solution of -4piezon T in a volatile solvent (isopentane) mas prepared and forced through the column under slight pressure. At the breakthrough point, the process was stopped and the column was quickly attached to a vacuum system, as shown in Figure 1. A slight vacuum (740-mm. pressure) was obtained with an aspirator or a welltrapped vacuum pump. The vacuum was applied alternately to each end of the column starting with short times (1 second) and working up to longer times (about 1 minute) in about five steps. The pressure was then decreased to about 700 mm. and the above process repeated. The pressure was reduced at each step by about 30 mm. until a pressure of approximately 100 mm. was reached; then the column was left pumping for about 10 minutes. While the exact concentration of Apiezon T on the glass beads and walls of the metal tube is unknown, a highly
satisfactory performance of the column probably indicates a uniform deposition of the substrate in the column. The use of alternating flux of solvent and substrate inside the tube precludes preferential concentration of substrate in one part of the column. The performance of two columns prepared by the technique described was essentially identical. This was in sharp contrast to previous experience with coated glass-bead substrate preparation using conventional procedures. To test the column, a mixture of saturated hydrocarbons (C, to Clo) was separated on the column under programmed temperature conditions, using a Wilkens Model 600 Hy-Fi gas chromatograph with flame ionization detection. Eleven symmetrical peaks, evenly spaced in the Cg to Cla range, were obtained in less than 60 minutes for a mixture that contained 11 hydro-
carbons. The chromatogram is shown in Figure 2. LITERATURE CITED
(1) Frederick, D. H.,Cooke, W. D.,
Michigan State University preprints, ISA Proceedings, p. 21, 1961 Internatjonal Gas Chromatography Symposium. (2) Frederick, D. H.,Miranda, B. T., Cooke, W. D., ANAL. CHEM.34, 1521 (1962). (3) Hishta, C., Messerly, J. P., Reschke, R. F., Fredericks, D. H., Cooke, W. D., Zbid.,32, 880 (1960). (4)Lijinsky, W., Domsky, I., Mason, G., Ramahi, H. Y., Safavi, T., Zbid., 35, 952 (1963). IHOR LYSYJ PETERR. NEWTON
Rocketdyne Division of Yorth American Aviation, Inc. Canoga Park, Calif.
WORKsupported by the U.S. Department of Interior, Office of Saline Water, Contract 14-01-0001-332.
Factors Affecting the Determination of Hydroxyproline SIR: Using a modification of the Neuman and Logan (6) procedure for the determination of hydroxyproline, we have investigated the rate of color development and the stability of the color. By a selection of factors, a combination is suggested which produces a color that is very stable for at least 4 hours. The influence of various factors has been investigated by variation of the basic method. Modifications previously published have been incorporated in this method. They include the use of a higher concentration of copper sulfate ( I ) , the use of 40' C. for oxidation and subsequent destruction of peroxide (4, and the use of a higher concentration of propanol ( 3 ) . EXPERIMENTAL
Procedure. Two milliliters of hydroxyproline standard (5 to 50 gg. per ml.) or 2 ml. of distilled mater are placed in 25-ml. volumetric flasks. The following reagents are added: 1 ml. of 0.05.11 copper sulfate, 1.5 rnl. of 2X sodium hydroxide [recommended procedure (a)] and 1 ml. of 6% hydrogen peroxide. The contents are mixed gently. The flasks are held firmly in racks fitted with spring clips. Oxidation is carried out in a water bath at 40' C. for 10 minutes, and the peroxide is then destroyed by vigorous shaking. The flasks are cooled in ice and water and 2 ml. of 6.5N sulfuric acid and 5 ml. of 3% p-dimethylaniinobenzaldehyde are added. The color is developed in a water bath at 70' C. for 50 minutes [recommended procedure (a)]. The flasks are again 950
ANALYTICAL CHEMISTRY
cooled, then rewarmed to 20' C. Irecommended procedure (b) 3. Absorbances have been read in 1-cm. cuvettes at 560 mp in a Unicam SP500 spectrophotometer. Recommended Procedure. I n view of the results obtained two alterations to the basic method are recommended :
Table I. Rate and Intensity of Color Development Obtained by Variations of Basic Method, Using 30 pg./ml. of Hydroxyproline
Maximum Time, Variation absorbance minutes Temperature of color development 60O
70" 80 407, (v./v.) Prop-
anol Residual sulfuric acid 0.4N 0.8N 1.2hT
0.748 0.791 0.810
105 50 28
0.670 0.831 0.865
150 50 28
0.700 0.798 0.832
70 27 16
0.774 0.864 0.890 0.872 0.806 0.627
32 35 42 50 70 90
20% (v./v.) Prop-
Normality of added sodium hydroxide 0.5N 1.ON
l.5N 2.ON 3.ON 4.0N
(a) Changing the normality of sodium hydroxide t o 1.5N and therefore changing the heating time a t 70" C. t o 42 minutes (Table I). (b) Dilution to 25 ml. with 40% propanol immediately on cooling the solution to stabilize the color. Variation of the concentration of the sulfuric acid was predetermined so that after neutralization by sodium hydroside the residual normalities in the reaction mixture during color development were 0.4, 0.8, and 1.2A7. The residual sulfuric acid concentration of the basic method is 0.8N. RESULTS AND DISCUSSION
Rate of Color Development and Destruction. Color development and destruction are accelerated by increasing the temperature (Figure 1, Table I), by decreasing the concentration of propanol, and by lowering the pH (Table I). Stability of the Color at 20' C. On cooling t o 20" C. a secondary color rise occurs before the color eventually fades. By dilution with various combinations of reagents i t has been found that the secondary color rise is greatest and most prolonged when the concentrations of p-dimethylaminobenzaldehyde, propanol, and acid are all high (Figure 2, A and B). High acid in combination with low p-dimethylaminobenzaldehyde and propanol concentrations promotes early color fading (Figure 2E). Stabilization of the color for at least 4 hours can be achieved by a 1 in 2
0.800
-
0.540
0.600
-
0.500
0.400-
0,460
O.*OO
tt
L
0
Figure 1. ment
11
I
3
2 TIME (HOURS)
0.420
L
0.360
4
0
I TIME
2 (HOURS)
4
3
The effect of temperature on color developFigure 2. The effect of varying concentrations of acid, propanol and p-dimethylaminobenzaldehyde on color siability at 20" C.
Concentrationof hydroxyproline 30 pg/ml.
dilution with 40% propanol (Figure 20). Water is less effective (Figure 2C). The use of volumetric flasks instead of test tubes for the color development enablw rapid dilution to stabilize the color. Secondary Color Rise at 20" C. Bergman and Loxley (2) observed a change with time in the maximum absorbance peaks; so we investigated the secondary color rise using a Beckman DK2 recording spectrophotometer. A change in the maximum absorbance from 555 to 560 mp was observed, but the rise was mainly due to a n increase in absorbance.
A.
E. C.
D. €.
15 pg./ml. of hydroxyproline; color undiluted
A 30
pg./ml. of hydroxyproline; color diluted 1 in 2 with 3% (w./v.) p-dimethylaminobenzaldehyde and 0.8N sulfuric acid in 40% (v./v.) propanol 3 0 pg./ml. hydroxyproline; color diluted 1 in 2 with 40% (v./v.) propanol A 30 pg./ml. hydroyproline; color diluted 1 in 2 with distilled water 0 30 pg/ml. hydroxyproline; color diluted 1 in 2 with 0.8N sulfuric acid
There is a continuing small development of the color at 20" C. which is offset at 70" C. by the rapid color destruction. Development of the color a t 20" C. may be demonstrated by heating the oxidation product with the acid at 70" C., then cooling before adding the pdimethylaminobenzaldehyde a t 20" C.
The color in this case takes 2 hours to reach its maximum. When color development a t 20" C. was applied to standard hydroxyproline solutions, using an optimum heating time of 16 minutes with acid, the absorbances, read at 560 mr, were higher than with color development at 70" C. (Figure 3). However, when the
Table II.
Effect of Hydrolysis of Gelatin by Hydrochloric Acid and Sulfuric Acid on Calculated Per Cent of Hydroxyproline (5 ml. of acid used) Gelatin, mg. 2 5 10 20 50 100 200 Dilution factor 10 25 50 100 25 500 1000 Normality of salt 3.0 1.2 0.6 0.06 0.03 0.3 0.12 Hydroxyproline, 8 . 8 5 10.60 11.65 12.70 13.60 13.75 13.75 % {E%h 9.90" 12.50 13.65 13.88 13.96 13.75 14.25
Separation into alcoholic and aqueous layers occurred. ~
Table 111.
IO
30
50
HYDROXY PROLINE
(P (3 /mi) Figure 3. curves
0
Standard hydroxyproline Color developed at 70' C. Color developed a t 20' C.
Effect on Absorbance of Addition of Sodium Chloride and Sulfate to Hydroxyproline Standard (30 pg./ml.) Normality of salt in hydroxyproline standard Salt 0 0.25 0.5 1.0 1.5 2.0 2.5 Absorbance, microns Sodium chloride 0.431 0.407 0.383 0.360 0.341 0.332 0.321 0.435 0.405 0.383 0.358 0.339 0.330 0.316 Sodium sulfate 0.431 0.433 0.432 0.430 0 . 4 11 0.399 0.357" 0.435 0.434 0.435 0.423 0.408 0.397 0.353' a
Sodium
3.0 0,309 0.306 0.308O 0.304"
Separation into alcoholic and aqueous layers occurred. ~
VOL 36, NO. 4, APRIL 1964
~~
951
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. I n 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. I n 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).
