434 - 7-flow injection analysis

8/19/2019 434 - 7-Flow Injection Analysis

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(FIA)

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By Assoc. Prof. Dr. Jaroon Jakmunee20 September 2012


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Wet Chemical Analysis

•

(wet

chemical analysis)•

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.......


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ANALYSIS COST OF ANALYSIS

• ccuracy• Precision•

Sensitivity• Selectivity

• Chemical(s)• Chemical reaction

•

Timing• Instrument

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• operating cost• maintenance cost


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•

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(turbulence

flow) =>

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(carry over)


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•

(equilibrium)

•

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......

• steady state ...


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OVERVIEW

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1.1. Basics, 1.2. Principles, 1.3. Methods & Applications. 1.4. Instruments & Components

Flow Injection (FI), the first generation of FIA techniques, is the onemost widely used. In its simplest form , the sample zone (red) isinjected into a flowing carrier stream of reagent (blue). As the injectedzone moves downstream, the sample solution disperses into reagent,

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and the reagent. A detector placed downstream records a change ofcolor or of another parameter as it changes due to the passage of thederivatized sample material through the flow cell ( Ruzicka & Hansen 1975).

J. Ruzicka & E.H.Hansen, Anal. Chim. Acta , 78, 145 (1975) J. Ruzicka & E.H.Hansen, “Flow Injection Analysis” 2 nd ed. J. Willey, N.Y. 1988


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1.1.1

• eristaltic um

SINGLE STREAM MANIFOLD SIMPLEST , MANUALLY OPERATED SYSTEM IS COMPRIZED OF:

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•manually operated two position injection valve •manifold of connectors tubing and reactors• flow through detector

Basic FI instrument furnished with a tungsten light source andspectrophotometer, is well suited as a training tool in an undergraduate laboratory or for assay of small sample series.


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1.1.2.

TRAVEL TIME

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Peak height is the most frequently used response for construction of a calibrationcurve. Depending on the flow rate and reaction rate this readout is often availablewithin less than 30 seconds after sample injection. With a sampling frequency of upto 120 s/hour, thousands of samples are analyzed within a week in routineLaboratories, where FI system is usually coupled with an autosampler. Peak width(W) is readout for FI titration, while peak area (A) is used infrequently.


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1.1.3.

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1 1 4


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1.1.4.

Since many assays use several reagents that must be added in a given sequence,FI systems use multichannel pumps that propel carrier stream along with reagent

streams. This allows reagents to be added continuously to injected sample at adesired concentration, so that reactions can be carried out in sequence as thesample zone passes through the first and second reactor. A majority of FI systemsuse peristaltic pumps that allow flow rates of carrier and reagents to be controlled

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,

diameter. A typical flow rate is 0.5 -1,5mL/min of individual streams, while aninjected volume is selected in a range of 25 to 100 µ µµ µ L.Multistream FI system are routinely combined into multichannel systems, whereeach channel is dedicated to a different chemical assay. Typically a three channelsystem allows , phosphate, nitrate and nitrite to be analyzed simultaneously in

water and soil sample extracts.Yet another advantage of multistream FI systems is their versatility, that allowsautomation of solvent extraction, dialysis, and gas diffusion based assays.


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Fully automated FI analyzer furnished with an autosampler

1.1.5.

pump, injection valve and an integrated manifold with a z-type flow through cell. For spectrophotometic measurements, the flow cell isconnected by fiber optics to a tungsten lamp and

a scanning spectrophotometer.

A@540nm 8

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The attached readout shows a calibration record of 0,2,5 and 8ppm nitrate, followed by aroutine run of nitrate assay in soil samples using cadmium

reduction column and sulfanilamide reagent.seconds

0

2

1 1 6


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OPTICALFIBE

1.1.6

SAMPLE

MIXINGCOIL #2

WASTE

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MIXINGCOIL #1

FLOW CELL INJECTION VALVE

SAMPLE LOOP


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PRINCIPLE

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1.2.1.

This section is designed to provide an understanding of processes that yield FI response curve, and to offer tools for optimizing sensitivity,detection limit and sampling frequency of flow injection based assays.

We begin with definition of three cornerstones on which allflow injection techniques are based:

•sample injection •controlled dispersion•reproducible timing

and will continue with examples how these parameters are controlled

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,

manifold configurations. While single reagent assays can be performedusing the simplest, single stream manifold, it will be shown why a majority of FI techniques use multistream manifolds, where several reagents aresequentially merged with a carrier stream that moves the injected sample

zone through the manifold and a flow cell. Note that discussion in the following sections deals with a single stream system. For multistream systemsD-values have to be corrected by dilution caused by additional streams.


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SAMPLE INJECTION CONTROLLED DISPERSION REPRODUCIBLE TIMING

Sam le in ection rovides the initial C o

1.2.3.

square input serving as a staring pointfor initial concentration ( C o ) and startuptime. T

o

Controlled dispersion takes place as the sample zone moves downstream through the manifold.This process forms a well defined concentration gradient that can be viewed as continuum of elements of fluid with different concentrations, where the highest one ( C max ) corresponds to peak maximum.Since it is convenient to locate peak maximum, most FI methods use this element of fluid as a readout In order to optimize a given assay it useful to know how much the sample has been diluted in the FIsystem and how much time was available for chemical reactions to proceed. Therefore the dispersioncoefficient has been defined a s D= C o / C max allowing the degree of sample dilution

SAMPLE

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C max

T max

o e es ma e . m ar y s e me e apse rom e momen o n ec on

T o

to the moment of peak maximum Tmax.

Reproducible timing of sample travelfrom injection to detection yields repeatable value of T max .

DISPERSED SAMPLE ZONE


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1.2.5.


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1.2.5.

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Flow Injection response is a result of two processes, both kinetic in nature: the physical process of dispersion of the sample zone and the chemical process of formation of a detectable species. These two processes occur simultaneously and they yield , together with the dynamic characteristics of the detector the FI

response curve. Note that the reaction product (yellow), is gradually formed at theinterface between the sample zone (red) and carrier stream of reagent (blue).


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1.2.7.


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As the injected zone (A) moves downstream, it dispersesforming a concentration gradient that can be viewed as

composed of a continuum ofconcentration segments of individual concentrations C.Of these segments, the onesituated at peak apex ( C max ) isthe one on which peak height measurement, and calibration,will be based.

0 max

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flow injection systems are designed to yield

Note that for D=2 a sample segment situated atop the peak, has been diluted to half of its original concentration, by carrier solution .

dispersion of the injected sample

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When the distance between injectorand detector is minimized in a single

sufficiently large sample volumewill produce a “square” peak thatwill have a horizontal “steady state” section with C max concentration

situated at its top.Systems with limited dispersion are designed for automation of all reagent based assays. or time

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•conductivity measurement •for direct sample injection in high sensitivity ICP and AA based assays •for bioligand interaction studies by surface plasmon resonance (BIA) •for functional receptor binding assays on live cell for drug discovery.•pH measurement

Note that in a multistream system, D value is always > 2 as the flowrates of at confluence points have to be taken into account.

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By adjusting the volume of injected sample, of the volume of reactors between injector and flow cell andof flow rates in single or multistream

,to a medium value. Resultingconcentration gradient will have a form of a smooth peak that will be only slightly skewed.

Systems with medium dispersion are designed for automation of all reagent based assays . Note that in orderto reach high sensitivity of a colorimetric assays:

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•sample volume should be maximized •reagents should be added by reagent streams via confluence points •long path flow cell should be used •dispersion coefficient should be adjusted to between 2 and 5

NOTE: Sensitivity and detection limit of reagent based assays canbe further enhanced by Bead Injection Technique .

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By decreasing volume of injected sample, and by increasing the volumeof conduits between injector and flow

cell, dispersion can be increasedto a large value. Resultingconcentration gradient will have a form of a smooth long peak that willhave an exponentially decreasing tailingedge. In order to obtain very large Dvalues a mixing chamber should beintegrated into flow manifold.

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If the volume of a mixing chamber dominates the volume of the flow channel the resulting concentration gradient will have a exponentially decreasing trailing edge if the system is operated t continuous constant flow rate.Systems with large dispersion are used for process control monitoring when extensive sample dilution is required and for automated titrations.

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1.2.11.

Increasing the injected sample volume increases peak height, until a steady state plateau is reached.Up to D =2 value (in a single stream system), peak

volume. The sample volume needed to reach 50% of the steady state depends on the volume, geometry and flow rates in the channel between injector andflow cell. For conventional FI systems this value is

around 50 µ µµ µ L, for micro SI systems as low as 5 µ µµ µ L .If the radial mass transfer is incomplete, the resulting peak shape is composed of two exponential curves,

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mixing, gradients are reshaped and follow erf function, ultimately approachingGaussian shape ( see Section 0.2.2. ).Changing injected sample volume isversatile, and convenient tool for adjusting dispersion coefficient and foroptimizing the sensitivity of flow injection based assays.

Recording shows traces obtaining by injection bromothymol blue solution into 0.5mmI.D tubing, 20 cmlong, at a flow rate of 1,4mL/min in a single stream system . Spectropotometry at 620nm.

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.0 A 516nm567nm

40

5 0

µµµµ L BTB

Selection of injected sample volumes is a powerful tool for optimization of all FIAtechniques. It allows :

ce

.5

.0

.

10

20

30•. Selection of sensitivity and detection limit(as shown here on spectrophotometry of a dye) • Identification of the linear range of a detector • Automated dilution of sample material

Volume of a sample solution injected intoconventional FI system is accomplished bymanually changing volume of the sample loop.

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.100 200 300 400

TIME, sec

in Sequential Injection sample volume is

determined by the volume of stroke reversal of a syringe pump. This allows automated selection of injected volumes by means of software control.

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Increasing length of a tubular channel decreases peak height while peak shape undergoes a change

rom asymme r ca o symme r ca s ape.same time the resident time of the peak maximum increases with the distance traveled and the peak base broadens. This is the principal limitation ofFI based on constant forward flow, as the constant flow rate limits the incubation time for chemical reaction to about 20 seconds, with a total conduitlength of about 250cm, and combined flow rates

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.

1,4mL/min, sample volume of 60 µ µµ µ L in a way described in the previousslide).The use of programmable, instead of continuous flow, allows incubationtime to be prolonged by stopping the flow , and speeding up the system wash by accelerating the flow . Programming the flow makes SequentialInjection technique more versatile than FI.

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INJECTOR INJECTORDETECTOR DETECTOR

Since all chemical reactions are time dependent, reproducible timing of sample handling operations is critical to success of flow based chemicalassays, as in this format reaction equilibrium is not necessarily achieved.

At continuous flow the time intervalavailable for chemical reaction to

Stop flow allows longer reaction timewithout penalty of dilution, thus yielding

STOP

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a e p ace s e ne y near ow

velocity and is limited by the lengthof conduit between point ofinjection and detector. Althoughlonger tubes allow longer reactiontime the yield is offset by dilution

due to increase in sample zonedispersion.

higher sensitivity. It saves reagents and

generates less waste than continuouspumping. It allows miniaturization byminimizing the length between injectorand detector. It provides information onreaction kinetics through reaction rate

measurement.


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IDEA

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1.2.15.


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DETECTORThe stop flow mode isbased on arresting a selected

portion of the sample zone in thedetector. Provided that the reactiondid not reach e uilibrium while the

zone was on the way to detector,reaction rate curve will be recordedwhile the reaction product (yellow)is being formed in the detector.

Next, flow is resumed and

reacted sample zone is flushed outof the detector, while the baselineis restored.While stop flow technique has been

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use n ormat, t s cu t to

carry out reproducibly whenperistaltic pumps are used propelcarrier and reagent streams.Syringe driven systems either FI orSI are reliable and their use in

stopped flow mode is highlyrecommended.

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Since the analyte (red) disperses as thesample zone travel downstream, the thusformed concentration provides numerous

BLANK

can be recorded. This can be in followingwaysZone sampling relies on diverting adesired diluted section from themainstream by a valve into a secondarymanifold for further processing•Electronic dilution is based readoutobtained at the tail section of the peak,rather than on eak maximum. This is

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DELAY TIME

useful, when high analyte concentrationcauses readout to be out of detectorrange .•Stop flow is the most useful and effectiveapproach for reaction rate based assays .

S REPRESENTS SAMPLE PROFILE

I IS INJECTION POINT. DELAY TIME DEFINES SELECTION OF GRADIENT PORTION.

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ANALYTE

Delay time between sample injectionand commencement of the stop flowperiod determines which section ( )

BLANK

DELAY TIME

o e samp e zone w e arres e nthe observation field of a detector ( )for reaction rate measurement. Sincethe analyte (red) disperses within thereagent stream (blue) on the way tothe detector while the product (yellow)is being formed, it is essential thatthe delay time is perfectly reproducedfor each assay . In the example shown,

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slopes since tail sections of thesample zone are more diluted, whileshorter delay times ( up to peakmaximum) will yield steeper slopes. Inthe absence of analyte horizontal(blank) line will be observed.

1.3.1.


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Glucose + 2H 2O + O2 Gluconic Acid + H 2O2

2 H 2O2 + 4-Aminoantipyrine + p-Hydroxybenzene Sulfonate Quinoneimine Dye + 4H 2O

GLUCOSE OXIDASE

PEROXIDASE

nzyma c assay o g ucose, mon ore a nm is carried out by reaction rate measurement during a stopped flow period lasting 20 seconds while data are collected for construction of a calibration curve.Using a single stream flow scheme, FIAlab 2500 andassociate software series of standards containing 500,1000, 1500, 2000 and 2500 ppm glucose were injectedyielding response curves shown on the right.

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Note that the travel time between injector

and detector has been minimized in order to carry our the reaction within the flowcell, while the pump has been stopped.

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A majority of FI assays are carried at continuous flow, when carried and reagents are pumped simultaneously at a constant flow rate. Sample is injected into carrier stream of water (or appropriate buffer) while reagent streams are added at confluence

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. :

•even addition of reagents to entire sample zone length •steady baseline•minimized carryover •simplicity of operation and transparency to user.

The main drawback of FI is continuous reagent consumption and waste generation

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Almost all FI instruments employ multichannel peristaltic pumps to move carrier and reagent

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( R1,R2) streams that merge at confluence points ( ) where reagent merges with sample

zone. Sample is injected by means of a two position injection valve with a fixed injectionloop. The valve is furnished with a bypass (not shown) that allows carrier solution to passthrough the valve, while the sample is being filled into the loop. The pump moves solutionscontinuously in forward direction, thus providing a repeatable time frame for samples andstandards as they are serially injected. In this way all samples and standards are processed inexactly the same way and the standards yield a readout used for construction of a calibrationcurve.

1.3.4.


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Three stream FI manifold whereat the first confluence point ( )molybdate is added to stream of water,

a carr es n ec e samp e o p osp a eto be analyzed. At the second confluencepoint ( ), ascorbic acid is added to formphosphomolybdenum blue. Sincemolybdate/ascorbic acid mixturedecomposes rapidly, forming a blueproduct, these reagents must be storedseparately and added sequentially to thecarrier stream. Use of water as carrier

A@720nm

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limit for assay of phosphate in waterbased samples.

This method, yields samplefrequency of 80 s/h and is one of the mostfrequently performed FI assays. It is evenmore effective when miniaturized into SI- LOV format, as waste generation andreagent consumption is reduced 50 times.

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Agricultural and environmental analysis of water, soil and fertilizers for assay ofammonia, nitrate, nitrite, phosphate, chloride, iron , chromium ( IV and VI) andcyanide are routinely carried in test laboratories on a very large number ofsamples by FI. These reagent based assays are all based on spectrophotometricdetection in VIS region and their limit of detection is adjusted by selectingreaction conditions and the length of flow cell path. A comprehensive review

detection limits for nitrate 0.1 µ µµ µ M, sulfide 1,2mM and phosphate 0.05 µ µµ µ M.Trace analysis of metals by atomic spectroscopies (AA, HGAAS, ICP and ICP-MS) uses FI as a “front end” sample processing system in two ways. The mostsignificant method is based on hydride generation, that converts target analytesinto volatile metal hydrides, leaving matrix interferences in a sample solution.Three monographs and a large number of papers deal in detail with FI basedhydride generation ( see FI based separations). Yet another, simple use of FI is inassaying sea water, saline, fertilizers and serum, i.e. samples that cannot becontinuousl um ed into nebulizers of AA or ICP instruments as hi h content of

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water soluble salts will crystallize and block the nozzle. By injecting small, welldefined sample volumes into carrier stream of water this problem is eliminated.Pharmaceutical and enzymatic assays with UV-VIS or fluorescence detection isyet another area, where FI is routinely used in a large scale for quality andprocess control. These application are reviewed and summarized in monographsof Catalyud and Trojanowicz.

Calatayud J.M.: (Ed.), Flow Injection Analysis of Pharmaceuticals,, Taylor & Francis, London, 1997.Trojanowicz M.: Flow Injection Analysis, Instrumentation and Applications, World Scientific Ltd., Singapore, 2000.

J. Atienza, M.A.Herrero, A.Maquieira and R. Puchades, Critical Rew. Nal. Chem 22(5) 331-344 (1991)

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Multistream FI systems are ideally suited for automation of all separations based on partition between two phases. This section deals briefly only with

•gas/ liquid separations and•solvent extraction

while ion exchange, sorbent extraction and other microcolumn based

separation and conversions (enzymatic, redox etc) are too numerousto be reviewed here.An excellent , detailed review of FI based separations is found in thefollowin mono ra hs:

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Fang Z-L.:, Flow-Injection Separation and Preconcentration, VCH Verlagsgesellschaft mbh, Weinheim, 1993.

Trojanowicz M.: Flow Injection Analysis, Instrumentation and Applications,

World Scientific Ltd., Singapore, 2000.

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Hydride generation based Atomic Spectroscopies are routinely used for trace analysis ofAs, Bi, Ge, Hg, Pb, Se, Sn and Te, while assay of volatile compounds of Ag, Co, Cu, Ni, andZn has been reported in research publications. Advantages of hydride generation:separation of the trace metals from complex matrices, analyte enrichment, fast reaction

on FI based –hydride AA assay of bismuth. By combining an acidified sample stream witha strong reducing agent (sodium borohydride), hydrogen and metal hydride is rapidlyreleased and the gaseous phase is separated with aid of purging gas ( air or argon) and swept into the detector. Atomic absorption spectroscopy , coldvapor atomic absorption spectroscopy , inductively coupled

plasma spectroscopy , as well as inductively coupled plasma mass spectrometry have been used s detectors

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Fang Z-L.: Flow Injection Atomic Spectrometry, Wiley, Chichester, 1995.Sanz-Medel A.: (Ed.), Flow Analysis with Atomic Spectrometric Detectors, Elsevier, Amsterdam, 1999 Burguera J.L: (Ed.) Flow Injection Atomic Spectroscopy, Marcel Dekker, New York, 1989

The key component of the

design is the gas-liquid separator.

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There are two types of separators: gas expansion separator and membrane separator.Gas expansion separators are most frequently used, as they are robust and easy to construct and maintain. The entire separator, or at least its vertical tubular body is made of glass, and often partially filled with large glass beads, as hydrophilic surface of glass assists in gas liquid separation. Carrier/hydrogen/hydride stream is confluencedwith purging gas ( air, nitrogen, or argon) that sweeps the liquid within the separator andcarr es t e re ease vo at es nto t e etector. e eve o qu n t e separator s maintained by external pump. Gas expansion separators are operated at high flow rates; combined flow of sample, reagent and carrier is up to 15mL/min and purging gas flow rate of 30mL/min is not unusual. Membrane separators rely on gas diffusion through ahydrophobic membrane and offer higher sensitivity at lower flow rates, since their internal gas volume is much smaller. When integrated with a flow cell for cold Hg assay, they offer an excellent sensitivity and detection limit ( Fang 1988 ) .

Membrane separator.

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Gas expansion separator

Fang, Z.-l.; et.al., Anal. Chim. Acta 1988, 214, 41-55.

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In a two stream FI system, sample containing carbonate (or dissolved carbondioxide) is acidified, releasing carbon dioxide , that diffuses across a siliconerubber made membrane from a donor ( blue) to an acceptor (green) streamchanging color of an acidobasic indicator, monitored at 430nm (Baadenhuijsen1979). Membranes made of Teflon are hydrophobic, with up to 50%porosity,

forming an air gap between carrier and donor stream through which gases likeammonia, sulphur dioxide, chlorine, ozone or volatile compounds rapidlypermeate into an acceptor stream where they are detected by means of asuitable rea ent. Flat late diffusers, as the one shown above are eas to

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assemble. The drawback hydrophobic membranes is that they can be fouled by

surfactants that destroy the air gap barrier. When miniaturized and integrated witha fiber optic detector, placed into acceptor channel, a “sandwich cell” constructionallows increase of sensitivity of an assay.Another, innovative approach to gas separation is gas pervaporation , that offers arobust alternative to gas diffusion in parallel plate diffuser (Castro 1998)

H. Baadenhijsen & H.E.H. Seuren-Jacobs, Clin. Chem. 25, 443, (1979) •M. D. L. de Castro & I. Papaefstathiou, TRAC, 17, 41, (1998)

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This flow cell design uses a bifurcated optical cable toilluminate a white surface and to collect reflected light as it passed twice through the monitored aqueous layer.

,

liquid stream, or if furnished with a gas permeable membrane,(M) mounted between two spacers (A,B) it is useful to monitor volatile species emanating from a donor stream.Note that Teflon membrane may be furnished with an opening ( ) situated downstream from the fiber, to alleviate pressure differences between acceptor and carrier streams. Note that stopping the flow of acceptor (indicator) stream allows accumulation of analyte and increase of sensitivity of measurement. (Pavon et. al. 1992)

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AB

M

J.L.P.Pavon et.al. Anal. Chem. 64, 923 (1992)C. G. Pinto, M. E. F. Laespada, J. L. P. Pavon and B. M. Cordero Analytical applications of separation techniques through membranes Lab. Autom. Inf. Managem., 34(2) 115-130 (1999)

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Two stream manifold forautomated solvent extraction. Sample(S) is injected into a moving carrierstream of water (AQ), which is merged(a) with an organic phase (ORG) andpumped through a Teflon madeextraction coil (b). In separator (c) theaqueous phase is discarded intowaste, while organic phase is led into aflow cell. Detail showing circulation of extracted dye within segment oforganic phase (Nord & Karlberg 1984),as it moves through a Teflon tubing, provides

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.This method, applicable to assay of hormones, pharmaceuticals and

numerous hydrophobic compounds, (Karlberg & Thelander 1978), revolutionizedsolvent extraction technique, that up to that time was mostly carried manually.Miniaturization and automation of solvent extraction minimizes exposure toharmful solvents and reduces consumption of reagents and generation ofhazardous waste.

B. Karlberg & S.Thelander, Anal. Chim. Acta 98, 1 (1978) L. Nord & B. Karlberg, Anal. Chim. Acta, 164, 233 (1984)

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a b

HEAVIER THANWATER

ORGANIC PHASELIGHTER THANWATER

b

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Choice of materials for manifold components and their orientation is critical because

aqueous phase (aq) adheres to glass, while organic phase adheres to Teflon.In segmentor organic phase enters through a glass fitting and adheres to Teflon tubing (1).In separator a thin Teflon strip (3) serves to guide organic phase through a glass made T piece. In themembrane separator Teflon made membrane allows only the organic phase to penetrate throughhydrophobic pores, while aqueous phase is discarded.Karlberg B. Pacey C.E.: Flow Injection Analysis, A Practical Guide, Elsevier, Amsterdam, 1989.Fang Z-L.:, Flow-Injection Separation and Preconcentration, VCH Verlagsgesellschaft Weinheim, 1993.


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INSTRUMENT

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1.4.1.


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In the early days from 1974 up to mid 80’ a vast majority of Flow Injection instruments was “homemade” from components found in the lab or purchased piecemeal. This was because most researchers found challenge and joy in innovative design of their own systems and also because the commerciallyavailable systems were quite expensive. With advent of computers, however, a significant change took

lac inc oftwar b cam a k com on nt of a cc f l d i n. Initially , in research laboratories, and especially in Academia , a whole generation of graduate students became victim of necessity to create “home made” software, while their supervisors became in turnvictims of their former graduate students, who left behind software bundles, that no one could unravel It is not a trivial task to design and to write software package that does control instrument functions,that does provide flexible timing of events, and controls peripherals such as spectrophotometers,external pumps and valves while collecting and evaluating data in a real time.

Today versatile software is commercially available that accommodates peripherals added to coreInstrument. Such open architecture allows FI instrument to be assembled for virtually any research taskor a specilaized assay. For advanced detectors ( AA, ICP), “patches” are available that allow to bridge thegap between FIAlab or LABview software and detector with proprietary software drive. Therefore it is no

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. ,would be writing of a personal version of a word processing or slide presenting program.For routine, serial assays such as soil water or environmental analyses, a several commercial instrument packages from FIAlab or Lachat Instruments is available.All commercially available FI instruments were recently reviewed (Smith 2002), including prices, special features and available peripherals.

J.P.Smith & V. Hinson-Smith, Anal. Chem. 74, 385A ( 2002)

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Single channel, three stream FI system, with two reaction coils and fixed volume loop injection valveis the configuration, most frequently used for automation of reagent based chemical assays. While

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continuously pumping FI systems were in the past operated manually, and their response was recordedon a chart , modern systems use automated two position injection valves, and computer controlledperistaltic pumps as well as computerized data collection.Since UV-VIS spectrophotometry is the most frequently used detection technique, fiber optic flow cells with a 10mm optical path coupled to software controlled solid state spectrophotometer are nowcommon, replacing earlier designs with filter photometers. For teaching and single purposeassays, where a single wavelength is sufficient light emitting diodes offering yet another practical

alternative.

1.4.3.


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Integration of manifold components allows miniaturization and optimization of flow channel dimensions in order to minimize sample and reagent consumption. Integration of valve with the sam le rocessin channel and a flow cellwas originally suggested as a tool for miniaturization of Sequential Injection technique ( See Section 2). In FI format such “lab-on-valve” platform is used to streamline manifold components ( valve, tube fittings, confluence points and flow cell), while reactor coils #1 and#2 are mounted externally.The advantage of this construction is that it makes function

of the manifold transparent to the user, and for routine assays provides a format that is easy to reproduce, so thatwhen a standard serial assays ( such as phosphate, nitrate etc.) is optimized on one instrument, it can be transferred to other

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.The FI-LOV configuration shown here is designed for two reagent assay using 50 cm and 100cm long reaction coils,fiber optic flow cell with 10mm light path and 50 µ µµ µ L sample Injection loop. Eight roller four channel peristaltic pump is used to fill sample loop, to propel the carrier stream and two reagent streams.

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Replacing peristaltic pump with four channel syringe pump is a logical extension of FI-LOVinstrument development. Advantages of using syringe pumps for FI applications have beenrecognized by Japanese researchers long time ago (Yoza 1977) and the use of Multisyringe Flow Injection Systems (MSFIA) has been proposed in numerous publications (Cerda 1999).However, use of peristaltic pumps for FI applications is deeply entrenched, and it is likely to

, , ,

replacement of peristaltic tubing. Yet, an instrument build around individually driven syringe pumps combined with solvent resistant LOV module has following advantages:•resistance to corrosive chemicals •precise control of liquid delivery and manipulation •capability of programmable flow, including

stop flow FI for reaction rate measurement.

The main drawback of using multiple syringesis mechanical complexity, as compared to the

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conventional FI system. Also microSI instrument,is far less complex as it operates with a only a single pump and a single valve. Indeed, unless all four pumps will be run in a fully synchronized and automatically cycled mode, the flow programming of this novel instrument configuration will be a challenging task.

Yoza N., Ishibashi K., Ohashi S. J. Chromatography134, 497 (1977) Cerda V. et. al. Talanta 50, 695 (1999) Miro M., Estela J.M., Cerda V., TRAC 21, 199 (2002)

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For teaching applications one may wish to construct a simple robust inexpensivesystem, with replaceable components. Such instrument does not have to becom uter controlled if it uses eristaltic um and two osition manuall o eratedinjection valve . An interesting alternative to peristaltic pumping and valve injectionis the use of solenoid driven pumps (1.4.6) that, however, need a simple software and computer control for flow rate selection and sample injection.For research applications there are almost infinite combinations possible ofavailable components. To begin with, the most important is the choice of software,

as it has to be compatible not only with the instrument, detector and otherperipherals, but also with the user itself. Buying valves, pumps etc and connectingthem with a tubing is the easiest step. To make these components work in concert is quite another matter. The key to success is in designing simplest possible system

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w t sma est num er o components an t en s mp y t urt er. emem er t at:

“Once you exhausted all possibilities, there is a simple solution highly visible toeverybody else, but you “( Murphy’s Law).The most practical way to approach construction of a research instrument is topurchase a core unit, driven by software with and open architecture and to adddesired peripherals as the project gradually develops. Make sure that the peripherals

you intend to use are compatible with the software before purchasing the core unit.

1.4.6.

Peristaltic pumps are still the most frequently used drives for FI systems, since they generate


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continuous flow in any desired number of parallel channels. while the flow rates can be easily adjustedby rotation rate and I.D of peristaltic tubing. It is important to use a pump furnished with at least eightrollers, in order to generate a flow with small regular pulses – as otherwise resulting irregular flow ratewill affect dispersion and repeatability of assay. Contributing factor to popularity of peristaltic pumpingis its apparently low cost, although cost of peristaltic tubing exceeds many times the price of a pumpover its lifetime. The largest drawback of peristaltic pumping is due to elasticity of peristaltic tubing as

,

analyzer.Stepper motor driven syringe pumps generate highly reproducible flow that can be computer controlledin a programmable way. They cover a very wide range of flow rates as the piston speed and syringe sizecan be varied. They are durable and chemically resistant, their only drawbacks being cost and inabilityto generate continuous flow beyond the capacity of the syringe – that has to be refilled.Solenoid activated micro pumps generate flow by delivering well defined pulses the frequency and

volume of which controls the flow rate. A typical FI pulsed flow system (Rangel 2005) used 8 µ µµ µ L pulses in three stream, three pump system generating flow between 0.48 to 1.92mL/min., depending on pulsingfrequency (60 to 240 pulses/min). The weakness of this truly innovative approach is durability of thesepumps that must generate about 300.000 pulses/day while exposed to aggressive chemicals.

Table of Contents Santos J.L.M, Clausse, A.,Lima J.L.F.C., Saraiava M.L.M.F.S.,Rangel A.O.S., Analyt. Sci. 21, 461 (2005)

Solenoid Pump.

Peristaltic pump


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Peristaltic pump

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1.4.7.


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While I.D. of 0.5mm to 0.8mm is typical for majority of FI and SI systems, there is a widevariety of tubing materials available for constructing reactor coils and connection lines.Teflon and Peek are the most frequently used polymers . Stainless steel is yet anothermaterial that has advantage of heat conductivity gas impermeability and surface

.

transparent and often availablecolor coded, so that tubing I.D. can be identified at glance.Connectors made of colored coded polymers are fitted with ferrules that are designedto grip tubing while the connector nut is being tightened. Since all FIA systems operateat a low pressure, there is not necessary to use connectors designed for HPLC. It is,

however very important to use nuts, ferrules and fitting from a single manufacturer asproducts from different sources are often incompatible, resulting in a leak.

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Tubing connectors, ferrule and T-connector

Teflon made reactor coil . Heated reactor coil with temperature controller.

1.4.8.


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LOADLOOP

Two position, six port injection valve with a fixed loop is the most frequently used tool for injection of well definedsample volumes. Volume of the external loop (shown above) can is selected between 20 and 100 µ µµ µ L by changing the length and I.D of the loop tubing. The valve can be switched from load to inject mode manually or automatically and theloop can be filled either manually by syringe, or automatically from an autosampler by means of a pump (above). It is important to keep the length of the conduit between sample container and port #4 as short as possible in order to

save sample material, and to avoid sample to sample cross contamination. Introducing air bubble and wash betweensamples is useful, but requires exact timing so that the injected volume is air free and contains undiluted sample.

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Six port multiposition valve combined with a stepper motor driven syringe pump is the key component of all Sequential Injection systems ( See Section 2). It allows injected volumes to be chosen at will, and at a selected flow rate. This injection mode is an ideal tool for automated optimization of FI and SI based assays.

1.4.9.


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Sample volume and reaction time are the mostimportant parameters of flow injection experimentalprotocol. By changing injected volumes and reaction times sensitivity and detection limit of reagent basedassays can be adjusted to desired level.

Since conventional FIA employs a two position valve furnished with fixed sample loop volume, injected volumes cannot be automatically selected bya computer.Variable volume injection removes this limitationallowing automated optimizationof assay parameters. The key difference is in thatthe injection system based on a multiposition valve and the volume of injected sample iscontrolled b a s rin e um .

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Injected volumes are controlled by high precision syringe pump that aspirates selected volume ofsample solution from sample cups, while the central port is connected to port #4. (The auxiliarypump serves to transport sample solution from sample cup just past port #4, whenever nextsample change is to be injected).The volume of sample solution to be injected is determined by: •the volume of the reversal stroke of the syringe pump, and

•the volume of the forward stroke of the syringe pump, when central port is connected to port #2 .

FLOW

1.4.10.


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AB

INJECTING AN ENTIRE SAMPLE VOLUME

If selected volumes of reversal and forward stroke areidentical, not all sample material will be injected intothe sample processing manifold, because the sample forms a concentration gradient ( A) in the sample

D

.double of average flow velocity, sample zone occupies in the holding coil twice the length of aspirated volume (B)the upstream end of sample zone being diluted bycarrier solution. Thus, if entire sample is to be injected into the sample processing channel, theforward stroke should be at least twice of the reversalstroke volume . (C).

INJECTING ONLY PART OF SAMPLE

Smaller volumes of the forward stroke can be, injected into sample’

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,must be flushed to waste (through port # 1) in order to avoid carryover of sample material remaining in the holding coil into the next sample processing cycle.

DILUTING SAMPLE ( D ).

If sample is to be diluted prior to injection into the sample processing manifold, a desired portion of sample solution that has been aspirated by flow reversal, adjacent to the valve is directed via port #1into waste, and than a selected section of the remaining diluted sample zone is injected into the sample processing manifold.

1.4.11.


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Fiber optics and solid state spectrophotometers revolutionized theway in which all FIA techniques are carried out, since this technology allowed optimization by bringing light and collecting data from any

.

Sequential Injection, also more traditional FI systems benefit from versatility and robustness of fiber optic technology. A typical system comprises a“z-type” flow cell connected with quartz fibers to a spectrophotometer anda tungsten or deuterium lamp. For a single purpose systems, a light emitting diode is mounted directly onto the flow cell.

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Ocean Optics

Spectrophotometer and a Tungtsen light source.

Z-cell with 10mm

light path

Z-cell with 10 cm light path.

Th i i l i l l d i FI li i ld b i k l d


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There is a vast experimental material accumulated in FI literature, yet it would be mistake to conclude

all has been done already and therefore a further original research cannot be done in this area. Therecent work of Brazilian ( Lapa 2002) and Portugese ( Rangel 2005) teams on pulsed flow FI is an outstanding example of an innovative research, that opens a novel, practical way to miniaturization ofFI systems. Their work has a special significance, since downscaling of FI to sub microliter level , although much tried within last ten years, has not gained acceptance, as it failed to becomeapplicable to real life assays. Indeed it is puzzling , why almost all microfluidic systems described in µ µµ µ TAS literature so far, have been designed to function on continuous flow basis, while their proponents rediscover well known limitations . The central problem, mixing of sample with reagents at conditions of stabilized laminar flow remains unsolved. Attempts to use osmotic or electrophoretic pumping fail, due to different electrolytic properties of sample and reagent materials, or because the conduit walls become fouled by real life samples.Microreactor technology , that aims at exploring novel ways how to synthesize small amounts of rare chemicals, or to study flow through reactor design in microscale is a research field closely related to FI

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technology. (Haswell & Skelton,2000 ). In appropriately scaled version, and carried out within robust

conduits made of steel, glass or Teflon, using syringe pump drive it will benefit from“technology transfer” of solvent extraction, ion exchange and gas pervaporation (Castro 1998),techniques originally designed for FI. A joint meeting of “Flow Analysts” with “MicroreactorSynthetists” would surely not only be only inspiring, but will also advance progress of both fields.

S.J.Haswell & V. Skelton, TRAC, 19, 389 (2000) M.D. L de Castro & I. Papaefstathiou, TRAC, 17, 41, (1998

Lapa R.A.S, Lima J.F.L.C, Reis B.F., Santos J.L.M. Zagatto, E.A.G. Anal. Chim. Acta. 466, 125, (2002)

Santos J.L.M, Clausse, A.,Lima J.L.F.C., Saraiava M.L.M.F.S.,Rangel A.O.S., Analyt. Sci. 21, 461 (2005)


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Conventional flow injection is a mature technique, that has a wide range of applications,described in over 13.000 publications. It provides an unprecedented versatile samplehandling, along with strict control of reaction conditions. It has been applied as a front endto practically all spectroscopic and electrochemical detectors, of which UV-VISs ectrosco Atomic Absor tion and Inductivel Cou led Plasma S ectrosco are mostprominent examples.

The chief advantage of FI is the transparency of its experimental setup, where sampleinjection and movement through reagent addition and product detection follow a simpleroute, traveled by means of continuous flow. That allows automated assay to be carried outeven without computer control, since it is the flow generated by a pump, along withsample injection, that provide strict time framework for reaction conditions. Such controlof mixing and timing allows reagent based assays to be carried reproducibly, even ifchemical reactions involved do not reach completion.While manually operated experimental setup would be, understandably, frowned upon bywell heeled technician armed with PC and autosampler, it should be remembered thatmanually operated FI has been a workhorse of serial assays in developing countries, and itis in any setting the best tool for teaching of principles of flow analysis, as it allows

t d nt to rc iv th int r la of kin tic of h ical di r ion and ch mical

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reactions without unnecessary distraction provided by software or PC.Advances in computerization has enhanced FI mainly through automation of datacollection and of calibration routines, while majority of commercial analyzers still usescontinuous flow platform, where computer control has nothing to offer. Yet, continuousflow operation is the main drawback of conventional FI as is consumes reagents, andcreates chemical waste continuously, from the moment of instrument startup, even whenno samples are being injected – and analyzed.

The unique feature of all flow injection methods is the well defined concentration gradient


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The unique feature of all flow injection methods is the well defined concentration gradient

formed when analyte is injected into a carrier (or reagent ) stream. Surprisingly, this featurestill remains relatively unexplored, although it offers opportunity to automate:•analyte dilution •optimization of analyte / reagent ratio •titration Stopped flow FI format exploits concentration gradients through exact timing of microfluidic

operations, controlled by computer and programmed through dedicated software. Stopped- flow gradients are ideal for enzymatic assays since they allow automated selection of a properreagent/analyte ratio for reaction rate measurement of either substrate concentration orenzymatic activity. Stopped flow FI should be carried out using syringe pump, or solenoidactivated micro pump driven systems, as elasticity of peristaltic pump tubing makes selectionof reagent/analyte ratio difficult to maintain as the flow rate changes during the day.In the future choice between FI mode or microSI mode will be mainly a matter of a personalpreference. Since FIA technology is already fully computerized, and since advantages ofcomputer control of microfluidic manipulations, are now recognized, the deciding factor might

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more complex. This apparent drawback is , however, much offset by unprecedented savings of

time, of reagent consumption and of waste generation and versatility of programmable flow.For a researchers microSI is the way to proceed, as it offers unexplored avenues for noveldiscovery. Bead injection (BI) and SI Chromatography are a stellar examples of avenues thatopened new approaches to enhancement of immunoassays, trace analysis, pharmaceuticalassays, drug discovery and cell biology.


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434 - 7-flow injection analysis

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