Determination of Trace Quantities of Thiourea and Cysteine by Hydrogen Overvoltage Measurement on Platinum in Dilute Sulfuric Acid Paul E. Holland,’ J. T. Peeler, and A. J. Wehby National Center for Urban and Industrial Health, U.S . Department of Health, Education, and Welfare, Public Health Service, Cincinnati, Ohio This paper presents a simple method, requiring relatively inexpensive equipment, for the determination of trace quantities of thiourea and cysteine. The standard curve is a plot of hydrogen overvoltage, as measured in 0.3N sulfuric acid on a platinum electrode, against the log of the concentration of these compounds. Both compounds can be detected at 10-8M and can be quantitatively determined between lO-7M and 5 x lO-4M. Trace quantities of cystine and methionine can also be detected by the method; however, the precision is only good enough for qualitative work. The overvoltage change is interpreted as the result of a change in the outer potential of closest approach on an inactive platinum electrode.
FOR
electrochemists have regarded the sensitive poisoning effects of solution impurities as undesirable because of the uncertainty they caused in their electrode kinetic measurements. Juda, Frant, and Kramer (I, 2) and Moulton et al. (3)recognized the possibility of utilizing the phenomenon and accordingly studied the effect of large nitrogen molecules and reduced sulfur compounds on hydrogen overvoltage on platinum electrodes in dilute sulfuric acid. They found the electrodes to be most sensitive to reduced sulfur compounds, with detection limits as low as 1 ppb for thiourea and cysteine. They also found that they could detect the bacterium Escherichia coli at densities of 5.0 X lo4organisms per milliliter, and they attributed this to a detection of the sulfur in the organisms. Numerous other studies-e.g., (4-6)-have presented the quantitative effects of various types of solution impurities on hydrogen overvoltage. If a reproducible and sensitive technique employing the phenomenon could be developed, it might present solutions to such diversified problems as monitoring air or water pollution and monitoring sulfhydryl group exposure in a protein denaturation. The object of this study was to develop a technique whereby reduced sulfur compounds, such as thiourea, cysteine, cystine, and methionine, could be determined.
C U R R E N T SOURCE AI
R2
L
EXPERIMENTAL
A simplified diagram of the apparatus is shown in Figure 1. The working and auxiliary electrodes were made from 25-mil platinum wire sealed in soft glass 1 Present address, Richardson and Holland Corp., 1001 John Street, Seattle, Wash. 98109. (1) W. Juda, M. S. Frant, and D. N. Kramer, Science, 146, 521 ( 1964). (2) W. Juda, H. C. Petrow, and A. Clark, Final Report, Contract PH-86-66-144, U. S. Public Health Service, 1967. (3) D. M. Moulton, A. Clark, W. Juda, and J. Meany, Final Report, Contract DA-l8-035-AMC-404(A), U. S. Army, 1964. (4) J. OM. Bockris, Chem. Rev., 43,546 (1948). (5) J. O’M. Bockris and B. E. Conway, Trans. Faraday SOC.,45, 989 (1949). (6) A. N. Frumkin in “Advances in Electrochemistry and Electrochemical Engineering,” Vol. 3, P. Delahay, Ed., Interscience, New York, 1963, part 11.
I
LE‘
TO RECOROER
1’
1.
YEARS,
Apparatus.
RESPONSE B I A S
Vi
FRITTED DISC
50 m l T E S I SOLUTl ON
S T I R R I N G BAR E . L E C T R O C H E M l C A 1 CELL
Figure 1. Schematic diagram of circuit and electrochemical cell E1 = Working electrode, EZ = auxiliary electrode, Ea = hydrogen supply, E4 = referenceelectrode; Rl = 2K,RP = 250K,Ra = 10K; S1 = current reversal switch, Sz = current on-off switch; V, = 22.5 volts, Vz = 1.5 volts
tubing. The electrodes were roughened with a file, heated in a gas-oxygen flame until the surface of the platinum reached the fusion temperature, and subsequently cooled in air. The prepared soft-glass electrodes were connected to extended-tip, 7/25 glass joints with polyethylene tubing. Numerous electrodes were prepared, and the one that gave the most consistent, sensitive readings in a 10-eMthiourea solution was chosen as the working electrode. The apparent surface area of this electrode was 0.034 cm2. The potential on the working and auxiliary electrodes was measured with respect to a saturated calomel reference electrode acquired from the Instrumentation Laboratory of Cambridge, Mass, The circuit shown in Figure 1 provided for a potential measurement between either the working or auxiliary electrode as a cathode and the reference electrode. This response potential was measured on the 200-mV scale of a Heath Malmstadt-Enke pH recording electrometer. The readings were not corrected for potential drops resulting from the resistance of the supporting electrolyte because the same current was used for all experiments, and hence, these drops remained constant. A voltage bias was placed in series with the response potential to allow for positioning the potential reading on the recorder. The double-pole single-throw switch (SI) reversed the current and the electrode being measured, and the single-pole switch (SZ) opened and closed the circuit. A 22.5-volt mercury cell connected in series with a 250K variable resistor and a 2K constant resistor controlled the current between the working and auxiliary electrodes on the closed circuit. The applied current for all of the data VOL. 41, NO. 1, JANUARY 1969
153
E X C I T A T I O N SIGNAL
-
T I M E (MINUTES)
IO
0
T I M E (MINUTES)
Figure 2. signals
Excitation and overvoltage response (7)
reported was 0.4 mA-an apparent current density of 11.8 mA/cmZon the working electrode-and was measured with a Class I milliammeter from the API Instruments Co. The electrolytic cell was made from an outer 45/50 groundglass joint coupled, through a fritted-glass disk, with a side arm to accommodate the reference electrode. The inner 45/50 joint was provided with three 1/25 glass joints that
would accommodate the two working electrodes and a hydrogen supply. Procedure. Test solutions were prepared by serial dilution with 0.3N H2S04 of freshly prepared working standards. The 0.3N H2S04was prepared each week from triply-distilled water. The experiment began by placing the electrodes in 50 ml of freshly-made test solution, which was then saturated with hydrogen. A diagram of the excitation signal and a typical response signal can be found in Figure 2. The potential of the auxiliary electrode with respect to the reference electrode was measured on the open circuit. After 2 minutes the circuit was closed and the cathodic potential on the auxiliary electrode was measured. The current remained on for 1 minute before it was reversed and the circuit subsequently opened. The potential of the working electrode was measured on the open circuit. After another 4 minutes, the circuit again was closed and the cathodic potential of the working electrode was measured. This time the current remained on for 1.2 minutes before the current was reversed and the circuit opened. Here the cycle repeated with the potential measurement of the auxiliary electrode on the open circuit. The device was allowed to pass through one cycle before overvoltages were recorded. The overvoltage measurement discussed in this paper was the difference between the potential on the working electrode as a cathode 5 minutes after the current reversal and the equilibrium potential of the same electrode on the open circuit. Three readings were taken for each solution and triplicate solutions were tested for each concentration studied. Each overvoltage value given in the results was the average of the three readings taken on the individual solution. The anodic current density exceeded the limiting current of hydrogen diffusion to the anode, thus making the anode an -100,
---
CONFIDENCE I N T E R V A L S
-301 -8
-7
-5
-6
-4
L O G (MOL ES/L I T E R TH I OU RE A
-tour -REGRESSION LINE CONFIDENCE I N T E R V A L S
LOG (MOLES/tITER
CYSTINE)
-I
I I I111111 I I I111111 -6 -5 LO C (MOL E S/L I T f R CY S T E IN f )
0
ANALYTICAL CHEMISTRY
-4
-4
LOG (MOL E S/1
I 1 E R ME 1H I ON I N E )
Figure 3. Standard overvoltage curves for thiourea, cysteine, cystine, and methionine with corresponding 95 154
I I I1 IUU
REGRESSION L I N E CONFIDENCE I N T E R V A L S
-5
-6
-7
I I I111111
---
---
- 30- 8
-e
prediction intervals
/..
-40
A 0
-60
>
-REGRESSION LINE A A A INVERSE CONFIDENCE I N T E R V A L S ---PREDICTION INTERVALS
E
Y
0 >
-50
I I1111111
I I1111111
-7
-8
I I1111111
-6
1 IIllllU
-5
-4
LOG (MOLES/LITER C Y S T E I N E )
Figure 4. Standard curves for straight-line portions of cysteine data with corresponding 95 prediction intervals and inverse confidence intervals
oxygen electrode. The positive potential of this electrode desorbed or oxidized impurities and adsorbed oxygen (7, p 254); hence, the time of current reversal, making the electrode a hydrogen cathode, resulted in a practically instantaneous reduction of adsorbed oxygen and commencement of hydrogen and sulfur compound adsorption. Test solutions were well stirred throughout the experiments with a Teflon magnetic stirring bar, and the temperature of the solutions was controlled at 25 =t 1 “C. Switching operations were automated with a synchronous electric motor.
RESULTS The effects of thiourea, cysteine, methionine, and cystine concentration on overvoltage are shown in Figure 3. Each line is the result of a least squares polynomial regression assuming the model 7 =
Po
+
PI
log c
+
* *
.
+ Pdlog +
E
(1)
7 = overvoltage (millivolts)
c
Pi c
= = =
concentration (molesiliter) ith regression coefficient (i = 0, 1, . . . , n ) random error
(95z confidence interval about the mean of nine readings). Each experimental point is the average of three overvoltage readings on a solution; for practical purposes these readings were identical. The degree of the polynomial chosen was the lowest value for which the lack of fit could not be shown to be significant-i.e., the model was one of many that would adequately describe the data. For the case of cysteine, the lack of fit was significant for first, second, and third degree polynomials, so the third degree regression line shown does not adequately represent the data. Linear regressions were calculated for the straight-line portions of the cysteine curve between lO-7M and 10-6M and between 10-6Mand 5 X 10-4M, and for the straight-line portion of the thiourea curve between 5 X lO-+Mand 1 0 - 4 ~ . Those for cysteine can be found in Figure 4. The 95z prediction intervals for the overvoltage at a particular concentration for the mean of three overvoltage readings were calculated by the method described by Graybill (8), and 95z inverse confidence intervals about the concentration for the mean of three overvoltage readings were calculated by the method described by Ostle (9). Figure 3 shows only the forward prediction intervals, whereas Figure 4 shows a comparison of both the forward prediction and inverse confidence intervals for the two straight-line portions of the cysteine data. Table I gives the statistical quantities obtained from the polynomial and linear regressions performed. The sR2 is the residual mean square as calculated by Ostle (9). The Fl values calculated for Bartlett’s test (IO) indicate that for each case the variances at each concentration could not be shown to differ and, hence, could be pooled. The s p 2is the pooled variance. R 2 is the multiple correlation coefficient and represents the fraction of the sums of squares (SS) of deviations, which is explained by the model. FZ was calculated as in Ostle ( 9 ) and indicates whether the lack of fit of the model is significant. The residual mean square can be partitioned into two parts representing the experimental error ( s p 2 )and the SS of deviations due to lack of fit. If F2 is significant, as it was for cysteine, it means that the SS due to ~
The overvoltage of the electrode in pure supporting electrolyte 5 minutes after the current reversal was 37.02 f 0.03 mV (7) P. Delahay, “Double Layer and Electrode Kinetics,” Interscience, New York, 1966.
(8) F. A. Graybill, “An Introduction to Linear Statistical Models,” Vol. I, McGraw Hill, New York, 1961. (9) Bernard Ostle, “Statistics in Research,” 2nd ed., The Iowa State University Press, Ames, Iowa, 1963. (10) W. J. Dixon and F. J. Massey, “Introduction to Statistical Analysis,” 2nd ed., McGraw Hill, New York, 1957.
Table I. Statistics from Regression Analysis.
Chemical Thiourea
Range of data (moles/liter) 10-8-10-4 5
Cysteine
x 10-5-10-4
10-8-10-4 10-7-10-6 -~
10-5-5
x 10-4
3.09 2.96 2.84 1.11 1.38 10.2
sP2(mV2) 2.66
R2 0.998
0.70
0.996 0.994
8.31
0.992 0.980 0.956
bo
0.517 (7, 201)
1.76 (4, 15)
1.236 (7, 256)
16.37~ (4, 16)
193.0
243.4
50.0
2.9
-357.6 179.7
-44.0 158.5
28.8
1.5
-215.0 -145.9 -87.7
-23.9 -12.3 -4.4
1.130 1.96 -1.3 (7, 256) ( 5 , 16) 4.34 3.58 0.933 2.305 1.78 -83.4 -5.8 Methionine 10-8- 10-4 (7, 256) (6, 16) = Residual mean square; sp2 = pooled variance; R 2 = multiple correlation coefficient; Fl = Bartlett’s F ratio; F2 = lack offit F ratio; bj = regression coefficients (i = 0, 1, 2, or 3). * Degrees of freedom associated with FI or F2 shown below F value. c Significant at the level of a = 0.01. Cystine
10-8-10-4
s.2 (mV2)
VOL. 41, NO. 1, JANUARY 1969
155
-1
0 > Y
0 >
-8
-7
-6
-5
-4
-20 -
LOG (MOLES/LITER THIOUREA)
Figure 5. Standard curves for thiourea with overvoltage readings taken over different time intervals
the lack of fit are larger than the spz. The bi’s are the estimates of the regression coefficients shown in the model of Equation 1. Figure 5 is representative of the standard curves that are obtained if different time intervals are used for the overvoltage readings. These do not represent regression lines, but represent lines drawn through actual data points. The change of overvoltage with respect to time for several thiourea concentrations, used to find the values in Figure 5 , is shown in Figure 6. In all the experiments with concentrations less than 10-aM, the open circuit potential of the electrode remained at the equilibrium potential of a hydrogen electrode. At thiourea concentrations above 10-3M, there was a substantial increase in the overvoltage; furthermore, the equilibrium voltage of the electrode shifted toward a more positive value. Quantitative measurements of this phenomenon were inconsistent and, hence, are not reported. While preparing the electrodes, platinum heated to the fusion point and cooled in air was observed to be more sensitive to solution organics than platinum that was roughened by filing, Platinized platinum electrodes were not sensitive to solution organics at the concentrations studied in this paper. DISCUSSION
The results show that thiourea and cysteine can be determined by hydrogen overvoltage measurement. The third degree regression line with its corresponding prediction intervals for thiourea in Figure 3 and the linear regressions on the straight-line portions of the cysteine data in Figure 4 could serve as useful standard curves. These curves indicate that the detection limit for these compounds is on the order of 10-8M and that the quantitative sensitivity is on the order of lO-’M. The results on methionine and cystine, however, indicate that although these compounds could be detected by the method, they could not easily be determined. The slight slope of the methionine line, indicating the insensitivity of the electrode to this compound, and the high residual mean square for cystine, caused by varying experimental conditions, resulted in the low concentration precision for these two compounds. Nevertheless, the regression lines give an indication of the trend in the data. 156
ANALYTICAL CHEMISTRY
0.
’
I
1
I
I
The characteristic sigmoidal shape of the regression lines for cysteine and thiourea is similar to the sigmoidal shape of standard colorimetric curves over wide concentration ranges (11, p 6-18). In this case, the linear portion of the curve is often used for a standard. When linear regressions were carried out on the cysteine data, the residual mean square was substantially reduced and this resulted in narrow inverse prediction intervals on the concentration. Thus, by choosing a linear portion of the standard curve, the precision of the technique for cysteine determination was substantially increased. This increase would not have occurred, however, if the polynomial regression had adequately fit the data, as was the case for thiourea. The inverse confidence intervals for the linear regression models practically coincided with the forward prediction intervals. It is assumed that for practical purposes when the data fit the model and when the residual mean square is small, the forward prediction intervals can be used to approximate the inverse intervals. Accordingly, the intervals shown in Figures 3 and 4 indicate the confidence one can have in a concentration predicted from the mean of three overvoltage readings. The detection limit of 10-8M and a quantitative sensitivity of lO-’M are comparable to the sensitivity of other trace electrochemical methods. Laitinen (12) has reviewed the quantitative limits of a number of these methods. The sensitivities ranged from 10-4Mto lO-5M for ac polarography to IO-SM to 10-IOM for anodic stripping. The electrochemical methods whose sensitivity correspond to that of hydrogen overvoltage were square wave, second harmonic ac, and taste polarography; linear sweep, staircase, and derivative voltammetry ; and coulostatic and chemical stripping analysis. The primary advantages with the overvoltage method are its simplicity and its economy. Also of interest is the fact that the method is sensitive to organic sulfur compounds. The primary disadvantages are its nonselectivity and its imprecision; (1 1) L. Meites, “Handbook of Analytical Chemistry,” McGraw Hill, New York, 1963.
(12) H. A. Laitinen in “Trace Characterization Chemical and
Physical,” W. W. Meinke and B. F. Scribner, Eds., National Bureau of Standards Monograph 100, Government Printing Office, Washington, D. C., 1967.
the method is not as precise as the others in its sensitivity range. It may be possible to increase the sensitivity, precision, and selectivity of the method. For example, greater sensitivities can be realized by taking readings over longer time intervals. This is because the linear portion of the standard curve shifts toward lower concentration ranges as is shown in Figure 5 . The reason for this behavior can be better understood by studying both Figures 5 and 6. The overvoltagetime relationship in Figure 6 suggests an inverted decreasing exponential curve approaching an asymptote. Deviations in the linearity of the overvoltage-log concentration curves for low concentrations evidently result from taking readings from this decreasing exponential portion of the time curve. Equally important, the exponential portion of the curve is probably controlled by the kinetics of adsorption and by the limiting mass transfer of the compound from the bulk of the solution to the electrode. If this is true, the readings taken from this portion of the time curve will be subject to such experimental variables as stirring rate and other diffusion parameters. Hence, monitoring overvoltage readings over longer time intervals increases the sensitivity by improving the precision of the method at lower concentrations, as well as by shifting the linear portion of the standard curve toward these ranges. The sensitivity of the method can also be increased by developing more sensitive electrodes. The electrode used for these experiments, with a detection limit of 1O-SM for thiourea and cysteine, does not necessarily represent the most sensitive one, In fact, every electrode prepared by the technique described in the methods section gave a different type of response. An empirical search for more sensitive electrodes and a better theoretical understanding of the adsorption process of organics on platinum would allow for the determination of the ultimate sensitivity of the method. Possibly the sensitivity of the electrodes to organics is related to the space available for organic adsorption on the electrode and this space corresponds to the inactive area of a platinum electrode. Frumkin (6, p 358), when considering the effects of I- on the anodic ionization of Ha on platinum electrodes, hypothesized the existence of an active surface, This surface was the part of the electrode surface on which the kinetics of the ionization depended. Anion adsorption caused a reduction in the active surface and, consequently, a reduction in the electrode activity. Suppose that this active surface can also be defined on the platinum cathode and that this surface is the one determining the equilibrium potential through the charge transfer reaction H30+ e- = Hads HzO. According to Franklin (13), the equilibrium potential of the electrode will be determined by the magnitude of Hads which will be called the active adsorbed hydrogen. Because, for the cases studied in this paper, the organic adsorption did not affect the equilibrium potential but did affect the overvoltage, the adsorption was probably taking place on the inactive surface of the electrode. This would explain why the very active platinized platinum electrodes were insensitive to the organics and why the organic adsorbents did not affect the equilibrium potential of the electrodes. Hence, the search for electrodes sensitive to chemicals like cysteine and thiourea could be reduced to a search for electrodes with relatively large inactive areas. The precision of the method can be improved by obtaining more experience in the measurement of overvoltage and by
+
+
(13) T. C. Franklin and R. D. Sothern, J. Phys. Chem., 58, 951 (1954).
using more precise instruments. In preliminary experiments, for example, an intermittent rather than a continuous application of current increased the precision of overvoltage measurement because it tended to minimize both the potential dependent adsorption and the anodic dissolution of the electrode. The increased precision in the overvoltage measurement of cysteine over thiourea, as revealed by comparing their pooled variances, is believed to be the result of another experience factor. Further, the relative standard deviation ( l O O d s X ? ) for cysteine of 0.42% at 7 = 200 mV was on the same order of magnitude as the 0 2 % relative standard deviation on the 200-mV scale of the recorder used. Hence, for this case, more precise instrumentation would have to be used to detect the increased precision of a more refined method. There is also a possibility of increasing the selectivity of the method. Preliminary experiments showed that 0.1N 2-amino-2-hydroxy-methyl-1,3-propanediol (tris) buffer adjusted to a pH of 8.0 with 5N H804 masked the sensitivity of the electrode to such large nitrogen-containing compounds as 100 ppm bovine serum albumin, but did not mask the sensitivity to thiourea, cysteine, or cystine. This was not the case with these compounds in 0.1N HzS04. Moreover, when the solution of albumin was heated for 5 minutes in boiling water and subsequently cooled, the overvoltage reading was considerably higher than for the 0.1N tris control. This suggests the possibility of following thermal protein denaturation by the sensitive detection of unmasked sulfhydryl groups. Thus, by using another supporting electrolyte, not only was the selectivity increased but the possibility of additional uses for the device was revealed. The overvoltage can be both conveniently and theoretically plotted against the log of the concentration. There are at least four variables associated with the adsorption of organics on electrodes that can affect hydrogen overvoltage (7, pp 26973). They are the potential (p2) in the outer plane of closest approach to the electrode referred to the potential of the bulk of the solution, the hydrogen coverage of the electrode, the energy of adsorption (AGO) for hydrogen on the electrode, and the resistance of the adsorbed film to hydrogen penetration, At concentrations above 10-3M, both the change of active hydrogen coverage and the penetration resistance affected the overvoltage. The active hydrogen coverage was not affected at concentrations below 10-3M because the equilibrium potential of the electrode did not change. Because the active hydrogen was not affected and inasmuch as no abnormal increase in the overvoltage occurred, probably the resistance to Hz penetration was also unimportant below 10-aM. In this range, then, the primary adsorption variables affecting overvoltage were probably p2 and AGO. According to Delahay (7, p 268), pz is the controlling variable at electrodes with low hydrogen coverage. If the sensitive electrodes used in these experiments were inactive, as is hypothesized, then the active hydrogen coverage was probably low and p2 was the controlling variable. Frumkin (14, pp 96-7) found the plot of overvoltage against the log of the concentration to be linear for organic cations adsorbed on mercury. He stated that this supported the supposition that the cations shifted the potential; may be approximated for the work in this paper as equal to the p~ potential. Accordingly, the fact that the log plot is a reasonable function over the substantial concentration intervals studied in this work is (14) A. N. Frumkin in “Advances in Electrochemistry and Electrochemical Engineering,” Vol. 1, P. Delahay, Ed., Interscience, New York, 1961, part I. VOL. 41, NO. 1, JANUARY 1969
157
probably because cp2 was the controlling variable. If inactively adsorbed hydrogen affects the hydrogen overvoltage, however, these arguments do not hold, for both the AGO and the hydrogen coverage could be altered. The deviations from linearity, such as those at both lower and higher concentrations, may have been caused by any of the four mentioned variables, as well as by the particular form of the adsorption isotherm for the sulfur compound on the electrode.
ACKNOWLEDGMENT
We thank J. E. Campbell of the National Center for Urban and Industrial Health and Walter Juda of the Prototech Co. in Cambridge, Mass., for continued interest and helpful discussions. RECEIVED for review July 18, 1968. Accepted October 3, 1968. Mention of a commercial product does not imply endorsement by the Public Health Service.
Potentiometric Determinations of Calcium, Magnesium, and Complexing Agents in Water and Biological Fluids Bart van’t Riet and James E. Wynn Department of Chemistry and Pharmaceutical Chemistry, Medical College of Virginia, Richmond, Va. 23219 Calcium is determined accurately, even in multicomponent solutions, by direct titration with a solution of the tetrasodium salt of EGTA at a pH of about 8.5. A recording pH meter indicates a sharp upward break at the equivalence point, unaffected by a large excess of magnesium in the presence of citrate. Continued titration with tetrasodium EDTA of the same sample shows a break in pH at the equivalence point for magnesium. The limit of detection for both metals is 0.5 pg in samples up to 100 ml. More than 10 pg of EDTA or EGTA can be determined by addition of excess cadmium and back-titration with tetrasodium EGTA at pH 4.5 to 5.0. Reproducibility and accuracy are better than 10.5% for the samples containing more than 0.5 pmole of the metals or ligands.
DIFFICULTIES in end point detection of chelometric titrations in turbid or colored biological fluids can be avoided by potentiometric detection of the equivalence point. Schmid and Reilley ( I ) used a mercury electrode and some mercuric chelate in their samples of pure salt solutions for determination of calcium. This method, however, is limited by interferences in practical solutions-e.g., redox potential and chloride ion. Titrations of calcium plus magnesium can be done with the tetrasodium salt of EDTA under conditions that a change in pH indicates the end point. Schwarzenbach and Biedermann (2,3) did such titrations of pure salt solutions. Their results were not promising, and they discontinued this approach. A search for colored indicators was expanded and continues to the present. We shall present data showing why the pH change approach did not give better results. Conditions will be given under which sharp end points are obtained, even in urine and blood plasma, using readily available equipment. EXPERIMENTAL Apparatus. k e d s and Northrup microelectrodes, or any type of combination pH electrodes, are used in 50- or 100-ml beakers containing magnetic stirrers. The pH measurements (1) R. W. Schmid and C. N. Reilley, ANAL.CHEM., 29,264 (1957). (2) G. Swarzenbach and W. Biedermann, Helu. Chim. Acta, 31, 459 (1948). (3) A. M. Martell and Melvin Calvin, “Chemistry of the Metal Chelate Compounds,” Prentice-Hall, New York, N. Y.,1952, p 481.
158 *
ANALYTICAL CHEMISTRY
are made with a multispeed recording pH meter (Heath EUW 301-M). Either one of two Sargent constant rate burets is attached to the outlet which is only energized when the recorder operates. Reagents. Deionized distilled water is used in the preparation of the solutions which are kept in polyethylene containers. Ethyleneglycolbis- (betaaminoethylether) - N,N’ - tetraacetic acid (or EGTA, Eastman Cat. No. 8276) is dissolved by addition of an accurately measured volume of sodium hydroxide solution to make pH about 7.0. Its tetrasodium salt is prepared by addition of another identical volume of hydroxide, and a stock solution of 0.1M is made. Disodium ethylenediaminetetraacetate (Fisher Scientific Co.) is converted to tetrasodium EDTA with a calculated amount of 50% sodium hydroxide, and diluted to 0.1M. The proper stoichiometry is checked by titration of a standard calcium solution (see procedures). Trisodium citrate and cadmium nitrate, each about 5 x 10-3M solutions, are used. Procedures. CALCIUM.Samples preferably containing more than 20 pg of calcium are diluted to about 20 ml and adjusted to pH 8.0 to 8.6 with dilute strong acid or base. Urine and blood plasma can be titrated without further preparation. For poorly buffered samples-e.g., distilled water-and in the presence of an excess of magnesium, or when a precipitate is formed on adjustment of pH, the addition of 2 or more ml of 5 X 10-3Mtrisodium citrate is recommended. Samples containing a large excess of magnesium should be titrated in a pH region of about 8.2 or below. The titrant is 5 x to 10-3M tetrasodium-EGTA, added at a rate of 0.96 ml/min. During the titration of calcium, little change of pH is observed. The titration is discontinued after a sharp break occurs in the pH plot, and sufficient excess of titrant (about 0.5 ml) is added to allow precise interpolation. The sum of dead time and calcium content of water and citrate solution is obtained by performing a blank titration using the same amount of water and citrate as in the sample. The dead time is found by restarting the buret and recorder about 1 min after completion of the titration The distance on the plot before pH rises again represents the delay between start and response of the electrode. Blank and dead time can be determined with high precision using a high speed of the recorder. Standardization of tetrasodium-EGTA or -EDTA solutions is done by titration of a standard calcium solution ob-
