(9) Kolthoff, I. M., Livingston, R. S., Ind. Eng. Chem., Anal. Ed. 7, 209 (1935). Chem. (10) Lambert, R. H., Anal. 24, 868(1952). (11) Lein, A., Schwartz, N., Ibid., 23, 1507 (1951). (12) Marasco, M., Ind. Eng. Chem. 18, 701 (1926). (13) Mitchell, John, Jr., “Organic Analysis,” Vol. II, pp. 237-52, Interscience, New York, 1954.
(14) Moran, J. J., Anal, Chem. 24, 378 (1952). (15) blander, Z. physik. Chem. 129, 1 (1927). (16) Reich, H. J., Ungvary, R. L., Rev. Sci. Instr. 19, 43 (1948). (17) Reilley, C. N„ McCurdy, W. H., Jr., Anal. Chem. 25, 86 (1953). (18) Roe, H. R., Mitchell, J., Jr., Ibid., 23, 1758(1951). (19) Sandell, E. B., Kolthoff, I. M., J. Am. Chem. Soc. 56, 1426 (1934).
(20) Shrivastava, H., J. Indian Chem. Soc. 17, 387 (1940). (21) Siggia, S., “Quantitative Organic Analysis via Functional Groups,” p. 18, Wiley, New York, 1949. (22) Smith, D. M., Mitchell, J., Jr., Anal. Chem. 22, 750 (1950). (23) Underwood, A. L., Burrill, A. M., Rogers, L. B., Ibid., 24, 1597 (1952). (24) West, P. W., Ibid., 23, 176 (1951). Received for review June 10, 1957. Accepted June 17, 1958.
Ammonium Molybdate as Spraying Agent for Paper Chromatograms of Reducing Sugars H. EL KHADEM and S. HANESSIAN
Chemistry Department, Faculty of Science, Alexandria University, and Starch Products Co., Ltd., Alexandria, Egypt
Paper chromatograms of reducing sugars sprayed wth ammonium molybdate revealed spots which did not fade with time, while the background remained colorless if not exposed to strong light.
Aronoff
and Vernon (I) have suggested the use of 10% aqueous ammonium molybdate, concentrated hydrochloric acid, and ammonium chloride as a spraying agent for paper chromatograms of reducing sugars and easily hydrolyzable nonreducing disaccharides such as sucrose.
It
found that ammonium molybdate alone readily revealed reducing sugars and offered several advantages over the above reagent. The background of the chromatograms remained was
colorless instead of turning blue, the chromatograms were less sensitive to heat, so that they could be rolled and
put in
a
drying
oven
without acquiring
heat marks, the paper chromatograms did not deteriorate on keeping, and the spraying reagent could be stored unchanged for long periods. Paper chromatograms having spots containing 25 of each of the sugars—· arabinose, xylose, rhamnosé, glucose, mannose, galactose, fructose, sorbose, glucosamine hydrochloride, maltose, and lactose—wrere developed with the upper layer of a mixture of butanolethanol-water-ammonia (40:10:49:1) (2). The dry chromatograms were sprayed with 10% ammonium molybdate and heated to 100°' for 10 minutes. The spots revealed wTere first yellow, and after about 6 hours turned blue-gray on
colorless background. It has been reported (3) that they got decidedly darker and clearer on standing, wdiich is a big advantage over most of the available spray reagents that give spots which fade with time. The spots could be observed to greater advantage in daylight or under ultraviolet light. The background of the chromatograms remained colorless for several months, provided they were not exposed to strong light. a
LITERATURE CITED
(1) Aronoff, S., Vernon, L. P., Arch. Biochem. 28, 424 (1950).
(2) Hirst, E. L., Hough, L., Jones, J. K. N., J. Chem. Soc. 1949, 928. (3) Lamblou, M. G., Blouin, F., private communication.
Received for review April Accepted August 7, 1958.
10,
1958.
Solvent Extraction of Chromium with Acetylacetone JAMES
P.
McKAVENEY1 and HENRY PREISER2
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pa. The fact that the hydrated chromium(lll) ion is inert to chelate formation and solvent extraction under normal conditions has been used to effect the separation of chromium from aluminum, iron, vanadium, molybdenum, and titanium. The latter metals are separated from an aqueous solution at pH 2.0 by extraction with a 1 to 1 mixture of acetylacetone (2,4-pentanedione) and chloroform. After extraction of the interfering ions, the aqueous raffinate is refluxed with acetylacetone to convert the hydrated chromium(lll) ion to the chelate form.
The chromium acetylacetonate is then
extracted with a I to I mixture of The acetylacetone and chloroform. red-violet extract has been used for the determination of chromium above 0.20% in ferrous materials. Lower chromium contents are determined by the diphenylcarbazide procedure, following isolation with acetylacetone. is a continuation of the the solvent extraction of metal ions encountered in ferrous analysis. Although chromium acetylacetonate has been known since 1899 paper
This study
on
(6) the peculiar behavior of chromium toward solvent extraction wdth acetylacetone has only been recently noted (1). Direct shaking of a chromium(III) solution with a 1 to 1 mixture of acetylacetone and chloroform did not result in any extraction of chromium in contrast to the effect on such metals as aluminum (I II), iron(III), titanium (IV), molybdenum(VI), and vanadium(III), (IV) , and (V) (2) which were easily Present address, Crucible Steel Research Laboratory, Pittsburgh 13, Pa. 2 Present address, University of Arizona, Tucson 25, Ariz. 1
VOL. 30, NO. 12, DECEMBER 1958
·
1965
extracted. This was also difficult to explain in view of Steinbach’s work (4) which had indicated a 98% distribution of chromium into acetylacetone when chromium (III) acetylacetonate dissolved in acetylacetone was equilibrated with aqueous solutions of various pH values. A literature survey on the hydrated chromium(III) ion indicated that is was extremely inert to complex and chelate formation under normal conditions. This failure to extract chromium (III) under normal conditions is probably due to the extremely slow rate of chelate formation and the anomalous behavior of the chromium (III) ion has been used to advantage in separating it from such ions as iron(III), aluminum (III), and vanadium(V). APPARATUS AND REAGENTS
A Beckman Model G pH meter was calibrated at pH 4.00 with standard buffer solution obtained from the Fisher Scientific Co. A Beckman Model DU spectrophotometer was used to obtain all absorbance measurements in the ultraviolet and visible regions. It was equipped with interchangeable 1.00-cm. quartz and Corex glass absorption cells. The quartz cells were used from 320 to 500 µ and the Corex glass cells from 500 to 700 my. Acetylacetone (2,4-pentanedione) from Union Carbide Chemicals Co., stock grade, was purified by fractional distillation and the fraction boiling from 135° to 137° C. (745 mm. of mercury) was
used.
Chloroform from the Fisher Scientific Co., reagent grade C-298,
was
used
without further purification. 1,5-Diphenylcarbohydrazide (symdiphenvlcarbazide) solution was prepared as needed by dissolving 125 mg. of Fisher Scientific Co. reagent grade compound in 25 ml. of warm 95% ethyl alcohol. The solution w7as cooled and diluted to volume in a 50-ml. volumetric flask, using ethyl alcohol. NBS steel samples were from the National Bureau of Standards, the Department of Commerce, Washington 25, D. C. Chromium(lII) solution was prepared by dissolving exactly 1.1412 grams of Fisher Scientific Co. reagent potassium dichromate in 60 ml. of water. The dichromate was reduced by adding 10 ml. of concentrated nitric acid, 20 ml. of concentrated hydrochloric acid, and 10 ml. of concentrated sulfuric acid, and evaporating to fumes of sulfuric acid. The solution was diluted to 100 ml. with water in a volumetric flask. One milliliter contained 4.00 mg. of chromium. PREPARATION OF CHROMIUM(III) ACETYLACETONATE
The inertness of the chromium (III) ion to chelate formation indicated that elevated temperatures and a pH value of 7.0 were necessary to form the chelate. Because preliminary work had shown that acetylacetone forms a
1966
·
ANALYTICAL CHEMISTRY
Table
1.
Distribution of Chromium(lll) Acetylacetonate Aqueous
pH -0.80 -0.20 2.01 4.04 6.02
Layer, % 0.50 0.53 0.48 0.46 0.40
Organic Layer, % (Caled.) 99.50 99.47 99.52 99.54 99.60
water azeotrope (boiling point 94.0° C.), and that violent bumping of the aqueous and acetylacetone layers occurred during heating, a special arrangement was necessary to form the chelate. A reflux arrangement was used to prevent loss of the acetylacetone as the azeotrope and a stirring arrangement kept the aqueous and acetylacetone layers well mixed.
To carry out this procedure, 25.0 ml. of chromium(III) solution (4.0 mg. of chromium per milliliter) were transferred to a 250-ml. beaker. Then approximately 8.0 ml. of concentrated ammonium hydroxide were added to obtain a solution wffiich was just basic to litmus. An additional 2.0 ml. of ammonium hydroxide were added in excess and the mixture was transferred with washings to a 500-ml. roundbottomed flask. Twenty-five milliliters of pure acetylacetone were added and a magnetic stirring bar was carefully placed in the flask. A reflux condenser was attached to the flask and the flask and contents were heated with an electric mantle. A magnetic stirring unit -was placed directly under the heating mantle to control the rate of mixing of the organic and inorganic layers. After refluxing for 30 minutes, the flask, with condenser attached, was cooled in cold water. The contents of the flask were transferred to a 250-ml. separatory funnel and the lower aqueous layer was separated. The upper acetylacetone layer was transferred to a 300ml. Erlenmeyer flask. The aqueous layer was returned to the separatory funnel and backwashed with two 25-mL portions of chloroform wffiich were also combined with the acetylacetone extract. Three grams of anhydrous sodium sulfate were added to the Erlenmeyer flask and mixed with the organic extract to remove the water. The solution was filtered through No. 42 Whatman filter paper and the residue was washed with chloroform wffiich was also passed through the filter paper. The extracts were combined in a 250-ml. beaker and the solution was evaporated to a volume of about 50 ml. on a lowheat hot plate. After cooling, the solution was transferred to a 100-ml. volumetric flask and 25.0 ml. of acetylacetone were added, plus enough chloroform to dilute to volume. After examination of the aqueous phase for residual chromium [employing diphenylcarbazide (3) ], the extraction was found to be 99.54% complete. Thus, the organic extract contained a total of 0.09954 gram of chromium.
DISTRIBUTION OF CHROMIUM(III) ACETYLACETONATE
Ten-milliliter aliquots of sulfuric acid solutions, the pH values of which had been adjusted to various approximate values with ammonium hydroxide, wrere transferred to 50-ml. extraction bulbs. A 10.0-ml. aliquot of the chromium(III) acetylacetonate in 1 to 1 chloroformacetylacetone prepared as described above was added. The extraction bulbs were placed on a mechanical shaker for 30 minutes, and after extraction, the pH of the aqueous phase was measured or calculated for pH values below zero. Aliquots were also removed from the aqueous layer for chromium analysis. The organic matter in the aliquot was destroyed by adding 5.0 ml. of concentrated sulfuric acid, 10.0 ml. of concentrated nitric acid, and heating to fumes of sulfur trioxide. The diphenylcarbazide procedure using permanganate oxidation was used for the colorimetric determination of the residual chromium. Distribution data for chromium are listed in Table
I. DETERMINATION OF CHROMIUM IN FERROUS ALLOYS
Direct Analysis. Transfer up to 1 gram of ferrous material to a 400-ml. beaker. Dissolve with 50 ml. of 1 to 1 hydrochloric acid and 20 ml. of 1 to 1 sulfuric acid. Evaporate to a volume of about 25 ml. and oxidize with 50 drops of concentrated nitric acid. Evaporate to salt formation and cool. Add about 50 ml. of water and heat to boiling to dissolve salts. Filter through No. 40 Whatman paper and wash the paper with 2.0% sulfuric acid. This filtration usually removes most of the silica, tungsten, niobium, and tantalum. Evaporate the filtrate to a volume of about 25 ml. and cool. Add slowly from a buret about 20 ml. of concentrated ammonium hydroxide, mixing well between additions to prevent precipitation of ferric hydroxide. Cool the mixture in an ice bath before measuring the pH value and carefully continue the addition of ammonium hydroxide until Transfer the a pH of 2.0 is reached. solution to a 500-ml. separatory funnel and add 25 ml. of acetylacetone. After mixing, separate the lower aqueous mixture and return it to the original beaker, disregarding any entrainment of acetylacetone. Readjust the pH to 2.0 with ammonium hydroxide and return the solution again to the separatory funnel. Add 25 ml. of chloroform and mix the two solutions for 2 minutes. Allow the layers to separate and discard the lower chloroform-acetylacetone extract. Extract the aqueous solution again with 50 ml. of 1 to 1 chloroformacetvlacetone, discarding the organic extract. Transfer the aqueous raffinate to a 500-ml. round-bottomed flask and add concentrated ammonium hydroxide until the solution is basic to litmus, then Add 30 ml. of acetyl2.0 ml. in excess. acetone to the mixture, insert· a magnetic stirring bar and reflux with stirring
for 30 minutes to form the chromium(III) acetylacetonate. After cooling, transfer the mixture and washings to a 500-ml. separatory funnel. Without mixing, allow the layers to separate, and drain out the lower aqueous layer. Transfer the acetylacetone extract to another beaker. Return the aqueous solution to the separatory funnel and extract with 25 ml. of chloroform. Combine the chloroform and acetylacetone extracts. Discard the aqueous solution. Transfer the combined organic extracts to the separatory funnel and backwash four times with 50-ml. portions of 6.Y sulfuric acid. Transfer the lower organic extract, which at this point should be red-violet, to a 50-ml. volumetric flask containing 2.00 grams of anhydrous sodium sulfate. Extract the combined aqueous backwashings with 15 ml. of chloroform. Combine all organic extracts and dilute to volume with acetylacetone. Decant a suitable portion through No. 42 Whatman paper, and measure the absorbance at 560 µ. Determine the amount of chromium from a calibration curve prepared by taking aliquots of a chromium(III) acetylacetonate solution in 1 to 1 chloroform-acetylacetone [1.0 mg. of chromium(III) per milliliter] and diluting to 50 ml. with 4 to 1 chloroform-acetylacetone in a flask containing 2.00 grams of anhydrous sodium sulfate. Measure the absorbance at 560 mg where chromium(III) acetylacetone has an absorption peak (molar extinction coefficient 64.3). The 4 to 1 chloroform-acetylacetone is used as the reference solvent. =
This procedure on all samples containing in excess of 2.0 mg. of chromium. For low chromium samples the absorbance is too low for accurate measurement, necessitating the use of the diphenylcarbazide method on an aliquot of the organic extract. Indirect Analysis.
is suitable for direct analysis
Transfer the aliquot, which should not contain more than 0.30 mg. of chromium to a 125-ml. beaker, place on a steam bath, and boil off the excess solvent. Add 5 ml. of concentrated nitric acid to the dry residue and wet thoroughly. Take to dryness on the steam bath. Then add 5.0 ml. of concentrated sulfuric acid and take the mixture to strong fumes of sulfur trioxide. Carefully add several drops of concentrated nitric acid to the fuming mixture to destroy traces of organic matter. After cooling, dilute the solution to 100 ml. with water, mix, and transfer to a 400-ml. beaker with washings. After several minutes’ boiling, add about 8 drops of 0.LV potassium permanganate and continue boiling for another 10 minutes. If the pink color disappears) restore it with several additional drops of permanganate.
After boiling, add 6 ml. of 10 volume % hydrochloric acid (10 ml. of concentrated hydrochloric acid diluted to 100 ml. with water) to destroy the permanganate color. Boil an additional 5
Table II.
Analytical Data for Chromium
NBS. Sample No. 5K 8H 30d 32d
55d 61a 116a
123a 134 139 152 156
Type of Ferroalloy Cast iron Low alloy steel Low allov steel
(Cr-V) Low· allov steel (Cr-Ni) Open hearth iron Ferrovanadium Ferrotitanium® "
Stainless steel (18 Cr-11 Ni) High alloy steel
(W-Cr-V-Mo) Low alloy steel (Cr-Ni- Mo) Low allov steel (Sn-Cu) Low alloy steel Low allov steel
(Cr-MoAg) “
National Bureau of Standards Samples Cr, % Found
1.13, 1.16, 1.14
1.14, 1.08, 1.13
Cr, % Certificate 0.109 0.022 1.15
0.73,0.71,0.70
0.68,0.74
0.71
0.0057, 0.0055
0.005 0.68 0.23
Indirect
Direct
0.109,0.108,0.109 0.020,0.021,0.020
0.71,0.66,0.68 0.25,0.24,0.23 18.3, 18.0, 18.1
18.05
3.-79, 3.73, 3.77
3.73
0.58,0.57,0.58
0.55
0.054,0.051,0.052
0.050
0.43,0.43,0.41
0.45,0.42,0.44
0.43
0.97,0.99,0.99
1.06, 1.05, 1.03
1.00
"
(Cr-Ni-Mo) 159
on
Because of titanium hydrolysis above pH 1,50, 3 extractions
1.50 to
remove
iron and titanium.
minutes after color disappearance before cooling the beaker in cold water. Transfer the solution to a 250-ml. volumetric flask. Dilute to about 230 ml., mix, and add 5.0 ml. of diphenylcarbazide solution. Dilute to volume, mix, and allow to stand 20 minutes for color development. Determine the absorbance in 1.0-cm. Corex cells at 525 mg. The calibration curve is linear to 0.30 mg. of chromium per 250 ml. of solution. Water is the reference solvent. Table II lists analytical results obtained using both the direct and indirect method for chromium on various NBS samples. DISCUSSION
The data of Table I indicate that chromium (III) has practically a constant distribution ratio over a wide range of pH values. Because the chromium(III) could not be removed from an aqueous solution by direct shaking with acetylacetone, a modified method of extraction was developed in w'hich chromium acetylacetonate was preformed by heating acetylacetone and solution containing the hydrated chromium (III) ion prior to extraction with chloroform. Table I indicated that chromium (III) acetylacetonat e, when formed, is removed quantitatively. The inert behavior of the hydrated chromium (III) ion toward chelation with acetylacetone is characteristic of all its complex-forming reactions (-5). To counter the high activation energy of substitution, and form the chelate, it was necessary to reflux the chromium solution with acetylacetone. This inertness provides a novel way of separating chromium from the other ions. In the preliminary ex-
were
carried out at pH
traction procedure, ions such as iron(III), aluminum(III), titanium (IV), molybdenum(VI), vanadium(V), etc., are separated nearly quantitatively at pH 2.0 from the aqueous phase, wiiile the inert chromium(III) ion remains behind, plus residual iron, vanadium, and all the cobalt and nickel. The aqueous phase is then refluxed with acetylacetone and the chromium extracted with chloroform after cooling. The backwashing of the chloroform extract with 6 7 sulfuric acid removes residual iron and vanadium from the organic phase, permitting the spectrophotometric measurement of the chromium(III) acetylacetonate. This represents one of the few methods in which a complex of chromium(lll) is the basis of a color method. The usual colorimetric procedures for chromium depend upon the reaction of the chromate or dichromate ion. It may well be possible to obtain similar chromium (III) reactions with other reagents such as 8-quinolinol by the use of elevated temperatures and refluxing. The direct analytical procedure is ideal for chromium analysis in that there is no interference from any ion normally present in ferrous material. Because of the high stability of chromium acetylacetonate, a chloroform solution of this material can be backwashed many times with 6 * sulfuric acid to remove interferences. However, the method has the disadvantage of requiring more time than does the usual titration procedure for chromium in steel samples. The advantage of the acetylacetone extraction method is in its application to samples conVOL. 30, NO. 12, DECEMBER 1958
·
1967
taining high ratios of vanadium to chromium, or in samples containing extremely low concentrations of chromium. Samples with high vanadium content cannot be analyzed for chromium by current procedures without complex chemical separations. Samples with low chromium contents are
by the diphenylcarbazide colorimetric procedure. However, large amounts of iron and vanadium interfere in the diphenylcarbazide procedure and must be separated by ether extraction, and sodium hydroxide, respectively. Data in Table II indicate that acetylacetone can be used for the determination of chromium in a wide variety of ferrous alloys. The direct
usually
analyzed
procedure which utilizes the absorbance of the violet chromium acetylacetonate gives the most reproducible results when compared with the indirect diphenylcarbazide procedure. The indirect procedure is superior to the direct method in the low chromium range—i.e., below 0.20% chromium. However, it is also lengthy in that it involves destruction of the chelate prior to diphenylcarbazide treatment after the chromium has been isolated from all the interfering elements. It would be impossible to oxidize the chromium after the preliminary extraction of iron, vanadium, molybdenum, etc., because of the large amount of ammonium sulfate and organic matter in the aqueous phase. Also samples with high cobalt
and nickel contents would necessitate isolation of the chromium because of background color interference as these elements are not extracted with acetylacetone. LITERATURE CITED
(1) McKaveney, J. P., Freiser, Henry, Anal. Chbm. 29, 290 (1957). (2) Ibid., 30, 526 (1958). (3) Pigott, E. C., “Ferrous Analysis— Modern Practice and Theory,” 2nd ed., p. 156, Wiley, New York, 1953. (4) Steinbach, J. F., Ph.D. thesis, University of Pittsburgh, 1953. (5) Taube, H., Chem. Revs. 50, 69 (1952). (6) Urbaine, G., Debierne, A., Compt. rend. 129, 302 (1899).
Received for review February Accepted July 30, 1958.
7, 1958.
Voltammetric Determination of Histidine BRUNO JASELSKIS
Department of Chemistry, University of Michigan, Ann Arbor, Mich.
Milligram amounts of histidine have been determined amperometrically in the presence of small amounts of other amino acids, histamine, and protein hydrolyzates, using cobalt(ll) as a titrant. The determination is based on the formation of the polarographically active species, bihistidinatocobalt(ll), which is formed on the addition of cobalt(ll) to the histidine buffer solution at pH 8.0. direct
polarographic
deter-
mination of histidine is not possible The
as it is not reduced at the dropping mercury electrode. However, histidine can be determined indirectly by reacting with a reagent that is reducible with one that gives a reducible or product. The indirect determination of amino acids, by the reaction of phthalaldehyde which is reduced at the dropping mercury electrode, has been described by Norton and Furman (7). The application of the reaction which yields reducible products of amino acids has been described by Wiesner (12) for the reaction of quinone, and by Wenger, Monnier, and Feraggi (11) for the reaction of histidine with dinitrofluorobenzene. Amino acids have been determined polarographically as copper complexes by Jones (3), and histidine has been determined as a cobalt(II) complex by Roberts (9). The polarographic behavior of histidine with cobalt(II) has been studied by Millar (5), and with the other metal ions by Pleticha (8) and many others.
1968
·
ANALYTICAL CHEMISTRY
Most of the methods based on the reactions of the amino acids with the organic reagents are not specific and can be used only in the presence of one amino acid. The determination of amino acids as copper complexes lacks specificity because of the limited differences in the half-wave potentials. The determination of histidine as a cobalt(II) complex by Roberts using the maximum lacks specificity, and is dependent on the concentration of cobalt(II) and the presence of the other amino acids. In this paper the determination of milligram amounts of histidine is described in the presence of histamine, protein hydrolyzates, and other amino acids. Its determination is based on the formation of an anodic wave of the polarographically active species [bihistidinatocobalt(II) ] upon the addition of cobalt(II) to the buffered histidine solutions. The well defined one-electron anodic wave at —0.20 volt vs. S.C.E. has been recently reported (2) as resulting from the reac-
tion
Co(II) (hi)s
=
Co(III) (hi)2+ +
e
The amount of histidine is obtained from the voltammetric titration of histidine with the cobalt(II) standard
solution.
electrode with an agar potassium chloride bridge was used as a reference electrode. Capillary constants -were determined at a mercury column height of 420 mm. at —0.20 volt vs. S.C.E. in the phosphate buffer solution. The value of m (rate of flow of mercury) was 2.738 mg. per second, while the drop time was 3.56 seconds per drop. A Beckman Model G pH meter was used to measure the pH of the buffer solutions. The temperature was controlled to 25.0° ± 0.1° C. Reagents. A free base L-histidine was obtained from the Nutritional Biochemicals Corp. The purity of the histidine was determined by the nonaqueous titration method potentiometrically (6). These results were compared with the carbon, hydrogen, and nitrogen analyses. The other amino acids used for the interference studies were obtained from Eastman Kodak Co. and the Nutritional Biochemical Corp. Casein acid and enzyme hydrolyzates were obtained from the Nutritional Biochemical Corp. Gelatin, U.S.P. grade, was procured from J. T. Baker Chemical Co. and egg albumen from Baker and Adamson Co. The gelatin and egg albumen hydrolyses were carried out in a sealed tube at 105° C. in 6.0.Y hydrochloric acid for 60 hours as described by Smith and Stockwell (10). Test solutions of histidine and synthetic mixtures of amino acids were prepared by dissolving a weighed amount of amino acids in a supporting electrolyte buffer and diluting to a definite volume. The standard cobalt (II) sulfate solution was prepared from the anhydrous cobalt(II) sulfate. The concentration of the standard cobalt(II) solution was '
EXPERIMENTAL
Apparatus. The polarograms were recorded with a Sargent Model XXI A saturated calomel polarograph.
