Spectrophotometric Determination of Carboxylic Acids by the Formation of Hydroxamic Acids with Dicyclohexylcarbodiimide Yasuhiko Kasai, Takenori Tanimura, and Zenzo Tamura Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3- 1, Bunkyo-ku, Tokyo, Japan
A new method of spectrophotometric determination of carboxylic acids was developed. The unique feature of the method is the formation of hydroxamate from carboxylic acid and hydroxylamine by the aid of dlcyclohexylcarbodiimide (DCC) in a single step. Hydroxamate was made by adding DCC to the mixture of carboxylic acid and hydroxylamine in anhydrous or aqueous ethanol at 50 or 60 OC. Ferric hydroxamate was made with large excess of ferric perchlorate in ethanol solution of perchloric acid and was determined at 525 nm. The working curves obtained were linear between 0.25 and 2.5 micromoles of benzoic, caprylic, lactic, and p-nitrophenylacetic acid.
Feigl e t al. ( I ) first utilized the formation of ferric hydroxamate for detection of carboxylic acids. Later Hill and others ( 2 - 5 ) used the same reaction for the determination. Although hydroxamic acid could be formed from esters, anhydrides, and acid chlorides by a direct reaction with hydroxylamine, the formation of hydroxamate is not facile from free carboxylic acid under the same condition. Consequently, the conversion of carboxylic acids into their esters or chlorides to make an activated acyl group is necessary for the formation of hydroxamate. Since an anhydrous condition is required for both esterification and acid chloride formation, the application of this method to an aqueous specimen is cumbersome. One of the methods to make an activated carboxyl group is the application of DCC which was used by Sheehan and Hess (6) for peptide bond formation. Pesez et al. (7, 8 ) reported the application of DCC for the determination of aliphatic carboxylic acids through the formation of their methyl esters; however, the sensitivity was not satisfactory. Franzblau et a / . (9) found that hydroxamic acid could be formed from free carboxylic acid and hydroxylamine hydrochloride by using water-soluble carbodiimide in aqueous medium; however, the yield was not sufficient. Hoare et al. ( I O ) found that once formed hydroxamic acid subsequently reacted with excess carbodiimide following the sequence of Lossen rearrangement. Thus, the conditions have not been successfully established for the determination of free carboxylic acids. In our previous paper ( I I ) , the specific detection of several micrograms of carboxylic acids in solution or on paperor thin-layer chromatograms was carried out simply by the direct formation of hydroxamic acid from free carboxylic acid, hydroxylamine hydrochloride, and DCC. In this paper, the reaction conditions were investigated to provide the specific and reproducible method for the determination of free carboxylic acid in both ethanolic and aqueous solution.
EXPERIMENTAL Instruments. A Hitachi Recording Spectrophotometer Model EPS 3-T was used for spectrophotometric measurement and a Hitachi Spectrophotometer Model 101 was used for determination. Materials. Hydroxylamine perchlorate was prepared from hydroxylamine sulfate and barium perchlorate by the procedure of 34
Robson ( 1 2 ) . Benzohydroxamic acid was prepared by the procedure of Blatt ( 1 3 ) ;mp 125-128 "C. Caprylohydroxamic acid was given by K. Kobashi of the University of Toyama. p - Nitrophenylacetohydroxamic acid was prepared following the procedure of Wise et ~ l (14) . from ethyl p-nitrophenylacetate and hydroxylamine hydrochloride in alkaline medium. Yield 68%;mp 143.5-144 "C. Anal. Calcd for C~H8N204:C, 48.98; H, 4.11; N. 14.29. Found: C, 48.98; H, 4.23; N, 14.51. The other reagents were obtained from Kanto Chemical Co. Ltd., Tokyo. Reagent Solutions. 0.275M Hydroxylamine perchlorate solution. Hygroscopic crystals of hydroxylamine perchlorate (assay 95.2%) were weighed in a graduated cylinder with a glass stopper and a calculated amount of anhydrous ethanol was added. The turbidity was removed by filtration with a small amount of active charcoal. 0.092M Hydroxylamine perchlorate solution. This solution was prepared by diluting 0.275M hydroxylamine perchlorate solution threefold with anhydrous ethanol. Hydroxylamine perchlorate solutions are stable for at least one month at room temperature. 0.5MDCC solution. DCC was dissolved in anhydrous ethanol of calculated volume. 2.0-, 1.0-,and 0.5M Perchloric acid. Commercially available 70% perchloric acid was diluted to the designated concentration with anhydrous ethanol. The accurate content of commercial 70% perchloric acid was determined by titrating with 0.LV sodium hydroxide using phenolphthalein. 0.01M Fe(II1) solution. Ferric perchlorate, Fe(C104):;. 6H20, was dissolved in 2.OM perchloric acid. 0.02M Fe(II1) solution. Ferric perchlorate was dissolved in 0.5M perchloric acid. Procedure I ifor determination of the ethanolic samole). Take 1.0 ml of ethanolic solution which contains 0.25 to 2.5 micromoles of carboxylic acid and add 1.0 ml of 0.275M hydroxylamine perchlorate solution, then add 0.5 ml of 0.5M DCC solution. The reaction mixture is incubated for 30 minutes at 60 "C and, after cooling to room temperature, 1.0 ml of 0.01M Fe(II1) solution is added to form ferric hydroxamate. The total volume is adjusted to 5.0 ml with 1.OM perchloric acid, and absorbance is measured at 525 nm cs. the blank solution. Procedure I1 (for determination of the aqueous sample). Take 0.5 ml of the aqueous solution which contains 0.25 to 2.5 micromoles of carboxylic acid and add 3.0 ml of 0.092M hydroxylamine perchlorate solution. A 0.5 ml of 0.5M DCC solution is added and the mixture is incubated for 30 minutes at 50 "C. After cooling to room temperature, 1.0 ml of 0.02M Fe(II1) solution is added to adjust the total volume to 5.0 ml. Let stand for 5 minutes t o stabilize the absorption of blank solution. The absorbance is measured at 525 nm.
RESULTS AND DISCUSSION Method for Determination of Hydroxamate. It has been reported by several authors (2-5, 1.5) that the coloration of hydroxamate with ferric ion varies depending on acidity of the medium, composition of the solvent, molar ratio of ferric ion to hydroxamate, and the nature of the substituent comprising the hydroxamic acid. To determine the optimum reaction conditions of hydroxamate formation, the reliable method for the determination of hydroxamate was examined. The formation of ferric hydroxamate was reduced with increasing acidity in aqueous solution and the reduction was observed even in 0.01M perchloric acid (Figure 1). The significant effect of acidity on the absorbance in aqueous solution, however, was compensated by adding ethanol and
A N A L Y T I C A L C H E M I S T R Y , VOL. 47, NO. 1, J A N U A R Y 1975
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