method was applied to gelatin hydrolysates, values of only about 7% for hydroxyproline content were obtained compared with values of 14% using color development at 70" C. This emphasizes the importance of testing the method on gelatin hydrolysates as well as on hydroxyproline standard solutions. wrrole-2-carboxylic Acid. The spectrum a t 244 to 300 mp was observed after adding sulfuric acid to the oxidation product. il compound having a spectrum identical with pyrrole-2-carboxylic acid (PCA) formed slowly, reaching a maximum after 4 hours a t 20' C. Radhakrishnan and Meister (6) reported rapid formation of PCA during nonenzymic oxidation of hydroxy proline with strong acid. Bergman and Loxley ( 2 ) indicated a lower rate of color development with PCA than with their hydroxyproline oxidation product. In our own method the red chromogen
which developed after 1 hour's contact with acid had a 7% lower absorbance. These results suggest that the oxidation product is not PCA, but is convertible to it. PCA formation should be avoided by minimal delay in the procedure after adding the acid. Concentration of Sodium Chloride and Sodium Sulfate. The effect of sodium chloride and sodium sulfate was investigated by adding these salts to a standard hydroxyproline solution and also by hydrolyzing gelatin (2 t o 200 mg.) in 5 ml. of hydrochloric and sulfuric acids and then neutralizing (Table 11). The results (Table 111) show that sodium chloride has a much greater depressing effect on the absorbance than sodium sulfate. ACKNOWLEDGMENT
We acknowledge the advice and assistance of E. G. Cleary of the Depart-
ment of Physiology, University of Sydney, of Philip Stewart of Messrs. Davis Gelatine (Aust) Pty. Ltd., and of R. N. Beale of the Institute of Clinical Pathology, Lidcombe State Hospital. LITERATURE CITED
(1) Baker, L. C., Lampitt, L. H., Brown, K. P., J. Sci. Food Agric. 4, 165 (1953). (2) Bergman, I., Loxley, R., ANAL.CHEM. 35, 1961 (1963). (3) Hutterer, F., Singer, E. J., Zbid., 32, 556 (1960). (4) Leach, A . A., Biochem. J . 74, 70 (1960). (5) Neuman, R. E., Logan, M. A., J. Biol. Chem. 184, 299 (1950). (6) Radhakrishnan, A . N., Meister, A,, Zbzd., 226,559 (1957).
JEANETTE BLOMFIELD J. F. FARRAR
Children's Medical Research Foundation Royal Alexandra Hospital for Children Sydney, New South Wales, Australia
Improved Calomel Reference Electrode for Nonaqueous Titration of Halogen Acid Salts of Organic Bases James R. Deily and Leon Donn, Jefferson Chemical Co., Inc., Austin, Texas
whereby halogen acid of organic bases could be titrated with perchloric acid in a glacial acetic acid medium has been described by Pifer and Wollish (1). Their procedure involved the addition of mercuric acetate to tie up the halide as the undissociated mercuric halide thus freeing the organic base for titration. In their work they found that when the titration was followed potentiometrically with a glass and calomel electrode system, the calomel electrode was easily contaminated by the mercuric acetate, but could be rejuvenated by flushing it out with distilled water and refilling with fresh saturated potassium chloride. We found as they did that the fibertype calomel electrode was easily contaminated but we were not always successful in rejuvenating it as they described. In our experience the mercuric acetate interfered by giving erratic and generally high results. It was surmised that the erratic results were due to a great increase in the liquid junction resistance when the potassium chloride was replaced by mercuric chloride in the fiber wick of the electrode. The high results were attributed to the reaction between the mercuric acetate and potassium chloride to produce titratable potassium acetate. The electrode shown in Figure 1 was designed to avoid these difficulties. It was prepared from a Coleman No. 3-521 PROCEDURE
A salts
952
ANALYTICAL CHEMISTRY
r
-To
I+
PH. M a t e r
Soturotad
KCI Solution
Figure 1. Improved calomel reference electrode
calomel reference electrode which employs a plunger type potassium chloride reservoir. The reservoir, which may be unscrewed from the body of the electrode, was modified by removing the plunger and sealing a coarse-porosity
fritted glass disk into its lower end. A hot 3y0 agar solution, 1N in potassium nitrate, was poured into the modified reservoir until the constricted portion was nearly filled. While the solution was still hot, gentle air pressure was applied to the top of the reservoir to force the agar solution into the voids of the disk. Any of the agar which passed through the disk was wiped off with absorbent paper or a clean cloth. After the agar solution solidified, saturated potassium chloride was added, and the electrode was assembled. When not in use, the tip of the electrode was immersed in distilled water to avoid dehydration of the agar bridge. This improved calomel reference electrode has been successfully used in conjunction with a Beckman No. 41263 glass indicating electrode and an automatic recording potentiometric titrator to follow the titration of hydrochlorides of piperazine and of other organic bases in glacial acetic acid with 2-naphthalenesulfonic acid in the presence of mercuric acetate. The electrode has been found equally suitable for ordinary nonaqueous titrations of piperazine citrate, for example, where mercuric acetate was not required. LITERATURE CITED
(1) Pifer, C. W., Wollish, E. G., ANAL. CHEM.24, 300-6 (1952).
