Qua nt ita t ive Determina t io n of
G 1y oxy 1ic Acid
DAVID N. KRAMER, NATHAN KLEIN, and RONALD A. BASELICE U. S. Army Chemical Warfare laboratories, Army Chemical Center, Md. ,A simple quantitative colorimetric method for the semimicrodetermination of glyoxylic acid is based on the formation of the intensely colored 1,5-diphenylformazancarboxylic acid obtained from the mild oxidation of glyoxylic acid phenylhydrazone in the presence of excess phenylhydrazine. Beer's law is obeyed in the concentration range of 1 0-4 to 1O-6M glyoxylic With suitable modifications, acid. the method may possibly b e suitable as a general quantitative procedure for aldehydes. Alcohols, ketones, acids, and esters (excepting formates) do not interfere with the test.
W
studying the radiolysis of aqueous trichloroethylene solutions in these laboratories, i t was necessary to analj-ze for micromolar quant'ities of glyoxylic acid. The reported methods ( 4 , 5 ) were not suitable because they are insensitive and/or too tedious and time-consuming for analytical work. Feigl reports (3) a specific det'ection method for glyoxylic acid in micromolar quantities, which consist's of adding excess phrnylhydrazine hydrochloride to glyoxylic acid and heat'ing. The mixture is then made strongly acidic and an oxidizing agent is added. The appearance of a bright red color indicates a positive tcst. The authors herein report the drtails of a quantitative colorimet'ric method for glyoxylic acid which is similar to one reported by Tanenbaum and Bricker (8),as suggested by Schryver ( 7 ) and by Desnuelle and Naudet ( 2 ) , as a method for formaldehyde. Further investigations shoned that the procedure is not specific for glyoxylic acid, but n-ith suitable modifications it may possibly be a general method for the detection of aldehydes. An attempt was also made to clarify t'he structure of the color body obtained. HILB
APPARATUS A N D REAGENTS
Use a Perkin-Elmer Model 13-U Universal spectrophotometer with fused silica prism, double-beam optics, and a lP28 photomultiplier, or any other spectrophotometer having suitable sensitivity and reproducibility. Phenylhydrazine Hydrochloride Solution, 1% (approximately). Dissolve 1 gram of phenylhydrazine hydrochloride (Eastnmn 330) in 100 ml of distilled water. Prepare fresh daily. 250
ANALYTICAL CHEMISTRY
. Concentrated Hydrochloric Acid, Baker & Adamson, C.P. grade. Potassium Ferricyanide Solution, 1% (approximately). Dissolve 1 gram of potassium ferricyanide (B&A, C.P. grade) in 100 ml. of distilled water. Sodium Glyoxylate, 99% pure by carbon and hydrogen analysis (obtained through the courtesy of C. R. hlaswell, National Institutes of Health). Prepare a 10-3ilf solution and dilute appropriately with water. PROCEDURE
Prepare calibration curves in the following manner: I n a 15-ml. test tube place 2.5 ml. of known glyoxylate solution and 2.5 ml. of phenylhydrazine h j drochloride stock solution. Incubate the resultant solution in a 110' C. oven for about 5 minutes. Allow the solution to cool to room temperature. Add 2.5 ml. each of concentrated hydrochloric acid and potassium ferricyanide stock solution, mix well, and allow to stand 2 minutes. Transfer to a spectrophotometer cell and obtain absorbance at 520 mM, using n reference consisting of the test reagents with distilled water in place of the glyoxylnte sohtion. Glyoxylate concentrations in unknowns are determined by reference to a calibration curve. STUDY
OF
VARIABLES
pH Effect. As variations in p H of the reagent mixture result,ed in differences in the stability of the colored product, experiments v-ere conducted to determine t h e optimum pI-1 for the method. At p H 12, no ferricyanide was needed to produce a red color with glyoxylic acid; however, the reaction \\-as not quantitative and the color \vas unstable. At neutrality, there was no reaction. At p H 3, a precipitate was obtained and no color was formed. The optimum acidity, however, for obtaining reproducibly quantitative results $vas found to be a t pH 1.2 or below. The color was stable under these conditions for at least 2 hours. Effect of Oxidizing Agent Concentration. Following the procedure outlined by Feigl (S), t h e authors found t h a t using hydrogen peroxide resulted in a n unstable color regardless of peroxide concentrations. However, use of potassium ferricyanide in concentrations between 1 and 3%, gave a color which was stable and reproducible. The use of lY0 potassium ferricyanide solution was adopted, as
higher ferricyanide concentrations gave highly colored reference solutions. Effect of Temperature and Incubation Time. When the phenylhydrazine
and glyoxylate solutions were miued, heating for 3 t o 5 minutes in a 110' C. oven gave quantitative yields of t h e phenylhydrazone. Similar results mere obtained by permitting the solution t o stand at room temperature for approximately 30 minutes. Heating the phenylhydrazine and glyoxylate solutions a t 110" C. for more than 7 minutes resulted in loss of reproducible color densities. It kyas necessary to cool the mixture to room temperature prior to the addition of hydrochloric acid and potassium ferricyanide in order to achieve reproducible colors. Stability of Reagents. T h e phenylhydrazine hydrochloride solution darkened on standing for more t h a n 24 hours because of decomposition oi t h e reagent. T h e potassium ferricyanide became cloudy on standing but was found to be still usable. Similar observations n cre reported by Tanenbaum and Briclrer (8) in a method for formaldehyde using potassium ferricyanide and phenylhydrazine hydrochloride.. The above authors also found that the phenylhydrazine hydrochloride solution was usable after filtering off any turbidity formed on standing. It is recommended, holyever, that the phenylhydrazine solution be prepared fresh daily and the ferricyanide solution be prepared fresh iveekly. Because glyoxylic acid in solution oxidizes in the prescnce of air, stock solutions should be kept under nitrogen and refrigerated. RESULTS
The method described may be employed with a precision of &5% in the quantitative determination of glyoxylic acid concentrations ranging from 10-8 to 10-4X. K i t h suitable modifications, such as the use of proper solvents, it may be utilized as a general method for the quantitative determination of aldehydes. Tanenbaum and Bricker (8) report a method for the determination of formaldehyde using potassium ferricyanide and phenylhydrazine hydrochloride in basic media. Figure 1 shows the spectra obtained using the test procedure 011 formalde-
I3t
n
X = 6 X IO"M 0 :3
F i g u r e 1. Absorption spectra of the formazans of formaldehyde, glyoxylic acid, and glyoxal
0.8 I ' I r
H-C=O
h
x 10-o M H - C - C Z O
Table 1. Molar Extinction Coefficients for Formazans as Obtained with Various Aldehydes
bB
i-,
Aldehyde Formaldehyde Glyoxylic acid Glyoxal
\
Absorption Maximum, hlp 520 520
Molar Extinction Coefficients 8,750 17,870 34,800
375
Table II. Compounds Tested for Color Reaction with Aldehyde Reagent
Compound Glyoxylic acid Formaldehyde Glyoxal Acetaldehyde Benzaldehyde
p-Dimethylaminobenzalde-
WAVE LENGTH
h j de, g l o ~hc ) acid, and glyoxal. Nolnr extinction coefficients for the colored products of these three compounds are listed in Table I. Kumerous other compounds 11 ere tested using spot plate technique in ordcr to demonstrate the specificity of thc anal: tical method (Table 11).
my
H
hyde Blue Crotonaldehy de Red Furfuraldehyde Deep-red m-Nitrobenxaldehy de Blue n-Butyraldehyde Red o-Chlorobenzaldehyde Blue o-Hydroxybenzaldeh yde Purple hIethano1 ... Ethanol ... Phenol ... Acetic acid . . Oxalic acid ... Glycolic acid ... Pyruvic acid ... Acetone ... Urea ... Acetophenone Glucose ... Sucrose ... Aniline ... Chlorobenzene ... Nitrobenzene ... Ethyl acetate ... Methyl propionate ... Methylal ... Ethyl formate* Rrd Methyl formate* Red a Color formation indicates result; lack of color, negative resuKSitive b See discussion. I
DISCUSSION
Although numerous authors have reported tost procedures for formaldehyde utilizing phenylhj drazine and an oxidant, the nature of the colored procluct formed in the reaction appeared to be uiihnonn. The present authors have demonstrated that the colored products are formazans-in the case of glyoxylic acid, 1,5-diphenylformazancarboxylic acid. The production of the formazan proceeds in a number of steps. One probable sequence is sho\T-n below, [ O ] referring to the general oxidant:
Results" Red Red Red-bronn Red Blue-green
Folio\\ ing the formation of the hydrazone by the reaction of phenylhydrazine v i t h the aldehyde (step I), the oxidant produces the unstable radical A , , which in turn tautomerizes to the A 2 radical, which is resonance stabilized as shonn by Criegre and Lohaus ( I ) (step 2). Simultaneously, the excess phenylhydrazine is oxidized to the phenylhydrazyl radical, B (step 3). Radicals A2 and R then combine to give the formazan interincdiate (step 4), which is furthei oxidized, producing the formazan (step 5 ) . As a hydrogen atom on the carbonyl carbon is necessary for coupling to take place, ketones cannot yield the colored formazan. Formazans are intensely colored compounds that are moderately stable in acid media (6). As evidence for the proposed structure of the colored product, 1,6-diphenylformazancarboxylic acid was unequivocally synthesized by the method of 1-on Pechman (10). The spectrum and molar extinction coefficient of the synthesized formazan were identical with those obtained using
.
.
glyoxylic acid in the analytical method outlined. As further evidence, benzene diazonium chloride was substituted for the oxidizing agent in the test procedure. Identical results vere obtained with either procedure. The use of substitutcd phenylhydrazines results in different colored dyes. 2,4-Dinitro-, ' o-chloro-, p-nitro-, and p-broniophenylhS-drazines IT-ere used in place of unsubstituted phenylhydrazine. Substituted phenylhydrazones, however, are usually less soluble in the reaction mixture and hence are unsuitable in the analytical procedure outlined. The positive tests shown in Table I1 for ethyl and methyl formate require special mention. It has been reported (9) that formate esters in the presence of excess phenylhydrazine and a mild oxidizing agent yield 1.5-diphenylformazan. Therefore, all formates should give results identical with those obVOL. 31, NO. 2, FEBRUARY 1959
0
251
tained with formaldehyde. H o m v e r , the formate reaction is considerably slower than the reaction with formaldehyde. LITERATURE CITED
(1) Criegee, R., Lohaus, G., Ber. 84, 219
(1951).
( 2 ) Desnuelle,
I'., Saudet, ll., Bull. soc. china. Frunce 12, 871 (1945). (3) Feigl, F., "Spot Tests," T'ol. 11, p. 255, Elsevier, h e w York, 1954. (4)Johnson, G. R. A , , Scholes, G., Weiss, J.. J . Chem. SOC.1953, 3091. (5) Metzler, D. E., Snell, E. E., J . Am. Chetn. Soc. 74, 979 (195%). ( 6 ) Sinehnm, .\. K., Cheni. /?PI'S. 5 5 , 355 (1955 I .
( 7 ) Schryver, S.B., Proc. Roy. Soc. (Lond o n ) 82B, 226 (1910). (8) Tanenbsum, M., Bricker, C. E., ,%SAL. CHEJI.23, 354-7 (1951 1. (9) 1-on Pechman, H., Ber. 2 5 , 31i5 (1892). (10) Ibid., 27, 3'20 (1894).
RECEIVED for revieiv Xarch 26, 1958. .\ccepted September 23, 1958.
Gravimetric Determination of Zirconium in Titanium J. H. HILL and M. J. MILES Titanium Metals Corp. o f America, Henderson, Nev.
b Mandelic acid is a r a p i d and accurate reagent for determining zirconium in titanium alloys without interference from many metals. Zirconium tetramandelate can b e precipitated quantitatively from either hydrochloric or perchloric acid solutions. Iron, aluminum, vanadium, tin, copper, chromium, cobalt, magnesium, manganese, molybdenum, and nickel do not interfere when present in the amounts usually found in titanium alloys. Hafnium and niobium interfere, causing high results.
I
use of zirconiuni as a constituent of titanium alloys ha? iiwessitated a fast and accurate method of determining zirconium in samples containing a large proportion of tltaniuni. Methods using cupferron, selenious acid, phosphate, and phenylarsonic acid have been used for the determination of zirconium in ores and in steel ( 2 ) . Titanium reacts with each of these reagents and interferes extensively n ith the determination of zirconium by these methods. Kumins ( 7 ) discovered that mandelic acid is aliiiost a specific reagent for t h r quantitative precipitation of zirconium and hafnium. Other 11orkers (3-10) have studied the 'determination of zirconium with mandelic acid and its derivatives. Some of this work has show ii that the zirconium precipitates formed with p-chloromandelic and p-bromoniandelic acids are superior to zirconium tetraniandelate because they h a w higher molecular n rights, do not require a mandelate nash solution, and can be weighed directly. However, mandelic acid was chosen bccausc i t is readily available and somewhat Ion er in cost than the chloro and bromo dcrimtives. The chloro and bromo derivatives are so similar to mandelic acid in the reactions involved that they SCRCASED
252
0
ANALYTICAL CHEMISTRY
can probably be substituted for mandelic acid in the folloning procedure. Kumins ( 7 ) and Hahn (3-5) indicated that titanium and such common alloying constituents as iron, aluminum, vanadium, tin, copper, chromium, cobalt, magnesium, manganese, and nickel do not interfere with the determination of zirconium by precipitation with mandelic acid. Their interference studies were not repeated here, except that vanadium n a s used in larger quantities than used by Kumins to more closely approximate the composition of the common allo! 5 . Kuiiiins found that large quantities of sulfate interfere. causing lon results. Hydrochloric and perchloric acid solutions have been recommended in the literature as the best media for precipitating zirconipm mandelate. Although titanium in a 1 to 1 ratio did not interfere ?I ith the zirconium mandelate method, it did 11hen present in larger amounts. This interference was probably caused by hydrolysis of titanium(1V) during digestion of the zirconium mandelate and by occlusion of titanium in the precipitate. This interference may be overcome by dissolving the initial precipitate in 1 4 ammonium hydroxide, filtering to remove titanium hydroxide, and reprecipitating from acid solution. Because titanium(II1) does not hydrolyze as readily as titaniuni(IV), precipitation from solutions of titanium(II1) is recommended. Also, the color of the trivalent ion serves as a convenient indicator to shorn when the precipitate is adequately TT ashed after the initial filtration. T o overcome the interference from titanium. Suss ( 9 , 10) converted the initial zirconium mandelate precipitate to zirconium hydroxide using sodium hydroxide, dissolved the zirconium hydroxide in hydrochloric acid, filtered the acid solution to remove insoluble impurities, and precipitated the zirconium mandelate. The presence of
+
mandelate ion throughout this purification process requires delicate adjustment. of the hydrochloric acid concentration to dissolve the zirconium hydroxide wit'hout simultaneously reprecipitating zirconium mandelate. Any zirconium Inandelate precipitated upon the addition of hydrochloric acid would remain with the impurities filtered off in the next st'ep, causing 1017 results. Purification n-it'h amnionium hydroxide is preferable because zirconium mandelate dissolves in ammonium hydroside iyithout forming zirconium hydroxide. The Suss met'hod is limited to tit'anium metal and alloys containing from 0.05 t'o 10% zirconiuni and no provision is niadc for overcoming t,he interferences of sulfat'e and fluoride. Titanium ores and residues generally require fusion with potassium pyrosulfate. High zirconium alloys frequently require trentinent with hydrofluoric acid. The method described here provides a means for oi.ercoming interferences from sulfate arid fluoride so that ores, residues, and high-zirconium alloys can be analyzed. METHOD
Reagents. Mandelic Acid, 16% solution. Dissolve 160 grams of mandelic acid in 1 liter of water. Mandelic Acid K a s h Solution. Dissolve 2 grams of mandelic acid in 100 ml. of 1 9 hydrochloric acid. Amnionium Hydroxide Wash Solution. Dilute conceiit'ratecl ammonium 4 11-ith n-ater. hydroxide 1 Titanium Metal, zirconium-free sponge. Zirconium Xetal, U. S. Bureau of Mines, more than 99.5% zirconium. Procedure. Dissolve a 5.000-gram sample in 300 ml. of concentrated hydrochloric acid. Samples containing 507, or more of zirconium may not dissolve completely; in t h a t case, use a mixture of perchloric and hydrofluoric acids and evaporate to fumes of perchloric acid to drive off the hydrofluoric acid. Transfer a n aliquot containing from 0.2 t,o 0.15 grain of zir-
+
+
