waves at the dropping mercury electrode. Waves are obtained which are almost certainly due to the reduction of quinone structures. In the case of nitric acid-oxidized coal humic acids, = -0.5 volt are waves at about observed, which are probably coalesced waves due to the simultaneous reduction of both nitro and quinone structures. Certain polycyclic quinones and aromatics when treated with nitric acid in the same way behave in an identical manner. The coal acids give a second reduction wave at about = -1.5 volts, believed to be due t o reduction of carbonyl groups present in part as ketone groups in the original structure and in part as oxanthranol type of structures resulting from the tautomerism of hydroquinone type of primary reduction products of quinone structures.
(9) Kinney, C. R., Kerschner, P. M., F u e l 31,414 (1952). (10) Kinney, C. R., Ockert, K. F., ISD. ENG.CHEM.48, 327 (1956). (11) Kolthoff, I. M., Lingane, J. J., “Polarography”, 2nd ed., p. 686, Interscience, New York, 1952. (12) Zbid., p. 700. (13) Zbid., p. 708. (14) I b i d . , p. 754. (15) Love, D. L., thesis, Pennsylvania State University, 1955. (16) Lowry, H. H., ed;: “Chemistry of Coal Utilization, p. 349, Wiley, New York, 1945. (17) Zbid., p. 356. (18) Xleites, L., Meites, T., ANAL.CHEM. 20,948 (1948). (19) Montgomery, R. S., Holly, E. D., Goehlke, R. S., Fuel 35, 49, 56, 60 (1956). (20) Stone, K. G., Furman, S. H., J . Am. Chem. SOC. 70,3062 (1948). RECEIVED for revien- April 25, 1956. Accepted June 11, 1957 Division of Gas and Fuel Chemistry, Symposium on Bituminous Materials, 129th lleeting, ACS, Dallas, Tex., April 1956.
ACKNOWLEDGMENT
The National Aniline Co., Williamsport, Pa., kindly supplied the v a t dyes used in this work. LITERATURE CITED
(1) Ahmed, M. D., Kinnev, C. R., J . Am. Chem. SOC.72,55611950). (2) Charmbury, H. B.. Eckerd. J. W., La Torie, J. S.,‘ Kinney,’ C. R.; Zbid., 67,625 (1945). (3) Chowdhury, J. K., Biswas, A. B., J . I n d i a n Chem. SOC. 19, 289 (1942). (4) Cody, -4.F., Milliken, S. R., Kinney, C. R., ANAL.CHEM.27,362 (1955). (5) Furman, N. H., Stone, K. G., J . Am. Chem. SOC.70, 3055 (1948). (6) Gill, R., Stonehill, H. I., J . Chem. SOC.1952, 1860 (also Fig. 2). ( 7 ) Howard, H. C., Znd. Eng. Chem. 44, 1083 (1952). (8) Juettner, B., Smith, R. C., Howard, H. C., J . Am. Chem. SOC.57. 2322 (1935)
Paper Chromatography of Homologous Saccharides Selection
of Solvent Components and Solvent Proportions
JOHN A. THOMA and DEXTER FRENCH Department o f Chemistry, Iowa State College, Ames, Iowa
b A procedure i s described which allows the selection of solvent proportions of ternary systems producing the highest mobilities of homologous saccharides concomitant with their complete separation on chromatograms. The solvent components of the ternary systems are selected for desirable physical properties.
A
numerous papers h a r e described solvents for paper chromatography of carbohydrates, few (3, 8,9, 12) report the effects of varying either solvent components or solvent proportions on the mobility of carbohydrates on paper. The only published systematic procedure for the selection of solvent proportions for paper chromatography has been designed by Durso and Alueller (3). Their procedure embodies a statistical approach through which solvent proportions in the whole of the miscible region of ternary systems are studied. The method was designed primarily to find solvent proportions to separate sets of monosaccharides. One of the main objectives of chromatography of homologs is to effect clear LTHOUGH
resolution of as many members of the series as possible. Although solvents in a large proportion of the miscible region may be used to resolve monoand disaccharides, they fail to resolve the higher homologs because they streak. Solvent proportions satisfactory for resolution of higher oligosaccharides are limited to a small region (the “chromatographic” region). For this reason the procedure of Durso and hIueller is not readily applicable t o homologous series of saccharides. Therefore, a procedure for this purpose was sought Table I.
which would be simple, rapid, and systematic. Because of their flexibility, ternary rather than binary systems were selected for study. The three sets of solvent systems examined contained ethyl acetate, 1-butanol, and nitromethane as the water-immiscible componentJs in conjunction with a variety of components (referred to as carrier solvents) soluble in both water and the water-immiscible component, Muller and Clegg ( 1 1 ) have defined a
+
“diffusion” constant, D = 5 1IE
b,
Effect of Diffusion Coefficients on Ascent Rate of Solvents
D
Solvents Hz0 H20,(CH,)*?;CHO, hIeSO? H20,EtOH, PIIelS02
HzO, bH?.(CH&.COO, EtOAc HzO, HOAc, EtOAc HzO yridine BuOH H20: EtOH, duOH
=
Y
1000 rig of Water and Water-Immiscible Compounds
Av.Time, Hours,
7.2 4 8 4 8
to Ascend 20 Cm. 1.5 2 5 3 5
6.0
3.5
1. o
6.0 6.5
6.0
1.0
4.5
VOL. 29, NO. 1 1 , NOVEMBER 1957
Av. Relative Ascent Rates 1.00
0 59 0 43
0.43 0 33 0.25 0.22 1645
t o which the rate of capillary diffusion of a solvent is proportional while ascending in the paper where: y is the surface tension, 7 is the viscosity, g is the specific gravity, and a and b are constants of the paper. This function was originally intended to apply t o the solvent mixture. However, its value for the water-immiscible component alone can serve as a rough index to indicate relative solvent diffusion rates, since this component comprises approximately half of the mixture by volume. This effect is reflected in the relative rates of solvent ascent shown in Table I. Kitromethane was selected as a materimmiscible solvent because it does not increase the viscosity of aqueous solutions as much as the stronger hydrogenbonding hydroxyl or carbonyl compounds. For this reason, solvents containing this component may show unusually rapid rates of solvent ascent. The water-immiscible solvents ethyl acetate and 1-butanol were studied because of their previous extensive application in carbohydrate chromatography. I n this paper, chromatographic characteristics refer only to the ability of solvents to move and separate carbohydrates on chromatograms and exclude any other physical properties of the solvent mixture or the solvent components. For paper chromatography of homologous oligosaccharides, the solvent proportions for any ternary system were sought which would produce the highest mobility of the sugars on the paper with enough separation to discern discrete spots after development. The index of merit is the product of these factors, (Rfl) (Rf, - R f J , and served as a measure of these characteristics where: R f l = Rr of the monomer and Rf,, = Rr of the n-mer (n generally 3 or 4). This index of merit was observed experimentally to pass through a maximum as solvent proportions were varied.
C
Figure 1. Phase boundary of typical ternary system plotted as per cent b y volume
A . Water B . Water-immiscible component C. Carrier solvent 1-5. Solvent proportions used to determine “chromatographic” region 6-10. Solvent proportions used t o select system producing maximum index of merit in “chromatographic” region
alkaline copper reagent followed by phospbomolybdic acid (4). RJ values were measured rapidly by a device designed by Glazko ( 7 ) . PROCEDURE
The phase boundaries for the ternary systems were plotted as per cent by
volume on triangular coordinate graph paper. The per cent by volume for each component was determined by titration of varying levels of the two immiscible components with the carrier solvent until turbidity disappeared. Chromatograms, spotted with known reference compounds, were irrigated with five or six solvent systems selected
MATERIALS
Inulin oligosaccharides. Inulin was hydrolyzed in 0.0lX sulfuric acid for 15 minutes a t 70” C. Dextran oligosaccharides. Dextran mas hydrolyzed in O . 1 N sulfuric acid for 60 minutes a t 100’ C. Maltodextrin oligosaccharides. Glucose and C.P. maltose were obtained from commercial sources, maltotriose was obtained from salivary digests ( I S ) followed by charcoal chromatography ( I d ) , and maltoheptaoae was obtained by partial acid hydrolysis of beta Schardinger dextrin (6). Butyrolactone \\-as kindly supplied b y Anatra Chemicals. All other solvents were of reagent grade and commercial origin. Eaton and Dikeman No. 613 paper, equilibrated with the solvent vapor (2) a t 25’ 5 1.5’ C. for 2 hours, was used. Paper length was 20 cm. All sugars were developed with 1646
ANALYTICAL CHEMISTRY
It I
30
I
I
40 50 % WATER-IMMISCIBLE S O L V E N T
I 60
Figure 2. Relation of percentage of water-immiscible solvent to index of merit
A . Solvent system H20-pyridine-l-BuOH, 107‘ pyridine in excess of amount required for miscibility B . Solvent system H20-DhIF-MeS02, 5 % DMF in excess of amount required for miscibility
Table
II.
matographic characteristics (see Figure 2).
Effect of Solvent Proportions of Water-Pyridine-1 -Butanol Solvents on Rf Values of Maltodextrin Oligosaccharides
R, Values
Solvent Proportions Pyridine 1-Butanol 0.32 0.20 0.11 0.005 28 55 0.33 0.18 0.10 0,005 13 32 55 0 40 0 26 0 17 0 02 20 30 50 0 39 0 24 0 14 0 01 16 34 50 0 46 0 32 0 23 0 035 23 32 45 0 46 0 32 0 23 0 040 19 36 45 0 54 0 44 0 33 0 12 22 38 40 0 55 0 46 0 38 0 12 27 33 40 0.61 0.54 0.46 0.25 26 39 35 0.62 0.56 0.48 0.26 31 34 35 0 63 0 61 0 58 0 38 36 34 30 0 63 0 58 0 57 0 38 32 39 29 0 57 0 i6 0 76 0 71 50 29 21 0 76 0 76 0 i6 0 71 46 36 19 GI, Gs, Ga, and Gi represent, respectively, glucose, maltose, maltotriose, and maltoheptaose. GI
G3
G2
H20 17
G7
a t regular intervals along the phase boundaries, varying 15 to 207, in water content (see Figure l ) , and developed with copper reagent and phosphomolybdic acid. Visual observation of the developed chromatograms indicated regions where reasonable mobility of the monomer occurs accompanied with enough separation to discern discrete spots for some of the higher homologs. These chromatographic regions usually contained 35 to 607, of the materimmiscible component and 0 t o 15y0 carrier solvent in excess of miscibility (see below). If very volatile materimmiscible components are used, the
paper should be equilibrated with the vapors. Lines \T-ere then drawn parallel to the phase boundaries in the chromatographic regions, so the solvent mixtures nould contain 5, 10, or 15% carrier solvent in excess of miscibility (see Figure 1). Solvent proportions selected a t intervals along these lines, varying a t 5% levels of the 11-aterimmiscible component, \\-ere used to irrigate another set of chromatograms. The solvent proportions from the chromatographic regions which gave the maximum indices of merit were chosen as those giving optimum chro-
DISCUSSION
Figures 3 t o 5 demonstrate typical results when log RI/l - R, (6) was plotted against the number of hexose units per molecule as solvent proportions n-ere varied. R, values of the sugars for solvent proportions intermediate between those used could be closely approximated by linear interpolation. Solvent systems containing very basic nitrogenous carrier solvents destroyed reducing sugars during one or two ascents. However, they are satisfactory for nonreducing sugars. The length of time required for each solvent mixture to ascend the paper was noted. The average values for all proportions of each combination of solrents, together n i t h the rates relative to that of n-ater, are recorded in Table I. I n the systems studied, solvent proportions markedly above the phase discontinuity appeared to be of little value. K i t h larger amounts of carrier solvent, streaking occurred. For a given system, solvent proportions selected to give the same RI value for glucose were essentially identical in their ability to move the higher saccharides (Table 11). K h e n solvents used for chromatography contained more than 10 to 15%
\
\0
0.05 L
0
t 1
1
I
2
3
1
,
D
,
4 5 6 7 8 HEXOSE UNITS PER MOLECULE
I
9
Figure 3. Chromatographic characteristics of maltodextrin oligosaccharides in chromatographic region with water-pyridine-1 -butanol solvents
A . 26:39:35 B . 22:38:40 C. 19:36:45
D. 16:34:50 Reported as by volume
\
\
\D
1 2 3 4 5 6 7 8 H E X O S E U N I T S P E R MOLECULE
Figure 4. Chromatographic characteristics of reducing inulin oligosaccharides in chromatographic region using water-acetic acid-ethyl acetate solvents
A. 32.5:22.5:45 B. 28:22:50 C. 24:21:55 D. 20:20:60 Reported as 70 by volume VOL. 29, NO. 1 1 , NOVEMBER 1957
0
1647
1-
I .o
A iI
~~
1 2 3 4 5 6 7 H E X O S E UNITS PER M O L E C U L E
Figure 5. Chromatographic characteristics of dextran oligosaccharides in chromato-
A . 28:37:35 B. 23:37:40 C. 19:36:45 D. 15:35:50
Reported as ’?& by volume
1648
ANALYTICAL CHEMISTRY
cule A only in that it has an attached group X,then: RT In a/@ = Apx. where CY
=
partition coefficient of A
= partition coefficient of
I
1
2
1
I
1
3 4 5 HEXOSE UNITS PER M O L E C U L E
I
6
Figure 6. Chromatographic characteristics of dextran oligosaccharides with various solvents giving glucose Rl values of 0.44 2 0.02
graphic region using water-ethanol-nitromethane solvents
carrier solvent in excess of miscibility or large proportions of water, it appeared that partition chromatography was no longer completely operative. Two observations led to this conclusion. First, the linear relationship between log R,/(l - R,) and number of hexose units per molecule was no longer exactly obeyed (6); and secondly, spot elongation was dependent upon both solvent proportions and degree of polymerization of the oligosaccharides. Some variation in the size of the chromatographic region and considerable variation in tendency to streak were found as solvent components were varied. However, the slight spot elongation observed with the water-dimethylformamide-nitromethane systems can probably be attributed to nonattainment of equilibrium because of the rapid ascent, rate. Even for solvent systems differing in the components present, it was possible to find solvent proportions that gave essentially identical chromatograms (see Figure 6). When solvent systems were utilized which produced high R f values 0.8 to 0.9for the monomers-Le., little or no separation of the homologs occurred. For these reasons, it is doubtful that an ideal aqueous solvent system, one which would allow high R, values for the monomer concomitant with complete separation of the higher homologs, can be found. Derivatization offers one solution to this problem. Martin (IO) has shown that if a molecule B differs from a mole-
I
B
A p x = change in chemical potential
for transference of 1 mole of group X from the mobile t o the stationary phase. It is independent of the rest of the molecule
Thus, derivatization would increase the Rl values of the homologs by increasing their solubility in the mobile phase and thereby decreasing their partition coefficient. However, the A p z for group X (a hexose unit), which is instrumental in the separation of the homologs, remains unchanged. Chromatography a t higher temperatures (1) has also proved advantageous by increasing both the R, values and resolution of the sugars. Jermyn and Isherwood (9) have suggested that if two compounds have similar R f values, it is desirable to employ solvents that produce Rf values of 0.2 t o 0.3 for their separation. Thus, after a solvent has been selected for desired properties, the appropriate system for chromatography can be selected by varying solvent proportions along or just above the phase boundary. The research worker is offered a simple, rapid, and systematic method for the selection of the best solvent proportions of ternary systems for
A. Water:DMF:MeNOz 20:30:50 B. Water: EtOH :BtOH 20: 25: 45 C. Water:EtOH:MeNOs 19:36:45 D. Water:Pyr.:BtOH 19:36:45 Reported as yo by volume Chromatographic characteristics of all other systems were between systems A and D
chromatography of any homologous saccharide series. The solvent components of the systems are selected to accord v i t h experimental conditions. LITERATURE CITED
Alcock, bl., Cannell, J. S., Nature 177,327 (1956).
Aronoff, S., “Techniques of Radiobiochemistry,” p. 25, 1on.a State College Press, Ames, Iowa, 1956. (3) Durso. D. F.. Mueller. W.8 . . ~ X A L . CHEM. 28,’1366 (1956). ’ (4) French, D., Knapp, D. W., Pazur, ~
J. H., J. Am. Chem. Soc. 72, 5148
(1950). (5) French, D., Levine, AI. L., Paaur, J. H., Ibid., 71, 356 (1949). (6) French, D., Wild, G. hl., Ibid., 75, 2612 (19,52\. \ - - - - I
GLZko, A. J., Dill, W. A., .$SAL. CHEM.25, 1782 (1953). Jeans, -4., Wise, C. S., Dimler, R. J., Ibid., 23, 415 (1951). Jermvn. M. A , . Isherwood. F. A.. Biichkm. J . 44,402 (1949): Martin, A. J. P., Biochem. SOC.Symp. 3, 4 (1949).
Muller, R. H., Clegg, D. L., A N A L . CHEM.23,408 (1951). Partridge, S. hl., Westall, R. G., Biochem. J. 42, 238 (1948). Pazur, J. H., French, D., Knapp, D. W.. Proc. Iowa Acad. Sci. 57, 203 (1950). (14) Whistler, R. L., Durso, D. F., J . A m . Chem. SOC.72,677 (1950).
RECEIVED for review December 26, 1956. ilccepted July 8, 1957. Research supported by a grant from General Foods, Inc.
