Analysis of Binary Mixtures by Second-Order Differential Reaction Rates SIR: This communication clarifies a statement in a previous correspondence by Papa, Mark, and Reilley ( 5 ) . That communication stated that Siggia and Hanna (3, 6) used an expression of Lee and Kolthoff (4). The graphical method of Siggia and Hanna ( I , 3, 6, 7 ) does not require the Lee and Kolthoff equation. There is a relationship between Siggia and Hanna’s graphical approach and the Lee and Kolthoff approach because the equation used by Siggia and Hanna to plot their data is the classic secondorder equation (6). Lee and Kolthoff derived their expression from the same classic equation to describe mathematically the same chemical situation as Siggia and Hanna describe graphically, but the mathematical Lee and Kolthoff approach cannot be used with the Siggia and Hanna approach.
To analyze chemical systems with the Lee and Kolthoff expression, one requires rate constants which necessitates operation a t fixed conditions. The graphical method does not require the rate constant since the plot automatically takes this into account. This provides not only flexibility for wider applications, but also provides flexibility to choose analysis conditions particularly amenable to resolution and/or accuracy and precision. The niathematical “double point” technique ( 5 ) also circumvents the limitations of the “single point” techniques (4, 6). However, it should be pointed out that the graphical approach has an added advantage in that many data points are plotted which tends to eliminate error resulting from random scatter of the data.
LITERATURE CITED
(1) Block, J., llorgan, E. Siggia, S., AXAL.CHEM.35, in press. ( 2 ) Glasstone, S., “Textbook of Physical Chemistry,” 2nd ed., p. 1055, Van
Sostrand, New York, 1946.
(3) Hanna, J. G., Siggia, S.,Ss.41,. CHEK 34, 547 (1962). (4) Lee, T. S.,Kolthoff, I. M.,Ann. N.Y . Acad. Scz. 53, 1093 (1951). ( 5 ) Papa, L. J., Mark, H. B., Jr., Reilley, c . s., A S A L . CHEM. 34, 1513 (1‘362). (6) Siggia, S., Hanna, J. G., Ibid., 33, 896 (1961).
(7) Siggia, S.,Hnnna, J. G., Ibid., 35, in press.
SIDNEY SIGGIA
Olin Research Center NeJT Haven, Conn.
H. B. MARK California Institute of Technology Pasadena, Calif.
Zinc as Masking Agent in the Spectrophotometric Determination of (Ethylene dinitri1o)tetraacetric Acid in Urine SIR: ;in earlier paper published by Cherney et al. ( I ) described a method for the quantitative determination of ( e t h y l e n e d i n i t r i l o ) tetraacetic acid (EDTA) in urine based on the color of the chromium EDTA complex. dttempts t o apply this procedure in our laboratory demonstrated rather wide variations v i t h known amounts of EDT.4. The method requires that one posieqs not only the urine specimen uhieh is t o be analyzed but also an earlier EDT.1-free urine from the same individual mhich serves as a blank. Furthermore, urines that were EDTAfwe did not always give the same abwrbancc (blank) nith the color reagent en u hen obtained from the same individual. I t n as, therefore, impossible t o determine the EDT.4 content of an isolated urine Ypecimeii l x the Chwney procedure. If a means for masking out EDTA could be devised, it should then be possible t o set up a scheme of analysis in n hich each specimen might serve as its own blank. This would increase the utility of the determination. Investigation of a number of metal ions (31
slioned that zinc formed a sufficiently stable chelate with EDT.4 to mask completely the reaction between chromium and EDTA. The method decribed here is based upon this ma&ing effect. EXPERIMENTAL
Reagents. Potassium Dichromate Solution. -1 0.55% solution of reagent grade potassium dichromate in distilled water is used. This solution is stable indefinitely when stored in borosilicate glass bottles. Arsenious ilcid Reagent. Transfer 4.95 grams of reagent grade arsenious oxide to a 500-ml. volumetric flask. Add 3.5 grams of sodium hydroxide and dissolve in about 150 ml. of distilled water with warming. Neutralize with glacial acetic acid using phenolphthalein as indicator and then slowly add 200 ml. of glacial acetic acid with mixing. Cool, dilute to volume with distilled water, and mix thoroughly. This solution is stable indefinitely when stored in borosilicate glass bottles, Arsenious Acid-Zinc Acetate Reagent. Transfer 4.13 grams of reagent grade zinc acetate dihydrate to a 250-ml.
volumetric flask. Dissolve in arsenious acid reagent solution and dilute to the mark with the same solution. Mix thoroughly. This reagent is stable indefinitely when stored in borosilicate glass bottles. EDTA Standard Solutions. Transfer loo-, 200-, and 300-milligram portions of dried reagent grade (ethylenediamine)tetraacetic acid into 100-ml. volumetric flasks. Add one sodium hydroxide pellet per 100 milligrams of EDTA. Dissolve in distilled water and dilute to volume. This solution is stable indefinitely when stored in plastic bottles. Procedure. Prepare two tubes for each sample. Pipet 2 ml. of the urine specimen into each tube. Add 2 nil. of arsenious acid reagent to one and 2 ml. of arsenious acid-zinc acetate reagent t o the other and mix. This latter tube serves as a blank. Add 2 ml. of potassium dichromate solution to each and mix. Allow to stand for 35 minutes a t room temperature and read each sample against its corresponding blank a t 560 mp. Prepare standards as above but use 2 ml. of distilled water in place of the standard solution to obtain a blank. All measurements were made with a Coleman VOL. 35, NO. 3, MARCH 1963
403
Model 14 spectrophotometer using 19 X 105 mm. round cuvettes. The entire color reaction was carried out in these cuvettes to avoid transfers. DISCUSSION AND RESULTS
Hydrated chromic ion is of no value in this reaction because of the stability of the complex between chromic ion and water. Presumably E D T A can react with hydrated chromic ion but the reaction is very slow. -1time measured in days or weeks is required for attainment of equilibrium in systems containing hydrated chromic ion, EDTA, and water. This difficulty has been
Table 1.
Standard Curve in Water against Reagent Blank
Absorbance Without With zinc zinc
EDTA, mg.
0.114 0,225 0,424 0.617
1 2 4 6
Table 11,
Patient, A A
Sample
0:ooo 0.001 0.000
resolved by manufacturing unhydrated chromic ion in the presence of EDTLI by reduction of chromate, Although the chromium EDTA complex is more stable than the ZnEDT.1 complex under equilibrium conditions, the presence of a n excess of zinc ion competitively depresses formation of the chromium complex When one considers that n ater is also competing for the unhydrated chromic ions it can be understood that the net result of the various reactions is relatively complete inhibition of formation of the chromium complex. This is documented in Table I. Table I1 illustrates the degree of variability of the reaction of urine mith color reagent even when no E D T d was present. This requires that each urine specimen become its onn blank. Reference to the last three columns of Table I1 indicates that even nhen EDTA had been added to these same specimens, it could be masked by the addition of zinc ion. .Ipproximately 10 mg. of zinc are sufficient to irevent formation of the EDT.1-chromium complex nithin the range of the method.
Comparison of Results with Various Blanks
Urine alonea us. reagent blank
1 2
Milligrams per 100 ml. Urine with EDTA added Bmount us. reagent us. urineadded blank zinc-blank
31 34 41 41
90 82 72 74 172 176 163 183
88
E 4
67 146 59
...
143 116 113 115 268 251 380 248
94 88 79 82 169 164 140 182
These urines were originally devoid of EDTA.
Table 111. Effect of Time on Color Development a t 560 mp
Time, min. 1 3
; 10
Absorbance" Fresh Agedb chromic ion chromic ion 0 430 0 520 0 550 0 582 0 597
0 004
0 004
a 6 nig. of EDTA were present in both tubes. b Aged chromic ion was prepared by aging a mixture of equal parts of the arsenite and dichromate reagents for 3 hours a t 25' C.
404
ANALYTICAL CHEMISTRY
The resulting zinc complex is colorleia and does not interfere. It may be seen that the error when using a conventional reagent blank is extremely large when compared with that obtained using the recommended urine-zinc-reagent blank. In contrast to Cherney ( 1 ) IT? have found it convenient t o work a t room temperature rather than a t 100' C. Reaction times n-ere reasonably short and n-ere not critical. Table 111 shams the effect of time of reaction of EDT 1 with chromic ion and emphasizes the necessity of using fresh chromic ion. Recovery of knon-n amounts of EDT-1 added to various urine bpecimens is tabulated in Table IV. Kecoveries are excellent a t concentratirm below 200 mg. per 100 ml. and arc acceptable a t levels up t o 300 mg. per 100 nil. For urine lerels exceeding
300 mg. per 100 ml., it may be desirable to use a smaller aliquot of urine.
Table IV.
Absorbance 0.112 0.111 0.104 0.108 0.207 0.213 0,209 0.208 0.388 0.383 0,391 0.386 0.612 0 603 0 606 0 604
Recovery of EDTA from Urine
Recovered,
EDTB,
mg./100 ml.
Added 50.0 50.0 50.0 50.0 100.0 100.0 100.0 100.0
200.0 200.0 200 0 200 0 300 0 300.0 300 0 300.0
Mean recovery
m /O
Found
100.4 50.2 50.2 100.4 96.0 48.0 100.0 50.0 98.0 98.0 100.0 100.0 98.0 98.0 98.0 98.0 94.0 188.0 90.0 180.0 94.0 188.0 93.5 187.0 96.7 289.0 284 0 94 7 285 0 95 0 284 0 94 7 = 96.4 f 2.42%
Table V lists levels of EDTA found in hourly urines of a human subject receiving an intravenous injection of 3.0 grams of disodium EDTA adjusted to body pH and infused over a period of 28 minutes starting from time zero. Kormally, levels will not exceed these values and in many cases mill be considerably lover since the drug is rapidly excreted.
Table V.
Levels of EDTA in Urine of a Human Subject
EDTA, mg./100 ml.
Time, hr.
0 00 403 359 150 109 50.0
0 1
2 3 4 5
Since the color formed in this reaction does not follow Beer's law exactly, it is advisable to measure several standards and to construct a calibration curve. LITERATURE CITED
(1) Chernev, P. J., Crafts, B., Ihgermoser, H. H., Boyle, A. J., Harbin, R., Zak, B., ~ A LCHEM. . 26, 1806 (1954). ROBERT E. MOSHER PATRICI IJ. BGRCAR
Department of Research Providence Hospital Detroit, Mich. Department of Chemistry Wayne State University Detroit, Mich.
A. J. BOYLE
