V O L U M E 23, NO. 3, M A R C H 1 9 5 1 the organic phme of the twc-phase system of Lugg and Overell (2)contained insufficient formic acid to suppress the ionization of the organic acids and resulted in tailing and poor resolution. the formation of a Addition of enough ethyl to aingle-phase system produced the results shown in Table I. ACKNOWLEDGMENT
The author expresses his appreciation for the help given by Margaret L. Buch in the cvperimental development of the Chromatopack technique.
413 LITERATURE CITED
(1) Consden, R., Gordon, A. H., and Martin. A. J. P., B i o c h . J . , 38, 224 (1944). (2) LUgg, J. w. H., and Overell, €3. T., Australian J . SCi. Research, Series A,1, 98 (1948). (3) Mitchell, H. K.,and Haskins, F. A , , Science, 110,278 (1949). (4) Hitohell, H. K.,Gordon, H.. and Haskins, F. A., J . BioZ. Chem. 180, 1071 (1949). ( 5 ) Porter, W. L., Naghski, J . , and Eisner, Abner, Arch. Biochem., 24, 461 (1949). (6) Rockland, L. B., and Dunn, JI. S., Science, 109,539 (1949). RECEIVED July 19. 19jO.
Paper Chromatography of Organic Acids .1. B. STARK, A . E. GOODBAN, AND H. S. OWENS Western Regional Research Laboratory, .41bany, Calif. The study of the paper chromatography of organic acids was undertaken to provide a simple method for the rapid identification of organic acids in sugarbeet processing liquors. The use of several developing solvent mixtures has been investigated. The RF values for 18 organic acids were measured. The effect on RF values of hydration of paper, water content of solvents, temperature of development, and presence of inorganic acids was studied. In many cases an inversion of RF values was obtained by altering the composition of the developing solvent. The method is suitable for detection of many organic acids o r their salts in plant extracts or other biological media. The presence of acid impurities or their production as reaction products may he detected.
A
INVESTIGATION under way in this laboratory on changes in composition of sugar-beet processing liquors required a technique suitable for rapid and accurate identification of organic acids. The detection and identification of amino acids and other compounds by paper chromatography (3-6, 10, 11) suggested its application. During this work, methods were published for paper chromatography of acids in the form of derivatives (2, 6) or labeled with radioisotopes ( I ) . The authors have studied the behavior of several nonvolatile acids in various solvent mixtures containing formic or acetic acid (9). The method discussed here is similar to that of Lugg and Overell (8, 9) but offers some advantages in identification. It hss been used to identify some of the acids in sugar-beet processing liquors and to determint= the efficacy of fractionation proredures. ,"i
METHOD
The paper sheets, 40 X 57 cm., Whatman No. 1, were developed by an ascending technique similar to that described by Williams and Kirby (18). Glass jars, lo1/* X 18 inches, with glaas covers were used t o contain the solvent and paper. The sheets were clipped to a hexagonal rack made from 5-mm. glass rod. Only one solvent phase was present in the developing tank. The paper sheets were not humidified, nor was the temperature of the developing bath controlFd; hfwever, the laboratory temperature averaged about 25 * . 3 C. The papers were spotted with 0.003 t o 0.01 ml. of solution 3 cm. from the bottom and 2 cm. apart. The standard acid solutions were 0.075 N . Development time was usually 16 hours, but periods as short as 2 hours were also satisfactory. Following development, the papers were removed from the tank and either dried overnight in a hood or for 45 minutes a t 100" C . in a circulating air oven. Better definition of spots was usually obtained on papers dried a t room temperature; some acids are volatile or subject to decomposition a t higher temperatures. Occasionally it was found advisable to hydrate the paper by exposure to water vapor before drying was finished, to remove traces of some acidic substances that might be present or formed on heating the paper and solvent. The position of the acids on the chromatogram was shown by spraying the dried paper with
either bromocresol green or bromophenol blue, 400 mg. per liter in 95% alcohol. The indicator solutions were made slightly alkaline with sodium hydroxide. The acid spots were yellow against a blue or purple background. They were circled and the centers marked. RF values were calculated for the individual spots. Many of the solvents contained alcohols, and some esterification with formic or acetic acid took place. The small amount of esters present did not appreciably affect the RF values, but the reduction in acid concentration caused considerable tailing. This was not serious during a period of a week or two, and if the solvent was not replaced in this time, a portion wm titrated and make-up acid added. Every month the solvent should be replaced by fresh solution. The solutions were made up in stock bottles and the formic or acetic acid was added when the solvent was placed in the jar. Solvents should be checked for the prep ence of nonvolatile acids and purified, if necessary, before use. EFFECT OF CONDITIONS OF EXPERIMENT
Effect of Hydration of Paper, Temperature of Development, and Water Concentration. A preliminary study wm made of the effects of hydration of the paper, of changes in water concentration in the phenol-water-formic acid system, and of temperature during development in solvents F and G. These results are shown in Table I. In general, humidification of the paper has an influence similar to increasing the water concentration of the developer-that is, the RF values of acids such as citric and aconitic are increased, while those that are high generally remain practically unchanged. In most cases temperature changes have only a slight influence. Becauee of the slight effect of temperature change and humidification, the chromatographic papers were not humidified and development was a t room temperature. Acid Concentration. Several acids were developed with 75% phenol and 25y0 water containing 0.2, 1, and 2% acetic acid.
ANALYTICAL CHEMISTRY
414 Table 1.
Some Factors Affecting RF Value ( R F X 100)
.4cid Aconitic Adipic Citric Fumaric Glutaric Glycolic Lactic Maleic Malic Malonic Oxalic Methylene bis-N-pyrrolidone carboxylic Pyrrolidone carbox-
Hydrated Solvent Ga 250 250 350 C. C. C. 45 37 42 82 82 85 25 27 32 61 60 60 7R 7fi .. 78 ._. 56 60 58 70 72 75 50 58 50 41 43 42 47 49 48 21 21 21 85
87
86
h-onhydrated Solvent Fb 250 350
8 0 % " 70%d 34 44 82 88 30 19 56 61 79 81 fin 59 .. 76 ._ 76 55 59 44 36 50 46 25 15 87
86
C.
C.
71 82 35 78 79 54 64 38 46 56 11
65 80 35 76 77 54 67 37 46 51 7
64
66
Effect of Various Solvents. h good developing solvent for chromatography of organic acids should give a wide range of relative movement for the acids being studied, contain some water or possibly formamide, be stable over a reasonable period, leave only neutral or slightly acidic substances on the paper if the spots are to be detected with an acid-base indicator, and contain no colored residue that will obscure the spots. Several of the solvents tried may be classed as failures or partial failures on this basis. A list of t,hese should be of interest in preventing duplication of effort
87 85 86 59 63 86 86 68 71 72 72 72 61 65 90 90 87 84 95 91 94 67 50 49 42 56 65 59 16 24 21 14 25 26 24 Solvent G phenol, 3 grams; water, 1 ml.; 90% formic acid, 1%. Solvent F = isopropyl alcohol, 1 ml.; tert-butyl alcohol, 1 ml.; water, 1 ml: benzyl alcohol, 3 ml.; 90% formic acid, 2 % . , c Phenol, 4 grams: water, 1 ml.; 90% formic acid, 1%. d Phenol, 7 grams; water, 3 ml.; 90% formic acid, 1%.
1
-
Considerably less tailing and slightly higher RF values were obtained with 1 or 2y0 acetic acid. Later experiments using formic in place of acetic acid to repress ionization yielded still better chromatograms. The authors have used it exclusively, but not a t the high concentrations of 0.85 to 4.3 M used by Lugg and Overell (8). They allowed esterification equilibrium to be reached and used the organic phase for development. Effect of Inorganic Acids. The interference by inorganic acids is dependent on their concentration. RF values for hydrochloric, sulfuric, nitric, and phosphoric acids a t 0.05 and 0.1 N were determined in solvents B and D (Table 11). The inorganic acids moved about twice as far a t 0.1 N as a t 0.05 N . Phosphoric acid moved farther than the other acids, but in no case was the R p greater than 0.1. Inorganic acids tend to complicate chrcmatograms of organic acids, because in low concentrations they obscure those with low RF values and tend to increase the RF of some of the others. In high concentrations inorgapic acids may move a distance nearly equal to the solvent front, thereby ruining the chromatogram.
Table 11. hcids Aconitic Adipic Citrica, Fumaric Glutaric
RF X 100 for Organic Acids .4 79 84 33 84 80 60 75
69 88 34 85 85 57 76
51 64 6
41 51 5
45
B
C 76 92 28 89 89 62 83 46
70 9
Solvents D E 65
fi2 ..
83 24
65
80 23 74 77 48 61
35 45 8
36 43 4
75
80 48
F 69 86 35 78 78 54 64 85 44 53 10
57
69
52
56
60
78 76 88 17 65
87
52 69 86 13 60
58 66 85 12 59
64 72 86 24 64 34
G 36 86 26 63
1729" 91 42 48 18 84
86 81 66 95 95 Svrinnic 13 15 19 Tartaiic 66 52 71 Tricarballylic 49 Unknowna Composition of developing solvents: .4 = iso-octane, 4 ml.; 95% ethyl alcohol, 4 ml.; acetone, 1 ml.; 90% formic acid, 1%. B = chloroform, 2 ml.; 95% ethyl alcohol, 1 ml.; 90% formic acid, 2%. C = chloroform, 1 ml.; 95% ethyl alcohol, 1 ml.; 90% formic acid, 1 % . D = n-butyl alcohol, 5 ml.; benzyl alcohol, 5 ml.; water, 1 ml.; 90% formic acid, 1%. E = tert-butyl alcohol, 5 ml.; benzyl alcohol, 15 ml.; water, 2 ml.; 90% formic acid, 1%. F = isopropyl alcohol 1 ml: tert-butyl alcohol. 1 ml.; benzyl alcohol, 3 ml.; water, 1 AI., 90% formic acid, 2%. G = phenol, 3 grams; water, 1 ml.; 90% formic acid, 1%. 0 Acids found in sugar-beet processing liquors. 59 75
CA
fI
LACTIC
Figure 1.
Inversion of RF Values of Certain Organic Acids in Various Solvents A. B. F. G.
Iso-octane, alcohol, acetone Chloroform, alcohol Benzyl, isopropyl, t e r t - b u t y l alcohols Phenol
Solvents with a high proportion of low-molecular-weight ketones or alcohols with water give RF values over a narrow range near the solvent boundary. Acids developed with equal parts of isopropyl alcohol, tert-butyl alcohol, and water have RF values of 80 or higher. Acetone, water, and acetic acid in the ratio of 13:5:3 yield similar chromatograms. A mixture of benzyl and tert-butyl alcohols without water gives only slight movement. The addition of water is necessary to produce sufficient movement for the separation to be useful. In fact, the addition of water to all developers seems to be necessary, although preliminary experiments indicate that the water might be replaced with formamide in some solvents. Acetone or alcohol must be added to is* octane (2,2,4trimethylpentane) or chloroform to permit the solution of a small amount of water before they can be used to separate acids in a mixture. A solution of freshly distilled furfuryl alcohol and water, 8 to 2, forms very desirable chromatograms, but sufficient decomposition occurs in a day or two to leave a nonvolatile colored residue on the paper. Solutions of dioxane, acetone, and water, and of benzene, tert-butyl alcohol, acetone, and water, formed or contained nonvolatile acidic fractions that obscured the position of the test acids on the chromatogram. Solutions of carbon tetrachloride, water, and acetic acid, of xylene, water, and acetic acid, or of dioxane and toluene did not give sufficient movement of the acids, Collidine-lutidine-water mixtures did not adequately separate the acids. Similarly, n-propyl alcohol, ammonia, and water, recommended by Hanes and Isherwood (7), did not separate the acids with which the authors were concerned. The l a b ter solution should be of value in identifying volatile acids, aB the acids are present as the ammonium salts. Their position is indicated by spraying with ninhydrin. -4mixture of phenol and
V O L U M E 23, NO. 3, M A R C H 1 9 5 1 water forms a band of acid extending about one fourth the distance to the solvent front. In spite of this defect, a phenol-water solution is one of the best developing solvents. To eliminate obscuring of acids with RF values less than 0.25, the initial spots may be made about 10 cm. from the bottom of the paper. Table I1 gives the composition of several suitable solvent mixtures and the RFvalues for 18 acids in these mixtures. As shown in the table, the presence of several of these acids in sugar-beet processing liquors has been confirmed. The advantages of developing separate sheets with two or more solvents are apparent in Figure 1, where the movements of five acids are compared in four solvents. Lactic and succinic acids vary by not more than five units in any solvent. In separate runs the difference may be much less. With solvent F the succinic acid spot will be consistently higher than that of lactic acid. With solvent G the positions are inverted. This inversion of RF is noticed with several other acids investigated. Two-dimensional chromatography first with solvent F and then G does not resolve lactic and succinic acids as satisfactorily as separate one-dimensional chromatograms. When identifying unknown acids it is necessary to compare the unknown with a known acid on the same paper. Day-to-day variations of RFvalues may be as much as three or more points from the mean, but there is only a slight change in the RF for the same acid in different spots on a single sheet. Unknown materials should be run in two or more solvent mixtures. APPLICATION TO PLANT MATERlALS
The following general procedure is recommended in studies of acids present in plant extracts. The presence of acids in moder-
415 ate amounts, 0.5% in dry solids, may be demonstrated by re moval of cations with an ion exchanger and chromatographing the solution. To identify acids in lower concentrations, it is sufficient in many cases to adsorb the acids on an anion exchange resin, elute with ammonium hydroxide, remove ammonia on a cation exchanger, concentrate, and chromatograph the solution. In case one acid is present in a much greater amount than the others, it may be necessary to remove as much as possible of that constituent before chromatographing to prevent masking of other acids. The importance of checking results in more than one solvent cannot be overemphasized. LlTERATURE CITED
(1) Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. C., Haas, V. rl., and Stepka, W., J . Am. Chem. Soc., 72, 1710 (1950). (2) Cavallini, D., Frontali, N., and Tosohi, G., Nature, 163, 568 (1949). (3) Consden, R., Gordon, 8.H., and Martin, A. J. P., Biochem. J . , 38, 224 (1944). (4) Crammer, J. L., Nature, 161, 349 (1948). (5) Dent, C. E., Biochem. J., 43, 169 (1948). (6) Fink, Kay, and Fink, R. M., Proc. SOC.Exptl. Biol. Med., 70, 654 (1949). (7) Hanes, C. S., and Isherwood, F. A., Nature, 164, 1107 (1949). (8) Lugg, J. W. H., and Overell, B. T.,Australian J. Sei. Research, 1, 98 (1948). (9) Lugg, J. W. H.. and Overell, B. T.,Nature, 160, 87 (1947). (10) Partridge, S. M., Biochem. J., 42,238 (1948). (11) Vischer, E., and Chargaff, E., J . Bid. Chem., 168, 781 (1947). (12) Williams, R. J., and Kirby, Helen, Science, 107, 481 (1948) RECEIVED September 25, 1950.
Improved Techniques in Paper Chromatography of Carbohydrates ALLENE JEANES, C. S. WISE, AND R. J. DIMLER
Northern Regional Research Laboratory, Peoria, I l l . To provide a basis for the extension of paper chromatography to the separation of disaccharides and oligosaccharides, a study was made of the interrelationships among the degree of resolution of the sugars, their RF values, and solvent composition. In addition, a multiple development technique was worked out to improve the resolution of sugar mixtures while retaining all the components on the paper sheet. A self-supporting glass spiral for holding the paper permitted use of sheets of various widths when ascending movement of the solvent was employed, while for descending movement a glass support enabled use of lengths of paper greater than the height of the container. For detection of re-
T
HE use of paper chromatography for the separation of carbohydrates has dealt mainly with monosaccharides. I n extending the method to reducing disaccharides and oligosaccharides, it was found necessary to study a number of new solvent combinations and to devise a new multiple development technique in order to obtain adequate separation of most of the sugars involved. The search for suitable solvents was on an empirical basis a t first because insufficient information was available (10, 19) on the interrelationships of the solvent composition, sugar
ducing sugars on the chromatograms, an alkaline 3,5-dinitrosalicyIate reagent proved advantageous. Included in the study were six common monosaccharides, six reducing disaccharides, and a mixture of glucose, maltose, and oligosaccharides obtained by the action of malt a-amylase on amylose and presumably constituting the homologous series up to a degree of polymerization of at least ten glucose units. The relationships established provide a basis for selecting optimum conditions for the separation of a wide variety of reducing sugars. The techniques and apparatus described should prove useful in the application of paper chromatography to the analysis of other types of compounds.
structure, and degree of resolution. The present studies of such relationships provide a more logical basis for selecting the solvents most likely to give satisfactory separation of a wide variety of mono-, di-, and oligosaccharides. The multiple development technique was chosen as the best method that would improve separations yet maintain all sugar spots on the paper. Because both descending (Partridge, 13) and ascending (Williams and Kirby, 1 6 ) movement of the solvent have been used extensively, both techniques were included in this work.
