1806
ANALYTICAL CHEMISTRY
are shown in Table.< 1 and 11. In all case? recoverkc of 90% or better were obtained for both acids. I n Figures 2 and 3 are shown the titrations of mixtures of sulfuric acid-hydrochloric acid and sulfuric acid-nitric acid using Procedure R. The recovery values for these determinations are shown in Table 111. No data are shoxn for Procedure D, ivhich is a potentiometric modification of Proredure E. Somewhat less precise result,s were obtained by Procedure -1and these data are not reported. The use of Procedure A is recommended only in the presence of organic acid.*. LlTER.4TURE CITED
(1)
Belcher, €1.. Kapel. >I., antl Sutten, A . J., A d . Chiin. d c t a , 8, 148 (1953).
Blaedel, W.J.. Lewis. \V.R , and Thoinas, 6. W., . h . k r . . C k m f . , 24, 509 (1952). Carrihre, E., Bull. soe. c h i m . , 9, 626 (1942). Clarke, F. E., ANAL.CHEZI.,22, 553 (1950). Ibid., p. 1458. KBszegi, D., 2. anal. C'heni., 129, 218 (1949). Palit, S. R., INTI.EXG.CHEM.,ASAL.ED.,18, 246 (1946). Parke. T. V.. and Davis, IT. IT'.,A N . ~ LCHEM., . 21, 1570 (1949). Savich, K. K., Zaiodskaya Lab., 8, 10% (1939). Shkodin. A. AI., antl Iamailov, N. A . , J . Gen. Chein. 17.S.S.R., 20, 39 (1960). Takagi, K., J . C'hcm. SOC..Japaii. Pure C'item. S c c f . , 72, 364 (1951). RECEIVED for review J u n e 30, 1034. Accepted September 7 . 1954,
Determination of Ethylenediaminetetraacetic Acid as the Chromium Complex P. J. CHERNEY and BARBARA CRAFTS, W a y n e
University College of Medicine, Detroit, M i c h .
H. H. HAGERMOSER, A. 1. BOULE, and RAY HARBIN,W a y n e BENNIE ZAK,
The determination of ethylenediaminetetraacetic acid in urine was investigated to provide a simpre, rapid, and accurate procedure for a substance which is achieving increasing prominence as a therapeutic agent in the field of medicine. The high formation constant of chromium prevents the interference of other chelates; as chromium is in excess. The reproducibility and accuracy obtained with standards and urines with and without the addition o f various anions indicate that the method can be used to determine ethylenediaminetetraacetic acid in urine or water solutions in the concentration ranges shown.
T
University, Detroit, M i c h .
W a y n e University College o f Medicine, Detroit, M i c h .
HE increasing use of ethylenediaminetetraacetic acid (EDTA, Versene) in medicine for the treatment of essential hypertension ( g ) , heavy metal poisoning ( 2 , Q), and hemochromatosis ( I S ) , dissolution of urinary calculi ( I O , 1 1 ) )investigation of metal toxicity ( I ) , and the study of atherosclerosis ( I d ) has created a need for a method of determination. Because the urinary tract is the only route of excretion, all ethylenediaminetetraacetic acid being recoverable from the urine unchanged ( 6 ) ,a procedure that makes possible the quantitative measurement of this substance in urine is of obvious importance. Two procedures have been described. The first ( 5 ) emploved nickel t o complex the ethylenediaminetetraacetic acid, followed by quantitative determination of the bound nickel after the exces? metal had been precipitated by dimethylglyoxime. The second ( 8 ) involved preliminary removal of urates, after which a known amount of copper sulfate was added. Pyridine and rhodanid (thiocyanate) then reacted with the unbound copper to produce a spectrophotometrically measurahle substance which is extractable in chloroform. In the former mrthodphosphates interfere by combining with some of the nickel, giving high results. As phosphate is a common constituent of urine, it should be removed before nickel and dimethylglyoxime are added ( 5 ) . The latter procedure, in addition t o a preliminary 24-hour precipitation with ammonium sulfate and ammonia to remove interfering urates, involves a chloroform extraction of the pyridine-thiocyanate-copper (excess) reaction mixture and a 2-hour wait for full development of color. Plumb et al. (7), in their determination of the displacement series of metal complexes of ethylenediaminetetraacetic acid, have investigated the formation and propertie. of a chromium-
ethylenediaminetetraacetic acid comple\. a purple substance that is quantitatively measurable (9). Because of the analytical possibilities in the determination of chromium by ethvlenectiaminetetrancetic acid, it was decided to investigate the reverse procedure. The present report describes a method for the determination of ethylenediaminetetraacetic acid in urine by the rapid production and spectrophotometric measurement of this stable, reproducible complex. The formation constant of chromium with this chelating agent is sufficiently high t o preclude the possibility of interference by any of the urinary constituents (9). Therefore, none of the cationi or anions present in normal or pathological urine need be removed, and a simple, rapid and accurate procedure is available for. the quantitative determination ethylenediaminetetraacetic acid a? it is excreted in the urinr. REAGEYTS
-411 chemicals used were of analvtical reagent grade unless otherwise specified. Sodium Chromate Solution Weigh out 5 gramq of sodium chromate, dissolve in distilled n-atw, and dilute to 1 liter in a volumetric flask. Ethvlenediaminetetraacetic acid Standard Solutions. Keigh o u t 100, 200, and 300 mg. of dried analytical reagent ethylenediaminetetraacetic acid. Add one sodium hydroxide pellet per 100 mz. of ethvlenedinniinptctraacetic acid. dilute to about 75 ml., a;d dissol4e. Dilute to the mnrli in k 100-ml. volumetric flask. .4rsenious Acid, 0.2A17.Weigh out 9.8910 grams of arsenious acid, add 7 grams of sodium hydroxide, and dissolve in about 300 ml. of distilled water x i t h warming. Seutralize with glacial acetic, dilute to almost 600 ml., and lowly add 400 ml. of glacial acetic acid with thorough mixing. Dilute to the mark in a 1liter volumetric flask. APPARATUS
Coleman Junior spectrophotometer, 1Iodel 6s. Reckman spectrophotometer, Model DU. Aargent XXI polarograph. PROCEDURE
Preparation of Pure Chromium-Ethylenediaminetetraacetic Acid Complex. Place 0.1 mole (29.2 grams) of ethylenediaminet,etraacetic acid in 800 ml. of cold m-vater in a 2-liter Erlenmeyer flask, and 10 grams of chromic oxide dissolved in 100 ml. of cold water and 400 ml. of ethyl alcohol. Bring the mixture slowly to a boil with constant, stirring. Evaporate the solution to approximately 200 ml., filter, and pour t,he concentrated solution rrith stirring into 1 liter of cth?-l nlcohol. Filter off the precipitate
1807
V O L U M E 2 6 , NO. 11, N O V E M B E R 1 9 5 4 Table I. Recovery of C h r o m i u m w i t h Ethylenediamiketetraacetic Acid Sample 1
3
3
.f 0 7 8 9 10 11 12 13 14 16 16 17 18 19 20 21
tr
Chromium, A I g J 1 0 0 MI. Present Found 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
054 064 054 024 054 054 054 089 089 089 089 089 089 089 125 125 125 125 125 125 125
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
053 054 054 053 053 054 034 088 089 090 087 090 040 088 127
126 125 128 126 126 128
Deviation
Recovered,
hfg./100 ail. - 0 001 0 000 0 000 - 0 051 - 0 051 0 000 0 000 - 0 001 0 000 f 0 001 - 0 002 t o 001 t o 001 - 0 001 t o 00% +0 001 0 000 + 0 003 0 000 t o 001 c 0 002
70 98 100 100 98 98 100 100 98 100 101 97 101 101 98 101 100 100 102 100 100 102
1
0 0
1
1 0 0 8 0 0
7
0 0 8 5 8 0 5 0 8
5
1.20-
E
+ Figure 1.
1.10-
Diffusion Current
with a Uiichner funnel and wash with absolute ethyl alcohol. -4 chromium content of 14.41% was found by analysis. Preparation of Calibration Curve. Pipet 2.0 ml. of each of the st.andard ethylenediaminetetraacetic acid solutions into 19 x 105 mm. Coleman cuvettes. Add 2.0 ml. of the sodium chromate solution with mixing, followed by 2.0 ml. of the arsenious acid reducing agent. Place the tubes in a rack in a boiling water bath for 5 minutes, remove, cool, and read the absorbance in the spectrophotometer a t 580 mp against a blank made by substituting 2.0 ml. of distilled water for the standards and treating in the manner described. Analysis of Urine Sample. Pipet 2.0 ml. of the ethylenediaminetetraacetic acid-containing urine int'o 19 X 105 mm. cuvettes, add 2.0 ml. of sodium chromate and 2.0 ml. of arsenious acid, a?ti heat and read as above. Use an aliquot of the same patient s urine before treatment, as a blank and treat in the same nianiirr a the s n m p l ~ . DISCUSSION
Polarographic investigation of t,he ethylenediaminetetraacetic acid-chromium complex dissolved in neutral 0 . l X potassium chloride and deoxygenated with hydrogen shows a two-wave polarogram. The diffusion current of the first wave is 7.6 Ha., while that of the second wave is 17.8 Ha., slightly more than twice as large as the first (Figure 1). It had previously been found that with trivalent chromium in 0 . 1 S potassium chloride as supporting cAlectrolyte, the height of the second wave is somewhat greater than twice the height of the first wave, apparently because of the simultaneous discharge of hydrogen ion Kith the chromous ion, and this is probably JThat occurs here. I n any event, the polarogram indicates that chromium is present in the trivalent. state, as was previously known ( 7 , 9) the first wave corresponding to the formation of divalent chromium and the second indicating the production of metallic chromium, Pfibil and Klubalova ( 8 ) found t h a t the formation of the chromium complexonate was not reproducible when reduction took place with hydrochloric acid, ethyl alcohol, oxalic acid, or zinc amalgam. They showed that reduction of chromate was best acconipliahed with ethylenediaminetetraacetic acid itself, using a trace of manganese as catalyst. They used some neutral solutions and some acidified with acetic acid. However, when arsenious acid in 40% acetic acid is used as the reducing agent, with heat to hasten the reaction, reproducibility is attainable. Table I shows t h a t several concentrations of chromium are recovered with excellent precision and accuracy. I n this case ethylenediaminet,etrascetic acid is present in excess.
I CQ-
a90t. 0
080-
z a 0.70-
m DI
0,60-
0 v)
050-
m 040-
030-
Ox)-
300
400
500 600 WAVE LENGTH
700
Figure 2. Spectral Curves w i t h ChromiumEthylenediaminetetraacetic Acid Complex
Figure 2 shows the spectral curves obtained with different concent,rations of pure chromium-eth> lenediaminetetraacetic acid complex using a DU Beckman spectrophotometer. Two sharp peaks, a t 395 and 580 mp, are evident. Measurement is feasible a t either peak, as both obey Beer's law over the ranges tested. This is graphically shown in Figure 3, where the absorbances obtained a t both wave lengths exhibit a linear relationship to different known concentrations of the complex formed. I n Figure 4 are the absorbance-concentration curves for ethylenediaminetetraacetic acid in water, in urine, and in urine with added 5 mg. of calcium. With the exception of one point on t,he curve for ethplenediaminetetraacetic acid in urine, the three lines can be superimposed. The chromium complex has a higher formation constant than any other metal which could be present in urine ( 7 , 9). Therefore, this colored complex is the only one which should form, as excess chromium is available for the reaction. The use of several different normal and pathological urines for standardization always gave the same absorbances, within es-
1808
ANALYTICAL CHEMISTRY
periniental error, as did pure standards. This fact enal~lesone to use the former for the calibration curve, provided that urine excreted prior to ethylenediaminetetraacetic acid administration is used as a blank for sample analysis. The blank for the ethylenedianiinetetraacetic acid determination contains excess reduced chromium, which lends a blue background to the purple complex. Holyever, this does not interfere in quantitation, as a straight-line relationship is always maintained regardless of whether t,he medium is water or urine (Figure 4). Table I1 shows good recovery from pure solution and Table I11 inc!ic>ates that recovery is equally good, when several different wines, both normal and pathological, serve as the medium. 04t. 0
Table 11.
Saniple
Recovery of Ethylenediaminetetraacetic Acid from Water EDT.4. Present 25
50 100 100 100 100
8 9 10 11 12 13 14
15 16 17 18 19 20
100 100 200 200 200 200 200 200 250 250 250 500 500 500
/ l o o Xf1. h K d 27 53 99 100 101 103 98 100 201 198
202 198 199 200 248 253 251 488 507 506
Deviation, ?/lo0 M I .
+2
5; 0 +1
+3 -2 0 S1 -2 +2 -2 -1 0 -2 +3
tl
- 12
+7
T G
z a
Reco\ ered,
m
"0
Is:
108
106 99
0
101 103 98 100 100 5 99 101 09 94.5 100 99 101.1 100.3 97.6 101 3 101 1
m
ion
03 -
0
=a 0 2 -
Versene in H20 e-- Versene in URINE CVersene
I
I.oo
0.90
E
/
a Oa70 .-t
Figure 3. Relation of Absorbance to Concentration
'9
I1
0 '
nig. per 100 nil. of ethylenediaminetetraacctic ac,id, which gives a deep purple color \Tith acetic acid, produced no vic;ible color change when the other acids were used. U-hen lower concentrations of these acids are used ( l . O S ) , the purple color appears, but is considerably less intense than that produced by acetic acid. Table IV s h o w per cent absorbance, taking as 100% the absorbance produced by the acet,icacid solution of arsenious acid. I t is evident that complete color formation by acetic acid is possible in the presence of phosphate, sulfate, or chloride, which are the principal anions of urine. -4lthough low concentrations of the individual anions tested without acetic acid present will give full color formation, the
Sample 1 3
3
4 During the investigation of this complex, it was noted that acetic acid consistently produced the most intense color. It was then decided to test the effect on absorbance of substituting various mineral acids for the glacial acetic acid. With comparable amounts of sulfuric, perchloric, nitric, hydrochloric, and phosphoric acids, the reduct,ion of chromium was almost iristantaneous. Acetic acid reduces chromium at a slow rate and requires treatment in a boiling water bath for several minutes to achieve maximum absorbance. The standard solution of 300
3 4I 5 I 6 I 7 ' 8 mg VERSENE per I O ml
Figure 4. Relation of Absorbance to Concentration
Table 111. I 0.8 I.c (mg./ml x 3 )
2
t Cat+
in URINE
6 7 8 9 10 11 12 13 14 15
16 17 18
Recovery of Ethylenedianiinetetraacetic Acid from Urine EDTA, RIg./100 Jf1. Present Found 46 3 e0 47 0 00 50 48 5 102 100 95 100 100.5 100 101 100 102 5 100 197 200 204 200 193 200 194 200 211 200 307 300 308 300 30.5 300 310 300 302 300
Deviation,
Xlg./lOO All. - 3 .5 - 3 0 - 1.5
+ z o - 0 0
+ 0 5
+ 1.0 + 2 5 - 3
t S - 7 - 6 +I1
- 3 + 5 + 1 +10 + 2
~ e ~ ~ , . e ~ e d ,
7% 93 94 97 102 95 100 101 102 98 102 96 97 105 94 101 101 103 100
5
5 5 5 5
5 5
3 8
1809
V O L U M E 2 6 , NO. 1 1 , N O V E M B E R 1 9 5 4 Table IV.
Belknap, E. L., I d . M c d . a d Sicig., 21, 305 (1952). Bersin, Th., and Schwars, H., S c h w i z . M e d . Wochsciii.., 33, 765
Effect of Different Acids on Absorbance
(1953).
Deviation Absorbance, .4cid UsedQ .4bsorbance (Absorbance) % Acetic, (407,) 0 60 . .. 100 Sulfuric 0 28 -0 32 46 7 0 20 - 0 40 33 3 Sitric 0 24 - 0 36 40 0 Hvdrochloric Pdrchloric 0 31 -0.29 31 7 Phosphoric 0.44 -0.16 73 4 Acetic-HaPOa (1 : 1) 0 59 -0.01 98.4 Acetic-Hap04 (3: 1 ) 0 61 +o. 01 101.6 f0.02 103.2 .kcetic-HzSO4 ( 3 : 1) 0 62 Acetic-HC1 ( 3 : 1) 0 62 +0.02 103 2 12 Acidity of all solutions u-as 1.0.V and arsenious acid concentration \\*as
Bessman, S. P., Reid, H., and Itubii:. 11.. M e d , A W L . Dipf, Columbia. 21, 312 (1952). Darhey, h.,A N ~ LCHEM., . 24, 3 7 3 ( 1 9 5 2 ~ . Foreman, H., Vier. AI., and Magee, .\I.. J . H i d . C'ltem., 203, 1 0 G (1953).
Plumb, 11. C., Martell, -4.E., arid Reriworth, F. C . , J . P / i / s . "2 Colloid Chern., 54, 1208 (1950).
Popovici, .4.,Geschichter, C . I:., and Rutin. 11..Bull. Grorgc~ O / C / LL7nia. M e d . C'ejder. 5, 108 (1951). I'fibil, I{., and Kluhaloi-a, ,J., Collection C'zcchosZor. C ' h e m . C'om/ ~ C ( I ? L . T . . 15, 42 (1950). Suby, FI. I...J, CroZ., 6 8 , 9 0 (1952). Suhy. H I.. Alhright, F., Wayne. J.. and Denipsey, E.. l t j j d , ,
0.7.v. b
Based on acetic acid solution absorbance as 1007c.
prmence of mixed and variable amounts of several of the anions makes acetic acid appe:tr to be the best choice.
6 6 , 527 (195%). Till, H. S.. B r o w n , 13. H., Zlntki*, .X.. Zak, B., 11yer3, ( + , 13.. and Bol-le. 1.*I.. A ? r a . J . C'Zirb. Path.. 23. 1226 119531. (13) Kishinsky, H., IVeiiihcrg, T., Prevoqt, E. AI.. Buagiii, H.. and AIiller, 31. J.. J . La/,, C l i r ~ .M e d . , 42, 530 (1953). ~I
ACKNOWLEDG31ENT
The ethylenediaminetetraacetic acid used in this esperimcntation was 1-ersene, supplied by the BersTvorth Co., Framinpham, Mass. LITERATC-RE CITED (1)
Bauer, R . O., Iiollo. E'. lt., Spooner, C., and Woodman, E., Federation Proc., 1 1 , 3 2 1 (1952).
R E C E I V E for D review April 2 2 , 1854. Accepted d l i g r i e t 6. 1954. Presented before the Division of Biological Chemistry a t the 124th AIeeting of the AMERICANCHEMICAL SOCIETY, Chicago, Ill. Work slipported in part by the Receiving Hospital Research Corp., t h e Detroit Cancer Institute, a n d in part by institutional grants t o tlie Detroit Institute of Cancer Research and Wayne University College of Medicine from t h e .kmrrican Cancer Society, Southeastern Michigan Dirision.
Standardization of Titanous Solutions against Potassium Dichromate RAYMOND H. PIERSON and E. ST. CLAIR GANTZ Analytical Chemistry Branch,
U.S. N a v a l Ordnance
Test Station, Inyokern, China Lake, Calif.
Coniniercial supplies of titanous solutions usually contain iron as an inipurit? Consequently, if such solutions are standardized against strong oxidants, the , d u e for titanous content will be in error. -4 con\enient method f o r determining the iron content and making the necessar? corrections has been de?eloped. The use of titanium h>dride, which has a negligible iron content, is ad\ocated for preparing titanous solutions. On the basis of the present findings, potassium dichromate is re-established as a convenient and valid reagent for standardization of titanous solutions.
.
T
I T B S O C S solutions are po~vcrfulreducing agents widely used in titrimetric analysis (1-10). They have been preparcd and standardized in numcrous ways. The purposes of this paper are: to advocate the use of titanium hj,clride, TiH, (obtairi:i~ble from 31etal IIydrides, Inc., Beverly, Mass.), for preparation of t,itanous solutions; to describe a convenient and accurate method for determining the iron content of titanous solutions; to furnish a correction procedure based 011 tlie determination, tlicxhy nxiking tlie direct standardization against dirhrom:ite valid; arid to readvocate the use of pot,assium dichromatc in st:tndardizing titanous solutions. Preparation of titatiou? solutions from titanium hydride [as previously recomnicndcd ( 6 , I O ) ] offers two advantages over the lower cost and use of commcrcially available solutions-much frrrdoiii from iron contc'n-which greatly outweigh the slightly greatcr time rcquired in ni:tkiiig the standard titanous solutions from the hydride. ond atlvant~ngc~ has probably not been as widely rrcogiiized a s the first. For example, Lamond recent1)- reported ( 4 ) that d l *upplic,.s of titmious chloride or sulfate tested i n his laboratories c.ontainrii significaut amounts of iron. However, in the 1almrator:- in which the a,uthorsare eniploved, so hit ion^ prepared
from the hydride have cont:iiiied extremely small, actual1)- insignificant amounts of iron. For some kiiida of work the presence of a moderate amount of iron in the titanous solution would not be objectionable; in othcr caws it would be. If a titanous solution that contains a siyiiificaiit amount of iron must be used the role played by tht: ferrous ions determines the method chosen for standardization. Depending on the I I I R I ~ I I C I ' i j i which the titanous solution is to be used, these ferrous ions ma!- be eit,her active or inactive. The determiriation of nitroglycerin in propellants ( 1 1 furnishes x gaod csample of the latter case which is the onc usually encountered. Sitivglycerin is reduce:l by an excess of ferrous solutidn to j.ield ferric ions, which arc then tit,ratetl by the standard titanous solution. The ferrous content of the titanous solution is inactive, so that a standardization i? required which yields a value of the titsnous solution excluding its ferrous content. I t is on the basis of the above situation that Lamond has niised olljections to the usc of potaisium dicliromate in the (direct) standardization of t:tanous solutions. He gives three altcrnativei for avoiding the error due to iron: ( l i preparation of ironfree titanous solution; ( 2 ) standartlizing "against potsssium dichromate through ferrous ammonium sulfate" using ammonium thiocyanate as indicator; and ( 3 ) standardization against some standard other than dichromxtP. Purifications of titanous solutions (made from impure rengeriti) as described by Lamond are time-consuming arid are believed l)y the authors of this paper to be unwarrant,ed as long as titanium hydride of high quality is available. Corrections for iron content are either negligihle or very small when thc hydride is used. ADVANTAGES O F POTASSIU3I DICHRO3IATE IN STAVD.ARDIZISC. TITANOUS SOLUTIONS
Daily, rapid, and accurate :tandardization is essential for many itpplirntions of titanous roliition, I)crauie its strength gradually
