ete rmination lorobis(ethylen SATORU EGASHIRA Department o f Chemistry, Faculty of Science, Kyoto University, Kyoto, Jopan
Ib A new method for the spectrophotometric determination of highly functional organic acids in the presence of monobasic acids is based on the facts that chromium(ll1) hat a strong affinity for carboxylate ion and complexes of monobasic acids are extractable in organic solvents. cis-Dichlorobis (ethylenediamine)chromium(ll1)chloride was used as the reagent. The method was applied to the determination of acids in the ion exchange ehromatographic effluent of organic acids.
very small amounts of substances exist in a mixture, and it is difficult to separate them from each other by classical means, chromatography is generally used. Liquid chromatograpy is often used when a quantitative separation is desired. In liquid chromatography, the amount of substances in each fraction of the effluent must be determined. This determination is usually made by a simple, Eghly sensitive colorimetric method. However, because of the low reactivity of the carboxyl group of organic acids, no sensitive reagent for colorimetry haa yet been found. Therefore, organic acids in the liquid chromatographic effluent have been determined chiefly by titrimetric methods (1, 6). However, these methods are difbcult to uBe when the acids exist in the form of salts, and when buffer solutions are used in elution. The author devised new spectrcphotometric method to overcome these difEculties. It has long been known that chromium(II1) has a strong affinity for anionic oxygen, such as that of the carboxyl group, and forms a stable complex with it. This phenomenon, known aa a masking effect in inorganic analysis, constitutes an interference in the detection or determination of chromium in the presence of certain organic substances. The present method is based on this phenomenon. When organic acids are treated with q u o or halogented complexes of tervalent chromium such as HEN
I) e ANALYTICAL CHEMISTRY
chromium(I1I) chloride, stable complexes are formed which are resistant to hydrolysis during such rather drastic treatments as extraction, acidification, or precipitation. Therefore, various methods can be safely applied to separate from such chromium complexes the complexes which are formed by the treatment of samples containing organic acids. cis-Dichlorobis ( e t h y l e n e d i a m i n e ) chromium (111) chloride [cis-(CrCl$n& CIHzO] was chosen as the reagent, because it gives a high color and its preparation is relatively easy. When this reagent was mixed with carboxylic acids in water and heated at loBo C., complexes were formed. It was presumed that chlorine atoms in the coordination sphere of the complex cation had been replaced by carboxylate anion in this reaction. After the completion of the reaction, the reaction mixture wm treated with ammonium thiocyanate to convert the excess reagent into a thiocyanato complex, which was extracted with an organic solvent. Chromium in the aqueous layer was determined spectrophotometrically with disodium (ethylenedinitrilo)tetraacetate (EDTA) according to the method of Cellini and Valiente @) It waa found that 1 to 10 Mmoles of organic acids could be determined with an error of &5% by this method. This method was applicable when the acids to be determined were in the form of free acid or salt. It w ~ galso applicable to the determination of organic acids in the chromatographic effluents eluted with buffer solutions that do not react with the reagents.
.
EXPERIMENTAL
A Beckman Model DU quarta spectrophotometer was used. Reagents. 3% cis-dichlorobis(ethvlenediamine) chromium(II1) . , chloh d e "solution. ' 0.5M ethylenediamine-hydrobromic acid buffer solution of pH 7.5. 20y0ammonium thiocyanate solution. 1-Butanol-ethyl acetate mixture (2: 1, v. to Ye)* O.6M tartaric acid solution.
1.5% disodium ethylenediamine tetraacetate [disodium (ethylenedinitri1o)tetraacetateJ (EDTA) solution. cis-Dichlorobis ethyl en e d i a m i n e 1 chromium(II1) c oride was by the hydrolysis of tris(et ylenediamine)chromium(III) chloride [(Crena)C&-3.SH20 ] by concentrated hydrochloric acid, T r i s (ethylenediamine)chromiq(III) Chloride. Pfeiffer's method (4) was partially modified. One hundred grams of finely powdered anhydrous chromium alum was prepared by heating cr talline chromium alum at 120' This was mixed well with 30 grams of diatomaceous earth, and the mixture placed in a 500-m1. roundbottomed flask equipped m t h a reflux condenser. Ninety milliliters of ethylenediamine monohydrate was added and spread over the contents offlask by shaking. The flask was heated for 6 hours on a boiling water bath. Sometimes the ethylenediamine boiled vigorously in the early part of the reaction, so it was necessary to use L highly efficient reflux condenser. After the flask had been cooled, the reaction mass was dissolved in 350 ml. of warm water (about 50' C.). After the solution had been cooIed, the insoluble residue was atered off, and 100 grams of ammonium chloride was added to the filtrate. Tris(ethy1enediamine)chromium(III) chlonde separated as ellow crystals. These crystals were colicted by filtration and washed with saturated ammonium chloride solution, a small amount of water, and last with 95%*ethanol. The yield was 75 gTams (50% of theoretical,). Dichlorobis(ethy1ene d ia m 1 ne) c h r 0 mium(II1) Chloride (8). Twenty-five grams of tris(ethy1enediamine)chromium(I1I) chloride and 28 mi. of concentrated hydrochloric acid were mixed in a 500-ml. flask equipped with a reflux condenser and heated on B boiling water bath for 30 to 40 minutes until the reaction mixture became reddish purple. Then the flask was cooled in cold water, and 50 ml. of95% ethanol was added. After standing at room temperature for 24 hours, the crystals were collected on a glass filter, and the cryatals of ethylenediamine dihydrochloride were removed by washing with 50% ethanol. The yield was 12 grams (65% of theoretical). Ten grams of crude salt waa dissolved in 30 ml. of hot concentrated hydro-
L
XPrepared
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-
chloric acid and the solution was filtered with a glass filter. Thirty milliliters of water was added to the a t r a t e and cooled in an ice bath. The crystals were collected on a lass filter and washed with 6N h droch?oric acid, 50% ethanol, and 9 5 6 ethanol, successively. Procedure. Five-tenths milliliter of 3% cis-dichlorobis(ethy1enediamine)chromium(III) chloride solution and 0.5 ml. of the buffer solution were added to 1 ml. of the solution containing 1 to 10 pmoles of organic acid and heated for 5 minutes on a boilkg water bath. Then 0.5 ml. of 20% ammonium thiocyanate solution waa added, and again heated for 5 minutes. After rapid cooling in a cold water bath, the vessel was allowed to stand a t room temperature for more than 15 minutes. Then the solution waa extracted twice with 3 mi. of 1butanol-ethyl acetate mixture. Sometimes a precipitate formed during thiocyanate treatment; however, this precipitate was soluble in an extracting solvent. To the residual aqueous solution, 0.5 ml. of 0.6M tartaric acid and 1 ml. of 1.5% EDTA solution were added, and the mixture was heated for 10 minutes on a boiling water bath. After the solution was cooled, i t was diluted to 10 ml. with water and the absorbance of the sample us. a reagent blank was determined spectrophotometrical1 a t a wave length of 543 mp. A d e n t i c samples containing various amounts of each acid were treated by the above method and the calibration curve was prepared.
Table I. Color Yield of Organic Acids
Absorbance er pmole Acix O.oo00 0.0212
Acid Acetio Aspartic Benaoio Carbonic Citrio, Formic karic Glutamic Lactic Glycine
0.0000
O.Oo46 0 0668 I
o.oO0o 0.0123 0.0219 0.0076
0,0032
P
d
,3
c .2
.I
I
I
1
6.0
7.0
8.a
1 9.0
10.0
Pk RESULTS AND DISCUSSION
Color yield of organic acids is, as shown in Table I, largely dependent on the chemical structure of the acids. Unsubstituted monobasic acids and salicyclic acid gave no color. Reaction of cis-Dichlorobis (ethyl-
Absorbance er pmole Acii
Acid Maleic Malic Malonic Oxalic 2-Oxoglutaric Pyruvic Salicylic Succinic Tartaric
Figure 1. Effect of pH on reaction of cis dichlorobis(ethy1enediamine)chromium(lll) chloride with organic acids
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0 Tartaric acid X Oxalic acid
0 Succinic acid --Blank
0.0144
0.0375
0.0212 0.0300
0.0241
0.0056 o.oO0o 0.0192 0.0614
formed between organic acids and cis-dichlorobis(ethy1enediamine)c hromium(II1) chloride so resembles that of the reagent itself that it is very difficult to determine these complexes directly by spectrophotometry. Therefore, when this reaction is applied to the determination of organic acids, the excess reagent must be removed before the amount of the complex formed is determined. Thiocyanate complexes of chromium (111) were generally extractable with organic solvents such as 1-butanol, 2-butanol, or ethyl acetate, in the presence of free thiocyanate. cisDichlorobis (ethylenediamine)chromium (111) ion and its aquo iom were readily converted into cis-diisothiocyanatobis (ethylenediamine) chromium (111) ion, { cis-[Cr(NCS)zena]+), by treatment with ammonium thiocyanate. In the present method, the excess reagent was removed from the reaction mixture by extraction with organic solvent after treatment with excess ammonium thiocyanate. When the concentration of ammonium thiocyanate was too low in this treatment or when the time of heating and of standing a t room tem-
enediam,ine)chromium(III) Chloride with Acids. As shown in Figure 1, this reaction was affected by the p H of the reaction mixture. Tartaric acid gave an optimal color yield, when the p H of the reaction mixture was 7.5. At this pH, the longer the time of heating, the higher the color yield. However, when the time of heating was longer than 5 minutes, a precipitate insoluble in extracting solvent was formed, and blank values became too high and unsteady. The pH for optimal color yield for the other acids was a little different from 7.5, but this pH was taken in all cases for the sake of convenience. Halogens, perchlorate, and bases did not affect this reaction, so they could be used in chromatographic elution. Sulfuric and phosphoric acids aa well aa highly functional organic acids, react with the reagent, so that they must not be present. Removal of Excess Reagent. The absorption spectrum of the complexes
IO
Figure 2. bromide
20
30
40
SO
60
70
80
90
JOO
Fraction Rumber Ion exchange chromatography of organic acids on cblvmn of Dowex 1 1. Pyruvic II. Maleic 111. Succinic, malls, and tartaric IV. 2-Oxoglutaric and oxalic V. Fumarie
VOL 33, NO. 13, DECEMBER 1961
1929
perature was too short, the blank value became higher, but the color yield was not so much influenced. Too high a concentration of ammonium thiocyanate caused a slight loss of the complexes of the acids to be determined. More polar soltents were more effective for extraction of thiocyanato complexes. However, too high polarity of solvent increased the loss of the arid complexes. When the ratio of 1-butanol to ethyl acetate was more than 2 to 1, the blank value decreased markedly, but the color yield was very low and fluctuated. Complexes of monobasic acids, especially those of fatty acids, were readily extractable by the abovementioned solvents. Therefore, these acids were not determined by this method. On the other hand, polybasic acids or other highly functional organic acids could be determined in the presence of monobasic acids. Color Development with EDTA. Cdlini and Valiente (2) reported that the color developed by the reaction of chromium with EDTA was constant a t pH 1.5 to 4.0. The author, however, found that in the reaction of chromiumorganic acid complex with EDTA, the color yield was markedly dependent on pH, and was optimum at p H 2.8.
I
Color Yield and Structure of Acids. Table I shows the absorbance per 10 pmoles of organic acid8, when the final volume was 10 ml. When an acid reacts in the molar ratio of 1 t o 1 with cis-dichlorobis(ethy1enediamine) c h r omium(111) ion, the absorbance per 1 pmole of the acid should be 0.0193. Most of the dibasic acids gave values very near to this, while the value for hydroxy acids was always abnormally high-for example, the value for tartaric acid was three times as high. Most of the monobasic acids gave no or very weak color in this method. However, spectrographic studies showed that reaction between these acids and the reagent was quantitative. In the case of lactic acid, for example, a molar reaction ratio of 1 to 1 was found by spectrophotometric experiments. The low color yield obtained in the rase of these acids may, therefore, be attributed to the high extractability of complexes of acid into an organic solvcnt used in the extraction. Application to Chromatography. Figure 2 shows a liquid chromatogram of organic acids. A sample solution containing pyruvate, oxalate, succinate, malate, tartrate, 2-oxoglutarate, maleate, and fumarate was applied
. -
tometric Determination ot
to the top of a 1 X 25 em. column of anion exchange resin. Dowex 1 X 8 bromide (200- to 40O-mesh), was eluted with piperazine-hydrobromic acid buffer solution (pH 5.0, bromide, ion concentration 0.05M) at a rate of 1 ml. per minute. The volume of each fraction was 5 ml. The acid content in each fraction was determined by the spectrophotometric method mentioned above. ACKNOWLEDGMENT
The author thanks Ryutaro Tsuchida of Osaka University and Shoao Tanaka of Kyoto Cniversity for thcir continuous guidance and encouragement throughout this work. LITERATURE CITED
(1) Bush, H., Hurlbert, R., van Potter, R. J. B i d . Chem. 196, 717 (1952). (2) kellini, R. F., Valiente, E. .4., Anales real 80:. fis. y qu?ni. (biadrid) 51B
,e%@.
RECEIVED for review January 30, 1061.
Accepted July 25, 1981.
.
JBMANN KORKISCH and G. E. JANAUER Analyfical Institute, University o f Vienna, I X . Wuhringerstrasse 38, Vienna, Austria
b A sensitive and accurate method is described for the spectrophotometric determination of microgram amounts of thorium using the azo dye Solochromate Fast Red. This dyestuff reacts with thorium in hydrochloric acid-methanol solutions to form an orange complex which shows maximum absorption at 490 mp. Beer's law is valid over a range from 0.1 to 20 pg. of thorium per mi. of measuring solution. The molar absorptivity of the thorium-dyestuff complex is 13,970. Only a very small number of foreign ions interfere; hence, this method can b e expected to find general application, as in the determination of thorium in minerals and rocks.
research work on the application of azo dyes for the spectrophotometric determination of thorium and uranium in mixed solvents has REVIOUS
1930
ANALYTICAL CHEMISTRY
proved a number of azo dyes of the Solochrome class to be suitable for the determination of uranium(1V) and (VI) (5, 6). Solochromate Fast Red is also a very sensitive and rather specific reagent for thorium. Because of its low solubility in water, a series of organic solvents was investigated for use in the spectrophotometric determination of thorium. The best results were obtained in a methanolic medium in which the solubility of inorganic salts is also sufficiently high. All the interfering ions can be separated from thorium by anion exchange methods (4, '7) developed in this laboratory, which ensure quantitative removal of practically all elements. In many cases separation will not be necessary, as the common interferences of small amounts of iron(I11) and copper(I1) are eliminated by addition of ascorbic acid, which does not interfere under the conditions applied here (Table 11).
The determination of thorium by Solochromate Fast Red is as seneitivc! and accurate as the extensively used Thorono1 method (@, yet Y, La, Pr, and Nd do not interfere, and this reagent is much less expensive than Thoronol. EXPERIMENTAL
Reagents. STANDARDTHORLUM SOLUTIONS. Thorium nitrate was transformed t o the chloride by repeated evaporation with 6N hydrochloric acid. The thorium chloride was dissolved in 1N hydrochloric acid. This solution, containing 3. mg. of thorium per ml., was used as a stack solution and standardized spectrophotometrically ( 2 ) . By dilution with 1N hydrochloric acid, standard solutions of lower thorium concentrations were prepared. DYESTUFF SOLUTIONS. Solochromate Fast Red 3 G 200 (C.T. Nordant Red 19) (250 mg.) of the formula 6-amino-4chloro-1-phenol-2-sulfonic acid -+ 3methyl-1-phenyl-5-pyrazolone
