Analysis of Mixtures of Sugars and Mixtures of Amino Acids by Dialysis Based on Differential Kinetics SIDNEY SIGGIA, J. G O R D O N HANNA, and NICHOLAS M. SERENCHA Olin Research Center, Olin Mathieson Chemical Corp., New Haven 4, Conn.
b The differential rate approach for the analysis of mixtures using dialysis through a thin-layer film has been applied to the analysis of mixtures of sugars and mixtures of amino acids. The rates of diffusion of the various compounds studied are sufficiently different that standard first-order rate plots show a linear portion for each component in two- and three-component mixtures. Using the graphical extrapolation method, the concentration of each component in the mixture is calculated. of sugars are usually separated by paper or column chromatography and the separated spots or fractions evaluated ( 5 ) . Gas chromatography has also been used following methylation of sugars (1, 7 , 9 ) . Mixtures of fructose and glucose have been resolved on the basis of the reaction of fructose with anthrone at room temperature, while glucose does not react until the temperature is raised to 100' C. ( 2 ) . A differential reaction rate method has been described for the determination of fructose-glucose mixtures in blood and serum (10). This latter method involves the spectrophotometric determination of the blue color developed with ammonium molybIXTURES
-20-
X 4
E
0" -2.2
-
-2.4-
i
26-0
Figure 1. mixture
638
IO0
t,MlnUte6
200
I
300
Dialysis of dextrose-fructose
ANALYTICAL CHEMISTRY
date. Sucrose-fructose and sucroseglucose mixtures can also be analyzed by this method. Mixtures of amino acids are separated by paper and column chromatography (8) and ion exchange chromatography (3, 8, 13, 14). Mixtures of amino acids that yield volatile aldehydes can be oxidized and separated in a gas chromatography unit (15). Gas chromatography has been used also to separate derivatives of amino acids (6, 11, 16). Craig and Pulley (4) have shown that thin-layer film dialysis permits effective separations of a variety of sugars, and that standard first-order kinetic rate plots show straight lines for the dialysis of pure sugars. I n a previous publication (12) demonstrating that chemical kinetic principles can be applied to
Table I.
physical processes, Siggia, Hanna, and Serencha showed that dialysis can be used to analyze two- and three- component systems of a variety of compounds. The method has now been applied to the quantitative analysis of mixtures of sugars and mixtures of amino acids. Thus dialysis, in addition to giving information concerning relative molecular size, makes available a practical method for the gathering of quantitative data for mixtures of these compounds. EXPERIMENTAL
Procedure. T h e procedure is substantially t h a t described previously (12). The total initial concentration of sugars or amino acids was approximately 0.02 mole. Deionized water was
Mixtures of Sugars
2'c %B %A Found Present Found Present Found Present Mixture" ... ... 75.2 74.9 24.8 25.1 1. A. Galactose ... ... 25.3 25.6 74.7 74.4 B. Dextrose ... 75.8 76.1 24.2 23.9 2. A. Galactose ... 27.4 27.9 72.6 72.1 B. Fructose . . . 73.6 74.1 26.4 25.9 3. A. Mannose B. Galactose ... ... 28.1 28.3 71.9 71.7 4. A. Fructose ... ... 74.2 74.0 25.8 26.0 B. Dextrose . . . ... 77.0 78.1 23.0 21.9 5. A. Sucrose ... ... 26.7 22.4 73.3 77.6 B. Lactose . . . . .. 69.0 70.5 31.0 29.5 6. A. Sucrose B. Maltose ... ... 23.1 24.0 76.9 76.0 7. A. Lactose B. Maltose ... ... 26.0 25.0 74.0 75.0 8. A. Mannitol ... ... 76.5 76.0 23.5 25.0 B. Sorbitol 50.2 53.4 3 4 . 5 3 3 . 0 15.3 13.6 9. A. Mannose B. Galactose C. Fructose 51.4 51.8 40.1 34.4 8.5 13.8 10. A. Mannose B. Galactose C. Dextrose 52.3 53.6 24.6 32.0 23.1 14.4 11. A. Mannose B. Fructose C. Dextrose 54.3 56.2 32.1 32.2 13.6 11.6 12. A. Galactose B. Fructose C. Dextrose 53.7 49.5 37.5 36.3 8.8 14.2 13. A. Sucrose B. Lactose C. Maltose a Relative rates of dialysis of components in mixtures are in order A > B > C.
Table
Mixture”
I 0
200
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300
t, M i n L tes
Figure 2. Dialysis nilic acid mixture
of glycine-anthra-
used as solvent in the case of the sugar mixtures. Aliquots of the outside solution were removed, and the amount dialyzed at the different time intervals was determined by drying and weighing the removed samples. I n the case of the amino acid mixtures, a 1 to 1 mixture of deionized water-pyridine was used as solvent, and the aliquots removed were titrated i)otentiometricallv in pyridine, using standard 0.OlN XaOH. A
RESULTS AND CISCUSSION
The mixtures of sugars analyzed contained compounds of the same molecular weight, making it possible to determine the total dialyzed at cach time interval by simply weighing the dried material from the aliquots removed. Tables I and I1 show the results obtained for mixtures of sugars rind amino acids, respectively. Two- and three-component mistures could be analyzed. A plot of the data for the misture of dextrose and fructose is shown in
1. A. B. 2. A. B. 3. A. B. 4. A. B. 5. A. B. 6. A. B. 7. A. B. 8. A. B. 9. A. B. 10. A. B. 11. A. B. 12. A. B. 13. A. B. 14. A. B. 15. A. B. C. 16. A. B. C.
Glycine €-Leucine Anthranilic acid p-Aminobenzoic acid Glycine Anthranilic acid Glycine p-Aminobenzoic acid Aspartic acid p-Aminobenzoic acid Glycine Aspartic acid Aspartic acid Anthranilic acid Glycine 4-Aminobutyric acid 4-Aminobutyric acid p-Aminobenzoic acid 4-Aminobutyric acid Anthranilic acid 4-Aminobutyric acid Aspartic acid 4-Aminobutyric acid -Leucine €-Leucine p-Aminobenzoic acid €-Leucine Anthranilic acid Glycine -Leucine Anthranilic acid Glycine -Leucine p-Aminobenzoic acid
II. Mixtures of Amino Acids %A Found Present
...
...
64.8 40.6 23.1 30.7 10.5
64.4 38.4 23.6 30.1 10.2
35.2 59.4 76.9 69.3 89.5
35.6 61.6 76.4 69.9 89.8
... ... ... ...
... ... ... ...
11.3
12.1
88.7
87.9
...
...
19.9
21.2
80.1
78.8
...
9.0
14.1
91.0
85.9
...
... ...
15.8
16.5
84.2
83.5
...
...
22.5 63.0 24.0
21.8 61.1 21.5
77.5 37.0 76.0
78.2 38.9 78.5
... ...
...
...
...
15.2 21.4 19.8
15.9 18.6 19.9
84.8 78.6 80.2
84.1 81.4 80.1
... ...
... ... ...
22.2
19.6
77.8
80.4
.,.
...
72.5
71.8
27.5
28.2
...
...
20.9 62.0 5.6
22.9 62.9 5.4
79.1 38.0 33.2
77.1 37.1 33.2
...
...
61.2
61.4
4.7
7.8
38.5
35.7
56.8
56.5
Figure 1, and a plot for the mixture of glycine and anthranilic acid is shown in Figure 2. Figure 3 is a plot of the three-component mixture of mannose, fructose, and destrose. The observation of Craig and Pulley (4) that single reducing sugars give a break in the plot of the logarithm of per cent remaining us. time was not confirmed under the conditions used here. KO attempt was made in this study to control the porosity of the dialysis membrane; therefore its selectivity for the different isomers or conformational forms of the same sugar may not be sufficient to produce the break in the line. This was fortunate in this case, because breaks in the straight-line plots for single components would have complicated the interpretation of the plots for mistures.
(1) Bishop, C. T., Cooper, F. P., Can. J . Chem. 38,388 (1960). (2) Bonting, S. L., Arch. Biochem. Biophys. 52, 272 (1954).
...
...
...
Relative rates of dialysis of components in mixtures are in order A > B
LITERATURE CITED
Figure 3. Dialysis of mannose-fructose-dextrose mixture
%B %C Found Present Found Present
...
>C
(3) ~, Buchanan. D. L.. Markiw. R. T.. ANAL.CHEM. 32, 1400 (1960).‘ (4) Craig, L. C., Pulley, A. O., Biochem. 1, 89 (1962). (5) Hough, L., “Methods of Biochemical Analysis,” I. D. Glick, ed., p. 205, Interscience. New York. 1954. (6) Johnson, D.E., Scott,’ S. J., Meister, A., ANAL. CHEM.33, 669 (1961). (7) Kircher, H. W., Ibid., 32, 1103 (1960). (8) . . Lederer, E., Lederer, M., “Chroma,tography,” ’Chap. 30,’ Elsevier, Amsterdam, 1957. (9) McInnes, 8. G., Ball, D. H., Cooper, F. P., Bishop, C. T., J . Chromatog. 1, 556 (1958). (10) Papa, L. J., Mark, H. B., Jr., Reilley, C. X., ASAL. CHEM.34, 1443 (1962). (11) Saroff, H. A., Karmen, A., Anal. Biochem. 1, 344 (1960). (12) Siggia, S., Hanna, J. G., Serencha, E.SI.,ANAL.CHEM.35, 365 (1963). (13) Simmonds, D. H., Rowlands, R. J., Ibid., 32, 259 (1960). (14) Spackman, D. H., Stein, W. H., Moore, S., Zbid., 30, 1185, 1190 (1958). (15) Zlatkis, A., Oro, J. F., Kimball, A. P., Ibid., 32, 162 (1960). (16) Zomzely, C., Marco, G., Emery, E., Ibid., 34, 1414 (1962).
RECEIVED for review October 18, 1963. Accepted December 26, 1963. VOL. 36, NO. 3, MARCH 1964
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