in 20 foils (4.8 ng), S in 16 (4.6 ng), Mn andFe in 11 (2 ng) as aerosol deposits from a single-orificeimpactor, are completely enveloped by the beam. In the caseof large homogeneous samples, the positioning facilitiesare only manipulated to make sure that no beam hits the frameon which the sample is suspended.

73

In the case of selfsuporting samples there is no needfor backing material. However, even in this case the samplematerial (usually solid) has to be mounted on the grame inorder to be expossed to proton beam. Since in many casesrather thin samples (o5_20yum) are required cutting bymicrotone is often used. In order to efficiently _use microtone the material to oe investigated must be firstembedded into the araldite.

Very often used araldite is AY103 with hardenerHY956. The only contaminants in araldite interfering withtrace element analysis are Cl and Br. For nonconductingsamples, for example biological material, it is necessaryto evaporate an aluminium lager (5-10 nm) to prevent chargingof the target and to reduce heating of the sample.

3.2 Target uniformity and homogeneityTarget uniformity and homogeneity should be checked

for any adapted procedure of target preparations. This isneeded especially when the beam spot size is smaller thanthe target area.

Let us describe uniformity test and homogeneity testas performed by Kivits (1980) for their sample preparationprocedure.

They tested the uniformity and homogeneity of targetsprepared using a solution consisting of Mn, Cu and Y (asnitrates) each 5 g/1. For testing they used a 2-mm diameter3.05-MeV proton beam, scanning along a diameter of, thetarget. Since rotational symmetry may be assumed, this scan-ning gives the radial distribution of the elements over thetarget. Results are given in Table 3.1. The normalised x-raypeak intensities of the three elements show standard devia-tions of about 6%. In calculation of the mean value, theposition at r = -13.^ mm is not taken into account becausethis spot is just at the edge of the wetted area, as wasobserved after irradiation on the basis of the colour change.The areas normally analysed with PIXE have diameters rangingfrom 5 to 15 mm; for such large areas the authors claim anon-uniformity of lower considerably than 6%. Inhomogeneity,i.e. deviation from a constant composition of elements, isclearly demonstrated in the last two columns of Table 3.1. Thestandard deviations for the ratios Cu/Mn and Y/Cu are 2.3%and 3-3% respectively, including the spot at -13.14 mm.

3.3 ReproducibilityFor any adapted method of target preparation the

reproducibility should be tested. This is usually done bydividing a larger sample into a number of subsamples andthen preparing the number of targets following identicalsteps in target preparation. Results obtained by Wheeleret al., (1974) from the measurements of some 50 samplesof human blood serum are presented in Figure 3.6.

The distribution of concentration values obtained forCa, Fe, Cu, Zn, Br, and Rb shown, together with their

74

Table 3.1

Results of scanning the target accross a diameter(27 mm) with a proton beam of 2 ram diameter,

as reported by Kivits (1980)

r. ______Uniformity test__________Homogeneity test(mm)

11.87.63.80.0

-8.4-13.4

Mn K

1 .10

0.920.980.981.020.84

1 .00

±0.07

Cu K

1.05

0.931 .01

0.971 .040.83

1 .00±0.05

CY K

1 .02

0.891 .04

1 .02

1.030.84

1 .00±0.06

Mn K

0.3350.3490.3580.3470.3560.345

0.348±0.008

CCu K

0.352

0 .3490.3730.3800.3590.370

0.364±0.012

Standard deviations obtained by least squares fitting toa normal distribution. The results show that trace elementscan be measured at the 1 ppm level (as seen in Cu) withan accuracy of - 15%. The greater uncertainty in determiningBr concentrations is consistent with the findings thatBr volatilizes even at low beam intensities.

Kivits (1980) has also tested the reproducibility ofhis method of target preparation from homogenized fish meal.For this purpose 1 gram was introduced into a 10 ml Formvar-dioxana solution. The average number of .x-rays per secondand per nA, with attendant relative standard deviation, wasdetermined for some elements (P, S, Cl, K, Ca, Fe): forP K 26.8 ± 5.3%; for S K 25.8 - 9.2%; Cl K 29.2 - 5.1%;K K 28.6 - 9.7%; Ca K.^59.5 - 4.7%; Fe K^ 2.1 - 11.3%. Sta-tistical errors are negligible. This means that the irre-producibility over the ten targets in "the amounts of materialirradiated is at most about 5%, assuming that the elementsphosphorus, chlorine and calcium are homogeneously distributedover the sample. The high relative standard deviation forthe other elements is largely due to the inhomogeneity of theoriginal sample. The contribution of sample inhomogeneity tothe P, Cl and Ca concentration is shown by the relativestandard deviation of the intensity ratio between the K x-raysof an element and those of calcium in each target: P K/Ca K^ =0.45 - 5.4% and Cl K/Ca K^ = 0.49 - 5.5%. The relativestandard deviation observed indicates for these elements acertain sample inhomogeneity, too. This implies that the ir-reproducibility of the amount of material irradiated issubstantially less than 5 per cent.

75

-40 -20 0 20 40 -40-20 0 20 40

DEVIATION (%)

Fig. 3.6 Distribution of concentration values measuredfor Fe, Cu and Zn in human blood serum byFIXE (after Wheeler et al., 197*0.

3.4 Effect of irradiationSeveral investigators have studied the effect of

irradiation on the sample. Probably the most detailed studyhas done by De Rooij et al., (1981) and we shall reportsome of their findings.

Important parameters for losses of elements duringirradiation are the volatility, and the thermal and radiationstability of the analytes. Other parameters are the volatilityof the radiolytic and thermolytic products of the analytes.With respect to the thermal and radiation stability, thebeam intensity and irradiation time are important parameters.

In the study by De Rooij et al., (1981) the effect ofthe several parameters was investigated by repeated bombard-ment with 3-05 MeV protons of the same target. The elementalconcentrations were determined for each of these measurements.To obtain an idea about the effect of volatilisation lossesin inorganic compounds, they investigated the behaviour ofinorganic chlorides and bromides. For these experiments thebeam intensity was kept constant at 50 nA/cm2 . The initialconcentrations of th-e halogenides were taken as 7.5 ug/cm2 .Table 3.2 shows the losses after 15 minutes irradiation forvarious chlorides and bromides. There are considerable dif-ferences in percentage loss between the halogenides; theselosses occur usually in the first 8 min of the bombardment.Two typical curves showing the time-dependent behaviour areshown in fig. 3.7. In these cases, the amount of copper andrubidium remained constant (-2%) during irradiation. Thelosses of the halogenides observed in our experiments are ofthe same order as those observed by Ishii et al., (1975)under comparable conditions.

The second parameter investigated by De Rooij et al.,(1981) was the concentration of the volatile element. Theexperiments, were repeated for CuBr2, with concentrationsranging from 5 to 20 ug/cm2 bromine. All curves had a similarshape, showing a plateau after about 10 min. The plateau cor-responds to a bromine decrease of 13% - 1%, independent of

76

Table 3.2

Percentage loss of Cl and Br for several compounds. Beamintensity 50 nA/cm2 , halogenide concentration 7.5 ug/ctn2 ,

proton energy 3-05 MeV and irradiation time 15 min.(after De Rooij et al., 1981)

Compound Binding energy(eV/atom)

Percentage loss

CuBr-NaBrRbBrCsBrZnCl2CuCl2KC1BaCl2

3.43.73.94.22.43-94.45.0

1466-9

205205

10-12

IS

Fig. 3-7 Percentage loss of bromine for rubidiumbromide and copper bromide on Selectronfilters, as a function of the irradiationtime. The bromine concentration was 7.5the beam energy 3.05 MeV and the beamintensity 50 nA/cm2 . (After de Roo'ij etal., 1981).

the initial concentration. The effect of beam intensity onlosses was checked for CuCl2(7.5 pg/cm2 chlorine), as is shownin fig. 3.8 for 50 and 100 nA/cm2 beam intensities.

As it is seen from the Figure 3.8 substantial volatili-zation may occur during the irradiation. Similar effects havebeen reported by Alexander et al., (1974), see figure 3.9.

77

100-

20

Fig. 3.8 Percentage loss of chlorine for copperchloride on Selectron filters, as a functionof irradiation time. The chlorine concen-tration was 7.5 /ig/cm2 , the beam energy3.05 MeV and the beam intensities 50 and100 nA/cm2 . (After de Rooij et al., 1981).

Q.Q.

zluooo

80706050,80'70601412IO8&4./.2

I

SEAWATER

-t1

K -

-i Co -

3 Cu _

Fig. 3.9

50 IOO 150 200 250 300 350

PROTON BEAM INTENSITY (nA)

Concentrations of different elements asdetermined from the bombardment of seawater targets by 3 MeV protons as afunction of beam intensity.

Concentrations of different elements were determined from thebombardment of sea water targets by 3 MeV protons as a functionof beam intensity. The results show the variations of K, Cuand Br concentrations with the beam intensity, with Br varyingsharply from 5 to 15 ppm.

In order to reduce these losses De Rooij et al., (1981)have considered surface treatment of the target by addition offixative agent or introduction of a shielding layer. In theirstudies dioxane was used as a fixative; this partly dissolvesthe selectron support, resulting in a collapse of the pore

78

Fig. 3 .10

DIOXANE

UNTREATED

FORMVARIN 100ml

j OOXANE

0 2 ' 4 ' 6~~TOTAL NUMBER OF X-RAß DETECT-ED (106 COUNTS)

The effect of different target treatmentson the loss of chlorine for copper chlorideon Selectron filters. The chlorine concen-tration was 7.5 ug/cm2 , the beam energy3.05 MeV and the beam intensity 40 nA/cm2.The Cl K X-ray intensity is shown per amountof 5x105 x-rays detected. (After de Rooijet al. , 1981 ).

structure, with inherent inclusion of the elements. A shieldinglayer of Formvar may be introduced on both sides^, by dispensinga solution of Formvar-dioxane onto a target. The Formvar layerwas expected to prevent volatilisation of the elements dueto its low permeability. The effect of different targettreatments on the volatilisation of chlorine from a CuCl2target is shown in fig. 3-10, with a beam intensity of 40nA/cm2 . Similar results were obtained for beam intensitiesof 25, 50 and 100 nA/cm2. The beam intensity usually usedby us is less than 100 nA/cm2 . As can be seen, the surfacetreatment is most effective for short analysis times (-«v-S min).For a beam intensity of 40 nA/cm2, the collection of 10"counts takes about 500 s.

Huda (1975) has investigated the effect of beam ofcharged particles on thick dried plasma pellets.

In a thick biomédical sample 2.5 MeV protons arecompletely stopped in a distance of about 10 mg/cm2. The beamin these experiments covered an area of~0.13 cm2 and thereforea beam of 50 nA delivers about 125 mW to the sample mass ofsome 1.3 mg. Under vacuum conditions the temperature wouldbegin to rise at over 1O3 C/min and samples of low thermalconductivity become damaged by charring. Apart from loss ofvolatile elements, concentration of trace elements has beenobserved due to loss of the low Z organic matrix and thereis also the possibility of contamination of the acceleratorsystem.

The charge deposited onto a thick biomédical target,which is a good insulator, must leak away to the backingmaterial which consisted of a pure aluminium foil. The

79

surface of the sample can thus get charged up to a highvoltage during the proton bombardment, attracting freeelectrons in the sample chamber which in turn gives rise toelectron bremsstrahlung x-rays.

Charge build up is also observed on the hair samplesunder proton bombardment. This appears to depend on thethickness of the hair, among other factors. It results inthe presence of a high background in the spectrum, due toperiodic discharging and should be avoided. For thick targetsthis can be accomplished by blending with graphite. For thintargets it may be necessary to coat with very thin layersof carbon or other suitable conducting material.

Other techniques of avoiding the problem are alsorecommended here. It is possible to place a heated tungstenfilament near the sample. The emitted electrons preventcharge buildup. It is also possible to arrange the apparatussuch that the sample is actually irradiated within theFaraday cage, and the beam enters via a small hole; the cageis differentially pumped and the pressure maintained atabout 10-2 torr. The effect of this is to remove charge fromthe sample by carrying it on gas molecules.

3.5 Internal standardsInternal standards are materials added to sample

under investigations in known chemical forms and pre-determined concentrations. Very often is it useful toknow the shape of the x-ray spectra for the targets ofknown composition. This is required when the compositionof sample is being guessed without the aid of a computer.In order to help workers in the field of proton-inducedx-ray emission spectroscopy the following three figuresare shown :

Figure 3.11 is the x-ray spectrum obtained by protonirradiation of target with 9^9 ppm Hg (L-lines)and 65.5 ppm Y (K-lines); Cu and Pb are presentas contaminants.

Figure 3-12 shows the iodine lines; ratio of I/Yconcentration was 25 .

Figure 3-13 shows the x-ray spectrum from Ni(50 ppm)to Pb (952 ppm) target. All three targets wereprepared on Al-FormvarR backing by drying afew drops of solution.

Determination of relative elemental sensitivities forany system based on the detection of characteristic x-raysrequires the step of preparation of standards. Differentauthors use different approaches for this measurement. Forexample, in the work by Kubo''22 the following chemicalswere chosen for a standard solution: Ca(NC>3)2 4H2Û, Fe(N03)39H20, Cu(N03)2 3K20, Sr(N03)2, AgN03 and Pb(N03)2. The Fe,Pb, Ca, Fe, Cu, Sr, and Ag compounds were dissolved in 200 cm3doubly distilled water. A small amount of standard solutionwas pipetted onto the backing foil which was placed on themicrobalance, and the target tare weight was then determined.

80

200 400 600 800CHANNEL NUMBER

1000

Fig. 3.11 Mercury spectrum (Yttrium is theinternal standard).

400 600 800CHANNEL NUMBER

1000 1200

Fig. 3 - 1 2 Iodine spectrum.

81

Pb(952ppm)

Al-FORMVARBACKING

400 600 800CHANNEL NUMBER

1000 1200

Fig. 3.13 Spectrum of lead(nickel is added as dopant).

" Ä 5 Ö i b T BOO loi»CHANNEL NUMBER

1200

Fig. 3.14 X-ray spectrum of Fe-Cu-Zn-Pb-Hg-Sr-Ysolution (all elements 1000 ppm) as aresult of irradiation with 3 MeVprotons. All elements 1000 ppm, Al-formvar backing.

82

These values were then used to obtain absolute concentrationsof trace elements in biological samples.

Usually an element which does not appear in samplesto be studied is chosen as an internal standard,. This isoften yttrium. Before measuring the samples, a set of stand-ards which contain the known rations of yttrium and otherelements needs to be measured. For example, when thesolution Fe-Cu-Zn-Pb-Hg-Sr-Y (all elements 1000 ppm) was usedfor the target preparation, and the target irradiated with3 MeV protons, the spectrum shown in Figure 3.14 resulted.Intensities of peaks corresponding to different elements arenot all the same because of the differences in:

1 .. Detector efficiencies for different energies2. Different cross sections for the production of

x-rays .The method of internal standards relies on the mass

absorption coefficients cancelling out of the intensityratio equations , so that

VJs = K WVWtswhere I , Wt = integrated intensity, weight of component

P P p in the sampleI , Wt = integrated intensity, weight of internal3 s standards

K = constant which depends on p and s, but isindependent of the matrix

K can be found by calculating the slope of the graph ofintensity ratios vs weight ratios. In our calculations a'modified least squares linear fit is assumed, with theintercept forced to be the origin.

In the work by Roelandts et al., (1978) the internalstandard technique was adopted in order to minimize targetinhomogeneity effects and to correct for beam currentfluctuations. Silver was selected for this purpose, asthis element is very rare in usual samples. Its K,*, lines(K i = 22.1 keV, K«c2 = 21.9 keV) were used as internalmonitors .

A 500 /ag Ag/ml stock standard solution was preparedby dissolving AgNU3 in water. A few drops of dilute nitricacid were added for stabilization.

Individual stock standard solutions ( 1000 jug /ml) ofeach element of interest (Y, La, Ce, Nd , Dy, Ho, Tm andLu) were prepared by dissolving accurately weighed amountsof "Spec pure" oxides in nitric acid, evaporated nearly todryness and taken up in very dilute nitric acid. By suitabledilutions of the stock solution, in 10 ml -graduate flasks,a range of required calibration standard solutions was made.1 ml of Ag internal standard solution was added by pipetteinto each 10 mi-graduate flask before dilution to volume.

Aluminized mylar foils (0.5 mg/cm2 mylar, 0.4 mg/cmzaluminum) inserted between two rings made of aluminum wereadopted as support materials. Aluminum helps the preventionof charging of the samples.

83

Calibration targets were prepared by pipetting aliquotsof 200 ul of the calibration standard solutions of Y(+Ag)and the individual REE(+Ag) onto these mylar disks and evapo-rating to dryness, exposed to the air. Afterwards they wereplaced in an oven at 40 C and finally stored in a desiccator.Thus five calibration targets were prepared for Y and in-dividual REE: 5 ,Ug, 10 ;ig, 20 >ig, 50 ;ug and 100 ;ag, each ofthem containing 10 ;ug Ag as an internal standard element.

Buso et al., (1982) have used tellurium as an internalstandard for determination of selenium in biological samplesby PIXE.

15-

Fig. 3 -15

200 400 600 800m(Seyng

1000

Selenium determination at nanogram levelwithout organic matrix (after Buso et al..1982).

The first step of target preparation was the destructionof the organic matrix. The destruction was done in non-oxidative way, unlike usual methods, to allow the subsequentprecipitation of selenium, which can take place only inreducing medium.

Tellerium (600 jug) was used both as internal standardand as coprecipitant. The suspension obtained is then filteredon 1 cm2 millipore filter. Proton beams damage milliporefilters even at very low current; therefore it was neces-sary to support these filters with a suitable backing duringirradiation. Whatman filters were chosen for their good beamresistance and low background. First the authors have studiedthe efficiency of the precipitation at nanogram level withoutorganic matrix.

Some results obtained by Buso et al., (1982) are shownin Figure 3-15. The reported errors are due to countingstatistics. Similar correlations were obtained with differentquantities of tellurium in the range 200-1000 jug; 600 jug waschosen as a good compromise value, taking into accountcounting rate and target resistance.

The efficiency of the technique with organic matrix isshown in Figure 3-16: 1 cm3 serum were doped with increasing

84

TOO 2 0 0 3 0 0m(Se)/ng

400

Fig. 3.16 Efficiency of selenium precipitationwith organic matrix (1 cm3 serum), seetext; after Buso et al.. 1982).

quantities of selenium (50-400 jug) and then treated aspreviously described. The efficiency measured with and withoutorganic matrix is the same, it means that the recovery ofselenium added is not affected by the organic matrix. Onecan therefore use Figure 3-15 as calibration curve forselenium determination with this preconcentration technique.

3.6 Examples of sample preparation for PIXE3.6.1 General

Target preparation for irradiation by chargedparticles often requires that the sample be solubilized.For most biological substances, the two common proceduresfor organic matter destruction are wet digestion and dryashing. The advantages and disadvantages of these twotechniques have been researched by many. The best and mostcomprehensive work on this subject is by Gorsuch (1970).For routine plant analysis and most other biologicalmaterials, the dry ashing technique has been used suces-sfully by many researchers. Losses of elements or contami-nation have been minimal. High wall ashing vessels andthe placement of the vessel up off the floor of the mufflefurnace are essential parameters necessary to minimizepotential losses of the more volatile elements (Jones, 1976)

Thin specimens may be prepared directly from wetplant or animal tissue by various physical methods. Valkovicet al., (1974) crushed tissue in water and stored theresulting suspension until the cells were dissociated.However, the suspension was not particularly homogeneousand consistency for supposedly equivalent targets was notgood. Jolly and White (1971) have described a nebulizerwhich directs a fine mist of droplets from sonicated mate-rial towards a backing, on which a thin film builds up.

85

Campbell et al., (1975) have prepared slices of 10 /amthickness by cutting with a stainless steel blade using afreezing microtome; freezing to enable slicing is prefera-ble to embedding with a hardening agent (paraffin) fromthe contamination aspect. Slices of diameter 5 to 10 mmadhere satisfactorily to carbon foils.

Several workers have made thin targets by depositinga very small mass (£1 mg) of lyophilized or ashed mate-rial on a backing foil, adhesion being effected by addinga drop of wetting agent or glue. This approach has alsobeen used, by various authors, for specimens such as finelypowdered rock, and seafloor sediments.

Such targets have thicknesses of the order of 1 mg/cm2and there are thus no x-ray self-absorption problems.Although one might expect the powder produced by lyophiliza-tion or ashing to be sufficiently homogeneous that 1 mgsamples would be representative of the bulk, is notnecessarily so. For example, the National Bureau of Standardswarns users of its standard trace element reference mate-rials SRM 1571 (orchard leaves) and 1577 (bovine liver) that0.25 g is the minimum mass of powder that will afford arepresentative sampling of the bulk.

There are several methods of disintegrating cells,among which ultrasonic irradiation has been accepted ascertainly one of the most effective. The disruption ofliving cells by ultrasound usually depends on the soundintensity in the medium being maintained above a criticalvalue sufficiently high to induce the phenomenon! knownas "cavitation". Cavitation is a complex process whichmay be summarized as follows. When a liquid is subjectedto sound intensity above the critical value the transportof the sound waves through the liquid causes very rapidalternations of pressure, resulting-in the formation oflarge masses .of minute gas-filled bubbles. These grow andpulsate through several sound cycles until a criticalsize is reached, the magnitude of which depends on thefrequency of the sound. At this stage the bubbles dis-integrate implosively, giving rise to intense local shockwaves, high instantaneous temperatures and to micro-streaming of the liquid around the points of collapse.The streaming around bubbles produces high shear gradientswhich are responsible for the degradative effects oncells.

The preference for the particular method of targetpreparation depends on many factors, most of them beingsubjective in nature. One should keep in mind that thereis no universal prescription and that usually severalmethods will give equivalent results. This is indicatedalso in Figure'3»17 which shows the x-ray spectra- fromthe targets of microorganisms, mold, which was harvestedby the collection on filter paper. Spectrum A resultsfrom a direct exposition of such a filter paper withproton beam. Because of problems with insulating backingsSchotchR tape is used as a conductor (spectrum B). Thishas introduced bromine as a contaminant. Spectrum C wasobtained by ashing filter paper with harvest and subsequentdeposition of ash on ScotchR tape. The last spectrum (D)

86

Mn Cu«ft1C*

1C*.o2-

iio'-8«a

V)

Si#^lO2

1C*

id-

10-0

TARGET PREPARATIONTECHNIQUES

Sr

a 4MO 400 $00 KO K

CHANNEL NUMBER

Fig. 3.17 X-ray spectra from the same biologicaltarget prepared on different backings(see text).

is obtained by irradiation of harvest which was transferredfrom filter paper onto Scotch^ tape. With careful analysis,and taking care of background radiation, information onthe concentration of essential trace elements (Cr, Mn, Fe,Ni, Cu, and Zn) was obtained in all the cases.

3.6.2 Aqueous samplesWater and some other liquid samples can be prepared

by drying a few drops on an appropriate backing when chargedparticles are used for excitation. This method is not goodenough for the sample excitation with x-ray tube or radio-active sources, or when concentrations smaller than 0.1 ppmneed to be measured. In cases like this preconcentration ofelements to be analyzed should be determined. This is usuallydone

1 . By evaporation2. By complexing the metals with ammonium pyrrolidine

dithiocarbamate and extraction with methyl isobutylketone (the concentrations can therefore be dopedwith an internal standard and evaporated on anappropriate foil)

87

3. By complexing some elements with oxine and absorbingthe dissolved complexes on activated charcoal(Vanderborght and Grieken, 1978).

There are some other procedures described in more detailin the chapter on trace elements in water.

Very often ammonium-1-pyrrolidine dithiocarbamate isused as chelating agent. It forms insoluble coordinationcomplexes with more than 30 transition metals (for details,see Elder et al., 1975).

De Rooij et al., (1981) have described the method forvarious target preparation techniques for liquid samples.

This method can be shortly described as follows:a) Selectron cellulose-acetate foil (5 mg/cm2) is

employed as a supporting material;b) The aqueous sample is diluted with ethanol 1:1 (v/v);c) Selectron filter is rotated at 18 000 rpm;d) 50 ul of the solution is dispensed as quickly as

possible (4 0.2 s), yielding a wetted area of 570 mm2 ;d) The target is air dried at room temperature.Sometimes, two-phase systems like emulsions and sus-

pensions have to be analysed. Such systems may involve amore complex physical behaviour in target preparation. However,the physical behaviour of suspensions may be changed bychemical treatment into that of a homogeneous real solution.The advantage of thin targets is that the intensity of theproton beam, striking the target, can be measured.

Several authors use external beam for the analysis ofliquid targets. For example in the work by Tsang-Lang Linet al., (1979) Van de Graaff accelerator was used as a sourceof protons. The plastic target holder is mounted directly onthe extension tube of the Al chamber. Dry nitrogen is con-tinuously and slowly flushed in the volume between the exitwindow and the sample in order to suppress the x-rays ironargon in air. The argon K x-ray peaks, which were severeoriginally were supressed effectively.

Liquids are contained in Chemplex XRF sample cups witha 6.35 pm thick mylar film as cover. Protons panetrate themylar film and are then slowed down in the liquid. The x-rayswere detected by a Si(Li) x-ray detector which was placed ata distance of 8 cm from the sample. Detector signals wereamplified by an optical feedback preamplitier, a linearamplifier and men analysed by an MCA. A lead shield was usedto prevent the x-rays produced at the proton exit windowirom reaching the detector. Proton beam currents can bemeasured beforehand by placing a metal target at the samplesite to collect proton charges. Proton currents were extractedcontinuously from the isolated exit window which was servedas beam monitor during the measurement.

Mylar films of 6.35 urn thickness were strong enoughin tolerating proton bombardment up to total proton chargesof 10 uC. However, it became fragile and deformed at thebeam spot. A deformed surface will alter the absorption

88

factor of x-rays in the sample thus changes of the mylarfilm cover are necessary after it has been exposed to acumulative proton beam of 10 C charges.

!n literature, several techniques for depositing aliquid onto a support are described. A multi-drop techniqueis described by Camp et al., (1974). A quantity of 220 ul isdeposited in drops of 10-15 jal on a support, just enough towet the entire surface. The reported standard deviation ofthe composition over the wetted area is 3% when measured withPIXE using a 5-mm diameter proton beam. In our experience itis difficult to obtain reproducible results with thispreparation technique. This is probably due to ""fractionalcrystallisation or different migration speeds of the elementsin the supporting material (of. paper chromatography).

The preparation method of dropping a solution on aMylar foil of 10-jum thickness and drying in the open air atroom temperature tends to give non-uniform targets, as wasshown by Ishii et al., (1975).

Baum et al., (1977) describe a method using a deviceconsisting of 37 capillary tubes of equal and known volume.When dipped into a solution, the tubes fill by capillaryaction. The device is then placed on a filter, and the capil-laries drain, simultaneously wetting the entire filter area.Freeze drying is necessary because otherwise the compositionof different elements along a radius will not be constant dueto different migration speeds, as is mentioned by the authors.Target prepared according to this method are reported to behighly reproducible. However, besides the drawback of theneed for freeze drying there is another disadvantage: thecurling of the. filter paper after the wetting-drying process.This implies a deterioration of the target geometry withinherent variable x-ray attenuation, especially for low-Zelements. Practical disadvantages are the fragility and therather complex cleaning of the capillary device.

Let us describe in some detail/ method used by Kivits(1980) for target preparation for real solutions:

For target preparation of aqueous samples based onthe use of a support Selctron OE67 foils (Schleicher andSchull, Dassel, F.R.G.) as supporting material were chosen.The area density of Selectron is about 5 mg/cm2 , and elementalanalysis gave an overall composition of 032^4023. Byrapidly depositing the liquid on a rapidly rotating Selectronfoil one can achieve uniform distribution of the solutionover a constant area. It was found experimentally that thesolution was uniformly spread over the entire wetted volumeof the target. Besides the Selectron support, the authoralso investigated thinner supporting materials such asNuclepore and Formvar. These supports proved to be inap-propriate for the rotating-foil technique, because they arenot easily wettable.

The apparatus developed for the preparation of thetarget is shown in 3.18. A microdispenser is centered abovea rotating table on which a ring can be mounted. The outerpart of the Selectron foil is clamped into the ring. Theinner part of the foil which is also the area to be wetted,does not touch any part of the device. Air drying is applied

89

Fig. 3-18Apparatus for target preparationfrom liquid samples as described byKivits (1980):(1) Microdispenser,(2) height adjuster,(3) centering column,(4) tip of dispenser,(5) target (frame),(6) rotating table,(7) Perspex house,(8) motor.

before removal of the foil from the ring and is found to bevery effective in preventing curling of the foil.

Parameters that can be adjusted in order to obtainmaximum uniformity and homogeneity are the speed of rotation,the distance between the tip of the dispenser and the Selectronfoil and the speed of depositing the liquid. Moreover, theparameters of the liquid (e.g. the viscosity and surfacetensor) can be changed by the addition of certain chemicals.

The author has investigated aqueous samples, of coppernitrate (1 gram copper per litre). Aliquots of 50 ul weredeposited on Selectron foils; in order to visualize thedistribution of the liquid, methyl red was added. For targetsprepared with a rotation speed of 15.000 rpm the distributionof the liquid was found neither uniform nor reproducible.Moreover, part of the sample was lost by sliding over thefoil with a high speed and was found back on the edge ofthe apparatus. To investigate the influence of the surfacetension, different kinds of synthetic detergents and aliphaticalcohol were added. The addition of ethanol proved to bemost successful. Although addition of propanol decreases thesurface tension more than ethanol, the author has opted forthe latter, to avoid problems with precipitation of ionicconstituents. Under the chosen working conditions a volumeof ethanol equal to the volume of the aqueous solution gave,on visual examination, sufficiently reproducible uniformdistribution with 18.000 rpm. Another parameter investigatedis the rate of dispension. The best results are obtained byrapid dispensing (<0.2 s); slow dispensing results inring-shaped distribution.

Some information about the transport mechanism ofthe solution on the Selectron foil was obtained from theollowing experiment: 25 pi of red coloured solution was

sucked into a 50 _ul pipet followed by 25 jul of a yellowsolution. The resulting 50-jal volume was immediatelydispensed on a foil in order to prevent the solutionbeing mixed up. The resulting target showed the yellowcolour in the inner-circle surrounded by a red ring, with

90

a sharp borderline between the two areas. Moreover, goodrotational symmetry is clearly demonstrated. Thisphenomenon suggests a mechanism in which the firstfraction of the dispensed solution is absorbed in thecentre part of the foil, subsequent liquid fractions eachtime sliding over the previous fraction.

Measurements of the coloured area of 50-;ul targets(methyl red is used as dye) gave an average wetted area of570 mm2 with a standard deviation of 20 mm2. Moreover,addition of a dye has another advantage: after irradiation,the proton-beam-exposed area can be determined accuratelydue to a change in colour. This may, for instance be help-ful in outlining the proton beam.

Because of the constant amount of liquid deposited(50 pi), the reproducible wetted area (570 mm2), theconstant porosity of the Selectron foil and its low areadensity (5 mg/cm2), good analytical results are ensured.

Based on the results described above, Kivits (1980)adopted the following standard preparation procedure forreal solutions:

(a) Selectron is employed as a supporting material;(b) The aqueous sample is diluted with ethanol 1:1

(v/v) and coloured by methyl red;(c) Selectron filter is rotated at 18.000 rpm;(d) 50 ul of the solution is dispensed as quickly

as possible (4 0.2 s), yielding a wetted areaq£_5_70 mm2';

(e) The target is air dried at room temperature.The authors has also checked by PIXE analysis the

retentivity of the target for volatile elements such asCl and Br in different inorganic compounds (CuCl2, CuBr2>RbBr, RbCl). Storing the target at room temperature inthe open air for one week did not influence the amount ofanalyte in the target; neither did vacuum pretreatment of12 hours at 30°C.

The retentivity for CuBr2 targets was also checkedin a radioactive tracer experiment. CuBr2 activated vianeutron irradiation. The intensities of the °^Cu and 82ßrj^-rays were measured in aliquotsof 50 ul CuBr2 solutionand in targets onto which 50 p.1 was deposited. No significantdifferences were found. From this experiment the author has \concluded that the whole aliquot is retained by the supporr, (?)either in the dispensing step or in the drying step.

The retentivity of the targets prepared with thisradioactive CuBr2 solution was also checked for a prolongedstorage time. Targets were kept at 30 Ç in vacuum for 24hours and some targets were kept at 57 C in the open airtfor the same period. Both treatments gave no significant>:losse"s".

3.6.3 Biological samplesTarget preparations techniques for biological samples

have been discussed in many papers. For example, Jundt et

91

al., (1972) have used.Formvar^ backing and diffused protonbeam. Techniques they have used in preparing biologicaltissue samples include:

1. Samples deep-frozen, then sliced with a microtome.2. Washed, formalin fixed and paraffin embedded, then

sliced with microtome, after removing paraffin withan appropriate solvent, deposited on target backing.

3. Homogeneous solution in distilled water depositedon backing and dried.

Renan and van Rensburg (1980) have describedthe proceduresused for the preparation of sample from biological"material(laboratory animals). Liver and oesophagus samples wereobtained at autopsy; sections 1-2 mm in thickness were usedfor the trace element determinations. The tip of the middlelobe of the liver (after removal of the outer capsule), andthe epithelium of the middle third of the oesophagus wereanalysed in each case. Care was taken to ensure that con-nective tissue and visible blood vessels were not includedin the samples for analysis. All tissues analysed wereinfinitely thick in the sense that the projectile beam stoppedcompletely in the material; the section thickness used wasgreater than the range of protons in an organic matrix atthe energy used (approximately 15 mg cm~^ at 3 MeV). Althoughthe section's were prepared with as uniform a thickness aspossible, most of the x-ray yield originates from the outermostlayers of the specimen (where the production cross-sectionin greatest and x-ray absorption least); thus small inhomoge-neous patches will not greatly affect the accuracy of theanalysis.

Numerous investigators have cautioned againstcontamination in the analytical laboratory; the well knownwork in this area is that of Sansoni and lyengar (1978).The stringent protocol adopted should be based on theguidelines suggested by these authors.

In the work by Renan and Rensburg (1980) plasticworking surfaces and gloves were used throughout, as werePerspex knives for the dissection. Unfortunately theseknives were inadequate for the removal of the epithelialtissue from the. oesophagus and stainless steel surgicalinstruments were necessary. The contamination so introducedis probably not significant but the results for Cr in thisorgan should not be regarded as reliable. Immediately afterdissection, the samples were mounted on clean Perspexholders and freeze-dried at -50 C for 2^ hours. The sampleswere then stored in sealed plastic containers in a vacuumdesicator prior to analysis. The tissue samples were analysedintact, i.e. no ashing, grinding or acid digestion was foundto be necessary. As a consequence, differential losses ofelements and inhomogeneities due to fragmentation were keptto a minimum.

Chen et al., (1977) have measured nickel concentrationsin lung and kidney tissues. In their work samples (0.1 to0.5 g) of tissue were weighed in sterile, capped plastictest tubes. Concentrated nitric acid (Baker "Ultrex") wasadded, and the samples were heated to promote digestion.

92

Portions (10 ul) of the solutions were evaporated to drynessand analyzed directly in a 2-MeV proton beam. Proton-inducedcharacteristic x-rays, including those of nickel at 7.472 keV,were detected in a Si(Li) detector, and the nickel concentra-tions were determined. The quantitative calibration wereperformed and cross-check measurements by analyzing samplesto which known concentrations of nickel had been'added.

Hair is one of the biological materials often analyzedby PIXE. Analysis by PIXE is easiest in two cases; when thesample is extremely thin, and when it is infinitely thick tothe proton beam. Cases between these two extremes are analy-tically difficult, and it is recommended that sample preparationbe used to convert samples to one of the two forms. Thisapplies generally to biological samples, but specificallyto hair, which is neither infinitely thin nor infinitelythick for protons at energy 2-3 MeV and in addition isheterogeneous. Special sample preparation is therefore es-sential 'for quantitative work, except where specific infor-mation is required about the distribution of elements withinthe hairs, or much less rigorous quantitative data is required

Three possible methods of preparation can be recom-mended: (a) pulverisation and preparation of a thick target,(b) dissolution and evaporation on a thin backing, and (c)embedding and sectioning.

(a) Pulverisation is recommended to the level ofapproximately 50 /am. Various techniques are possible: (1)using liquid nitrogen and an ultrasonic vibrator, pulveri-sation in a teflon container with a teflon bail, or (ii)pulverising without freezing in an agate mortar.. In eachcase about 1% of reactor-grade pure graphite may be addedto avoid charging effects.

(b) The procedure of dissolving hair can be best donein hot concentrated nitric acid rather than concentratedalkali, but that the technique be checked for volatilizationof mercury (and possibly other elements) before it be usedon a large scale for determination of that element. Thesolution or a portion of it (with possible addition of aninternal standard) should be dried on a thin film of somepure resistant material. Hostaphan and Kapton are alreadyknown to be satisfactory though the problem of flaking needsfurther investigation. Any other material should be checkedcarefully for purity before use on an extended scale.3.6.4 Blood serum samples

Whole blood and serum samples are often investigatedfor trace elements using PIXE as the analytical techniques.We shall here describe some of the approaches taken inthese studies.

It has been demonstrated by Valkovic (1973) that theblood serum target might be prepared on A1 + formvar backingwith satisfactory reproducibility of results. In theirstudies the blood plasma was doped by 100 ppm solution ofyttrium.

Berti et al., (1971) have determined selenium in theserum. Palladium was chosen as internal standard for the

93

determination of selenium. The targets were prepared in thefollowing way: 1,2 cm3 of blood serum were doped with 100 ppmPd as PdCl2. Serum and internal standard were ultrasonicallymixed, dry-ashed in borosilicate glass beakers for about1 h at 60 C, ground into a fine powder using an agate mortarand then thoroughly mixed with 20% of nuclear graphite powder.

A self-supporting pellet was prepared from this mixtureby pressing at 4x103 kg/cm2 into a suitable die. The resultingpellets are 12 mm in diameter and have area density 50-60 mg/cm2 ,which constitutes infinite thickness for protons in our energyrange. By varying the graphite concentration in the mixture,it was found that about 20% allows the pellets to-withstand thebeam heating for the time necessary to reach the requiredsensitivity, (see also Calaritti et al., 1980; Perona et al.,1977).

One should take precautions to avoid contaminationof serum. How to do that is well described by Versiecket al., (1978): Venous blood should be taken with aplastic cannula trocar Olntranule 110 16; Vygon) and col-lected in high-purity quartz tubes (Spectrosil; ThermalQuarz-Schmelze)previously cleaned with twice-distilledwater, boiled in a mixture of equal volumes of nitric acid(min. 65%) and sulfuric acid (96%) (SuprapurR; Merck),rinsed again and finally steam-cleaned with triple quartz-distilled water. Transport of the samples should be limitedas much as possible and done in air-tight boxes.Spontaneous clotting can be allowed. Before the irradiation,further handling of the samples should be carried out ina dust-free room. After centrifugation the serum should bedecanted into thoroughly cleaned polyethylene containers,frozen and lyophilized.

A detailed description of serum preparation for ir-radiation by protons can be found in the paper by Bearseet al., (1974): Capillary pipets, rinsed in heparin andair-dried, were used to draw 0.1-ml samples of whole bloodfrom the sinus cavities of mice. Human blood samples weredrawn into 5-ml syringes, potassium oxalate was added, andthe samples were repipetted with an Oxford 0.1-ml autopipet.The samples were pipetted directly into 1-ml borosilicateglass beakers that had been previously weighed on a Torbalbalance to an accuracy of 0.2 mg. About 30 samples wereprocessed at one time. The filled beakers were arranged ona 7.6- by 15.2-cm borosilicate glass plate on the base ofa bell-jar system, and all beakers were repeatedly filledwith liquid nitrogen until the blood was thoroughly frozen.With the beakers still containing liquid nitrogen, the bell-jar was sealed and the system evacuated. The system waspumped for several hours, and an ultimate vacuum of about0.06 Torr was achieved. The beakers were then removed fromthe bell-jar and reweighed to obtain the dry weight of thewhole blood. The weight reduction from wet to dry blood wasabout a factor of 4.8. The samples were placed in TracerlabLTA-302 asher on a 7.6- by 15.2-cm borosilicate glass platewhich allowed the beakers to be on the diameter of the ashingchamber. The samples were ashed for 48-72 hours at a powerof 100 watts, a pressure of 0.9 Torr, and an oxygen flow of150-cm3/min.

94

The purpose of ashing is to increase the concentrationof the elements with Z £, 26 by removal of the elements H, C,N, and 0 which comprise the bulk of the blood mass. Targetswith a higher concentration of trace elements can then befabricated with a much smaller total mass. This decrease inmass reduces the amount of background due to secondary electronbremsstrahlung thus improving the detectability limit. It hasbeen shown that plasma ashing is the method of choice to keepmetal losses to a minimum.

After ashing, the samples were removed from the chamber,and 0.1 ml of a solution of 6 parts 1.8% HC1 and 4 parts of1000 ppm PdCl2 (for normalization purposes) was added to eachsample using the autopipet. A new disposable tip was usedfor each sample, and dissolution was effected by rapidinhalation and expiration of the solution, using the pipet.Occasionally, small back specks appeared in the solutionand, so long as they were few and small, they did not seemto affect the accuracy of the analysis. A 0.02-ml autopipetwas used to transfer that amount of the sample to the surfaceof a Formvar foil on which a still wet 0.02-ml drop of 3%NHi|OH solution had been placed. The NHi|OH neutralized theacidic blood solution which would otherwise have attackedthe foil. The samples dried on the foils within a diameterof 5-6 mm. They were stored and transported in a desiccatorand then transferred to the Los Alamos Scientific Laboratorytrace-analysis chamber which had been modified to hold six2.5- by 5-cm aluminum frames with 1,9-cm diameter holes.Larger frame holes were used so that the target and beam areacould be increased to à reasonable and practical size(governed by the size to which the liquid drops dried) withoutstray beam striking the frame. Even a beam 103 times smallerthan the primary beam would cause an appreciable backgroundspectrum upon hitting the frame because of the effectivelyinfinite thickness of the frame.

Formvar foils were used in this work because they wererelatively easy to prepare in quantity, withstood the requiredbeam intensity, and were tough enough to endure the abuse oftarget preparation and subsequent handling. Another importantreason for using Formvar was that it can be made very thin,thus contributing insignificantly to the secondary electronbremsstrahlung continuum. A Formvar solution was preparedwhich consisted of 16 grams of Formvar resin 15/95E (MonsantoPolymers and Petrochemicals Co.), 200 ml of methyl benzoate,480 ml of toluene, and 320 ml of ethanol. The foils weremade by placing a drop of this solution onto the surface ofdoubly distilled water. When the edges of the film began towrinkle, it was picked up onto one of the aluminum frames,with care that double layers were not formed. These filmswere measured, by weighing and by the energy loss of alphaparticles to be about 10 ug/cm2. The foils were preparedahead of time, allowed to dry, and stored in a desiccatorcontaining CaSO^ desiccant.

Blood consists of a liquid (serum) and cellular particles(e.g. erythrocytes and, lencocytes). In the target preparationscheme described by Kivits (1980) and De Rooij et al., (1981)cellulor particles are destructed allowing blood to be treatedas if it were a real solution. For this purpose, additon of

95

0.25 ml 1 M NaOK per ml blood gives a good solution, as couldbe microscopically observed. The method consists of followingsteps :

a) To the blood sample, NaOH is added (1 ml blood:0.25 ml 1M NaOH);

b) propanol is added, such that the ratio of pre-treated sample to propanol is 5:1 (v/v);

c) a Selectron filter is rotated at 18 000 rpm;d) 50 ul of the solution is dispensed as quickly as

possible, yielding a wetted area of about 10 cm2;e) the target is air dried at room temperature.To test the blood samples for their reproducibility

the amount of iron per unit area was measured by PIXE. Forthis purpose, ten targets were prepared from the blood ofone person. The reproducibility of the targets amounted toabout 1.6%.

Because of the low trace-element concentrations inblood, it could be favourable to use a thinner supportingmaterial. The peak-to-background ratio in a PIXE spectrumwill increase, for the same amount deposited per cm2,resulting in a lower detection limit and more accuratepeak-area determination. The quantity of blood on aSelectron foil of 5 mg/cm2 is about 2 mg/cm2 . The supportcontributes to the Bremsstrahlungs continuum for about 70per cent. If Formvar (0.2 mg/cm2) were used as support,this contribution would decrease to about 10%, providedthe wetted area is equal to that for Selectron. Sinceattempts to prepare reproducible targets with blood solutionsshowed rather good prospects with Formvar supports, Kivits(1980) has developed a technique based on their use:

a) to the blood sample, NaOH is added (1 ml blood:0.25 ml 1M NaOH);

b) propanol is added, such that the ratio of pretreatedsample to propanol is 25:1 (v/v);

c) a Formvar filter is rotated at 5,500 rpm;d) 50 /il of the solution is dispensed as quickly as

possible, yielding a wetted area of about 10 cm2 ;e) the target is air-dried at room temperature.

3.6.5 PIXE targets preparation for solid samplesDe Rooij et al., (1981) have descussed targets prepa-

ration for solid samples. Solid samples may be analysed assuch, or may be reduced to slices, powders or solutions. Ifthe material can be analysed in the as-received state, theanalyses are usually very rapid and convenient. However,since most solid samples are not homogeneous, problems mayarise in the target preparation with respect to a represen-tative bulk analysis. The representativity may be improvedby powdering of the solid, in order to obtain a homogeneoussample and to reduce the particle size. Addition of aninternal standard in the sample is then also feasible. Adisadvantage of powdering a sample is that such pretreatment

96

is time-consuming and may be an extra source of contamina-tion .

Powders may be spread into a thin layer, dry or asslurries. In the "dry technique" the powder can be dustedonto an adhesive surface, or can be spread out over a sup-port adding a fixative later. In practise it seems to bedifficult to obtain a uniform or thin layer of the powderby the "dry techniques" described. In the "slurry technique"commonly a small amount of powder and bonding material isslurried in an appropriate solvent. The slurry is spreadover a clean microscope slide. The film is floated off onwater, then scooped up and supported some other way. Thistechnique however, involves many manipulations, which mayintroduce sources of analytical errors.

De Rooij et al., (1981) have developed a thin-targetpreparation technique via a "slurry procedure" requiringless manipulations and therefore less time. For powderingthe solid, they use a "brittle fracture technique" similarto that of lyengar. Thin targets (~1 mg/cm2 ) have beenchosen, since for such targets the requirements of uni-formity and homogeneity are less stringent. On the otherhand, for a good representativity of the bulk composition,preparation of several targets for irradiation from thesame sample seems advisable. The steps involved are:

a) A solution of 5 g Formvar in 100 ml dioxane isprepared ;

b) powdered solid sample is mixed with this solu-tion (sample weight fraction about 10%) with theaid of a whirlimixer;

c) a Selectron filter is rotated at 3,000 rpm andsaturated with 0.5 ml water;

d) 0.2 ml of the powder-Formvar-dioxane mixture isimmediately dispensed on the wetted rotating filter;

e) the resulting Formvar foil, in which the solidsample is embedded, is pulled from the Selectronfilter.

The resulting foils have an area density of less than1 mg/cm2 , as determined with an at, -particle set-up. Thetarget preparation technique takes only a few minutes; thefraction of failures is less than 10%. The reproducibilityin the amount of material irradiated and the reproducibilityin the homogeneity of this material was tested using tentargets prepared from homogenised fish meal (elementsconsidered: P, S, Cl, K, Ca and Fe). The reproducibilityfor the amount of material as well as the homogeneity wasbetter than 5%.

Preparation of soil sample for analysis by PIXE isdescribed by Navarrete et al., (1976). First, soil wasdried and powdered to micro-millimeter fineness The targetused for 3-5 MeV proton was prepared by scattering a fewmilligrams of soil on 4 um Mylar film and using a dropof 0.1 ml of polyvinyl acetate diluted with acetone asadhesive. It was ascertained that the film of soil washomogeneous and adequately thin. Energy loss of proton beam

97

in the sample was negligible as to cause error in determi-nations. Aluminium absorber of 50 ;am thickenss was usedto decrease the low energy bremsstrahlung background.

Another target was prepared for bombardment using200 keV proton. The soil sample was mixed with high puritygraphite into 10:3 soil-graphite ratio and was compressedinto a diskshape with 0.5 mm thickness and 10 mm diameter.A Mylar film of 20 ^m thickness was placed in front ofthe Si(Li) detector as absorber to avoid the pilling upeffect of low energy x-rays due to the high concentrationof Al and Si.

In the destructive method, 10 g soil sample wasextracted with 50% HC1 and 50% HN03 and diluted to 100 ml.An aliquot portion of 0.4 ml of the extract was dried on4 jam Mylar foil.

Baker and Piper (1976) have described trace elementanalysis of marine suspensates. They have used a specialholder with a Nucleapore^ filter attached to the tip of thesyringe, and the necessary amount of water squeezed throughthe filter. Immediately after collection, the filters wererinsed free of sea salt by at least five flushes withfiltered, distilled water and then stored in sealed plasticPetri dishes. In the laboratory, the filters were dried atroom temperature and weighed to determine the total amountof suspensate collected. The filters were then mounted inthe XRF sample chamber between two pieces of Mylar" film,the top piece having a hole just smaller than the diameterof the sample filter so that the filter was held flat againstthe bottom piece without disturbing the collected particulatematter. This simplicity of preparation is particularlyadvantageous when dealing with very dilute suspensions,where the possibility of contamination is serious.

Separating the elements of interest from a largevolume into the -almost ideal matrix and volume of a resin-loaded paper disk has proven to be a versatile and effectiveapproach to trace metals determination by x-ray spectrography(Law and Campbell, 1974). These papers are composed ofapproximately 50% cellulose and 50% powdered ion-exchangeor chelating resin, providing a matrix of low-atomic-numberelements that have little effect on the x-ray determinationof most elements. Incorporation of the resin in a thinpaper disk provides a convenient media for handling smallquantities of resin and for supporting the resin in thex-ray spectrograph or energy dispersive system. Standardsand unknowns can be prepared on similar resin-loaded papers,providing a match that is often impractical to achieve indirect x-ray analysis.

The general analytical procedure consists of the fol-lowing steps (after Law and Campbell (1974):

1. Dissolution of the sample, or selective dissolutionof the element or elements of interest

2. Adjustment of pH, addition of complexing or maskingagents, or other chemical treatment that may be neces-sary to achieve the selectivity desired in the ionexchange process

98

3. Collection of the desired elements on a resin-loadedpaper disk by filtration or by suspension of the diskin the solution

4. X-ray determination of the elements on the driedresin-loaded disk, using disks containing knownquantities of the elements as standards.

The combination of ion-exchange separation and collectiongreatly extends the range of fluorescent x-ray spectrometry.The chemical preconcentration reduces or eliminates problemsof matrix correction, variations in physical properties of thesample, and increases sensitivity by several orders ofmagnitude. In addition, reliable standards are easily prepared.

4. SAMPLE PREPARATIONS FOR EXCITATION WITH RADIOACTIVESOURCES AND TUBE EXCITATION

In this chapter we shall describe sample preparationsfor analysis with x-ray fluorescence (radioactive sourceand x-ray tube excitation). Only some characteristicprocedures will be presented in the form as published by theauthors .

4.1 WaterMonitoring elemental composition of water is of great

importance to life. However this is a difficult problembecause of concentration levels of about 10~3 ppm for mostelements of interest. Many preconcentrations methods havebeen described when preparing water samples for analysisby x-ray emission spectroscopy. Sample preparation for theanalysis by tube system is often accomplished by precipitatingthe elements with the nonspecific chelating agent, ammonium-1-pyrolidine dithiocarbamate (APDC), and filtering throughmembrane filter.

Simplicity of this method permits processing of largenumber of samples in relatively short time. The bidentatechelating agent forms insoluble coordination complexes withmore than 30 transition metals: V, Cr, Mn, Fe, Co, Ni, Cu,Zn, Ga, As, Se, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, W,Te, Os, ir, Pt, Au, Hg, Tl, Pb, Bi and U. The alkali andalkaline metals are excluded. The high stabilities and lowsolubilities of the metal dithiocarbamate chelates aresufficient to permit quantitative recovery of many metalswith little or no pretreatment of the natural water at adithiocarbamate concentration of 10 M. The usefulness ofAPDC chelation is enhanced by its effectiveness over broadpH ranges.

Equipment for sample preparation consists of a Pyrexfilter holder and includes a reservoir, a 17-mm circularfritted glass support and a metal clamp. The filter paperdiscs often used are white 25-mm Millipore paper discs of0.45 urn pore size. After insertion of the discs and installationof the reservoir the solution should be filtered under suction.

99

Let us describe measurements done by Marijanovic et al.(1982). Initial pH of seawater was 7,96. Preparing a targetof solutions with known concentrations of elements it wasshown that the best preconcentration is obtained at pH 3.For particular elements complexation is possible over thewide pH range (2-14) but for simplicity and rapidity ofprocess it is necessary to complexate all elements at onepH value. It is shown that results obtained are differentand lower for other pH values.

Usually 200 ml of water sample is measured intosuitable container. The pH is adjusted by adding of HC1 orsolution of NH3 in water. Four ml of 1% - APDC solution isadded. Solution is prepared fresh daily by dissolving.ammonium-1-pyrolidine dithiocarbamate in distilled water.APDC is added to sample while mixing with a magnetic stirrer for20 minutes at room temperature to permit complexation.Suspension is filtered through a Millipore filter whichis then allowed to dry on clean absorbent tissue forseveral hours. In that way the uniform thin samples suitablefor x-ray analysis'were prepared.

X-ray spectrum from seawater sample shown in Fig. 4.1is obtained by using x-rays from Mo x-ray tube for excitation.Usual working conditions for Phillips x-ray apparatus ModelPW 1010/30 were 26 kV, 12 mA with x-ray beam passing throughZr, Ti and Mo filters to reduce its intensity. Si(Li) x-raydetector used has an energy resolution of 180 eV, at E =6.4 keV.Results are presented in Table 4.1. x

Similar procedure is also described by Elder et al.,(1975). A volume of water (usually 100 ml) free of particulatematter is measured into a suitable container. The pH can beadjusted, if judged necessary, by addition of a small amountof HC1 or HN03. A 1.0% APDC solution is prepared fresh dailyby dissolving ammonium-1-pyrrolidine dithiocarbamate in distilledwater and filtering through a prewashed glass fiber filter(Whatman GF/C or equivalent). A minimum of 2 ml of thissolution per 100 ml of water sample is introduced with thoroughmixing, and the sample is allowed to stand at room temperaturefor 20 min to permit equilibration. The suspension is filtered •through a prewashed membrane filter after which the filteris placed on a clean absorbent tissue and weighted around theedges with a plastic ring to prevent curling. The filter isallowed to dry for several hours then mounted on a targetholder for x-ray analysis.

About 30 min are required to reduce a natural waterspecimen to a form suitable for analysis. A portion of thistime is for the separation of the particulate matter fromthe aqueous phase. This may be accomplished by centrifuga-tion or by filtration through a membrane filter. If thelatter method is used, the particulate matter, if not toocoarse, may also be analyzed by XRF (Elder et al., 1975).

Leyden et al., (1975) have described the use of ionexchange resin for sample preparation. In their recoverystudies, the synthetic sea water doped with metal ions wasused. The purpose of this study was to determine the recoverycapability of the resin in saline solutions simulating seawater. The resin to be used was buffered to pH 6.0 by washing

100

CO•z.Lu

ArK,

CI

ÇaK ';

Br

FoK«x.

Cu

Ni '. zn'^V;./- ; Hg

. U

Rb -.\ • '.

BrK^--•~-Se • . •>»-.•:'

CHANNEL NUMBERFig. 4 .1 X-ray spectrum from seawater sample.

o«o

Table 4.1Concentrations of the elements in seawater obtained by analysing of samples complexated

at the different pH values

Element

VCrMnFeNiCuZnGeAsPbU

pH 2

1.51x10~30.29x10~30.69x10~30.29x10~3

0.69x10~3

pH 3

2.3lx10~30.68x10~30.39x10~32.5 x10~21.3 x10~33.1 x10~31.3 x10~30.68x10~30.59x10~31.68x10~31 .5 xlO

pH 1

1.86x10~30.1 x10~31.8 x10~31.6 x10~30.2 x10~3

1.1 x10~3

pH 5

8.5x10~3O.lxio"31.6x10~35-1x10~3

0.2x10~3l.lxlO"3

pH 6

31.0x10~30.5x10~32.2x10~32.8x10~3

0.2xlO~3

pH 7

7.0x10~30.5x10~31.7x10~35.9x10~3

0.1x10~3

0.3x10~3

pH 8 pH 9

2.6x10~3 5.110~3O.lxlO"3 0.3x10~30.8x10~3 0.9x10~30.3lx10~3 1.6x10~3

0 20 40 60 80 100 120CONCENTRATION'/pg-T1

Fig. 4.2 X-ray counts versus concentration ofsome elements in synthetic (afterLeyden et al.. 1975)-

with a 0.05 M sodium acetate solution to which sufficientHN03 had been added to obtain pH 6.0 by pH meter reading.The studies were conducted by placing 150 - 1 mg of the resinin 15-tnl beakers and adding approximately 2 ml of 0.7 M NaCl.This slurry was allowed to sit for 24 hours to equilibratewith the salt solution. The resin was placed in a column(4x0.5 cm) which had been previously f itted-with a glass wool plugThe resin in the column was washed with the pH „6 buffer untilthe emergent solution was pH 6. Each of several 500-ml plasticbottles were filled with the synthetic sea water, to these,various aliquots of the standard solutions were added. Thesynthetic sea water served as a blank. These solutions werethen passed through the columns containing the resin at aflow rate of *v 3 ml/min. After all of the solution had passedthrough the column, the bottle was rinsed with a 10-mlportion of the acetate buffer and this rinse was also allowedto pass through the resin bed. The resin was then transferredfrom the column into 30-ml sintered glass filtering cruciblesof fine porosity and washed with three 5-ml portions of theacetate buffer using suction. The resin was pelletized in a0.6-inch diameter hardened stainless steel die under a pres-sure of 15.000 pounds, then analyzed by x-ray fluorescence.The standard resin pellets were checked by grinding them andeluting the metals using HN03. The metal ions in the eluantsolution were determined by atomic absorption using a set ofstandards prepared independently of the synthetic sea water.In all cases, the concentration of the transition metalswere within B% of their calculated value. This test indicatedthat no significant error resulted from impurities in thereagents used to prepare the synthetic sea water solutions(Leyden et al., 1975). Fig. 4.2 shows x-ray counts versusconcentration of some elements in synthetic seawater.

In the sampling of seawater Leyden et al., (1975) 30liter Niskin bottles at depths above, in, and below theoxygen method. The water samples were drained into cleanplastic containers and then 4-liter subsampels were transfer-

103

red into 1-gallon plastic jugs equipped with 4-cm columnsof TEPA resin. The samples were allowed to pass through theresin columns by gravity feed until sufficient water hadbeen samples (either the entire 4 liters or the amount thatpassed in 48 hours). The glass columns were sealed withpara-film and rubber stoppers and transported to thelaboratory in that manner.

Seils and Tisue (1979) have described procedures forpreparation of samples from rain, snowmelt or lakewater.Here one is seldom limited by sample size. In other instances(sediment interstitial fluids) only small sample volumes areavailable, and some modifications of technique become- neces-sary. These two circumstances are treated separately. It is-demonstrable that the presence of cations other than H"1" orthe alkali metals may interfere with the determination byacting to hold back sulfate in solution, or by coprecipitatingwith BaSOij, thereby altering the stoichiometric relationshipbetween Ba and S on which the indirect method depends.

For these situations, the authors have described apretreatment of the sample with chelating resin. (The rainsamples analyzed did not require this conditioning).

(i) Procedure for samples and standards up to' 0.1 1 and15 umol S04=:

This procedure is applicable to sample volumes of 100 mlcontaining up to 15 umol SO^". For more concentrated samplesan aliquot portion containing 15 umol SO^" should be dilutedto 100 ml with 10 mM HC1. Reagents: 10 mM HC1; 0.5 M BaCl2î0.1% by weigh* "Tween-20" surfactant (Fisher Scientific Co.);10 mM HCl/10% methanol by volume; a'nd 6 M HC1. (All reagentsare prepared with Barnstead "Nanopure" deionized water, orequivalent, passed through a 0.22 jam final filter. Likewise,all samples are filtered through a 0.4 ^im membrane prior tofurther treatment).

A 100 ml aliquot portion of the filtered sample ispipetted into a 250 ml beaker. The pH is then adjusted """Oabout 2 by the dropwise addition of 6 M HC1 to eliminateC0o~ interference; pH paper is a suitable indicator. Whilestirring gently with a teflon-coated magnet, one then adds5 ml of 0.1% Tween-20, and 1 ml of 0.5 M Bad 2- The resultingmixture is stirred at 80 C for one hour, then cooled andallowed to stand overnight under a watch glass. It was foundconvenient to dislodge the precipitate from the beakers'walls by ultrasonication for 2-5 min, and then to suspendit by magnetic stirring. The precipitate is collected by pas-sing the resulting slurry through a 25 mm diameter, 0.4 urnNuclepore filter contained in a 5/8" ID glass Milliporevacuum filtration holder. To provide reliable sealing aroundthe inner circumference of the funnel's base, one may placea 5 urn Millipore membrane behing the Nuclepore filter. Thebeaker and filtration funnel are washed with several smallportions of 0.01 M HCl/10% methanol to complete the transferand to remove excess Ba+2. The collected precipitate isdried in clean air and mounted between 0.5 ml Mylar film ina cardboard slide holder.

If pretreatment to remove interfering cations isindicated by preliminary tests, the following technique is

104

applicable. Additional reagent: AG 50W-X8 cation exchangeresin, 100-200 mesh, hydrogen form (BioRad Laboratories),or equivalent, washed first with 6 M HC1, then with 10_mM HC1.Sometimes the resin absorbs small' amounts of S0n~ irreversibly.Samples containing 3 umol S0i|~ gave low and erratic recoveries.The difficulty may be overcome in the main by pre-equilibratingthe resin with a small amount of the sample being analyzed.However, in ultratrace analyses using the small spot geometryit is desirable to substitute Chelex-100 in the H^ form,since that material does not appear to absorb SO^" vide infra.

A 47 mm, 0.4 ;jm Nuclepore membrane is mounted under avacuum filtration funnel of >100 ml capacity then'coveredfor 5 min with ca. 1 g of prewashed cation exchange resinand 25 ml of the sample. Vacuum is applied and the resindrawn dry without washing. After changing the receiver, oneadds the sample (about 100 ml) and allows 5-10 min forequilibration with the resin while stirring occasionally.The sample filtrate is then drawn off by vacuum withoutwashing; the used resin is discarded. 100 ml of the filtrateis pipetted into a 250 ml beaker. The BaSOjj. precipitateprocedure described earlier is followed in subsequent steps.

(ii) Procedure for samples up to '10 ml containing lessthan 1 jumol SO^":

This procedure is applicable to sample volumes up toca. 10 ml containing 1 umol SO^"". The reagents are the sameas were described for the large spot procedure.

A portion of the filtered sample is pipetted into a50 ml centrifuge tube and adjusted to a volume of ca. 15 mlwith 0.1 M HC1. To it is added ca. 0.1 g of Chelex-100 resin,100-200 mesh, in the H+ form. (This reagent may be omittedin the absence of interfering cations.) Resin and sample areallowed to react for 30 min with occasional stirring, thenfiltered through a 25 mm diameter, 0.4/im Nuclepore membranebacked by a 25 mm diameter, 5 /im Millipore membrane. It isconvenient to collect the filtrate in a 50 ml centrifuge tube.The collected resin should be washed with amounts of 0.1 M HC1sufficiently small that the combined volume of sample andwash liquid is 25 ml.

To the filtrate one then adds 1 ml of 0.1% of 0.5 MBaClg- The resulting precipitate is digested for 1 hour at80° in a water bath, covered, and allowed to stand overnight.Prior to filtration, it is desirable to dislodge theprecipitate by ultrasonication for 2-5 min. It may then becollected on a 25 mm diameter, 0.4 jam Nuclepore membranebacked by a 5 pm Millipore membrane. To confine the precipitateto an area smaller than the x-ray beam in spectrometer, a1/4" diameter filter holder and funnel were used. To obtainleak free operations, the upper and lower portions areclamped together tightly by means of a screw clamp; the usualspring-leaded clamps do not prevent loss of precipitate aroundthe gasket seals.

Transfer and washing of the precipitate are completedby washing the centrifuge tube and filter with several smallportions of 10~2 M HC1 containing 10% methanol by volume.After drying in clean air, the filter containing theprecipitate is mounted for x-rây fluorescence analysis.

105

Let us also describe procedures used by van Griekenet al., (1982) for trace element analysis of water. Forthe suspended matter, simple filtration through e.g. a0.4 urn pore-size Nuclepore membrane was found to be asuitable collection step. For the dissolved material, thechoice of the preconcentration method is not an obviousone and many alternative procedures have been proposed,most of which are included in Bachmann's survey (1981) onpreconcentration methods.

Van Grieken et al., (1982) prefer following scheme:- the use of cellulose filters with 2,2' -diamino-

diethylamine (DEN) functional groups (Smits andvan Grieken, 1981), which allows direct enrichmentby a simple filtration step, leads to sub-ppbdetection limits of most cations, almost independentlyof the major ion composition, but implies thesynthesis of the DEN-filters;

- co-crystallisation on 1-(2-pyridylazo)-2-naphtol (PAN)(Vanderstappen and van Grieken, 1978) which is quitesimple and leads to enrichment factors around 1 CP anddetection limits _nea_r_0.1 jug/1, but suffers somewhatfrom collection yield"variations with speciation;

. - co-precipitation on ferric hydroxide (Chakravorty andvan Grieken, 1978) which allows a quantitativecompromise between high enrichment factors and highcollection yields;

- chelation by oxine and subsequent adsorption ontoactivated carbon (Vanderborght and van Grieken, 1978)which collects also quantitatively the colloidal andorganic forms of the trace metals, is dependent ofthe concentration and matrix composition and wasfound quite valuable in recent preconcentrationintercomparisons (Leyden et al., 1979); Smits et al.,1979), in spite of its being a two step procedure.

Numerous other methods are, of course, available inthe literature; each has specific advantages and drawbacks.Some authors prefer ion exchange material, in the column orbatch mode, e.g. silylated glass beads (Leyden et al.,- 1976)and Hyphan (Burba and Liesen, 1977), or commerically availableion exchange filters (Campbell et al., 1966), or they usecoprecipitation on sodium-diethyldithiocarbamate, ammonium-pyrrolidinedithiocarbamate or related compounds (Elder et al.,1975), or solvent extraction (Marcie 1976) or electrodeposition(Wundt et al., 1976) or other chelation and immobilisationprocedures (Knapp et al., 1975).

Besides multi-element analysis procedures, varioushighly specific single element or elemental species precon-centration methods are available. Most of the chemicalpreconcentration procedures combine the following advantages:they lead to thin homogeneous targets that are ideallysuitable for XRF and they allow very advantageous detectionlimits. On the other hand, the chemical speciation of theions to be determined and the matrix composition caninfluence the collection yield, particularly in relativelyconcentrated samples like waste water.

106

Simple evaporation or freeze-drying of the water matrixobviously collects all non-volatile element, independent ofthe chemical and physical speciation, even in very complexsamples like waste water and sewage sludge. For XRF analysis,the residues were.hitherto either strongly diluted with abinder, to reduce matrix effects at the expense of thesensitivity, or the analysis was limited to one type of watersample only. Via the automated spectrum evaluation andmatrix correction procedure highly variable evaporationresidues of sewage sludge and waste water can be analysed ina very simple automatic and versatile way (Van Dyck; 1980,19'82).

The sample preparation thus includes freeze-drying ofa 250 ml waste water volume, in the presence of 100 mggraphite if little dissolved or suspended matter is present,and with 100 ug Y as an internal standard. The residue ismore or less quantitatively transferred to a mortar,homogenized, and pelletized on a thin Mylar carrier for XRFanalysis.

4 .2 Liquid SamplesVarious target preparation techniques for liquid samples

have been described in the literature. These techniques arebased either on the use of liquid-specimen cells or on depositingthe liquid on a supporting material and drying.

For liquid-specimen cells it is fairly easy to producereproducible targets, but there are some practical problems(Kivits, 1980):

- the cell window must be uniform in thickness andimpervious to the constituents of the solution;

- the cell window must be thin enough to transmit boththe incident particles or photons and the inducedx-rays without appreciable attenuation;

- some liquid cells are difficult to fill properly orare leaky and easily become messy;

- some solutions are volatile and must be contained inclosed cells;

- liquid may be heated under irradiation causingexpansion and in closed cells distension of the cellwindow;

- some specimens may undergo radiolysis with possibleprecipitation.

Liquid-specimen cells are easy to use and attractivefor volatile analytes (e.g. Hg in water). However, the methodis not useful for detecting low-Z elements, because of theabsorption of x-rays by the cell window.

Another method is based on depositing the liquid on athin support. This has the advantage of a better peak-to-background ratio and will be discussed below. However, it ismore complex. Important criteria in the selection of a sup-port are (Kivits, 1980):

- homogeneity;

107

- uniformity and low area density to minimise x-raysscatter and absorption;

- low concentration of elements with atomic numberZ >11 ;

- resistant to chemicals, mechanical stress andradiation damage;

- retention of the material to be analysed during theentire procedure of preparation and irradiation, toprevent losses;

- wettable by liquids.The most commonly used support materials, in order of

decreasing thickness (mg/cm2) are: filter paper ( 10);Millipore (5); Nuclepore (1); Mylar (0.5-1); colldion (0.02)and Formvar (0.02).

In addition to real solutions, one is often dealingwith systems containing two phases, e.g. suspensions (solid-liquid) or emulsions (liquid-liquid). In general it isdifficult to prepare homogeneous and uniform targets fromthese samples, due to their complex physical behaviour. Inthe case of suspensions, the volume fraction or weightfraction of the solid material and its particle size distri-bution may be critical parameters in target preparation.

Jolly and White (1971) describe a method for preparinguniform film deposits (10-1000 jig/cm2) from solutions orcolloidal suspensions of micron-size particles. The suspendedor dissolved material is placed in a container of a nebuliserA compressed gas forces the liquid through a small hole toform a spray which in turn impinges with high speed on anobstruction and"thus breaks up into droplets of various sizesThe resulting mist is then allowed to deposit on a rotatingsubstrate. The particle size of the suspended material in theoriginal suspension and the microscopic size of the dropletsis crucial to the success of this method in obtaining a thinuniform deposit. Valkovic (1975) reports that the reproduci-bility of these targets will be questionable because thefinest droplets are swept along by the gas stream and do notimpinge on the obstruction. From the results of Wilknissand Bressan (1971) it may be concluded that the trace-elementcomposition may not be constant for all droplet sizes.

In addition this method involves dividing the liquidinto a super-fine mist and deposition onto the targetsubstrate at a rate equal to the rate of evaporation. Onecan have difficulties with residue recovery due to "mistbounce-off". This can be corrected by the use of internalstandardization however, the change of contamination is highand the rate of concentration is low with this method.Physical methods such as buretting onto Mylar and evaporationby heat lamp, produce surface gradients of elementaldistribution since the solubility of the dissolved constitu-ents varies greatly and the configuration of the water(droplet etc.) is constantly changing (i.e. the residue isa series of concentric closed contours). Brady and Cahill(1973) attempted to overcome this configuration change bythin film evaporation utilizing surfactants to reduce thesurface tension of the water. The authors have tried Vatsol

108

OT and surfactant 77 but evaporation still proved to bevariable since precise leveling of the substrate and auniform drying environment are critical. They also attemptedabsorbing the liquids into Cellex and/or Somar spectroscopicgrade powders followed by pelletization. This technique provedto be tedious and held merit only for the production ofstandards. In addition the sensitivity was greatly reduceddue to the high matrix to trace metal ratio, which increasesthe background with which the x-ray signals must complete.Residue recovery can be also a problem for thismethod if thecellulose is over-saturated initially.

Physical techniques are essentially abandoned becauseof successful chemical means of liquid target preparation.Briefly this technique involves the formation of water-insoluble metallic complexes and filtration as in the caseof suspended material. The flocculent distribution producedhas proven to be very uniform and the collection efficiencyhas been shown to be essentially 100% for the transitionseries. This simple method is sensitive down to one ppbas can be seen in the spectrum of a natural water sampleon the following page. The only critical phase of thepreparation (besides using clean active reagents and cleanglassware) is the complete separation of particu andsolute phases. The particulates can be analyzed separately(Brady and Cahill, 1973).

4.3 Solid SamplesSolid samples may be analysed as such, or may be reduced

to slices, powders or solutions. If the material can beanalysed in the as-received state, the analysis are usuallyvery rapid and convenient.

Kivits (1980) has shown that thin targets (-c 1 mg/cm2 )are most favourable, and solid samples may therefore bedivided into slices. This is for instance useful for analysisas a function of depth. One should take into account thatsurface treatment may result in unwanted contamination and/orin selective removal of certain constituents. Any surfacetreatment involving movement of a cutting tool across thesurface may result in smearing of soft constituents. Thiscauses a spectral enhancement for the smeared constituentsand attenuation for the covered constituents. The latter effectis more severe the higher the absorption coefficient of thesmeared material and the lower the x-ray energy of the coveredmaterial.

For an analysis of the bulk of a sample, the problemsdue to inhomogeneous solids must be solved. These may beovercome by powdering the solids, in order to obtian a homo-geneous sample and to reduce the particle size. Targetsprepared from homogeneous powders of small particle sizeare expected to be representative for the original sample.Addition of an internal standard in the sample is then alsofeasible. A disadvantage of powdering a sample is that suchpretreatment is time-consuming and may be an extra sourceof contamination. Thin targets for powdered samples may beprepared by spreading into thin layers, dry or as slurries(Kivits, 1980).

109

In the "dry technique" the powder can be dusted ontoan adhesive surface (e.g. Scotch tape), or can be spread outover a support, adding a fixative later. Rudolph et al.(1972) placed 0.3-0.5 mg of powder in a 4-mm diameter circleon a Formvar foil. Adherence is effected with polysterenedoped in toluene. Barnes et al., (1975) ashed whole bloodand mounted it on Kapton backings with 5 jul of albumin fixative

Sometimes a measured amount of powder is placed on aMylar film and distributed as uniformly as possible. A secondMylar film is then stretched over the powder and the firstfilm, thus enclosing the powder layer (Rinsvelt, 1977).

In practice it seems to be very difficult to obtain auniform or thin layer of the powder by the "Dry techniques"described.

In a commonly used slurry technique, a small amount ofpowder and bonding material is slurried in an appropriatesolvent. The slurry is spread over a "scrupulously" cleanmicroscope slide. The film is floated off on water, thenscooped up and supported in some other way. Suggestedsolvents are: amyl acetate, chloroform, dioxane anddichloroethane. Bonding materials recommended are: nitro-cellulose, ethyl-cellulose and polystyrene. The techniquesdescribed above involve many manipulations, which mayintroduce sources of analytical errors, such as contami-nation and loss of elements to be analysed.

Kivits (1980) has described a procedure for thepreparation of thin targets from the powdered materials.For the preparation of thin targets, the powdered samplesshould have particle sizes below about 10 pm. Fo=r samplesreceived as non-fine powders a pulverising technique is needed.

A "brittle fracture technique" (lyengar and Kasperek,1977) is often used to homogenise solid samples and toreduce their particle size. Particle size reduction isneeded to obtain homogeneous and thin targets.

In the work by Kivits (1980), use is made of aball in a vibrating Teflon vessel. Dry sample material isbrought into this vessel. The vessel is cooled in liquidnitrogen for a few minutes and then vibrated for 1 minuteat 3000 cycles per minute with a mechanical shaker,constructed in our workshop. If necessary, the cooling-shaking process may be repeated. Quantities of up to50 grams can be pulverised and homogenised as one batch.Soft tissues (human tissue, hair) can be pulverised andhomogenised using a Teflon ball or a high-purity quartzball. Hard samples (teeth, bone, nails) are treated usinga Teflon ball with a metal core or a tungsten carbideball. Wet samples such as human tissue are better pulverisedafter previous lyophilisation.

The following steps are included in the samplepreparation method used by Kivits (1980):

a) a solution of 5 grams Formvar in 100 ml dioxane isprepared ;

b) powdered solid sample is mixed with this solution(sample weight fraction about 10%) with the aidof a whirlimixer;

110

c) a Selectron filter is rotated at 3-000 rpm andsaturated with 0.5 ml water;

d) 0.2 ml of the powder-Formvar-dioxane mixtureprepared is immediately dispensed on the wettedrotating filter;

e) the resulting Formvar foil, in which the solidsample is embedded, is pulled from the Selectronfilter.

The resulting foils have an area density of lessthan 1 mg/cm2 , as determined with an «^-particle absorptionset-up. The target-preparation technique takes only afew minutes; the fraction of failures is less than 10 percent (Kivits, 1980).

Brady and Cahill (1973) used two techniques in theirwork. The first technique requires about 500 mg or more ofhomogeneous target material. Homogeneity is obtained viaashing, grinding, or lyophilization coupled with grindingfor all biological type materials. This powder is thenspread into a 1 1/4" die and pressed into a pellet with a20-ton hydraulic press. A cellulose binder must be mixedinto the target material prior to pressing for somematerials. This technique is easy but self-absorption mustbe dealt with carefully for such thick targets, which arehardly optimum for the lightest elements.

The second technique requires only a few milligramsof material but only works well for dense insoluble powders(e.g. many geologic type samples). It involves placingthe powdered sample temporarily into a turbulent suspensionsucceeded by rapid filtration onto a Millipore type filter.This method has resulted in quite good thin uniform targets.However the target must be coated with an acrylic sprayafter the mass of the deposition has been determined. Thisis to reduce flaking.

They have also tried to use static charge to holdthin layers of powder onto Mylar and also Mylar with anadhesive coating. These two methods have proven to beinconsistent as far as yielding reproducible depositions.There is a lower limit as to how thin one should make atarget and that is on the order of the mean particlediameter.

In the analysis of material by x-ray fluorescenceutilizing the powder method, particle size effects can bea cominant factor in an accurate and reproduciblequantitative chemical analysis with variations in particlesize often responsible for significant changes in theemitted intensity. In multiphase systems, as portlandcement, the intensities from several elements may allincrease, decrease, or one may increase while anotherdecreases as a function of particle size. Here is the samplepreparation method as recommended by ORTEC:

Prior to the analysis, a minimum grinding time wasestablished by grinding one cement sample in a Spex ShatterBox (Tungsten carbide mill) for 1, 2, 3, 4, 5, and 6 minutes(5 g cement + 100 mg Ivory Snow). The resulting powderswere pelletized at a pressure of 15 tons/sq in. and mounted

1 1 1

in the instrument and each analyzed for 200 seconds. Agrinding time of five minutes was established since theintensity vs. particle size was stabilized and formed aplateau at approximately four minutes. As a result of thegrinding curve, all samples for subsequent analysis wereground for the minimum grinding time plus one minute orfor a total of six minutes.

4.4 SoilTrace metal levels in soils are not only important from

a geochemical or pedological point of view, but also withrespect to environmental research, since atmospheric fallout,incidental contamination and deliberate waste dumping canbe reflected.

In their work van Grieken et al., (1982) have preparedthin targets as follows: Aliquots of 1 g of soil or rock aremixed with 7 ml of twice-distilled water and pulverized for1 min in a McCrone Micronizing mill, with corundum or agategrinding elements. Of the resulting suspension a 0.5 mlfraction is pipetted onto a Mylar carrier foil and dried at80 C. Also thicker pellets can be compressed to obtain bettersensitivities (for the high-Z-elements only), at the expenseof more critical matrix effect corrections.

Here again the variations in particle size can causeserious errors in quantitative analysis. The pellets from soilsamples can be prepared as follows:

Five grams of sample, plus 100 mg of calcium stéarateas a grinding aid should be ground in a Spex Sha'tterbox fora period of five minutes. The resulting powder should be placedin a die with a boric acid backing added and pressed at 15tons/sq. in.

4 .5 Geological SamplesThere is a large number of possibilities how to prepare

geological samples for the analysis by XRF. Here we shalldescribe some .

Description of the technique used by Elsheimer and Fadbi(AXRA 17) contains the following instructions:

Mix samples by the hand-rolling technique just priorto analysis because high density sulfide and sulfate mineralsmay segregate during mechanical mixing, causing as much asa 15% error. For samples known to contain 10% or less ofsulfur as sulfide or sulfate, take 100 mg for each analysis.Weigh 50-mg portions for samples having a sulfur contentsignificantly greater than 10%, known to contain large amountsof ferrous iron, or thought to contain elemental sulfur. Mixeach sample intimately on weighing paper with 200 mg ofCe(NH4)2(N03) 5(99.97%% pure, G.F. Smith Chemical Co.),prepared for use by grinding to 100 mesh and drying for1 hr at -C 85°C. Lightly mix into this mixture 150 or200 mg of pure quartz, depending on the sample size(100 or 50 mg). Transfer the resulting mixture to a 30-ml,

112

black-glazed porcelain crucible containing 1.7500 g ofdried

Thoroughly mix the contents in the crucible and carefullytransfer term to a 30-ml, preignited graphite crucible witha hemispherical bottom. Fuse the sample mixture for 15 minin a furnace at 900 C and cool the resulting bead in air.

Remove the bead from the crucible, weigh and place ina large steel ball mill (Spex Industries, Inc., Model No. 8001)containing two 1 /2-inch-diameter steel balls. Grind for 4 minand transfer the resulting powder to a boron carbide mortarcontaining a weighed portion of Chromatographie cellulose(Whatmin CF-I.I.) sufficient to bring the combined weight ofbead and cellulose to 2.4000 g. After mixing to a uniformconsistency, return the mixture to the ball mill and grindfor an additional 5 min. To insure uniform particle sizeand homogeneity, transfer the ground mixture to the mortaragain and hand grind for a few min. Split the finely groundmixture into approximately equal parts on weighing paperand prepare 2 pellets for XRF determination. Analyze eachsample or standard in duplicate.

In the measurements described by King et al., (1976)a modified direct dilution method was used in which thesample-to-binder ratio was increased from 1:1 (Fabbi andMoore, 1970; Fabbi, 1970; Fabbi et al., 1975) to 85:15;the binder is a Chromatographie cellulose. A mixture of 28%paraffin base powder and 72% methyl cellulose was used forthe backing material. By increasing the amount of sampleto be analyzed, it was possible to obtain sufficiently highx-ray intensities from the trace elements of interest togain better precision and sensitivity.

Based on the results of the binder and backingexperiment, Fabbi 's direct dilution sample preparation methodwas modified by grinding 850 mg of sample instead of 500 mgin a vial for 10 min using a mixer mill. A binder consistingof 150 mg of 200 mesh Chromatographie cellulose was mixedwith the ground sample in a mortar and pestle. The mixturewas transferred back into the vial, and ground for 5 min inthe mixer mill. The finely ground powder was then pressedinto a pellet at 30.000 psi, using a backing material whichis a mixture of 28% paraffin base powder and 72% methylcellulose (Mixture A).

The results of binder and backing investigations byKing et al., (1976) are summarized in Table 4.2. When sampleswere not diluted or diluted with too small an amount ofbinder, the vials were hard to clean. The sample loss wasgreat, and the ground rock powder either did not adhereor adhered poorly to the backing. With an increasingratio of cellulose binder to the sample and by usingmethyl cellulose or mixture A, as a backing material,the vials can be easily cleaned, and the pellets obtainedhad homogeneous, mirror-like surface. With mixture Aas a backing, the adherence was excellent, no crackingof surfaces or any other problems were encountered inthe sample preparation. Using aspirin as a binder, thepreparation was very cumbersome , and uneven shades ofdarkness appeared on the pellet surface. Therefore, it

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Table 4.2

Results of binder and backingexperiments by King et a l . , (1976)

Sample-to-binderratio Binder Backing Results

100:0

95:5

85:15

85:15

85:15

85:15

80:20

75:25

Chromatographiecellulose

Chromatographiecellulose

Chromatographie

Aspirin

Paraffin,spectroscopicallypureChromatographie

cellulose

Chromatographie

Methyl-cellulose

Methyl-cellulose

Methyl-cellulose

Mixture AX

Methyl-cellulose

Methyl-eelluloseM

Methyl-cellulose

Methyl-eellulose

Vial hard to clean.Sample does not adhere to backing.Pellet surface cracks or pops up.

Vial hard to clean.Sample does not adhere or adheres.poorly to the backing.

Vial easy to clean, homogeneoussurface. Sample adheres tobacking.

Vial easy to clean, homogeneoussurface. Sample adheres rigidlyto the backing.

Vial hard to clean. Heterogeneoussurface.

Vial easy to clean, heterogeneoussurface. Sample deposits onthe glass lens.

Vial easy to clean, homogeneoussurface. Sample adheres rigidlyto the backing.

Vial easy to clean, homogeneoussurface. Sample adheres tothe backing.

Mixture A = 28% paraffin and 72% methyl cellulose.

S Ça Ti V Cr Fe Ni Cu Zn Au Se Rb U Sr

I, h hh h h M

"atoSAMPLE :COAL (II/19)

Mo-TUBE(26 kV, 12mA)

CHANNEL NUMBER

Fig. H.3 X-ray spectrum from coal sample.

Cfi

is not feasible to use aspirin as a binder, even thoughit is preweighed and can be directly crushed- and mixedwith the sample.

ORTEC procedure for the preparation of geologicalsamples includes the grounding of samples (about 5 gramsof material) in a tungsten carbide mill and pelletizedunder a pressure of 15 tons per square inch. (This grindingcontaminated the samples with a couple hundred ppm oftungsten which complicated the spectrum in the region ofCu and Zn due to tungsten L-line interference). The standardrock samples (AGV-1, BCR-1, PCC-1, G2 and GSP-1) werepelletized without grinding. The pressed pellets weretransferred directly to the TEFA analysis chamber whichwas evacuated prior to analysis.

SUj

CONCENTRATION

Fig. 4.4 Calibration curves for Ca, S and Fe incoal matrix.

'Standard addition method is often applied to geologicalsamples when a number of samples with identical (or similar)matrix has to be analyzed. As an example Fig. 4.3 showselements which were measured in coal samples using Mo-tubefor the excitation. In the experiment large number of coalsamples from the same coal mine had to be analyzed. Foreach element of interest number of standards was preparedby adding to cool known concentrations of element. Theanalysis has performed after appropriate mixing and samplepreparation and the x-ray intensities were measured as afunction of concentration. This is shown in Fig. 4.4 forÇa, S and Fe. These calibration curves were than used inthe concentration asignments for unknown coal samplesfrom the same coal mine. There was no need to apply anymatrix correction calculations afterwords.

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4.6 Atmospheric particulatesX-ray emission spectroscopy is applied to elemental

analysis of air particulates in all three forms of sampleexcitation: radioactive sources, x-ray tube, and chargedparticle beams. Here we shall first describe some systemsusing radioactive sources.

The analytical system used by Rhodes (1972) consistsof the following main components: automatic sample andsource changer with capacities of 30 samples and four sources ,respectively with three annular radioisotope source as-semblies: Fe-55, 120 mCi ; Pu-238, 400 mCi; and Cd-,109, 12 mCi.

Specimens in the form of discs of 47 mm diameter werecut out of the original filter papers and measured in batchesof 27 unknowns and three standards. To obtain the bestexcitation efficiencies, the 17 elements to be determinedwere divided into three groups, each excited with a differentsource, namely: Ca, Ti, V with Fe-55; Cr, Mn, Fe, Co, Ni,Cu, Zn with Pu-238; and Hg, Pb, As, Br, Sr, Zr, Mo with Cd-109Each batch of specimens was counted with each of the threesources. The counting period was that required to accumulate200.000 counts in the reference peak, which amounted to about10 min/specimen with Pu-238 and Cd-109, and 5 min with Fe-55.

The detection limits quoted in Table 4.5 are based on10-min measurements of deposits on cellulose filters. Longermeasurements improve detection limits while the presence ofhigh blanks (e.g., Zn in glass fiber filters, Cl in PVCmembrane filters) worsen them. Concentrations up to about1 mg/cm2 can be measured without interelemer^t effects andnonlinearities. After special calibration even higher concen-trations can be measured accurately.

More detailed literature is available on the topic ofanalysis aerosol loaded filters.

The major problems are in the correction for x-rayabsorption by the aerosol matter and by the filter material,in which the particles always penetrate to a certain extent,especially when cellulose fiber filters are being used. Thelatter problems can often be reduced significantly byanalysing the filter in a sandwich geometry, i.e. folded intwo with the loaded side inwards (Van Grieken et al., 1982).

For quantitative XRF analysis, the aerosol particlesneed to be collected as a uniform deposit on a suitablefilter medium. A filter medium of Teflon (Registered trademarkof E.I. duPont de Nemours and Company, Inc., Wilmington,Delaware) is preferred by Dzubay and Rickel (1978) becauseof the high purity and minimal tendency of Teflon to reactwith gaseous pollutants. Polycarbonate is also fairly goodas a filter medium for the same reasons. Esters of cellulosehave a high purity but are only useful as filter media forapplications where the tendency to absorb moisture and toreact with certain gaseous pollutants is not of concern. Mosttypes of filters that contain glass or quartz contain largeamounts of impurities of many elements, seriously limitingtheir application to XRF analysis.

Teflon membrane filters are available in a variety offorms. A thin 1-jam pore size Teflon membrane that is bonded

117

Table 4.5DETECTION LIMIT FOR A SYSTEM USING

RADIOACTIVE SOURCES (AFTER RHODES, 1972)

Elément g/cm2 g/M3

Si 0.50 120P 0.30 70S 0.17 40Cl 0.13 30K 0.10 20Ça 0.08 20Ti 0.05 12V 0.03 7Cr 0.20 50Mn 0.12 30Fe 0.10 20Co 0.08 20Ni 0.06 14Cu 0.04 9Zn 0.03 7As 0.10 20Se 0.10 20Br 0.10 20Pb 0.16 40Rb 0.06 14Sr 0.05 12Zr 0.04 9Mo 0.03 7Ag 0.20 50Cd 0.15 35In 0.20 50Sn 0.15 * 35Sb 0.15 35I 0.15 35Ba 0.20 50

CSI, Austin data. Criterion: 3 SD of background. Based onfilter area of 420 cm2 (8 in. x 10 in filter) and airvolume of 1800 M3 (24 hr. Hi-volume sample).

to a polyethylene support net is available from MilliporeCorporation as FALP Fluoropore. It was reported that itscollection efficiency exceeds 99.9% for all particles inthe 0.03-/Jm to 1-jum range. A minor problem with this typeof filter is the difficulty of making a good vacuum sealin some types of filter holders. A Teflon membrane withouta support net is available from Ghia Corporation, Pleasanton,California. This membrane has an asymmetric pore sizedistribution with 1-um pores on one side and 10-^um poreson the other. When particles are collected on the 10-/im side,there is a tendency for submicron particles to penetrate intothe filter. As a result, the filter medium attenuates thefluorescent x-rays from the particles, making it difficultto obtain quantitative results. Such attenuation is mostpronounced for the lighter elemetns which emit soft x-rays.By collecting the particles in the 1-^m side, the particlesare collected on the surface, which eliminates theattenuation by the filter.

In a typical XRF spectrometer, the exciting x ray beamis nonuniform across the sample. Therefore, the aerosol

118

deposit must be uniform if an accurate analysis is to beobtained. An acceptable deposit can be achieved if a filterholder with a suitable aerodynamic design is used.

4.7 PlantsPreparation of plant material for the analysis by XRF

requires a preconcentration step: ashing. This is early doneby some of the methods described in chapter 2.

Here is procedure used by van Grieken et al., (1982)for the preparation of algae samples:

The algae samples were dried at 80°C for 24 h inplastic Petri dishes, then manually ground by means of anagate pestle and mortar, dried again for 24 h in an ovenat 80°C and finally grounded for 1 min in a McCrone MicronizingMill, which introduced minimal contamination. Microscopephotographs showed that the powder is quite homogeneouswith a grain size of only a few micrometers. About 15 mg ofthe powder was suspended in some bidistilled water. Thisslurry was pipetted onto a Mylar foil (<4 jjm thickenss),which was glued to a Teflon ring, fitting in the samplechanger of the XRF-unit. The target area within the Teflonring was 9-6 cm2 . The slurries were dried carefully at 80 C,which resulted in targets of approximately 1.6 mg cm~2 plantmaterial, homogeneous to within a factor of two or better.

4.8 TissuesMany authors have analyzed different tissues for trace

elements using x-ray fluorescence as on analytical technique.Here we shall describe only some work, because there are no"many variations in sample preparation techniques used.

Here we shall describe work by Forssen (1972) who didtrace element analysis of different organs on autopsy samples.In this study the subjects were victims of accidental andother sudden deaths from external causes. The bodies wereplaced as soon as possible in a room at +4°C and stored therefor 1-2 days before autopsy. From each body 43 differentorgan samples were dissected and placed in polyethylenecontainers. The samples were frozen immediately and keptat a temperature below -15°C until prepared for ashing. Fromthe larger organs 20-70 g samples were taken. The smallerorgans were used whole. Skin samples were taken from themiddle of the surface of the abdomen and fat from below thesesamples. Polyethylene is a suitable container material forsamples to be analyzed for trace elements, since the traceelement concentrations in polyethylene are usually lessthan 1 mg/kg. Quartz crucibles were used for drying and ashingthe organ samples. Contamination was minimized by using dif-ferent sets of crucibles Tor each organ (brain, liver, etc.).The crucibles were washed with 32% HC1 each time before use,and rinsed with a liberal amount of cold and warm water andfinally with distilled water. They were then put in an ovenfor about 20 minutes at 100°C, cooled in a desiccator, andweighed. If cracks appeared in the glaze, the crucible wasdiscarded. For dry weight measurements the tissues were

119

dried at 105°C in the same quartz crucibles in which theywere to be ashed. Drying and ashing were performed in anelectric quartz muffle furnace. The initial temperature of200 C was maintained for some hours, it was then raised to450 C for one night. After the crucibles had been cooledin a desiccator and weighed, the ash was stored in tightlystoppered plastic tubes.

The ash was ground in an agate mortar, which wascleaned between grindings with silicon dioxide. 30 mg ofwell-ground ash was spread evenly on round filter paper. Theexact area supporting the ash was delimited in the middleof the filter paper by attaching another covering filterpaper, with a central circular aperture 18 mm in diameter,to the supporting paper. The weighed ash was spread over theround area delimited by the covering filter paper. A thincondenser paper was placed on the ash, and the sample waspressed with a hydraulic press (10-15 tons), which attachedthe ash to the filter paper. 30 mg of ash was found to be asuitable amount for this method. The optimal diameter of thecircle for spreading this amount of ash is 18 mm and thisdiameter was used in the present study.

Contact between the sample and metallic matter wascarefully avoided. When a metal tool had to be used it wasmade of pure tantalum.

Another detailed description for tissue samplepreparation is presented by lyengar and Sansoni (1980).

Calcified tissues such as bone and tooth presentformidable difficulties, especially when homogenization inthe natural state (i.e. without ashing) is desired. Thisdifficulty is exemplified by the absence of a certifiedreference material for bone in its natural form. Anotherdifficulty is the variation in the mass of marrow indifferent types and parts of bone. According to lyengar andSansoni (1980) big samples of bone can be divided into smallpieces by cooling in liquid nitrogen, wrapping in PVCsheets and then fracturing with the help of a nylon hammer.The conventional methods of powdering bone using agate,ceramic, tungsten carbide or steel mortars is unsuitablefor trace element analysis. Both wet digestion and dry ashingat high temperature have only limited use for calcifiedtissues. Low-temperature dry ashing (100-150 C) is usedby some investigators because of its simplicity and wideapplicability for the non-volatile elements. Concerning theloss of volatile elements, the assessment is complicated bythe unknown biochemical binding of trace elements in bone,which is a unique biological material.

In a recent attempt, the brittle fracture techniquehas been used to pulverize small pieces of bone at liquidnitrogen temperature, using a teflon.covered metal ball,teflon vessel and a microdismembrator. Because of technicaldifficulties, teflon-covered metal balls were not entirelysuccessful, and had to be replaced by pure titanium balls.However, only 2-3 g of bone and single tooth samples can behomogenized by this method, which is therefore unsuitablefor handling the much larger amounts needed for the preparationof analytical reference materials.

120

A primary difficulty with soft tissues results fromthe presence of unwanted components such as connective tissue,capsule, skin, visible fat, blood vessels, nerves, hair (inskin sampling), glandular parts, GI-tract contents such asfood remains and faeces, residual blood, extracellular fluid,etc., which are intimately mixed with the sample material tobe prepared for analysis and are difficult to remove completely.

Blood-rich organs, such as liver, heart, spleen, kidneyand placenta, present more difficulties than other soft tissues.It is not possible to remove the residual blood completelywithout damaging in some way the originality of the parenttissue.

Sample preparation is much simpler, and more reliable,when sufficient material is available for multiple subsampling,which is normally the case for autopsy material but not forbiopsy material. In the former case, after shaving off theouter layer with appropriate instruments, subsampling may bedone either in the frozen state or after thawing. However,random subsampling in the frozen state does not permit theremoval of interfering components, such as residual bloodin blood-rich organs. Another problem is that, if frozentissue is handled with a stainless steel knife, contaminationfrom elements such as Cr, Co, Mn and Ni should be anticipatedbecause of the greater friction in cutting the frozen solid.

Biopsy samples can be obtained from a number of organsand tissues such as liver, kidney, muscle, prostate, skin,tooth and bone. Only a few miligrams of the sample materialcan be obtained by needle biopsies. One problem with suchsmall samples is the difficulty of removing fat,^blood andconnective tissue. For example, about 1% of blood can befound in muscle samples. Another difficulty is to determinethe exact weight of the sample if information is needed ona fresh-weight basis. This is generally done by weighing thesamples several times at short known intervals of time soonafter removal and extrapolating to zero time.

Biological fluids are susceptible to bacterial growthin the unfrozen state and therefore sample preparation shouldpreferably be finished within 24 h. Among the various biologicalfluids whole blood, serum, plasma, urine, milk and cerebrospinalfluid are easily accessible, other less common specimens inthis group include semen, tears, sweat, sputum and saliva.Samples from fluids such as synovial, amniotic, pancreatic,prostatic, bile and gastric juice require special procedures.Drying of biological fluids concentrates the residue by a largefactor - about 5 for whole blood, 12 for serum and 30 for urine.

Let us describe procedure to be followed for some fluids:Blood

Blood is conveniently obtained by venous puncture. Sincevarious considerations limit the widespread use of non-contami-nating needles made of alloys such as Pt-Rh for drawing blood,disposable needles and syringes are generally used. It isrecommended to collect the blood in 10 or 20 ml fractions suc-cessively using the same needle, and to discard the first twoor three fractions in order to limit contamination. However,

121

this method is not entirely satisfactory for eleme^3 such asCr and Mn.

In addition to the difficulties listed above, thepreparation of neonatal blood samples presents additionalproblems since only a small volume can be obtained becauseof certain paediatric considerations.

It is important to avoid haemolysis of blood samples,since elements such as K and Fe, which are present at ahigher concentration in erythrocytes, may affect the serumvalue depending upon the degree of haemolysis. Visiblehaemolysis begins at about 50 mg of haemoglobin per 100 mlserum. Use of a dry syringe, slow transfer to a dry testtube, and sufficient time for clotting are all necessary.The use of fresh blood, blood from the newborn and bloodwhich has not sufficiently clotted, increase the risk ofhaemolysis. Up to 0.3% haemolysis does not affect the Cu andZn values in serum.

Simply drying a 200 ul serum aliquot on a thin carrierat ambient temperature can be chosen as a sample preparationstep. This yields a preconcentration factor of about elevenand makes it possible to reach detection limits, for severalelements, quite well below the normal concentration level inthe serum. To correct for the distinct inhomogeneity of theserum deposits, scandium and yttrium spikes were added as earlyas possible in the sample preparation to serve as internalstandards.

Serum<r

Serum should preferably be separated from clotted bloodwithin one hour. The clotting of blood takes about 15 minat room temperature, but is delayed if siliconized glassware,teflon or PE-containers are used. Fractionation of blood bycentrifugation is done at about 3000 rev/min for 5 to 10 min.The contents of the tubes should be kept closed to preventcontamination and loss by evaporation. Recentrifugation ofthe separated serum is sometimes necessary to spin down theresidual erythrocytes. Disadvantages with serum are thathaemolysis is greater than in plasma and clotting releasesK from platelets. For this reason, normal K values are lowerin plasma than in serum. Zn may also be affected in the sameway.

In the work by Cesareo (1982) different kinds ofserum samples have been compared: -0.5-1 ml of liquid serumsamples, directly analyzed; -0-1-0.5 ml serum samples depositedon thin filters and dried; -0.5 ml of serum samples, ashedat about 500°C. The first preparation method, i.e. directanalysis of liquid samples, has been considered for completeness,since analysis of liquid samples gives rise to superior MDL.

In the second case, when the acqueous component ofserum is evaporated, metals, proteins and other organiccompounds remain in the filter, and fluorescent radiation isstrongly attenuated. In the last case, proteins and otherorganic compounds split, and an ash residue of about 1-3 mgremains, which can be concentrated in a thin substrate andoptimally analyzed.

122

For the analysis of iron in blood in toto, theconcentration of which is about 500 ppm, high sensitivityis not required. In this case, XRF measurements requireapproximately 0.1 ml blood deposited on filter paper.

PlasmaThe main difference between serum and plasma is the

presence of fibrinogen in the latter. Plasma can be obtainedby immediate centrifugation of heparinized blood, but thepresence of the anticoagulant is undesirable for traceelement analysis. However, it is possible to obta.in plasmawithout an anticoagulant if fresh blood is immediatelycentrifuged. The conditions are the same as described underserum.

ErythrocytesCareful removal of all the serum is necessary to separate

the erythrocytes. However, a certain amount of trapped serum(usually 5-8%) is unavoidable and needs correction. A check onthe haematocrit is recommended if erythrocyte values are usedto compute whole blood values, or vice versa.

UrineAccording to the procedure described by van Grieken

et al., (1982) sample preparation procedure involves ashing25 ml urine samples overnight in an automatic Tecator wetdestruction unit. The program consists of heating at 130 C(for 2 h), 150°C (1 h), 250°C (1 h) 350°C (1 h), 400°C(1 h) and 460°C (3 h).

(The final ashing temperature was chosen after recordinga thermogravimetric ashing curve for the urine residue. Sincea significant weight loss was observed just below 460 C,resulting in more advantageous XRF detection limits, and sincemost authors do not report elemental losses below 500°C (exceptfor, e.g., Hg, Se, As), the maximum ashing temperature wasset at 460 C).

After cooling, the residue is transferred to an agatemortar for homogenizing with an agate pestle. Between two thinMylar foils, the powder is then pressed into a pellet at7000 kg cm-2.

_pThe pellet, of about 60 mg cm thickness, is fixed ontoa Mylar foil in a teflon ring which fits into the XRF targetholder.Sweat

Sweat is usually collected either by the so-called armbag method using a PE-bag around the arm or from the wholebody collecting the free flowing drops from various pointsof the body. This is done in a specially created sweatingenvironment after the subjects have showered. However, col-lection of a valid sweat sample is difficult because ofnumerous contamination hazards. In addition, unpredictable

123

dilution, may occur since the profuseness of sweating variesgreatly in different parts of the body. The sweat collectedshould be homogenized by vigorous shaking and prepared im-mediately for analysis, it is necessary to centrifuge it inorder to obtain a cell-free sample.

5. STANDARDS

Whenever it is possible analytical methods should bevalidatedthrough the use of certified standards. In theirabsence, independent analytical techniques should be usedon a subset of samples in order to obtain a measurement ofaccuracy.

The number of institutions and agencies involved inthe manufacturing, testing and distribution of standardreference materials (SRM) is increasing. Here are someaddresses :

in the u.s.A: many standards are available from the U.S.National Bureau of Standards. In addition, the Federal WaterQuality Administration, now a part of the EnvironmentalProtection Agency, has made available a number of standardwater samples for a few elements. Rock standards are availablefrom the U.S. Geological Survey and include granites, basalts,and carbonates. ^

In Europe ,a number of standard reference materials canbe obtained from the Analytical Quality Control Services (AQCS)provided by International Atomic Energy Agency.

The purpose of AQCS is to enable laboratories engagedin the analysis of nuclear materials, radionuclides or traceelements for which nuclear methods may be used to their advantage,to check the quality of their work. Such a control is neces-sary since results of analytical activities may be the basisupon which economic, administrative, medical or legal decisionsare taken; they must, therefore, be documented to be suf-ficiently reliable.

Reliability of results is a function of precision(reproducibility) and of accuracy. The precision of resultscan easily be determined by internal measures. The determinationof accuracy, however, in most cases requires more detailedprocedures such as (LAB/243 Circ., IAEA, 1982):

- Analysis to be carried out by as many different methods,analysts and instruments as possible. In cases whereagreement is good, results are assumed to be accurate.

- Control, analysis of so-called certified referencematerial, i.e. material of certified qualitative andquantitative composition which is as similar aspossible to the materials to be analysed. Agreementbetween certified and observed values is then adirect measure of accuracy for the particular typeof analysis .

- Participation in an interlaboratory comparison.Samples used in such an intercomparison should be,

124

as far as is possible, similar in composition andconcentration to the samples to be analysed on aroutine basis. The agreement of results from aparticular laboratory with the most probable valueobtained from a statistical evaluation of allresults is a measure of the accuracy for the typeof analysis under investigation.

For practical reasons, most analytical laboratories arenot in a position to check accuracy internally, as

- frequently resources are available for only onemethod ;

- certified reference material, particularly in the caseof trace analysis, is not available and can be preparedby the institutes themselves only in exceptional cases;

- intercomparisons are organized rather seldom and manyimportant types of analysis have not been covered sofar.

To oversome these difficulties I.A.E.A. provides AnalyticalQuality Control Services (AQCS), which is involved in distributingcertified reference materials (CRM), reference materials (RM),and samples for intercomparisons (I).

Reference materials and certified reference materialsavailable in 1983 are listed in Table 5.1. Reference materialsmarked by "rm" are materials which have previously beendistributed as intercomparison samples, and which can bestored for reasonable time without appreciable change. Althoughthe concentrations of elements or radionuclides in thosesamples ar*e in most cases reliably established-by the resultsof the intercomparison, the materials are not issued as CRMs,either because they have not been analysed by a sufficientlylarge number of different analytical techniques, or becausethe individual intercomparison results are too divergent.

Certified reference materials marked "CRM" in the Table 5.1are rigorously analysed or calibrated by well known laboratories,normally by many different methods. Some CRMs are certifiedon the basis of interlaboratory comparison runs provided thatthe number of participating laboratories and of technique usedwas sufficiently big and the results were sufficientlycompatible. Each material is supplied on request with acertificate stating its composition, physical form, etc.

5.1 Standard SolutionsStandard solutions can be prepared in the laboratory and

in most cases the needs of analyst using x-ray emissionspectroscopy can be satisfied in such a way.

Sometimes standard solutions used for atomic absorptionspectroscopy are available and this might be of great value.There are many manufactures from which such solutions -arecommercialy available. As an example, we list on Table 5.2compounds and matrices used in preparation of AA standardsolutions by Hicol BV (P.O. Box 1 1 5 1 , 3000 BD Rotterdam,Holland).

125

toTable 5-1

I.A.E.A. Reference Materials and CertifiedReference Materials

SAMPLECODE

Nuclear

S-7

S-8

S-12

S-13

S-lk

S-15S-16V-SMOWSLAP

MATRIX

materials and stable Isotope

Uranium ore:PitchblendeUranium ore:PitchblendeUranium ore:PitchblendeUranium ore:PitchblendeThorium oreThorium oreThorium oreWater

Water

ELEMENTS ORNUCLIDES

REFERENCED

standards

U content

U content

U content

U content

Th and U contentTh and U contentTh and U contentRatio: 180/160; 2H/}tt

Ratio: 180/160; 2H/!H

CONCENTRATIONCR ACTIVITY

LEVEL

0.527% U30ß

o.mo% u.oftJ 8

0.011% U,0n3 o

0.039% U30a

Th content below 0.1%Th content below 0.5%Th content above 1%

180 , - 55.5%ol/

SAMPLESIZE

100 g

100 g

100 g

100 g

50 g

50 g

50 g

. 30 ml30 ml

CLASSOF

SAMPLE

CRM

CRM

CRM

CRM

RM2-'

RM2/

RM2-7

RM

RM'H = - 428%o

Table 5.1 (cont'd)

SAMPLECODE

MATRIX

Environmental iraterials

Air-3/1

Soil-5

SL-1

F-1

Simulated depositionon air filter (con-taining also somemajor constituentsof dust)Soil

Lake Sediment

ELEMENTS ORNUCLIDESREFERENCED

Feldspar

Trace elements: As, Cd, Co,Cr, Cu, Fe, Mn, Ni, Pb, Se,U, V, Zn

Trace elements: Al, As, Be,Br, Ce, Co, Cr, Cs, Cu, Dy,Eu, Fe, Ga, Hf, Ho, K, La,Li, Lu, Mn, Na, Nd, Pb, Rb,Sb, Se, Sra, Ta, Tb, Th, U,Yb, ZnTrace elements: As, Ba, Br,Cd, Ce, Co, Cr, Cs, Cu, Dy,Fe, Hf, La, Mn, Na, Nd, Ni,Pb, Rb, Sb, Sc, Sm, Th, U,Yb, V, Zn, Ti '

CONCENTRATIONOR ACTIVITY

LEVELSAMPLESIZE

CLASSOF

SAMPLE

Fe, Pb, Zn: 100-200.ug 6 filters CRMothers: 0.1-30 .ug (+ 6per one filter blanks)

natural content

natural content

U, K natural content

25 g CRM

25 g CRM

25 g RM

CO Table 5.1 (cont'd)

SAMPLECODE

Animal andA-11

A-12

A-13

V-8

V-9

MaterialsH -4

H-5

MATRIX

plant materialsMilk powder

Animal boneFreeze driedanimal blood

Rye flour

Cotton cellulose

for biomédical studiesMuscle

Animal bone (inclu-ding mineral andorganic components)

ELEMENTS OR CONCENTRATIONNUCLIDES OR ACTIVITYREFERENCED LEVEL

Trace elements: Ca, Cl, natural contentCu, Fe, Hg, K, Mg, Mn,Na, P, Rb, ZnSr-90, Ra-226 environmental levelsTrace elements- natural content

Trace elements: Br, Ca, Cl, natural contentCl, Cu, Fe, K, Mg, Mn,P, Rb, ZnTrace elements- natural content

Elements: Br, Ca, Cl, Cs, natural contentCu, Fe, Hg, K, Mg, Mn, Ka,Rb, Se, ZnTrace elements: Ba, Er, Ca, natural contentCl, Fe, K, Mg, Na, P, Pb,Sr, Zn

SAMPLESIZE

25 g

80 g

25 g

50 g

25 g

20 g(2 vials)

30 g(2 vials)

CLASSOF

SAMPLE

RM

RM2-7

RM2-7

RM

RM27

CRM

RM

H-8 Horse kidney Cd •«• ether trace elements natural content 30 g(2 vials) RM

Table 5.1 (cont'd)

SAMPLE

Materials of marineMA-A-1

MA-A-2

MA-M-1

MATRIX

originDried copepoda

Homogenized fishflesh

Oyster homogenate

ELEMENTS ORNUCLIDESREFERENCED

Trace elements: CdfCu, Fe, Hg, Pb, Zn,etc. and chlorinatedhydrocarbons

Trace elements andchlorinatedhydrocarbonsChlorinatedhydrocarbons


LEVEL

natural content

100.ug/g

natural contentlevel of ug/g

100 .ug/g

CLASSSAMPLE OFSIZE SAMPLE

30 g RM

30 g RM

30 g RM

M = "sample - RV-SMOW ,. t ,,. _ ,. ï8nll6n 2U.1U .. .———j-————————— (in parts per mil), where R denotes O/ 0 or H/ H, respectively.V-SMOW

2l According to the results of intercomparison the material will be classified as RM or CRM.3/ Referenced elements will be listed according to the results of intercomparison.

K)

Table 5.2Compounds and Matrices for Standard Solutions

Element Compound Matrix

Aluminium AlCl.,Antimony KSbO C^H^OgArsenic NaOH As203Barium BaCl2Beryl-lium BeSO^Bismuth BiO(N03)Boron H, BO,Cadmium Cd(NO,)2Calcium CaCl2f* A w A i* m i N H 1 f^(ö(M O }Lr C I X U UJ \"*i)lX'3V^s5\,l»\*'-j/,

Césium ' CsClChromium Cr(NC>3)_Chromium K2Cr20-Cobalt Co(N03)2Copper Cu(NO,)2Dysprosium DyCl,Erbium ErCl3Europium EuCl3Gadolinium GdCl3Gallium Ga(N03),Germanium GeGold H AuCl^Hafnium HHNCO^Holmium HoCl,Indium In (NO,),Iridium HglrClgIron Fe(NO,)_Lanthanum Lad,Lead Pb(NO.,)2Lithium LiClLutetium Lu20.jMagnésium MgCl2Manganèse Mn(NO->)pMercury Hg ( NO^ ) ?Molybdenum MoMeodymium NdCl,Nickel Ni(NO.,)2Niobium NbCNO-,),.

HC1H20HN03Ij r\H20H2SO^HN03HC1HN03HC1

6 H2°H20HN03H20HN03HN03HC1HC1HC1HC1HN03HC1HC1HN03HC1HN03HC1HN03HC1HN03H20HC1HC1HN03HN03HC1 •HC1HN03HNO

130

Table 5.2 (cont'd)

ElementOsmiumPalladiumPhosphorusPlatinumPotassiumPraseodymiumRheniumRhodiumRubidiumRutheniumSamariumScandiumSeleniumSiliconSilverSodiumStrontiumTantalumTelluriumTerbiumThalliumThoriumThuliumTinTitaniumTungstenUraniumVanadiumYtterbiumYttriumZincZirconium

Compound

PdCl2

H2PtClgKC1

RbCl

SmCl.ScCl"SeSi

NaCl

HgTeOg

SnCl

W

MatrixH20HC1H2°HC1H.OHC1

HC1H.OHC1HC1HC1

H20

H20H20

H.OHC1

H.OHC1HC1H2SOH20H.O

HC1HC1

ZrOCl, HC1

Available for example fromHicol BV, P.O. Box 1 1 5 1 , Rotterdam, The Netherlands

131

K)3

O4

K)3

I02

10'

Ni: Pb (1=20) on Al-formvor backingE * 3 MeV

Ni K-lines

l a -r, ß

200 400 600 800 1000 1200 1400

CHANNEL NUMBER

Fig. 5.1 X-ray spectrum from Ni:Pb (1:20)standard solution

Standard solutions should be used during the measurementsof relative efficiency of system for different elements, aswell when the internal standards case used. For example, Fig. 5.1shows x-ray spectrum of Ni :Pb (1:20) standard solution. Otherexamples can be found in the literature.

"Homogeneous" standard samples can by prepared in thisway:

A measured volume of a gravimetrically-prepared solutionstandard is micropipetted by the multidrop technique onto acellulose fiber or cellulose membrane filter so as to homo-geneously impregnate the filter. This is rapidly dried yieldinga deposit having a submicron particle size, a homogeneity of5% or better on a scale of 5 mm, and an over-all concentrationknown to better than 2%.

Thin laye-red sample can be prepared using Snap-on ringsample cap. First, one has to affix thin-film sample suport;then a slight concavity in the thin-film with a round-endedglass rod. Gentle heating will restore thin-film to originaltautness. Insert solution droplet, evaporate if desired andoverlay with another piece of thin-film of suitable gaugewith Snap-on ring.

132

5 .2 Reference MaterialsThere is a number of reference material available to

the analyst. They can be classified into groups, i.e.biological, geological, etc. reference materials. For example,biological reference materials for trace element studiesare currently being prepared and issued by a number of dif-ferent international and national laboratories. Very com-monly, when such materials are submitted as unknown samplesto a diverse group of analytical laboratories', the resultsreported cover a wide range - in some cases so wide thatno consensus values can be derived. In part this may be dueto the inexperience of some of the laboratories concerned.However, for some elements and some matrices, even well-experienced laboratories may have great difficulties inproducing mutually consistent results. Such problems, asevidenced by results for reference materials, are probablyalso very typical of what may be encountered in analysingany biological material. Thus they provide a very usefuldemonstration of the practical problems that analystsworking in this field must be prepared to face (Kosta, 1980).

Biological CRMs for trace element studies arepresently available from two main sources, the US NationalBureau of Standards (Office of Standard Reference Materials,National Bureau of Standards, US Department of Commerce,Washington, D.C. 21234, USA) and the IAEA (Analytical QualityControl Services, Laboratory Seibersdofr. InternationalAtomic Energy Agency, P.OB 100, A-1400 Vienna, Austria). Inaddition, one of the earliest and most important of allbiological CRMs, Bowen's kale, is still available from itsoriginator (Prof. H.J.M. Bowen, Department of Chemistry,Reading University, Whiteknights Park, Reading, Berks RG62AD, United Kingdom). Table 5.3 gives details of.allpresently available CRMs and the elements for which theyaretcertified; the concentrations of these elements (exceptnitrogen) and other details taken from the relevantcertificates of analysis or information sheets are givenin the Table 5.4 (Parr, 1980).

The data in Table 5.4 are listed in alphabetic orderof the chemical symbol of the element, and within eachelement, in order of decreasing value of the concentration.The number of available CRMs for each element can thus beseen at a glance as well as the range of concentration ofthe element.

Here we shall present spectra from some referencematerials obtained by sample irradiation with x-ray tube(Mo-anode).

Fig. 5,2 shows the x-ray spectrum obtained from theirradiation of animal muscle, IAEA H-4 standard referencematerial. The spectrum showed was obtained in few minutesmeasurement with a sample prepared in the form of formvar-tissue-formvar sandwich. Two measurements were performed;thin and thick target measurement. Concentration ratioswere determined from thick target measurement while theabsolute Zn concentration was determined in a thin targetmeasurement.

133

Table 5.3

List of presently available CRMs. See textfor addresses of suppliers (after Parr 1980)

Supplier MaterialNBS Oyster

Wheat flourRice flour

Brewers yeastSpinach

Orchard leaves

Tomato leaves

Pine needles

Bovine liver

Elements certifiedAg,Mn,Ca,As,Na,CrAl,P,As,Mg,Se,As,Sr,Al,Pb,As,N,

AsNaCdCaSe

AsPb,B,MnSrCaThAsRbCa

Na,

, Ca,, Mi,, Cu,, Cd,, Zn

, Ca,Rb,Be,, Mo,, Th,, Cr,. u,.. Ca,, Sr,, Cd,Pb,

Cd,Pb,Fe,Co,

Cr,Sr,Ca,N,u,Cu,

ZnCr,Th,Cr,

Rb,

Cr,Rb,Hg,Cu,

Cu,Th,Cd,Na,ZnFe,

Cu,UCu,

Se,

CuSeK,Fe

Feu,Cr,Ni,

K,

Fe

FeZn

, Fe, Hg, Sr, U,Mn, Na,, Hg,. K,

, Hg, K,ZnCu, Fe,P, Pb,

Mn, P,

, Hg , K ,

, Hg, K,

, Mg,ZnSe , ZnMn,

Mn,

Hg, K,Rb, Sb,

Pb, Rb,

Mn, P,

Mn, Mg,

IAEA Animal bloodFish SolublesMilk powder

Animal muscle

Human serumCopepodFish fleshWheat flour

Co, Cu, Fe, Mn, Pb, Se, ZnAs, Co, Cr, Cu, Fe, Hg, Mn, Se ZnCa, Cl, Co, Cu, Fe, Hgr K, Mn, Mg,Na, P, Rb, Se, ZnBr, Ca, Cl, Cs, Cu, Fe, K, Mg, Mn,Na, Rb, Se, ZnI (total and protein bound)Cd, Cu, Fe, Hg, ZnAs , Cu, Fe , Hg, Mn, ZnI

Bowen Kale As, Au, B, Br, Ca, Cd, Cl, Co, Cs,Cu, Fe, Hg, K, La, Mn, Mg, Mo, N, Na,P, Rb, Sb, Se, Se, Sn, V, W, Zn

As another example Fig. 5-3 shows x-ray spectrum obtainedby the irradiation of fish sample, IAEA A6 standard referencematerial. It should be mentioned 0.5 mg of material was usedin sample preparation, therefore there is a question of howrepresentative that is.

Figures 5.4 and 5.5 show the measured x-ray spectra fromthe irradiation of IAEA hair, HH1 standard with Mo-tube (26 kV,12 mA) using 3 filters. Targets were prepared on formvar backingand in the form of formvar-hair (or hair ash) - formvar sandwich.Thick targets (20 mg/cm2 ) were used for the determination ofelement concentration ratios, while thin targets (0.2 mg/cm2)were used for determination of absolute concentrations of Zn

134

Table 5.4

Elemental composition of some CRMs inorder of the chemical symbol of the element

(after Parr, 1980)

Element Material

AgAlAlAsAsAsAsAsAsAsAsAsAsAuBBBeBrBrCaCaCaCaCaCaCaCaCaCaCaCaCdCdCdCd

Oyster tissueSpinachPine needlesFish solublesOyster tissueOrchard leavesFish fleshRice flourTomato leavesPine needlesSpinachBowen's kaleBovine liverBowen's kaleBowen's kaleOrchard leavesOrchard leavesBowen's kaleAnimal muscleBowen's kaleTomato leavesOrchard leavesSpinachMilk powderPine needlesPine needlesOyster tissueWheat flourAnimal muscleRice flourBovine liverOyster tissueBowen's kaleCoperodBovine liver

Code

NBS-SRM-1NBS-SRM-1NBS-SRM-1IAEA-A-6NBS-SRM-1NBS-SRM-1

566570575

566571

IAEA-MA-A-2NBS-SRM-1NBS-SRM-1NBS-SRM-1NBS-SRM-1

NBS-SRM-1

NBS-SRM-1NBS-SRM-1

IAEA-H-4

NBS-SRM-1NBS-SRM-1NBS-SRM-1IAEA-A-1 1NBS-SRM-1NBS-SRM-1NBS-SRM-1NBS-SRM-1IAEA-H-4NBS-SRM-1NBS-SRM-1NBS-SRM-1

568573575570

577

571571

573571570

575575566567

568577566

IAEA-MA-A-1NBS-SRM-1577

C*

CCCRCCQCCCCICIICCRRRCCCRCCCCRCCCIPC

Cone .(ppm)

08705451413102000000046330244

41400300002090013500129004100410015001901881401243000

Error

.89

.5

.4

.5

.41

.27

.21

.15

.14

.055

.0023

.027

.07

•

.5

.89

.75

.27

1055

1514201812191933149

1789375

1541126>44

135

131"

1 1108

15

-7.5

. i

.7

.1

.8

.3

.0

.4

.2

.2

.9

.9

.3

.4

.8

. 1

135

Table 5 .4 ( c t n ' d )

ElementCdCdCdClClClCo

CoCoCoCoCrCrCrCrCrCrCrCrCsCsCuCuCu

CuCuCuCuCuCuCuCuCuCuCuCuFe

MaterialOrchard leavesWheat flourRice flourMilk powderBowen's kaleAnimal muscleAnimal wholeBloodFish solublesBowen's kaleRice flourMilk powderSpinach .Tomato leave'sPine needlesOrchard leavesBrewers yeastFish solublesOyster tissueBovine liverAnimal muscleBoven's kaleBovine liverOyster tissueAnimal wholeBloodSpinachOrchard leavesTomato leavesCopepodFish solublesBowen's kale

• Fish fleshAnimal musclePine needlesRice flourWheat flourMilk powderTomato leaves

CodeNBS-SRM-1571NBS-SRM-1567NBS-SRM-1568IAEA-A-11

IAEA-H-4

IAEA-A-2IAEA-A-6

NBS-SRM-1568IAEA-A-11NBS-SRM-1570NBS-SRM-1573NBS-SRM-1575NBS-SRM-1571NBS-SRM-1569IAEA-A-6NBS-SRM-1566NBS-SRM-1577IAEA-H-4

NBS-SRM-1577NBS-SRM-1566

IAEA-A-2NBS-SRM-1570NBS-SRM-1571NBS-SRM-1573IAEA-MA-A-1IAEA-A-6

IAEA-MA-A-2IAEA-H-4NBS-SRM-1575NBS-SRM-1568NBS-SRM-1567IAEA-A-1 1NBS-SRM-1573

C*CCCRIR

RRRCRCCCCCRCCRICC

RCCCPRRQRCCCRC

Conc .(ppm)

0.110.0320.029

908035001890

0.420.220.060.020.0054.64.52.62.62.120.710.690.0880.120.075

19363.0

4512.01211 .07.65.254.94.63.963.02.22.00.84

690

Error(%)

9-12214198.64.4

24231750206.5

1 17.7122.424391412

• 6.75.25.6

8.8178.39.15.2128.6188.3

101415203.6

136

Table 5 .4 ( c t n ' d )

Element Material

FeFeFeFeFeFeFeFeFeFeFeFeFeFe

HgHgHgHgHgHgHgHgHgHgHgHgHgI

IKKKKKKKKK

Fish solublesSpinachOrchard leavesBovine liverPine needles -Oyster tissueBowen ' s kaleFish fleshCopepodAnimal muscleWheat flourRice flourMilk powderAnimal wholeBloodFish solublesFish fleshCopepodBowen 's kaleOrchard leavesPine needlesOyster tissueSpinachBovine liverAnimal muscleRice flourMilk powderWheat flourHuman bloodserumWheat flourTomato leavesSpinachBowen 's kaleMilk powder .Animal muscleOrchard leavesBovine liverOyster tissuePine needles

Code

IAEA-A-6NBS-SRM-1570NBS-SRM-1571NBS-SRM-1577NBS-SRM-1575NBS-SRM-1566

IAEA-MA-A-2IAEA-MA-A-1IAEA-tf-4NBS-SRM-1567NBS-SRM-1568IAEA-A-11

IAEA-A-2IAEA-A-6IAEA-MA-A-2IAEA-MA-A-1

NBS-SRMr1571NBS-SRM-1575NBS-SRM-1566NBS-SRM-1570NBS-SRM-1577IAEA-H-4NBS-SRM-1568IAEA-A-1 1NBS-SRM-1567

IAEA-H-6IAEA-V-5NBS-SRM-1573NBS-SRM-1570

IAEA-A-11IAEA-H-4NBS-SRM-1571NBS-SRM-1577NBS-SRM-1566NBS-SRM-1575

C*

RCCCCCRQPRCCR

RRQPRCCCCCRCRC

RRCCRRRCCCC

Conc .(ppm)

565550300268200195"1156l6049.118.38.73.73.41

73.90.480.28^0.180.1550.150.0570.0300.0160.0140.00600.00250.000

0.0680.0029

446003560024300172001584014700970096903700

Error(%)

7.73.66.73.05.0175.2136.64.25.56.9

21

6.4188.57.3

179.7

3326171327122980

16430.70.85.35.83-72.06.20.55.4

137

Table 5.4 ( c t n ' d )

Element Material

KKLaMgMgMgMgMgMgMnMnMnMn

MnMnMnMnMnMnMnMnMnMnMoMoNaNaNaNaNaNaNaNaNiNiPP

Wheat flourRice flourBowen's kaleOrchard LeavesBowen's kaleOyster tissueMilk powderAnimal muscleBovine liverPine needlesTomato leavesSpinachAnimal wholeBloodOrchard leavesRice flourOyster tissueBowen 's kale •Bovine liverWheat flourFish solublesFish fleshAnimal muscleMilk powderBowen's kaleOrchard leavesOyster tissueMilk powderBovine liverBowen's kaleAnimal muscleOrchard leavesWheat flourRice flourOrchard leavesOyster tissueMilk powderSpinach

Code

NBS-SRM-1567NBS-SRM-1568

NBS-SRM-1571

NBS-SRM-1566IAEA-A-11IAEA-H-4NBS-SRM-1577NBS-SRM-1575NBS-SRM-1573NBS-SRM-1570

IAEA-A-2NBS-SRM-1571NBS-SRM-1568NBS-SRM-1566

NBS-SRM-1577NBS-SRM-1567IAEA-A-6IAEA-MA-A-2IAEA-H-4IAEA-A-11

NBS-SRM-1571NBS-SRM-1566IAEA-A-1 1NBS-SRM-1577

IAEA-H-4NBS-SRM-1571NBS-SRM-1567NBS-SRM-1568NBS-SRM-1571NBS-SRM-1566IAEA-A-11NBS-SRM-1570

C*

CCICRCRRCCCC

RCCCRCCRQRRRCCRCIRCCCCCRC

Cone . Error(ppm) (%)

13601120

0.0862001560"12801 1001050604675238165

1239120.117.51510.38.54.731 .00.520.382.30.3

51004420243023002060

828.06.01.31.03

91005500

2.90.2

133-25.17.07.35.61.52.22.93.6

174.42.06.98.09.75.912217.1

219.1

335.97.55.3106.17.3

192515181 13.6

138

Element Material

Table 5.4 (ctn'd)

CodeCone . Error

PpppPbPbPbPbPb

PbPbRbRbRbRbRbRbRbRbRbSbSbScSeSeSeSeSe

SeSeSeSeSeSnSrSr

Bowen 'sTomatoOrchard

kaleleavesleaves

Pine needlesOrchard leavesPine needlesTomatoSpinachAnimalBloodOysterBovineBowen 's

leaves

whole

tissueliverkale

Milk powderAnimalBovineTomatoSpinachOrchard

muscleliverleaves

leavesPine needlesOysterOrchardBowen 'sBowen 's

tissueleaveskalekale

Fish solublesOyster tissueWheat flourBovineAnimalBloodAnimalBowen ' sOrchard

liverwhole

musclekaleleaves

Rice flourMilk powderBowen 'sSpinachTomato

kale

NBS-SRM-NBS-SRM-NBS-SRM-NBS-SRM-

1111

NBS-SRM-1NBS-SRM-NBS-SRM-

IAEA-A-2

11

NBS-SRM-1NBS-SRM-

IAEA-A-1IAEA-H-4NBS-SRM-NBS-SRM-NBS-SRM-NBS-SRM-

1

1

1111

NBS-SRM-1NBS-SRM-NBS-SRM-

IAEA-A-6NBS-SRM-NBS-SRM-NBS-SRM-

IAEA-A-2IAEA-H-4

NBS-SRM-

11

111

1NBS-SRM-1IAEA-A-1 1

573571575571575573570

566577

577573570571575566571

566567577

571568

NBS-SRM-1570leaves NBS-SRM-1573

ICCCCCCC

RCCRRRCCCCCCCIIRCCC

RRICCRI

CC

4450340021001200451061

000523118181612T21 142003211

0000000

8744

.8

.3

.2

.97

.48

.34

1

1

55476447

.8

.9

.8

.7

.6

.8

238.3

241020 -

-7.3.5.1

.7

.45

.9

.07

.008

.07

.1

.1

. 1

.59

.28

.14

.080

.040

.034

.21

.9

1111

75018020418

.8

.5

.6.

.7

.3

.9

.0

24189. 1

241

1

273. 1

25211H

20.3.7

139

Table 5.4 (ctn'd)

Element Material

SrSrSrThThThThUUUUUVWZnZnZnZn

ZnZnZnZnZnZnZnZnZnZn

Orchard leavesOyster tissuePine needlesTomato leavesSpinachOrchard leavesPine needlesOyster tissueTomato leavesSpinachOrchard leavesPine needlesBowen's kaleBowen's kaleOyster tissueCopepodBovine liverAnimal wholeBloodAnimal muscleTomato leavesSpinachMilk powderFish fleshBowen's kaleOrchard leavesRice flourFish solublesWheat flour

Code

NBS-SRM-1517NBS-SRM-1566NBS-SRM-1575NBS-SRM-1573NBS-SRM-1570NBS-SRM-1571NBS-SRM-1575NBS-SRM-1566NBS-SRM-1573NBS-SRM-1570NBS-SRM-1571NBS-SRM-1575

NBS-SRM-1566IAEA-MA-A-1NBS-SRM-1577

IAEA-A-2IAEA-H-4NBS-SRM-1573NBS-SRM-1570IAEA-A-11IAEA-MA-A-2

NBS-SRM-1571NBS-SRM-1568IAEA-A-6NBS-SRM-1567

C*

CcCcccccccccIIcpcRRCCRQRCCRC

Cone .(ppra)

3710.4.0.0.0.0.0.0.0.0.0.0.0.

852158130

8986.625038.36312519.18.10.

36 '81712 •0640371160610460290203606

3

9

496

Error(%)

254182598542017201 1121210

103945177

12579

.7

.4

.2

.4

.1

.2

.9

.6

.6

.9

.7

.0

.9

.1

.2

.0

.4

Type of £ertified value specified by issuing organizationC = certified concentrationI - indicated concentrationP = probable concentration

derived by applicationof Chauvenet's outlier test

Q = probable concentration(preliminary value; nooutlier test applied)

R = recommended concentration

140

ANIMAL MUSCLE. hK

Br Rb

1Uj

CHANNEL NUMBERFig. 5.2 X-ray spectrum from animal muscle,

IAEA H-4 SRM.

CLKL Cuu Bru

Fe, Zn,

FISH, A 6

COMPT.Mo,

S

CHANNEL NUMBER

Fig. 5.3 X-ray spectrum from fish sample,IAEA A6 SRM.

141

HAIR.H-1

ikl

Su Ça

CHANNEL NUMBER

Fig I 5.4 X-ray spectrum from hair sample,IAEA HH1 standard.

and Cu. The results obtained are shown in Table 5.5. Errorsshown are statistical ones .

Fig. 5.6 shows x-ray spectrum obtained with the samesystem from SOIL-5 SRM.

A number of other standard reference materials has beenprepared by different agencies. Very active in this field isoffice of Standard Reference Materials in National Bureauof Standards (Washington, D.C. USA). As an illustration weenclose the description of NBS SRM No 1635 (trace elements insubbituminous coal) in Table 5.6.

This Standard Reference Material is intended for use inthe calibration of apparatus and the evaluation of techniquesemployed in the trace element analysis of coal and similarmaterials. The material should be dried without heat to constantweight before use.

The recommended procedures for drying are either vacuumdrying at ambient temperature for 24 hours, or freeze dryingin which the drying chamber is kept at room temperature. Themoisture content of this material is approximately 20%. Becauseof this moisture level, it is recommended that small individualsamples be dried immediately before use. Drying of large samplesmay result in a violent discharge of water vapor and resultantloss of sample. When not in use, the material should be keptin a tightly sealed bottle and stored in a cool, dark place.Long-term ( >1 year) stability of this SRM has not beenrigorously established.

The certified values given in Table 5.6 are based on atleast a 250-mg sample of the dried material, the minimum amountthat should be used for analysis. During the preparation

142

Ar Ca|Ka Ka

FeKB

HAIR,ASH,H-1

As.,Fe.-Ca.NLClL.Zn., Zn„ Pb, Br.. Pb. SruKa Ka Ka Ko ' <a Kfl La Ka I L0 IK

CHANNEL NUMBER

Fig. 5-5 X-ray spectrum from hair ash sample

to

Pb,CuK0GaKct AsKaAsK(fbL^bK<

zPKJZnK,

SOIL- 5

CHANNEL NUMBER

Fig. 5.6 X-ray spectrum from soil sample,IAEA SOIL-5 SRM.

Table 5.5Trace element concentrations in IAEA hair HH-1 standard

ConcentrationElement ppm wet weight

ZnCuNiCoFeAsBrCaClSPb

•

==========

180122H

1915

1981643

35823only

.3

.68

.02

.6

.31

.11

in

•(••*•-1-+•f•h

-h

+

-t-

+

ash

1%4.8%16.7%16%15.2%1 9 . 5%6.9%3.8%11%2%

possibcontamination

144

Table 5.6

National Bureau of Standards (Washington D . C . U S A )Standard Reference Material 1635

Trace Elements in Subbituminous Coal

Element

Arsenic3 >bCadmium0 >d'e

Chromium0 >eCopper3 '°'eLead°'dManganese3 'eNickel°'dSelenium3 'e

Content, ug'/g

0.420.032.53.61.9

21.41.740.9

± 0.15- 0.01- 0.3i 0.3- 0.2- 1.5- 0.10- 0.3

Element

Thorium0'6Uranium0Vanadium6'8Zinc°'d

ElementIronc'd'e'fSulfurf 'h

Content, ug/g

0.62 -0.24 -5.2 ±4.7 ±

wt %20.2390.33

0.040.020.50.5

- 0.005- 0.03

Methods of Analysis:a. Atomic Absorption Spectroraetryb. Photon Activationc. Isotope Dilution Mass Spectrometryd. Polarographye. Neutron Activationf . Spectrophotometryg. Flame Emission Spectrometryh. Gravimetry

The estimated uncertainty is based on judgment and representsan evaluation of the combined effects of method imprecision,possible systematic errors among methods, and materialvariabili ty for samples of 250-mg or more . (No a t tempt wasmade to derive exact statistical measures of imprecisionbecause several methods were involved in the determinat ionof most const i tuents . )

of this material crushed and ground coal was sieved througha 250 urn ( N o . 60) sieve and thoroughly blended in a V-typeblender . Samples for homogeneity testing were taken fromthe top , middle , and bot tom of three bulk containers ofcoal and analyzed by neutron act ivation analysis forscand ium, chromium, i ron, cobalt , ce r ium, and thor ium.Replicate analyses of 250-mg samples indicated the materialvariabili ty for these elements to be within - 2% ( re la t ive) .The homogeneity measurements were also per formed .

The concentration values for some elements were notcert if ied because they were based on a non-reference me thod ,or were not determined by two or more independent methods .

145

5.3 Other StandardsA large number of standard materials have been developed "

by different agencies. For example, Columbia ScientificIndustries (11950 Jollyville Road, P.O. Box 9908, Austin,Texas 78766) is marketing thin specimen x-ray spectrometrycalibration standards. Single element or multielement driedsolution deposits on filters; also deposits of sized pureminerals or sized quartz spiked with trace elements.

In addition this company's marketing standard referencestrips -for air particulate analysis: 8 x 3/4 in. glass fiberor other filter strips impregnated with dried solution and/orparticulate deposits. Sulfate, nitrate, chloride, lead andarsenic available as dried solution deposits: also nine tracemetals available spiked on sized quartz particulates. Ammoniumion is available on cellulose filters only.

Standard samples are prepared gravimetrically from purematerials under carefully controlled conditions. All proceduresare rigorously monitored to insure a high standard of purity,accuracy and homogeneity of the reference samples. Specialdeposition techniques and non-interfering additives have beendeveloped to insure homogeneity of the x-ray calibrationstandards. Most of the calibration samples offered have beenindependently tested in interlaboratory comparisons.

A number of geological material standards are available.For example, standards used in the work by Beitz et al., (1970)are listed in Table 5.7. The first six standards of this tableare official standards of the U.S. Geological Survey Washington,D.C. They are supplied in the form of powders. The standardsT1 to T3 were supplied by a university together with analysisdata; the standards T12, T13, and T23 were obtained by mixingthe first three standards.

The melts were obtained by mixing 0.7 g of analysismaterial, 2.975 g of lithium tetraborate, and 0.525 g oflanthanum oxide, yielding melts at the ratio of 1:5.

The lanthanum oxide is used to reduce the interelementeffects on the measurement result, because the element Lahas a high absorption capacity for the radiations of theelements. Its use was due to a request made by the universitywhich commissioned the investigations. Actually, the lanthanumoxide could be dispensed with, because modern computerprograms are capable, even without such a damping agent, tocarry out matrix corrections with a high degree of accuracy.Moreover, the element La reduces the intensities of thecharacteristic x-ray radiation to be measured.

let us discuss the dificult problem of trace elementanalysis of oils. The analysis usually involves the additionof an accurately known amount of an oil-soluble compoundof the element in question to an oil which contains one ofthis element. The resulting standard solution is then car-ried through all the steps of the method and the amount ofthe element added is determined. The same procedure is ap-plied to the "unknown" oil, and its content of the elementis determined by comparison.

The problem of finding suitable metallo-organic compoundsto be used as standards has increased as more additives have

146

Table 5.7

Geological Standards

Designationof standard

PCC-1

DTS-1

BOR-1

G-2

GSP-1

AGV-1

T-1

T-2

T-3

T 12

T 13

T 23

Compound% MgO

13

19

30

0

1

9

.562

.805

.281

.782

.957

.192

.110

0.9302

5

5

1

.160

.170

-785

.515

% CaO

0.53*1

0.158

6.952

1.988

2.03*11.982

15.880

3.210

2.190

9-560

9.035

2.715

*K20

0.019

0.023

1.681

1.510

5.188

2.898

2.650

5.310

1.810

3-995

3-730

5.075

*»,2o

O.C53

0.016

3.3131.1562.880

1.331

3.65

1.720

3-670

1.185

3-660

1.195

% MnO

0.122

0.126

0.176

0.037

0.011

0.098

0.210

0.170

0.060

0.205

0.150

0.115

%Ti02

0.023

0.023

2.231

0.531

0.699

1 .082

2.970

1.190

0.580

2.080

1.775

0.885

%P2o5

0.011

0.013

0.3630.111

0.285

0.187

0.980

0.120

0.290

0.700

0.635

0.355

*Slo2

11.870

10.155

51.185

69.192

67.278

58.99739-910

57.860

67.620

18.885

53-76562.710

* A1203

0.85630.552

13-657

15.315

15.115

17.011

11.660

16.180

11.960

13-920

13-310

15.570

8.5378.850

13-508

2.768

1.3316.801

12.630

9.950

3-610

11.290

8.135

6.795

been introduced and as a more exact knowledge of the traceconstituents of petroleum and its products has been needed.Commerical metallo-organic compounds available were found notto be satisfactory as standards because (Isbell et al., 1962):

1. Metallic and other contaminants are often present inthese compounds in the amount greater than that ofthe trace element to be determined in the petroleumproduct ;

2. Some of the compounds are not sufficiently solublein petroleum products;

3. Some of the compounds are sufficiently volatile inthat considerable loss occurs during (or even before)the analysis; and

4. When combinations of the elements (as their respectivecompounds) were dissolved in a single oil. thecompounds often gave incompatible mixtures.

Isbell et al., (1962) have selected from 150 prospectivecompounds described in their monograph the ones having suitablephysical properties as standards in the determination of thefollowing 24 elements: Al, Ba. B, Cd. Ca, Cr. Co. Cu, Fe. Pb,Li, Mg, Mn, Hg, Ni, P, K, Si, Ag, Sr, Sn. V, and Zn. In theirpaper Isbell et al., (1962) give methods for preparation ofthe compounds and for spectrographic and chemical analysis ofthe chosen standards.

Procedures are described for the preparation of stablesolutions thereof in petroleum oils. Xylene, together with2-ethylhexanoic acid, 6-methyl-2, 4-heptanedione, and 2-ethylhexylamine are used as additives to render the varioussamples soluble. The resulting solutions are all'compatiblewith each other, and as a result blends containing a knownamount of several elements can be prepared.

The U.S. National Bureau of Standards (NBS) hasundertaken the development of Standard Reference Materials(SRM) for trace metals in gasoline and fuel oil (Von Lehndenet al., 1974); certified values have been established forFe, Ni, Pb, V, and Zn in the fuel oil (SRM-1634). In addition,information values (non-certified) are given for As, Cr, Hg,and Mn.

Metallo-organic standards, specially prepared organicsulfonates in al oil base, are available commercially (Conostanstandards, produced by Continental Oil Company, P.O. Box 1267.Ponca City, Oklahoma 74601). Individual standards at 5.000 ppmmetal in hydrocarbon oil solution are available for each ofthe following elements: Ag, Al, B, Ba, Be, Bi, Ca, Cd, Co, Cr,Cu, Fe, K, La, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Sb, Si, Sn. Ti,V, Y and Z.

The matrix oil for these standards is a paraffinichydrocarbon oil with an 80 SSU viscosity at 100 F (37.8 C) anda flash point of 340°F (171.1 C). These standards are readilysoluble in ketones as well as aromatic and paraffinichydrocarbons without the aid of solubilizers.

In addition, blended standards are also available. Blendsof equal amounts of 12 elements: Ag. Al, Cr. Cu. Fe, Mg, Na,Ni, Pb, Si, Sn, and Ti are available in concentrations from

148

ool/i

Uzz<Xol_o»CLCOh-ZOo

CHANNEL NUMBER (energy)Fig. 5.7 X-ray fluorescence spectra from th'e

C-20 Conostan standard: (a) excited byCu filtered brerasstrahlung radiationfrom W anode; (b) excited by Mo-filteredradiation.

1C ppm to 900 ppm. Standards which are blends of equal amountsof 20 elements are also prepared. C-20 are 20-element standardswith calcium.

Fig. 5.7 shows the characteristic x-ray spectrum fromthe 3 MeV proton bombardment of Conostan D-12 target. Thetarget was prepared by simple deposit of a few drops on filterpaper.

Fig. 5.8 shows examples of the x-ray spectra of the200 ppm C-20 multielement Conostan standard obtained by thex-ray fluorescence (Ortec, 1975). The 0 to 10 keV of thespectrum excited with Cu filtered bremsstrahlung radiationfrom a W anode is shown in Fig. 5.7(a). This spectrum wasaccumulated in only 200 seconds with anode voltage of 20 KVand anode current of 20 uA. The 0 to 20 keV portion (Fig. 5.7(b)of the x-ray spectrum excited with Mo-filtered radiation,shows clearly defined peaks for Ca, Ti, V, Cr; Mn, Fe, Ni,Cu, Zn, and Pb in a counting interval of only 400 seconds.This logarithmic plot shows a uniformly increasing peakintensity for all of the elements except for Ca which is five

149

10"

c/>

10'

10'

i i I T i \ \ rTi Cr Fe Ni Cu

CONOSTAN D-12 STANDARD

400 800 IZOO

CHANNEL NUMBER

1600

Fig. 5.8 Characteristic X-ray spectrum obtainedby the bombardment of Conostan D-12target by 3 MeV protons. The targetwas prepared by depositing a few dropson filter paper.

times more concentrated, and Pb which is represented by itsL lines. The anode voltage in this case was 35 kV , while theanode current was 50

Smith et al., (1975) have analyzed diluted ^ConostanD-20 standard by x-ray fluorescence and atomic absorptionmethod for Fe , Ni. Cu , and V at the sub-ppm level. Theirresults indicate that precise analysis at low levels isstill not always possible.. The results they obtained areshown in Table 5.8.

Several manufactures are producing powdered standardsformulated from selective high-purity inorganic compounds.For example, CHEMPLEX industries (140 Marbledale Road,Eastchester, N.Y. 10707, U.S.A.) are marketing 53 differentanalyses in different matrices (graphite 74 jam/-200 mesh,zinc oxide, lithium carbonate) with concentrations 0.1,0.05, 0.01, 0.005 and 0.001 per cent.

Their product called 1-2-3 Powdered SpectroStandardis formulated to provide 1.23 wt.% of each of the 53 listedanalytes ; its matrix may be considered as the total combi-nation of the oxides of the constituent elements. PowderedSpectroStandard Sets are comprised of a series of standardsand a blank of a specific matrix as defined as the diluent.Individual standards of a. set contain the same indicated53 elements diluted to furnish the same concentration valuefor each analyte. The standards in a set differ from eachother by incremental concentration values of the analytes.Every set provides calibration function for the 53 differentanalytes extending from 0.1 to 0.001 wt.%. Powdered Spectro-Standards are furnished in 2 gram quantities contained inglass vials with plastic-lined screw caps to ensure air-tight protection against the environment.

150

Table 5.8Analysis of Conostan D-20 standard

(after Smith et al., 1975.)

X-ray fluorescence Atomic absorptionElement ——————————————————————————————————————————————————————

20 ppra expected 0.5 ppm expected 1.0 ppra expected 0.5 ppm expected

Ni

Cu

Fe

V

0.77 - 0.24

1.22 - 0.45

0.61 - 0.21

0.36 - O.I2»

0.45 - 0.12

0.66 - 0.26

0.34 - 0.10

0.26 - 0.16

0.88

0.94

0.95

1 .00

0.33

0.45

0.39

0.35

The concentration of the analyte of interest in aweighted powdered sample is increased by a known amount bythe addition of a weighted quantity of the powdered standard.The resulting mixture consists of the initial unknownconcentration of the sample analyte plus the known analytein the SpectroStandard . Analyte-line intensities are measuredfrom both the original sample and standard mixture and theconcentration of the analyte of interest in the originalsample is calculated from the following relationship:

where, W„ = sample weight(!X/IM) Css w _ standard weight_ ___________________X " W = standard mixture

1 + <WX/WSS) (1 " IX/IM) weight!„ = intensity of

sampleIM = intensity of

standard mixtureC„s = weight fraction

of analyte addedC„ = weight fraction

of analyte insample

It is presumed that a linear relationship exists betweenanalyte-line intensity and analyte concentration with thecalibration curve intercepting the origin. In low concentrationranges (0-5%) the calibration function is usually linear andthe application of a correction factor is rarely necessary.For higher concentration levels, linearity may be limited toa narow range and the amount of analyte added becomes critical.Experimental errors may become too large if an insufficientquantity of analyte is added; with excessive amounts, theapproximation of linear correlation may be poor. Depending onrequired analytical accuracy, successive additions of analyteto the sample or dilutions of the standard mixture with the

151

sample represent approaches to cover the anticipated concentra-tion range of analytical interest concurrent with consideringnonlinearity and the slope of the calibration curve. In thisregard, a series of standard mixtures containing known increasesin analyte concentration are developed according to the pre-described procedure in preparation of establishing a calibrationcurve. The curve is extrapolated to zero analyte addition todetermine the analyte concentration in the orginal sample.Alternatively, successive dilutions of the initially preparedstandard mixture with the original sample is also used togenerate a series of standard mixtures of known analyteconcentrations. The concentration of the analyte of interestin the original sample is determined by extrapolation ofthe calibration curve to zero dilution. A variation intreating the intensity measurement data involves the as-signment of relative intensity strength values using anarbitrary scale to the analyte of interest for each preparedstandard mixture. The concentration of the analyte in theoriginal sample is represented at the point where no furtherchange in intensity is indicated.

The admixture of the Powdered SpectroStandard to thepowdered sample is critical for exacting results inaccuraciesassociated with the standard mixture preparation procedureare reflected and significantly magnified in applying dilutioncorrection factors. Dilutions greater than a factor of 10frequently tend to produce inhomogeneous standard mixtures.The magnitude of error becomes obvious when c nsidering thatthe dilution factor (DF) is a function of the weights of thestandard mixture and the original sample:

WX + WSS WMDF s -i———?! = __M eq(5.DWY WYA A

where, Wy = sample weightWSS = sPectroStandard weightWM = standard mixture weight

CHEMPLEX industries are also manufacturing briquettedsingle-element and multi-element powdered standards.

Chemplex SpectroSet-up Standards are single analyte-enriched powdered briquettes used to establish optimuminstrumentation conditions, periodically verify equipmentperformance, and to serve as semi-quantitative referencematerials.

Each SpectroSet-Up Standard is formulated to contain1 wt.% of an analyte by homogeneous admixing of a chemicallystable inorganic compound with Chemplex X-Ray Mix Powder.The characteristic energy generated at the 1 wt.% concentrationlevel is ample to produce a distinct spectral line distributionand analyteline intensities for integrations to intensitycomparisons to "unknowns".

SpectroSet-Up Standards are formed into briquettesencased in pellet cups for ease of handling and protection.Individual SpectroSet-Up Standards of complete sets consisting

152

of 20 standards contained in a Chemplex XRS sample storagebox are available. The sets are custom assembled to containonly the elements of interest requested by the analyst.

Chemplex Multi-Element SpectroPellet Standards providethe analyst with a readily available, convenient anduncomplicated analyte-line intensity comparison method forassigning reasonable concentration values to as many as 53different elements in an "unknown" powdered specimen. Forqualitative analysis, "unknown" sample spectral distributionsmany be compared to those generated by the Multi-ElementSpectroPellet Standard to confirm the presence or absenceof a multitude of elements.

Each Multi-Element SpectroPellet Standard is formulatedwith chemically stable inorganic compounds and diluted with74 micron Powdered Graphite to a weight percent concentrationof 1 for each of the 53 different contained analytes. Underbriquetting pressure, firm durable pellets are formed encasedin Pellet Cups for ease of handling and protection.

Chemplex Organo-Elemental SpectroStandards are synthesizedfrom spectrographically pure starting reagents and diluted byweight to concentration with an exceptionally pure water-whitebase oil. They are readily miscible with each other for preparingspecial formulations, all petroleum products and a host of non-aqueous matrices for compositional compatibility to unknownsfor spectrochemical applications in varied fields of interest.

Chemplex SpectroStandards are manufactured withoutsolubilizers, in small batch quantities for extended shelflife, and packaged in 2 f1. oz. (60 ml) amber glass bottlesto minimize potential photochemical reactions. Single-elementSpectroStandards are formulated by weight to 5000 ppm. Forfuel and motor oil additive studies, a multi-element Spectro-Standard containing proportional concentrations of differentelements is furnished. SpectroStandard Base Oil is alsoavailable for in-lab dilutions to suit specific concentrationlevels or formulations.

The use of Organo-Metallic Salts in powdered formpermits the analyst to select the appropriate organic solventor diluent oil for preparing standard reference solutionsmatrix-matched to ten unknown. Certification of assayed metalcontent of each SpectroStandard allows for the accuratecalculation and formulation and formulation of standardreference solutions in the anticipated analyte concentrationrange of investigation.

Chemplex Organo-Metallic SpectroStandard Salts arespecifically suitable for all spectrochemical analyticalapplications requiring metal standards soluble in non-aqueous media. They are traceable to NBS values and areparticularly selective to the analysis of petroleum productsfor trace wear-metals, crude oils, synthetic oils, fuel andmotor oil additives, lubricating oils, food fats and oils,and greases with applications extending into other non-aqueous spectrochemical areas.

The list of Chemplex Organo-Metallic SpectroStandardSalts is shown in Table 5.9.

153

Table 5.9

Organo-Mettallc Salts used as standards

Cat. No.

70177013700570567020701870277021

702970267080

70197003

Metal

AgAl

B

Ba

Ca

Cd

Co

CrCuFe

HgK

Li

Organo-Metallic Salt Formula

Silver Cyclohexanebutyrate C,H (CH ) COOAgAluminum Cyclohexanebutvrate C,H (CH-)-COO -A10HDL-Menthyl Borate (CH )2CHCH(CH2)2CH(CH )CH2CHO B

Barium Cyclohexanebutyrate C,H (CH?)_COO „Ba

Calcium 2-Ethylhexanoate CH (CH,) CH(C H )COO 2CaCadmium Cyclohexanebutyrate C, H ( CH_ ) _COO _CdCobalt Cyclohexanebutyrate C,H (CH_).jCOO CoChromium (III) Benzoylacetonate C,HCCOCH:C(CH0)0 _Cro 5 3 3Copper Cyclohexanebutyrate C,H (CH )_COO CuFerric Benzoylacetonate C, H,.COCH:C(CH.JO „Fe6 5 3 3Mercury Cyclohexanebutyrate " C„H (CH_)_COO „HgPotassium Cyclohexanebutyrate C,H (CH ) COOKLithium Cyclohexanebutyrate C,H (CH ) COOLi

% Metal*

38.937.052.2728.8612.28

21.9311.839.7115.80

10.3537.2118.773.91

Cat. No.

7012

702570117028

70157082701170507038

7023

7030

Metal

MgMnNaNiPPbSiSn

Sr

V

Zn

Organo-Metallic Salt Formula

Magnesium Cyclohexanebutyrate . C,H (CH ) COO MgManganous Cyclohexanebutyrate C, H . ( CH„ ) ,COO _MnSodium Cyclohexanebutyrate C,H, . (CH_)-COONa6 11 2 3Nickel Cyclohexanebutyrate C,H (CH ) COO ?NiTriphenyl Phosphate (C,H 0),POLead Cyclohexanebutyrate C,H (CH?).,COO Pb

Octaphenylcyclotetrasiloxane cliRHu n°l»SiüDibutyltin Bia(2-Ethylhexanoate) CH (CH ) CH(C H )COO Sn (CH

Strontium Cyclohexanebutyrate C,H (CH_) COO «SrVanadium Oxobis-(1-Phenyl-1, 3-Butanedionate) C,HCCOCH:C(CH,)0 0VOo 5 3 2Zinc Cyclohexanebutyrate C,H (CH ) COO Zn

% Metal1*

6.7013-9611.9611.78

9-1937-9711.15

) CH 22.8520.56

13-0916.19

Theoretical Value based on the anhydrous salt.

Ulin

5.4 IntercomparisonsIn recent years many different intercomparison exercises

were organized; sometimes including only few laboratories andone method, sometimes including large number of laboratoriescovering a spectrum of methods. In this latter group studiesorganized by IAEA should be mentioned, especialy differentbiological reference materials for trace element studies. Resultsfor these materials indicate that only a rather small groupof the essential trace elements (Cu, Fe . Mn, Se and Zn) can bedetermined satisfactorily by most of the analytical laboratoriesthat reported results. For other important essential traceelements, however, such as Co, Cr, I, Mo and Ni (and'also forimportant toxic elements such as Cd and Pb) the results reportedgenerally cover such a wide range that it is impossible todeduce a meaningful "recommended value" for the element concerned.For other essential trace elements such as F, Si, Sn and V,there are too few data available by which to judge the reliabilityof presently available analytical techniques. These problemsdo not usually appear to depend on the analytical techniqueemployed. On the basis of these findings it is difficult to avoidthe conclusion that many of the data reported in the literaturefor trace elements in biological materials are of extremelydoubtful validity.

Parr (1980) has recently summarized situations withrespect to biological material. This is illustrated in Fig. 5.9and Fig. 5.10 representing the distribution of values obtainedfor cobalt in IAEA animal muscle and chromium in the samematerial. Different symbols refer to different analyticaltechniques used.

Results of different intercomparison exercises arepublished in the scientific literature. We shall here mentiononly some to illustrate the methodology used.

For example, Fukai et al., (1978) have reported resultsof intercalibration exercise on oyster homogeneate. Approximately2 t, gross weight, of oysters were collected, the soft part(»»140 kg) were dissected, freeze-dried, powdered and sieved.The sieved fraction, between 63 and 500 jura, was retained andthen mechanically homogenized. Precautions were exercised tominimize contamination of the sample during these operations ,although some degree of contamination was inevitable inhandling the large quantities of the material. Measurements ofseveral trace elements on random fractions of the sample showedthat homogeneity varied according to the elements measured,but the standard deviations of the measurements were normallyless than - 10% at sample sizes around 100 mg; for many elements,standard .deviations of less than - 5% were not uncommon. Changesof the trace element content during storage were tested formore than 2 years by repeated measurements and found negligible.

Samples were sent to 127 laboratories at world-widelocations. These laboratories are oriented to various aspectsof environmental studies, including océanographie and fisheriesinstitutions, universities, analytical centers, nuclear studycenters, etc. The laboratories were requested to carry outdeterminations for trace elements of their preference by usingthe normal procedures employed in their work. Altogether, theresults of 85 laboratories from 25 countries and of 2 IAEAlaboratories were made available by the end of 1977.156

«z

id-o o

14 7 31^ l 38 13 19 56 46 53 32 35 4923 22 24 39 9 36 5 44 4 6 26 27 43 25LAB.CODE NO.

Fig. 5.9 Cobalt in IAEA animal muscle, ug/kg dryweight. Each point represents the meanvalue reported by one laboratory (pointswith downward facing arrows representlimits of detection). These results havebeen arranged from left to right of thefigure in order of increasing value. Thedifferent symbols represent differentanalytical techniques (after Parr, 1980)

«fl

101H

10' l>9

t't"

1 7 7 16 42 29 13 51 24 32 23 53 47 49 2650 22 36 56 31 14 9 5 6 38 39 27 35 15 25

LAB.CODE NO.

Fig. 5.10 Chromium_in IAEA animal muscle, mg/kg dryweight. Each point represents the mean valuereported by one laboratory (points withdownward facing arrows represent limitsof detection). These results have been ar-ranged from left to right of the figure inorder of increasing value. The differentsymbols represent different analyticaltechniques (after Parr. 1980).

157

c/»oo

Table 5.10Ranges, overall averages and averages after applyingChauvenet's or Dixon's teat for intercalibratlonexercise on oyster hotrcgenate (after Fukal et al., 1978)

ElementsNo. of reportedRange reportedOverall average

Chauvenet's tesNo. of acceptedAverage (XK) in()ig/g-dry)

Dixon's testNo. of acceptedAverage ( ) in

results( )(yg g-dry)( ) (^g/g-dry)

tresultsacceptable range

resultsacceptable range

Cr300.301-751 - 2(50%)

251.2 - 0.1(8.3%)

281.5 - 0.2(13%)

Mn

190.68(2

1272(1

1769(2

091-110± 2.9%)

± 1.1%)

Î 2.9%)

Fe

510.21-2 800360 - 50(11%)

12302 - 6(2.0%)

52300 - 10(3-3%)

Co260.1.(3

220.(6

261.(3

1

29-5.70 - 0.30%)

16 - 0.03.5%)

0 - 0.30%)

Cu6655.311(2.

60322(1.

66311(2.

5-180- 99%)

Î 69%)

± 99%)

Zn

772.8-52 700(3-7%)

612 830(1.1%)

772 700(3.7%)

100- 100

i 30

- 100

(jig/g-dry)

Element

No. of reported results (M)Range reported (jJg/g-dry)Overall average ( ) ()ig/g-dry)

Chauvenet's testNo. of acceptedAverage ( )(pg/g-dry)

Cixon's test

in

No. of acceptedAverage ( )(>Jg/g-dry)

in



As

230.016-1631 7 - 7(41%)

1910.7 - 0.6(5.6%)

211 0 - 1(10%)

Se

210.05-3-92.0 - 0.2(10%)

212.0 - 0.2(10%)

212.0 - 0.2(10%)

Ag

280.0058-17.666.1 - 0.6(9-8%)

265.8 - 0.4(6.9%)

275.6 - 0.4(7.1%)

Cd

500.4-212.7 - 0.4(15%)

482.(4

492.(4

2 - 0.1.5%)

3^0.1.3%)

Hg

440.059-1-60.27 - 0.04(15%)

390.(5

390.(5

19 - 0.01.3%)

19 - 0.01.3%)

Pb

340.2-12.733-0 - 0.5(17%)

261.5 Î 0.2(13%)

322-3 io.3(13%)

( ) Results showing only detection limits of methods used are not included.( ) Associated uncertainties represent standard errors.

o\o Table 5.11

IAEA 1983 - 198»! PROGRAMME FOR INTERCOMPARISON RUNS

SAMPLECODE

MATRIX ELEMENTS OPNUCLIDES TO

EE DETERMINED

CONCENTRATIONOR ACTIVITYLEVEL

SAMPLESIZE

SCHEDULED YEAROF DISTRIBUTION1983 1981

(tentât.)

Nuclear materials and stable Isotope standardsS-17 Uranium ore U

(phosphate matrix)

S-18

S-19

S-20

SR-60

SR-70

SR-61

Low U content 25 g

Uranium ore U(phosphate matrix)

Medium U con-tent

25 g

1(2)

1(2)

RM

RM

Uranium ore U(phosphate matrix)

High U content 25 g 1(2) RM

Uranium ore Ra/U ratio and(phosphate matrix) U-235 content

Low U naturalcontent

10 g

Uranium dioxide U content and iso- Depl. UO-tcpic composition

10 g 1(3)

Uranium dioxide U content and iso- Depl. DO.topio composition

Uranium trioxide U content and iso- Depl. UO.topic compcsition

10 g

20 g K3)

SAMPLECODE

MATRIX ELEMENTS ORNUCLIDES TOBE DETERMINED


SAMPLESIZE

SCHEDULED YEAROF DISTFIBUTION1983 198J«

(tentât.)

SR-71 Uranium trloxlde U content and iso-topic composition

Depl. U0_ 20 g

SR-52 Uranovanadate U content 70-80% 50 g

SR-5*

SR-61

GISP

EnvironmentalW-3/2

U3Ö8

U3°8Water

materialsFresh water(containing alsoprincipal consti-tuents of freshwater)

Ircpurities

ImpuritiesI8o/I6o; VH

Trace multielementanalysis: Pb, Hg, Mn,Cr, As, Cd, U, V, FefNi, Zn, Co, Cu, Mo,Se , Ea , Au

4

total 2000 ppm 10 g running I RM

total 2000 ppm 10 g - I

under calibration 30 ml . running I running I

as for natural concentr. - 1(2)fresh water solution

to be di-luted to2 litreswith bi-distilledwater

SOIL-6

SOIL-7

Soil Sr-90, Cs-137, Mn-54,Pu-239

natural level

Soil Trace multielement natural content

250 g

25 g

1(2)

1(2)

RM

RM

(O

Table 5.11 (cont.)

SAMPLECODE

MATRIX ELEMENTS ORNUCLIDES TOBE DETERMINED


LEVEL

Animal and plant materialsV-10 Hay (powder) Trace multielement

analysis

Milk powder Sr-90, Cs-137, Na,K, Ca, Sr

Materials for biomedical_stuûiea

H-9 Mixed human diet Trace multielementanalysis

Materials _of marine originSD-N-1/1 Marine sédiment Natural sample for low-

level transuranicanalysis

Fallout level

SD-N-1/2 Marine sediment Trace multielementanalysis, U, Th andtheir decay products

Natural content

SAMPLESIZE

SCHEDULED YEAROF DISTRIBUTION1983 198t

(tentât. )

natural content 25 g 1(4) RM

environm. level 250 g running I RMnatural content

natural content 20 g 1(3) RM

100 g running I RM

25 g RM

SAMPLECODE

MATRIX ELEMENTS ORNUCLIDES TO

BE DETERMINED


SAMPLESIZE

SCHEDULED YEAROF DISTRIBUTION1983 1981

(tentât.)

SD-N-2

SW-N-2

AG-B-1

MA-M-2

Marine sediment Natural sample forlow-level trans-uranic analysis

Natural seawater

Seaweed

Mussel tissuehomogenized

Sr-90, Cs-137, trans-uranics

Neutron-inducedradionuclides andtransuranic elements

Trace multielementanalysis and chlori-nated hydrocarbons

Fallout level(lower activitythan SD-N-1)

Elevated falloutlevel- 1 2 - 110 -10 Bq.Kg

natural content100.ug

100 g

50 g

30 g

RM

50 g Running I RM

RM

RM

o.OJ

The results of the analyses, reported on 12 selectedtrace elements, Cr, Mn, Fe, Co, Cu, Zn. As, Se, Ag, Cd, Hg andPb, were treated statistically to deduce the "consensus values"of these elements in the sample. The results of selectedlaboratories were treated similarly to estimate "probableconcentrations". These "probable concentrations" agree withthe "consensus values" for most of the elements. On the basisof the "probable concentrations", ranges for acceptable valueswere estimated for each element. More than 80% of the resultsreported were acceptable for Mn and Cu by applying these ranges,more than 70% for Cr, Zn, Se, Cd and Hg, more than 60% for Fe,Co and Ag, while 59% were acceptable for Pb and only 43% for As.

The results are presented also in Table 5.10. Runningand future intercomparison runs organized by IAEA are listedin Table 5.11 and marked "I". The number in brackets after thissymbol indicates the quarter of the year in which the samplewill presumably be distributed. Participation is free of charge,but all participants will be requested to report their analyticalresults to the IAEA before the announced deadline, usually 3 to6 months from receipt of the intercomparison sample. About 6months after the deadline, a report containing the statisticalevaluation of the intercomparison data will be issued and sentto the participants.

Participating laboratories will be coded by number inthese reports and each participant will be informed onlyof his own code number. The remainder of the sample stillin the possession of each laboratory after the intercomparisonhas been completed, can be considered as reference material,provided it can be stored for a considerable period of timewithout appreciable change.

6. LITERATURE ON SAMPLE PREPARATION TECHNIQUES1. F.G. Adams, and R.E. van Grieken: Absorption correction

for x-ray fluorescence analysis of aerosol loadedfilters, Anal. Cehm. 47 (1975) 1767.

2. B.B. Agarwal, and S.F. Fish: Indian J. Technol., 10,(1972) 117.

3. Akaiwa, Hideo, Kawamoto, Hiroshi, Ogura, Kazuko, Tanaka,Kazuhiko: Preconcentration of trace chalcophile elementsby a zincon-loaded resin and its application to neutronactivation analysis, Radioisotopes 28 (5) (1979) 291-4.

4. C. Alper: Specimen collection and preservation, Chap. 14,Clinical Chemistry: Principles and techniques (Henry,R.J.et al., Eds), Harpen and Row Publishers, (1974).

5. F. Alt, H. Bernot, J. Messerschmidt, D. Sommer: Determi-nation of cadmium, lead and thallium in mineral rawmaterials after chemical preconcentration using differentspectrometric methods, Spektrometertagung, (Vortr.), 13(1981) 331-6.

6. F. Amore: Losses, interference and contamination in tracemetal analysis; some examples, NBS special Publication No422, Vol. 2, (1976), 661.

164

7. Analytical Methods Committee: Methods for the destructionof organic matter. Analyst 85, (1960) 643-656.

8. V.D. Anand, D.M. Duchmore: Stability of Cr ions at lowconcentrations in aqueous and biological matrices storedin glass, polyethylene and polycarbonate containers, NBSspecial publication No. 422, vol. 1, (1976) 611.

9. V.D. Anand, J.M. White, H.V. Nino: Some aspects ofspecimen collection and stability in trace element analysisof body fluids, Clin. Chem. 21 (1975) 595.

10. T.H. Arkley, D.N. Munns, and C.M. Johnson: Preparationof plant tissues for imicronutrient analysis. -Removal ofdust spray contaminants, J. Agric. Food Chem. 8, (1960)318-321 .

11. P.I. Artyukhin: New interpretation of the adsorption oftrace elements with precipitates of slightly solublesilver salts. Izv. sib. otd. akad. nauk SSSR, ser. khim.nauk 1 (1981) 36-43-

12. G.S. Assarian, D. Oberleas: Effect of washing procedureson trace element content of hair, Clin. Chem. 23, (1977)1771.

13. L.T. Atalia, C.M. Suva, F.W. Lima: Activation analysisof as in human hair. Some observations on the problemof external contamination, An. Acad. Bras. Cienc. Riode Janeiro, 37, (1965) 433-

14." K. Bächmann: CRC Critical Reviews in Anal. Chem., 12,(1981) 1-67.

15. E.T. Baker, and D.Z. Piper: Suspended particulate matter:collection by pressure filtration and elemental analysisby thin-film x-ray fluorescence, Deep Sea Res., 23, (1976)181 .

16. Bank, L. Harvey, Robson, John, Rigelow, R. James,Morrison, John, Spell, H. Larry, Kantor, Raphe:Preparation of fingernails for trace element analysis,Dep. Pathol. , Med. Univ. South Carolina, Charleston.

17. B.K. Barnes, R.M. Coleman, G.H.R. Kegel, P.W. Quinn,N.J. Rencricca: Adv. x-ray Anal. 18 (1975) 343-

18. J.E. Barney: Determining trace metals in petroleumdistillates, (an acid extraction technique), Anal.Chem., 27 (1955) 1283.

19. J.E. Barney, and G.P. Haight: Determining trace metalsin petroleum distillates, (efficiency of recoverymethods), Anal. Chem., 27, (1955) 1285.

20. L.C. Bate: The use of activation analysis in proceduresfor the removal and characterization of the surfacecontaminants of hair, J. Forensic Sei. 10, (1965) 61.

21. F.E. Beamish, Talanta 14 (1967) 991.22. R.C. Bearse, D.A. Close, J.J. Malanify, C.J. Umbarger:

Phys. Rev. A7 (1973) 1269-23- Behne, Dietrich: Sources of error in sampling and sample

preparation for trace element analysis in medicine. J.clin. chem. clin, biochem. 19 (3) (1981) 115-20.

165

24. L. Beitz, L. Müller, R. Plesch: Analysis of geologicalsamples by means of the SRS Sequential x-ray spectrometer,Siemens Analytical Application Note No. 194.

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