complexometric titrationofcalciumand ...768 jones&mcguckin clinicalchemistry otherreports...

Complexometric Titration of Calcium and

Magnesium by a Semiautomated

Procedure

James D. Jones and Warren F. McGuckin

A semiautomatic procedure for the direct estimation of both serum calcium andmagnesium is described. The indicator used, Eriochrome blue SE (EBSE), has theadvantage of stability and freedom from interference by naturally occurring sub-stances. The procedure requires 3-5 mm. per sample and is very reproducible. Thevalues obtained agree with those obtained by back-titration of excesschelator and areconsistently 0.3 mg./100 ml. less than those obtained by a standard oxalate precipita-tion method.

C OMPLEXOMETRIC TITRATION s of calcium and magnesium in serum with

the aid of metallochromic indicators have been proposed by a numberof investigators. The advantages over other methods are increased

accuracy, speed, and simplicity. However, the detection of the endpoint is a potential source of variability and error. The indicatorsused are frequently unstable and can be masked by proteins and

naturally occurring ions. The method for direct titration of calcium

in serum with EDTA at pH 12.5 described in this report appears to befree of interference from other substances found in the serum. Theindicator is stable, and the time required per sample is 3-5 mm. The

results are very reproducible, since the detection of the end point is re-

corded automatically with respect to time. The distance, on the re-

corder chart, from start to extrapolated end point is proportional to

the concentration of calcium in the sample.

The results we have obtained are in general agreement with those in

From the Section of Biochemistry, Mayo Clinic and Mayo Foundation, Rochester, Minn.The authors wish to express their appreciation to Philip Burnett and Victor Johnson for

competent technical assistance and to Geigy Dyestuffs, Division of Geigy Chemical Corpora.tion, for generous gifts of Erioehrome blue SE and CHEL DE.

Received for publication Sept. 3, 1963.

767

768 JONES & MCGUCKIN Clinical Chemistry

other reports on coniplexometric methods (1-4) (0.3 mg. of calcium

per 100 mg. of serum less than determined by the modified Clark and(‘ollip procedure). The values are constant in samples of serum that

are highly pigmelited, contam abnormal proteins, or are moderatelylipemic. The determination by this method requires only one quanti-

tative transfer, and results agree well with values obtained by back-titration of excess ethylene glycol i)is_($_amnloethyl ether)-N,N’-tetraacetic acid (EG TA).

This method has now replaced a modified Clark and Collip deter-mmation for routine use in our laboratories and a preliminary studyof serum calcium ill a mixed normal population has been initiated. Ananalogous procedure for magnesium also is described.

Experimental

Apparatus

A titrator (Model SE*) with a Farrand interference filter (608 m,15 m half bandwidth, 47% transmission) is coupled with a constant-rate buret (Model C, 1 ml./min.*) and a logarithmic recorder (ModelRL,* with 2_ill./min. chart speed). The chart drive-motor of therecorder is activated simultalwously with the buret. A 1-ml. syringepipet for standards and samples and 30-ml. Pyrex beakers are theonly glassware neede(l.

Reagents

All solutions, with the exception of the indicator, which is stored inbrown glass, are stored in tightly closed polyethylene bottles. De-ionized water is used for all solutions.

Standard calcium solutions For the stock calcium standard,2.4970 gm. of primary standard CaC0:, dried to constant weight at1100, is transferred with 100 ml. of 1120, dissolved in 50-60 ml. of NHCi, and diluted to 1 L. with water, which gives a final concentration ofi: mg. of calcium per milliliter.

Standard magnesium solution Tile stock solution is 5.0678 gm. ofMgSO4 . 7H.,O diluted to 500 ml. with water, which gives a final con-centration of 1 mg. of Mg per milliliter. Working standards are pre-pared by diluting 75, 100, and 125 ml. of stock calcium and 20 ml. ofstock magnesium solutions to 1 L. to give solutions containing 7.5, 10,and 12.5 mg. of calcium and 2 mg. of magnesium per 100 ml. Two ad-

E. H. Sargent & Company, Chicago, Ill.

-A

Fig. 1. Titratioti curve.

vol. 10, No. 9, 1964 SEMIAUTOMATED CA AND MG 769

ditiollal magnesium standards are prepared by diluting 10 and 30 ml.of stock magnesium solution, each with 100 ml. of stock calcium solu-tion, to 1 L. to give solutions which contain 1 and 3 mg. of magnesiumand 10 mg. of calcium per 100 ml., respectively.

Other reagents The indicator is Eriochrome blue SE (EBSE)(Geigy Chemical), 0.380 gm./i00 ml. 1120. The titrating reagent is0.9922 gm. of EDTA, (disodium ethylenediaminetetraacetate dihy-drate, Baker Analyzed) per liter of water. Concentrated NaOH solu-tion is diluted to 2.4 N. Buffer, pH 10.1, contains 67.5 gm. of reagent-grade NH4C1 and 570 ml. concentrated NH4OH (c.p.) per liter of 1120.

Procedure

Calcium

One milliliter of each calcium standard (7.5, 10, and 12.5 mg./100 ml.,all with 2 mg. of magnesium per 100 ml.) is delivered to separate beak-ers, diluted to 15 ml. with deionized water, and mixed with 2 ml. of 2.4N NaOH and 3 drops of indicator.

The prepared sample is placed in position in the titrator (set at“spectro’’), the stirrer is activated, the recorder is set at “stalid by’’turned to “CK” (check), and the pen is adjusted to 100 on the scale

(extreme right) by use of the displacement knob. The recorder is seton ‘‘my’’ (millivolts) and the pen is adjusted to zero (extreme left) liv

Direct io I

of pen

B

use of the variable range knob. The titration tlmeti is started by turning

the button marked “automatic” on the buret. After the end point is

passed (Fig. 1), both buret and stirrer are shut off, the pen is raised,and the analysis is completed by drawing one line Oil tile slope (A in

s- s Calcium

o 0 Magnesium

/

//

//

/

3 4 5 6 7 8 9


Fig. 1) and allother on the final line (B in Fig. 1) at the end of the

titration. The distance, in centimeters, from the origin to the interceptof these two lines is proportional to the calcium concentration. Thetitration values, in centimeters, are plotted against the concentrations,in milligrams per 100 ml., of the standards (Fig. 2).

12

10

00

4

2

cm.

Fig. 2. Standard curve.

One-milliliter samples of serum are titrated in exactly the samemanner as the standards. The serum calcium concentration is deter-

mined by interpolation from the graph prepared from standards (Fig.2), or t.he concentration of EDTA can be adjusted so that the measureddistance in centimeters, when multiplied by a suitable factor (we ad-just the solution so that the factor is 2), gives the concentration ofcalcium in the serum in milligrams per 100 ml.

Since a syringe pipet is used for standards and samples, a directcomparison is made; however, if samples containing less than 0.05mg. of calcium are to be analyzed-for example, 0.5 ml. or less of se-rum-a standard curve including this concentration is necessary. Analternative procedure is to use an internal calcium standard. Deliveryof air in lieu of titrant or failure to eliminate the “slack” in the chartdrive mechanism of the recorder are the most common sources of errorin the determination.

Vol. 10, No. 9, 1964 SEMIAUTOMATED CA AND MG 771

Magnesium

One milliliter of each magnesium standard (1.0, 2.0, and 3.0 mg./100ml., all with 10 mg. of caicium/100 ml.) is delivered to separate beak-ers, diluted to 15 ml., and mixed with 2 ml. of pH 10.1 buffer and 3 dropsof indicator. The titration values obtained at pH 10.1 minus the valuefor a 10 mg./100 ml. calcium standard titrated at pH >12 are plotted,in centimeters, against the magnesium concentrations. The value formagnesium in a serum sample treated in a similar fashion is obtained

by interpolation from the standard curve.

Additional Procedures

Measurements of pH were made with the Metrion 1)11 meter* withthe Hyalk glass electrode. Trichloroacetic acid filtrates were preparedas described by Malmstadt and Hadjiioannou (5) and were titratedwith the aid of 650-ms and yellow cutout filters.

The back-titration procedure of Yarbo and Golby (6) was modifiedto allow detection of the end point with the titration assembly de-scribed. Titration of excess CHEL BEt (EGTA) at pH >12 was donewith standard calcium solution and Calcoi4 as indicator for calcium,and titration of excess EDTA at pH 10.1 was done with standard cal-cium for total magnesium and calcium with Eriochrome black T asindicator. Serum was ashed by placing 1 ml. of serum and 2 ml. of amixture of HNO3 and HC1O4 (concentrated acids, 1:1) in a 50-mi.Erlenmeyer flask and evaporating the mixture slowly to dryness on ahot plate. The ash was dissolved in 1 ml. of 0.5 N HC1 and the solutionso obtained was used for analysis.

In the modified Clark and Collip procedure used in this study, 2 ml.of serum, 2 ml. of 1120, and 1 ml. of 4% (NH4)2 C204 were mixed welland allowed to stand for 3 hr. at room temperature. The resulting pre-cipitate, removed by centrifugation at 1325 RCF for 30 mm., was re-suspended in 4 ml. of wash solution (327 ml. of EtOH, 327 ml. of diethylether, 327 ml. of 1120, and 27 ml. of cone. NH4OH) and recentrifuged.The entire washing procedure was repeated, the supernate was poured

off, andthe precipitate, which was dried for 6 hr. at 55-60#{176}C., was dis-solved in 2 ml. of N HOSO4, heated in boiling water for 3 mm., andtitrated with 0.01 N KMnO4. Normal values for serum calcium foradults by this procedure are 9.2-10.4 mg./100 ml. (7).

*Coleman Instruments, Inc., Maywood, Ill.

tGeigy Chemical Corporation, Ardsley, N. Y.J. T. Baker Chemical Co., Phullipsburg, N. J.

Calcium

The indicator, 3- (5-chloro-2-hydroxyphenylazo) -4, 5-d i hy dr oxy-iiaphthylene-2,7-disulfonic acid (EBSE), used earlier by Flaschkaand associates (8) is sensitive to calcium and magnesium, is water-soluble, is stable in alkaline and neutral solutions, and meets the gen-eral requirements for an indicator (9). The differences in absorbancefor the dye at pH 12.5 and 10.1 in the presence of calcium and mag-

nesiuni with and without au excess of El)TA are shown in Fig. 3. Thequantity of indicator used does not affect the value obtained for eithercalcium or magnesium but does determine the height of the titrationcurve (total change in absorbance).

The specificity of a complexometric titration is determined to agreat extent by the pH at which it is performed. Figures 4 and 5 illus-trate that it is necessary to add more NaOH thaii is required to attain

S

‘1

0Sb00S

0

I0SI

Wave length,millimicrons


Results and Discussion

Fig. 3. Change in absorbance

with and without excess chelator.

a p11 greater than 12 in the determillation of calcium. Tile 1)reakS inthe curves depicting pH occur at a lower concentration of NaOH thando the breaks in the curves showing titration value. This observationis similar hi both instances (standards and serum samples) and maybe

Vol. 10 No. 9, 1964 SEMIAUTOMATED CA AND MG 773

7.-

-

5r‘3

-0- -o -- - -0- ---0--- -o,0 12.5

0’ 0

12

I I I

I 2 3 4 5

Ml. 2.4W NoOH

7

12.5

o

12

I I I I2 3 4 5

MI. 2.4N NoOH

Fig. 4. (top). Effect of concentratioa of NaOH on pH and on result of titration of stand-

ard solutions. Changes in pH are shown by open circles and broken line, which correspond toscale at right. Changes in titration values are shown by solid circles and solid line, which cor-respond to scale at left. Fig. 5. (bottom,). Effect of concentration of NaOH on pH and on

result of titration of serum. Changes in pH are shown by open circles and broken line, which

correspond to scale at right. Changes in titration values are shown by solid circles and solidline, which correspond to scale at left.

cI

0

#{188}

24

20

16

12

8

4

0

Fig. 6. Effect of dilution

of serum on determination of

calcium with EDTA. Eachline represents dilutions of

different serum.

MI. serum


the reason for poor reproducibility noted in some other complexo-metric methods. This is undoubtedly a reflection of a change in ap-

parent stability constant of the calcium-dye complex with varyingionic strength of the solution (10). The titration value obtained in thepresence of a great excess of NaOH is not altered, as has been reportedwith murexide as the indicator (11). increasing the quantity of thebuffer does not change the calcium-plus-magnesium value.

One criterion that has been used in evaluation of methods is theeffect of dilution of sample on the value obtained. A plot of quantityof sample against titration value ideally should give a straight linewith the intercept at zero. This was found with our method both instandards and in samples of serum of varying calcium content (Fig.

6). In the procedure described by Malmstadt and Hadjiioannou (5)

a correction is needed since the intercept of standards and of serum isnot zero. The inability of the procedure described here to measure lowconcentrations accurately is indicated by the point representing 0.5 ml.of a serum containing 7.4 mg. of calcium per 100 ml. Recoveries of cal-

28

cium added to serum are 100 ± 0.5%. Error in the analysis of samplescontaining a low concentration of calcium, less than 0.05 mg., is elimi-nated either by adding an internal standard or by preparing a newstandard curve in this range of calcium concentrations.

*Added as KH,P02.

I Normal serum range, 3.2-4.3 X 10” mg. in this size sample.


The precision of the method is excellent. Titration values, in centi-meters, are presented in Table 1 ; these values multiplied by a factor of

2 represent milligrams of calcium per 100 ml. of the respective sam-ples. Reproducible values were obtained when the calcium concentra-tion of the pooled serum sample used in Table 1 was determined re-peatedly over a period of 15 days (N = 39, = 9.59 mg./100 ml., S. B.= ± 0.044).

Phosphate does not appear to have any significant effect on the anal-ysis as performed on serum (Table 2). This also is indicated by thereproducibility of the shape of the titration curve obtained, in which no

lag (indicative of a poor end point) was noted with these samples.This is in contrast to a direct titration of calcium in urine in which theend point, however slowly it is approached, is not readily detected re-

gardless of the indicator used. Cyanide, fluoride, and triethanolamine,commonly used masking agents, had no effect on values obtained, evenin severely hemolyzed samples; this indicates a lack of masking effecton the indicator by other ions (9).

A comparison of the modified Clark and Cohlip method and the di-

Table 1. PRECISION OF TITRATION OF CAI.cluht BY EDTA

Sample* (mg./100 ml.)

Titration ,alues (em.)

Repiwates Mean

Standard, 7.5 mg./l00 ml.

Standard, 10.0 mg./100 ml.

Standard, 12.5 mg./100 ml.

Serum (pooled sample)

3.76, 3.74, 3.74

4.99, 5.00, 4,98, 5.00, 4.97

6.20, 6.23, 6.20

4.80, 4.79, 4.80, 4.80, 4.81, 4.81,4.80, 4.80, 4.81, 4.78, 4.80, 4.85

375

4.996.21

4.80

5One milliliter.

Table 2. EFFECr OF ADDED PHOSPHORUS ON DETERMINATION OF CALCIUM ay EDTA

Phosphorus

added*

(mp. x 10-2)

Calcium

water standar4

(mg. )( 10’)Poole

(mg.

d serumt

)< 102)

0 10.0 9.6

5 10.0 9.6

10 10.0 9.6

15 10.0 9.5

20 9.9 9.8

50 9.8 9.8

5Corrected values.


rect titration method in over 200 samples indicated that the new meth-od gave values that were consistently 3% lower. To allow a directcomparison, the values shown ill Table 3 are corrected by this factor asare the values obtained by the complexometric procedure applied toTCA filtrates (Table 4). The procedure with TCA filtrates necessi-tated inclusion of TCA in the standard solutions in preparation of thestandard curve, and the values obtained appeared somewhat morevariable, as reported by Sadek and Reilley (12).

The new method also was tested by the comparison of two complexo-metric titrations: direct with EDTA and back-titration of excesschelator with standard calcium (Table 5). The values obtained agreereasonably well and again illustrate that a lower value is obtained bythis method than by the modified Clark and Collip procedure.

Table 3. RESULTS BY CLARK AND C0LLIP METHOD AND BY DIRECT EDTA TITRATION

(lark a,Id (olliI.

EDTA* (rny./100 ml seru,n)

Eriochrorne

(mg./100 ml. serum) blue SE Calco,,.

10.02 9.88 10.14

9.71 9.67 10.01

10.10 10.05 10.309.96 9.80 9.92

9.14 9.01 9.30

9.45 9.54 9.499.83 9.92 10.00

9.64 9.63 9.629.83 9.92 10.11

9.96 9.97 10.10

Correetetl values.

Table 4. RESULTS BY CLARK AND COLLIP METHOD AND BY EDTA TITRATION OF TCA FILTRATES

(lark aad Collip

EPTA* (mg./l00 ml. serum)

Erioel, rome

(my./100 ml. seru,n) blue SE Call-on

9.71 9.75 9.91

10.08 10.15 10.22

9.96 9.96 9.91

9.14 9.29 9.32

9.83 9.75 10.229.64 9.71 -

9.83 9.74 9.96

9.96 9.89 9.85


Table 5. RESULTS (No.1100 ML. SERUM) BY CLARK AND COLLIP METHOD, BY DIRECT TITRATION

WITH EDTA, AND BY BACK-TITRATION OF EXCESS EGTA

Clark and Collip* EDTA EGTA

10.4 10.0 10.0

9.6 9.4 9.55.6 5.4 5.3

10.2 9.8 10.0

10.1 9.8 9.7

9.9 9.5 9.710.3 10.2 10.2

9.5 9.3 9.29.9 9.7 9.6

10.5 10.1 10.1

9.6 9.4 9.410.2 10.0 10.0

5Values represent routine determinations done in the clinical laboratory.

Since higher values are ol)tanled by titration of oxalate in the Clarkalid Collip procedure, the following experiment was performed. Cal-cium was removed from serum by precipitation with oxalate for 6-8hr. The supernate thell was used as the source of oxalate to precipitatethe calcium from a water standard, and a blank from each “calcium-free” serum was diluted with an equal amount of water. The finalpermanganate titration values obtained on the precipitates from the

samples of serum and from the standards, minus the blank values andexpressed as milligrams of calcium per 100 ml. are shown in Table 6.The calcium standard in water consistently gave correct values (10

mg./100 ml.) OIl permanganate titration when the oxalate was in wa-ter. However, when the source of oxalate was a calcium-free serum, aconsistently higher value resulted. In similar experiments, these prep-arations yielded values of 9.7-9.8 nig. of calcium per 100 ml. whenmeasured by direct E1)TA titration of HNO1-HClO4-digested oxa-lates.

Since it has been demonstrated (13, 14) (and also reported to us byM. Power in a personal communication) that the total calcium contentfound in a sample, when determined by permanganate titration of anoxalate precipitate, is a functiomi of a number of phenomena (including

time allowed for precipitation of the oxalate, inclusion of oxalatesother than of calcium, amId soluhility of calcium oxalate itself), varia-tion may he expected. The data shown in the preceding tables illus-

trate that, in a comparison of methods, the reference method may besubject to considerable criticism.


Table 6. EFFECT OF SEaUM-OXALATE PREPARATION ON PRECIPITATION OF CALCIUM AS OXALATE

FROM WATER STANDARD

Sample No.

Calcium (ng./100 ml.)*

Original serum

Water standard

Exp. 1 Exp. 2

Standard

1

2

3

4

56

7

8

9

10

11

Water sol.

ammonium oxalate

-

10.16

9.8710.08

10.2110.40

7.71

10.24

9.76

7.29

9.89

9.98

10.35

9.95

-

10.35

10.35

10.35

10.35

10.28

10.28

10.3510.40

10.28

10.38

5By permanganate titration, corrected for blank (see text).

To evaluate the effect of serum proteins on the method, calcium-free

serum was prepared by passing a sample of serum over a chelatingresin (is) (Dowex A1*), which removed! all of the measurable cal-cium. This preparation, when added back to either serum or stand-ards, did not affect the analysis in any detectable manner. Since stand-ards added to serum also are recovered quantitatively, standards inwater are used exclusively in the routine determination. The proce-

dure has been applied successfully to serum from patients with jaun-dice, myeloma, and lipemia. Highly lipemic serum must be extractedwith a twofold volume of diethyl ether saturated with water prior tothe titration. The values obtained in these instances agree with thoseobtained on samples ashed with HNO.-HClO4.

Magnesium

The standard curve obtained in the titration of magnesium is shownin Fig. 2. The curve is not straight and does not have a zero intercept.The curve is not affected by calcium concentration in the range 0-20 X10 mg. amid maintains a similar shape with serum samples, as deter-mined by analyzing different volumes of a single serum. The use ofEriochrome black T for the combined calcium-plus-magnesium titra-

The Dow Chemical Company, Midland, Mich.


tion with either Calcon or EBSE for calcium alone yields a similar

curve.The direct titration (with EBSE at pH 10.1) of a solution contain-

ing only calcium gives a value 0.15 cm. greater than that found withcalcium alone at pH >12. This “initial blank” does not appear to re-

main constant with increasing concentrations of magnesium since thestandard curves do not remain straight. The effect on the end point ofincreasing the temperature of the titration mixture has not been eval-uated (9).

Although Lewis and Melnick (14) reported that titrations of cal-cium at pH 12.5 with Calcon or Cal-Red indicator agree with those atpH 10 with Eriochrome black T, results similar to those observed withEBSE were observed with these indicators.

The curve, which breaks at about 5 X 10_2 mg., is obtained by ana-lyzing water solutions of magnesium or magnesium plus calcium orstandards to which serum free of calcium and magnesium has beenadded. Since the curve from 5 X 10-2 mg. to higher concentrations ofmagnesium would extrapolate to a zero intercept, and since the titra-tion values for magnesium in serums to which varying known amountsof magnesium have been added plot as a straight line, it is necessary to

use the standard curve in calculating recovery values of magnesiumadded to serum. Recovery of magnesium from serum was 97-102%and 101-104% for direct titration and back-titration procedures, re-spectively.

Values obtained on serum by the back-titration procedure consist-ently were 0.4 mg./100 ml. lower. Manipulations to resolve the ap-parent discrepancy have failed to yield a satisfactory explanation.Possibly a more accurate value would be obtained by adding an inter-nal magnesium standard (0.05 mg.) but since this would entail a sec-ond quantitative transfer, the direct titration as described earlier is

preferred for routine use. Thus, the value for serum magnesium ob-tained by this procedure, although it appears to agree with those ob-tained by other methods (16-18), may not be the true value.

Prior to adopting the method described herein, a number of methodsfor the estimation of serum calcium were investigated, including high-and low-temperature flame photometry, colorimetric-complex forma-tion, complex formation, and other complexometric methods. Precisionand agreement with the modified Clark and Collip procedure were noteasily realized in the majority of those methods tried, and, while thehigh-temperature flame photometric procedure was acceptable, it was


sufficiently more time-consuming to niake questionable whether itwould be possible to process efficiently the large number of sampleshandled routinely in our clinical laboratory.

An extensive study of tile variations in concentration in serum of

calcium, magnesium, and phosphorus in normal persons has been ini-tiated; prelimillary data on 40 normal adults indicate calcium to be9.47 ± 0.26, and magnesium, 2.38 ± 0.14 mg./100 ml. (means ± S.D.).

References

1. Eldjarn, L., Nygaurd, 0., and Sveinsson, S. L., Seandi,,ar. ,J. Cli,,. 4- Lab. Invest. 7, 92(1955).

2. Bowden, C. H., and Patston, Valerie J., J. Cli,t. Pathol. 16, 18 (1963).3. Watson, D., and Rogers, J. A., Gun. Chint. Aeta. 8, 168 (1963).4. Lumb, G. A., Gun. Chint. Acta. 8, 33 (1963).5. Malmstadt, H. V., and Hadjiioannou, T. P., Clip. Chew. 5, 50 (1959).6. Yarbo, C. L., and Golby, R. L., Anal. Chew.. 30, 504 (1958).7. Power, M. H., Toogood, Ruth, and Adamson, Lucille, Clin. Chem. 2, 278 (1956).8. Flaschka, H., Abd El Raheein, A. A., and Sadek, F., Z. Physiol. Chem. 310, 97 (1958).9. Welcher, F. J., Analytical Uses of Ethylenediawineteira4eeiie Acid. Van Nostrand Co.,

Inc., Princeton, 1958.10. Brush, J. S., Anal. Chew. 33, 798 (1961).11. Kenny, A. D., and Cohn, V. H., Anal. Chem. 30, 1366 (1958).12. Spdek, F. S., and Reilley, C. N., J. Lab. 4’ Glii. Med. 54, 621 (1959).13. Maclntyre, 1., Biochern. J. 67, 164 (1957).14. Lewis, L. L., and Melnick, L. M., Anal. Chew. 32,38 (1960).15. Walser, Mackenzie, Anal. Chein. 32, 711 (1960).16. Hasselman, J. J. F., and Van Kainpen, E. J., Clin. Chin,. Ada. 3, 305 (1958).17. WaIlacli, Stanley, (‘ahill, L. N., Rogan, F. H.,and .loiies, H. L., J. Lab. 4- Gun. Med. 59,

195 (1962).18. Stewart, W. K., Hutchi,i.son, Frederiek, and Flemming, Laura W., J. Lab. 4- Clin .Med.

61, 858 (1963).

complexometric titrationofcalciumand ...768 jones&mcguckin clinicalchemistry otherreports...

Documents