Paper Chromatography of a Petroleum Porphyrin Aggregate H. N. DUNNING Petroleum Experiment Station, Bureau o f Mines,
U. S.
Department o f the Interior, Bartlesville,
Okla.
JACK K. CARLTON' University o f
Arkansas,
Fayetteville,
Ark. for spectrophotometric studies. The groups vere investigated further by paper-sheet chromatographic methods to estimate their purity and t o identify them with the groups isolated in the preliminary paper-strip and -sheet methods. Two groups of porphyrins Tyhich moved more rapidly through the paper columns had spectra of the etio (3, 5 ) type. The remaining groups, including the major constituent, had spectra of the phyllo type.
Paper-strip and paper-sheet chromatographic methods have been developed to separate the porphyrin aggregate of a California crude oil into several groups. The first two groups are characterized by spectra of the etio type, in which peak I11 (532 m p ) is stronger than peak I1 (565 mfi). Peaks I (620 mp) and IV (500 mp) are displaced toward longer and shorter w-ave lengths, respectively, in groups 1 and 2. The other groups have phyllotype spectra; peak I1 is stronger than peak 111. The wave length of the main fluorescence band changes about 2 m p toward shorter wave lengths proceeding from group 1 to 6, in line with the changes in the absorption spectra.
MATERIALS
The chemicals used in extracting the porphyrin aggregate from the crude oil have been described (2, 3 ) . The specifications of the materials used in the separation procedures are listed in Table I. The crude oil from which the porphyrin aggregate u a s isolated v a s a heavy, asphaltic oil from the North Belridge field, Kern County, California (2).
T
presence of remnants of plant and animal life in petroleum was established several years ago (IS),and a quantitative method for the extraction of the porphyrin contents of crude oils was published recently (6). This method, with modifications in the colorimetric procedure ( S ) , has been used to determine the porphyrin contents of various petroleum extracts (2, 4). These investigations indicated that the porphyrin aggregates were of a heterogeneous nature, as would be predicted from their biological origin. ,4 better knowledge of the nature of these porphyrins HE
Table I. Material
EXPERIMENTAL METHODS
Paper-Strip Method. The ascending method of development was used because of the simplicity of the apparatus employed and the better definition of the zones. The apparatus consisted of a glass tube, 7.5 cni. in inside diameter and 61 cm. in length, mounted vertically with the lower end immersed in the solvent container. The upper end was sealed by a waxed cork fitted with a metal support for the pa er strip. A 50-cm. strip of paper, to which the sample had been acfded, was suspended above the developer. Twelve to 24 hours were required for the system to reach equilibrium. Then the paper was lowered and development proceeded until the solvent front reached the desired height. Freshly prepared samples of 4 t o 10 pl., containing about 0.5 y of porphyrin aggregate in chloroform solution, were put on the paper strip a t a reference mark near the bottom.
Specifications
Source
Paper, strips Paper, sheets PaDer. sheets Paper pulp Petroleum ether
m:liatman T5 hatman Munktells Whatman Merck & Co , Inc.
Chloroform
llr+llinckrodt Chem.
Methanol Benzene 2,4-Lutidine 2 6-Lutidine 2:4,6-Collidine
Slerck & Merck & Eastxnan Eastman Eastman
Specifications No. 1, 1-inch roll KO.1, 16 X 19inches Chromatographic, 18 X 18 inches Ashless cellulose powder, coarse Reagent grade, boiling range 3060' C. Analytical reagent grade
GO.
Co., Inc. Co., Inc. Kodak Co. Xodak Co. Kodak Co.
Reagent grade Reagent grade Technical grade Practipal grade Technlcal grade
-
would allow more exact evaluation of their significance in the origin of and exploration for petroleum. Paper chromatography has proved to be a rapid, effective method for resolving naturally occurring mixtiires of porphyrins. However, this method has been used primarily for the separation of carboxylated porphyrins and their esters which are of clinical importance (1, 8-11). Much of the porphyrin aggregate of crude oils consists of decarboxylated porphyrins ( S ) , for which established methods are generally inapplicable. Paper-strip and -sheet chromatographic methods have been developed to allow the separation of the porphyrin aggregate of a California crude oil into five groups of porphyrins. This aggregate was derived principally from the nickel-porphyrin complex, of which this oil is a relatively rich source ( 2 , 3 ) . Larger quantities of the aggregate n-ere chromatographed on paper pulp t o obtain sufficient quantities of these, and two additional groups, 1
Present address Georgia Institute of Technology Atlanta, Ga
Paper-Sheet Method. A new method of paper-sheet chromatography was developed because saturation of the available cabinet was difficult with the solvents employed and several hours normally are required to attain equilibrium. The apparatus consisted of a large bell jar, inverted in a frameTTork. A vacuum desiccator lid was used for the cover, and the ground surfaces were lubricated with high-vacuum silicone grease. The opening in the standard-taper joint of the desiccator lid was fitted with a vented cork. Two 6-inch wooden disks were supported in the bell jar by means of a 3/4-inch dowel rod centered in the cork. The samples were placed on a reference line near the bottom of the paper sheet about an inch apart; the same spotting methods and concentrations were used as with the paper strips. The paper then was tacked t o the wooden disks and placed in the jar containing the solvent, and the system was rapidly evacuated. The solvent was allowed to boil briefly, the chamber sealed by rotating the sleeve on the desiccator lid, and development allowed t o proceed.
A second apparatus of similar design also has been used in the paper-sheet studies. This apparatus allowed the sheet to be lowered into the solvent after equilibrium v a s established. Therefore, it was used for both vacuum saturation studies and studies in which the atmosphere w,s saturated by allowing the closed system t o stand for seveial hours.
1362
V O L U M E 28, NO. 9, S E P T E M B E R 1 9 5 6 Paper-Pulp Method. The columns were packed by filling with dry cellulose powder and frequently tamping the powder tightly in place. The one-piece columns consisted of three sections: an upper section 4.1 by 44 cm., a center section 2.5 by 44 cm., and a lower section 1.1 by 25 em. The column was prewet with petroleum ether and, in the early studies, the sample was introduced in a petroleum ether solution. However, as the porphyrin aggregate was not entirely soluble in this solvent, quantihtive studies were complicated by this method. I n the final separation,,paper pulp was added to a chloroform solution of the porphyrln. Then the chloroform was evaporated, with constant mixing, to leave the sample on a small amount of paper. This impregnated paper pulp was tamped tightly in the top of the prewet column and the column developed with a 0.1 volume % solution of 2,4-lutidine in petroleum ether. This mixture also was used in the strip and sheet work, after it was found to give optimum resolution of the aggregate. The porphyrin zones on the various chromatograms were detected by their fluorescence under ultraviolet light. Except for this necessary illumination during location of the zones, the chromatograms were developed in darkness. The zones on the strips and sheets vere barely detectable after the paper vias dried. Therefore, their location and movement,s were determined in the apparatus with a cathetometer, or immediately upon removal from the apparatus.
RI Values. Rates of movement are expressed relative to the rate of movement of a pure sample of etioporphyrin I1 determined under identical conditions. The reproducibility of this relative rate was considerably greater than the individual RJ values in t,he various systems used and between the two laboraCories involved. The R , values were observed to be relatively insensitive to small differences in amounts of porphyrin in the spots, but varied somewhat with ditferent methods of obtaining saturation in the cabinet. The relative values were insensitive to differences in concentration, types of paper, saturation methods, and operators. The average R , value of the etioporphyrin I1 sample, with the petroleum ether-lutidine solvent and the cabinet allowed to become saturated by standing overnight, was 0.56 =k 0.04. Approximat,e R f values and porphyrincontents of the zones were determined by colorimetric analysis of each of the effluent fractions from the paper-pulp columns. Quantitative measurements of the porphyrin contents of the zones were accomplished by combining the effluent fractions to form the various groups and applying the colorimetric method of Dunning, Moore, and Myers (3). RESULTS AND DISCUSSION
-4 petroleum ether-2,4-lutidine mixture 15 as found most effective for the separation of the porphyrin aggregate. Mixtures containing 0.05, 0.1,0.2, and 0.3 volume yo of lutidine were tested The mixture containing 0.1 volume % lutidine was optimum, Mixtures in which 2,6-lutidine or 2,4,6-collidine was substituted for 2,A-lutidine were used without materially altering the results. Hexane was a satisfactory substitute for petroleum ether and because of its more constant composition is preferred. Although not so efficient as petroleum ether-lutidine mixtures, petroleum ether-chloroform mixtures also were effective as developers. Several compositions of the mixture r e r e tested and a 3 volume yG solution of chloroform mas the most effective. Several zones were obtained, but separation was not as good as with the petroleum ether-lutidine mixture and the leading zones were difficult to detect ..inother deterrent to using chloroform is that the porphvrins are less stable in chloroform than in the other solvents (3). Other developers that were tested, together with a brief statement of their effects in resolving the mobile zones, are summarized in Table 11. The systems listed in Table I1 were evaluated for their effectiveness in resolving the mobile porphyrins. However, part of the porphyrin aggregate is not moved by the petroleum etherlutidine mixture. Lutidinc, saturated with water and in the
1363 presence of ammonia vapors, was effective as a developing solvent for this part of the porphyrin aggregate. With the 0.1 volume % solution of 2,4-lutidine in petroleum ether, and paper strips, four distinct zones were observed, in addition to a portion that did not move from the point of application. The rates of movement of these zones, relative to the movement of a pure sample of etioporph n 11, were 1.0, 0.8, 0.7, and 0.5. There was an indication of%Irther resolution in the leading and trailing zones, but the concentrations were too low to make positive identifications. Similar results were obtained by the paper-sheet method. The use of sheets instead of strips allom the simultaneous separation of several samples. Also, because the experimental conditions are identical for any one sheet, the movement and separation of different samples may be compared accurately.
Table 11. Developers for Paper Chromatograms Components Major Butanol Butanol Water 2.6-Lutidine 2,l-Lutidine Water Methanol Methanol Petroleum ether Water
Volume Minor ratio Acetic acid 9 to 1 Water Saturated Butanol Saturated Water Saturated Water Saturated 2,l-Lutidine Saturated Water 10 to 1 None 1 to 1 Methanol hlethanol and 5 , 5 , and 3 2,l-lutidine
Effects Too strong Too strong Too weak Too strong Too strong Too strong Good movement. no resolution Too strong Good movement, no resolution Good movement no resolution
The behavior of the porphyrin aggregate was compared to that of several pure porphyrin samples in this manner. The esterified forms of coproporphyrin I and uroporphyrin I, the acidic form of these porphyrins, and the acidic form of mesoprophyrin IX did not move perceptibly with the petroleum ether-lutidine solvent The dimethyl ester of mesoprophyrin I X had an RI value of 0.04. I n the upper region of the chromatograms, the esterified and unesterified portions of the porphyrin aggregate moved identically, having zones with rate values relative to etioporphyrin I1 of 1.0, 0.8, 0.7, and 0.5. Homver, near the origin, the esterified porphyrin aggregate formed a zone not observed with the unesterified aggregate. This is attributed to the presence of porphyrin containing a single carboxylic acid group. The high rates of movement of the four leading zones indicate that they contain decarboxylated types of porphyrins. The portion of the aggregate remaining a t the origin is indicated to contain carboxylic acid groups. The porphyrin aggregate was chromatographed on paper pulp to obtain samples large enough for further studies. The petroleum ether-lutidine mixture used in strip and sheet Iyork was used as the developer. I n the final column chromatogram, 28 ml of solution containing 140 p.p.m. of porphyrin aggregate was fractionated. A total of 1800 ml. of eluate m m collected in 9-ml. portions over a period of 72 hours with an automatic fraction collector. Every fifth fraction was concentrated to an absorbance of about 0.7 to 620 r n w and 8 p1 of each was spotted on a paper sheet for chromatographic study of purity and R , value. Figure 1 illustrates the rate of movement of the several fractions on paper sheets relative to the movement of etioporphyrin 11. These studies shoTv that the fractions form six main groups Accordingly, the fractions \yere recombined to form the indicated groups. The seventh group remained a t the top of the pulp column. It was extracted with ethyl ether and separated from impurities of similar adsorption characteristics by extraction into 10 weight 70hydrochloric acid, neutralization to a pH of 5.2, and extraction by ether. In this manner, a homogeneous portion was obtained which contained about half of the porphyrin in group 7. This portion of group 7 will be referred to as group 7a.
ANALYTICAL CHEMISTRY
1364
The rates of movement of groups 1,3,4,5, 6, and 7 on the paperpulp column, relative to the rate of group 2 which had been observed to move a t the same rate as etioporphyrin 11, were 1.1,0.9, 0.8, 0.65, 0.45, and 0.0, respectively. The solutions containing the porphyrin groups were diluted to have an absorbance of 0.7 a t 620 mp. Then 8 pl. of each solution was spotted on a pape sheet and their movement, relative to etioporphyrin 11, measu ed for accurate establishment of rates of movement. The absorption spectra of the seven groups were determined with a Beckman D U spectrophotometer. The wave lengths of maximum absorption and fluorescence (under ultraviolet light) were determined with a Hartridge Reversion spectroscope, with which the bands can be determined more precisely, although not necessarily more accurately, than with the spectrophotometer. Average values of the properties of the porphyrin groups are summarized in Table 111.
9
Table 111. Properties of Porphyrin Groups Isolated by Paper-Pulp Chromatography Movement (Rd. to Etio 11)
Fractions (Incl.) 1- 12 13- 27 28- 40 41- 70 71- 97 98-20;
Group
Spectral Type (6)
% of Porphyrin Aggregate
...
Total
7.9 16.1 14.2 21.6 12.6 16.0 7.6 96.0
Remained at top of column and was extracted later.
1.2
.4 0
50
100 150 FRACTION NUMBER
The spectra of the groups are shown in Figures 2 and 3. The solutions were diluted to one tenth of the concentrations used for determinations in the visible region for measurements a t wave lengths below 470 mp. The absorbance peaks of porphyrins are designated I, 11, 111, and IV, proceeding toward shorter wave lengths through the visible region. The first two groups (Figure 2) have spectra of the etio type (6); peak 111 is stronger than peak 11. This type of spectrum is typical of animal porphyrins and a few plant porphyrins. The spectra of the other groups (Figures 2 and 3) are of the phyllo type, as peak I1 is stronger than peak I11 in each spectrum. This type of spectrum is typical of plant porphyrins in general. The etio-type spectra (groups I and 2 ) have peak I displaced about 2 mp toward the red and peak IT’ about 2 mp toward the blue, compared to the same peak in the phyllo spectra (groups 3 to 7 ) . The fluorescence of porphyrins irradiated by ultraviolet light affords an extremely sensitive means for their detection and, to a limited extent, for their identification. Porphyrins in neutral or slightly basic solutions typically have a strong fluorescence band a t about 620 to 630 mp and secondary bands a t longer wave lengths (19, f4). Fluorescence may be measured a t concentrations far below those detectable by absorbance studies ( 7 ) . The Hartridge revision spectroscope, calibrated Kith a mercury lamp, was used to locate the position of the major fluorescence band in the groups of porphyrins separated by paper pulp chromatography. The positions of the main fluorescence band and of the four absorption peaks in the visible region m-ere determined several times and the results averaged. Although it is difficult to locate an unsymmetrical peak-e.g., peaks 2 and 3-exactly with this instrument, the differences among corresponding peaks of the several groups were ieproducible. The locations of these bands are summarized in Table IV. These results show a progressive decrease in the wave length of peak I and the increase in wave length of peak IV from group 1 through group 5, in agreement with the spectrophotometric measurements. Corresponding shifts of the fluorescence band corroborate these slight changes. This kind of spectral change generally indicates a simplification of the side chains attached to
200
Figure 1. Rate of movement of porphyrin fractions on paper sheet relative to movement of etioporphyrin I1 Rf of etioporphyrin I1 0.56
Direct comparison of the R J values with those obtained in earlier paper-strip xork is invalidated by differences in concentration of the samples as well as impurities. However, relative observations indicate that groups 2, 3, 4 to 5 , and 6 correspond to the paper-strip zones in order of decreasing rate of movement. Group 1 moved faster than did a sample of pure etioporphyrin 11. Because previous work showed there were present decarboxylated porphyrins which moved a t various rates, this is entirely possible. However, the faster rates may possibly be due to the presence of solubilizing impurities in the aggregate, which increase the rates with TThich its zones move (4).
WAVELENGTH,
Figure 2.
rnp
Absorption spectra of porphyrin groups In 15 ml. of chloroform
V O L U M E 28, NO. 9, S E P T E M B E R 1 9 5 6 the porphyrin ring or ring closure as in derivatives of deoxophylloerythrin (6, 7, 19, 14). The spectrum of the etioporphyrin I1 sample was not detectably different from that of group 1. The higher R f values of the more mobile groups are attributed to the presence of more, or larger, aliphatic side chains in the porphyrins of these groups. The
1365 R , value of group 7a indicate that the porphyrin involved is a monocarboxylic porphyrin, probably deoxophylloerythrin, which Tyas identified in oil shale extracts by Treibs (IS). CONC LUSlOIv S
Paper-strip and paper-sheet chromatographic methods have been developed to separate the porphyrin aggregate of a California crude oil into several groups. Columns packed with paper pulp were used to prepare large enough samples of seven such groups for further study. The absorption spectra of the porphyrin groups have been investigated. The first two groups are characterized by spectra of the etio type, in which peak I11 (532 mp) is stronger than peak 11 (565 mp). Peak I (620 mp) and peak I V (500 m p ) are displaced toward longer and shorter wave lengths, respectively, in groups 1 and 2. The other groups have phyllo-type spectra; peak I1 is stronger than peak 111.
Table IV. Wave Lengths (nip) of Absorption Bands and >lain Fluorescence Band of Porphyrin Groups in Chloroform Absorption spectrum of porphyrin group 7 I n 40 ml. of chloroform
decarboxylated animal porphyriiis-e.g., niesoetioporphyrincontain four methyl and four ethyl side chains. The decarboxylated plant porphyrins contain three ethyl side chains and either four (pyrroetioporphyrin) or five (phylloetioporphyrin) methyl side chains or an isocyclic ring ( 5 ) . Therefore, the high Rj values of groups 1 and 2 indicate that the etio-type spectra of these groups are due t o the presence of decarboxylated porphyrins similar to the etioporphyrin I1 sample used as a standard. Porphyrins of the "animal" type sometimes occur in plant systems ( 7 ) . Therefore, the identification of etio-type porphyrins in the aggregate is not positive proof that animal matter was involved in the genesis of the crude oil. Correlations of data show that groups 3 through 6 contain decarboxylated porphyrins, presumably of the phylloetioporphyrin or deoxophyllerythroetioporphyrin type. No attempt has been made to distinguish among the spatial isomers of the various types of porphyrins. The early paper-sheet chromatographic investigations indicated the presence of a porphyrin species containing one carboxylic acid group. Therefore, group 7a (from the acidic portion of the aggregate) was investigated by the method of Nicholas and Rimington (10). The solvent was 2,4-lutidine saturated with water used in the presence of ammonia vapor. Saturation of the atmosphere was obtained by allowing the sytems to stand for 16 hours. The results are s h o m in Figure 4. The R J values of the standards (coproporphyrin, uroporphyrin, and mesoporphyrin) are in good agreement with data reported in the literature (8, 10). The R, value of group 7a shows that this component of group 7 consists of porphyrins containing one carboxylic acid group. This corroborates observations nith the esterified porphyrin aggregate. Peaks I and I V in the spectrum of this group were located a t longer and shorter wave lengths, respectively, than the corresponding peaks in the middle groups. The paper chromatograms show that group 7a contains a carboxylic acid group not present in the earlier groups, supporting the assumption that groups 1and 2 contain more, or larger, aliphatic side chains or side chains of a more aliphatic nature than do the middle groups ( 3 to 6). The locations of absorption bands, their relative intensities, and the
Absorption I11 53?.8 566.5 533 6 566.4 533.7 565.6 536.6 565.3 535.4 564.9 535.9 565.3 533.5 565.2
I
Group
Figure 3.
620.3 619.9 618.9 618.3 618.2 618.1 618.7
0
I
2
3
4
5
CARBOXYL
Fluorescence (Main) 620.9 620.5 019.7 619.3 619.2 619.3 620.0
IV
I1
499.9 500.5 501.2 502.4 502.5 502.6 501.5
6
7
8
9
10
GROUPS
Figure 4. Relation of RJ values to number of carboxyl groups in paper-sheet chromatogram
The wave length of the main fluorescence band changes about 2 mp toward shorter wave lengths proceeding from group 1 to 6, corroborating the changes in the absorption spectra. Representative samples of the porphyrin groups have been compared by paper-sheet chromatography. These studies verify the separations accomplished and give rates of movement relative t o that of etioporphyrin 11 of 1.18, 0.99, 0.85, 0.74, 0.62, 0.53, and 0.00 for the seven zones. Correlations of spectral and chromatographie data indicate that the etio-type spectra of groups 1 and 2 actually represent the presence of porphyrins similar t o etioporphyrin 111, rather than pyrroporphyrin, a plant porphyrin having a spectrum of the etio type.
1366
ANALYTICAL CHEMISTRY
Paper-sheet chromatography, using a water-saturated lutidine solvent in the presence of ammonia vapors, and spectral measurements, show that group 7a (a major portion of group 7) consists of monocarboxylic porphyrins similar to deoxophylloerythrin. ACKNOWLEDGMENT
The authors are indebted t o Samuel Schwarta, University of Minnesota, who furnished the samples of coproporphyrin and uroporphyrin, and to Aleoph H. Corwin, Johns Hopkins University, who furnished the sample of etioporphyrin. LITERATURE CITED
(1) Chu, T. C., Green, A. A., Chu, E. J.. J. BWZ. Chem. 190, 643 (1951). (2) Dunning, H. N., Moore, J. W., Denekas, M. O., I d . Eng. C h m . 45, 1759 (1953). (3) Dunning, H. N., Moore, J. W., Myers, A. T., Ibid., 46, 2000 (1954).
(4) Dunning, H. N., Rabon, Iiancy, Ibid., 48, 951 (1956).
(5) Fischer, H., Orth, H., “Die Chemie des Pyrrols,” vol. 11, Part 1, F‘yrrolfarbstoffe, Akademische Verlagsgesellschaft, Leipsig, 1937. (@ Groennings, S., ANAL.CHEM.25, 938 (1953). (7) Lemberg, R., Legge, J. W., “Hematin Compounds and Bile Pigments,” pp. 71-9, Interscience, New York, 1949. (8) McSwiney, R. R., Nicholas, R. E., Prunty, F. T. G., Biochm. J. 46, 147 (1949). (9) Nicholas, R. E. H., Rimington, C., Ibid., 48, 306 (1951). (10) Nicholas, R. E. H., Rimington, C., S c a d . J. Clin. Lab. Invsat. 1, 12 (1949). (11) Rrtppoport, D. A., Calvert, C., Loeffler, R. K . , Gast, J. H., ANAL.CHEM.27, 820 (1955). (12) Stern, A., Molvig, H., 2. phusik Chem. A-175, 38 (1936). (13) Treibs, A., Ann. 517, 172 (1935). (14) Vannotti, A., “Porphyrins,” pp. 18-25, Hilger & Watts, London, 1954. RECEIVED for review January 16, 1956. Accepted May 23, 1956. Division of Petroleum Chemistry, 129th Meeting, ACS, Dallas, Tex., April 1956. Work conducted by J. K. Carlton at the University of Arkansas under contract with the U. s. Department of the Interior.
Selection of Solvent Proportions for Paper Chromatography Ethyl Acetate-Acetic Acid-Water System DONALD F. DURSO
and
WILLIAM A. MUELLER
Cellulose and Specialties Technical Division, Buckeye Cellulose Corp., Memphis, Tenn.
Variation of composition within the system ethyl acetateacetic acid-water has been related by statistical methods to the effect on the rate and the degree of separation of monosaccharides. By selection of acetic acid content and ethyl acetate-water ratio, solvents may be tailored to suit the mixture to be separated. The chromatographeris relieved of guesswork by being able to calculate spot position, distance between spot centers, and time to accomplish the separation without danger of losing the most mobile component. Because one-phase systems far removed from the two-phase region are used, temperature changes do not affect the results.
viscosity. hmong the possibilities for doing this was increasing the temperature or varying the nature of the solvent. More recently, Muller and Clegg (7) reported that solvent front movement rate was related to the diffusion coefficient of the components. This value was defined as the ratio D = r/vd, where
surface tension viscosity d = specific gravity
y = 7 =
This useful relationship indicated that other physical properties of solvent components could be selected in order to obtain a high development rate, which led to the exploitation of the conclusions of Jermyn and Isherwood. Evaluation of 10 common solvents by their diffusion coefficients indicated that ethyl acetate, acetic acid, and water should be a promising mixture.
SINCF
,its first adaptation to sugars by Partridge and Westall (8) paper chromatography has received wide application in the study of carbohydrate polymers and derivatives. Despite the many references to be found in this field, only a limited number present the results of investigations on the separating power of solvent systems (9-5). A new worker must usually choose between a solvent producing a high degree of separation a t a slow rate or one which moves the sugars rapidly while sacrificing separation. A systematic method for developing a solvent combining high rate and high degree of separation was sought in this investigation. Two previous findings served as the basis for this work. Jermyn and Isherwood ( 5 )reported that improved separation for pairs of sugars was obtained by use of three-component solvents, which produced R, values of 0.2 for sugars. This conclusion indicated an optimum range in the ratio between rate of sugar movement to rate of solvent front movement. These authors also noted that rate of development was inversely related to solvent viscosity. If the best separation were to be obtained by a solvent which must travel five times the desired displacement distance of the sugars, then an obvious method for obtaining an analysis in a reasonable time xould involve lowering solvent
EXPERIMENTAL
From a three-component plot of molar compositions, 12 mixtures were so chosen that the results could be expressed a8 functions of the composition variables. To this end the factorial, a statistical design, was employed to determine the number of runs and to evaluate the significance of results and factors
Table I. Solvent Mixture 1
2 3
4
5
6
7 8 9 10 11 12
Composition of Test Solvent Mixtures Acetic acid 15 35 55 75 15 35 55 75 15
35 55
75
Mole % Ethyl acetate 57 43 30 16.7 45.5 35 24 13.3 34 26 18
IO
Water 28 22 15 8.3 39.5 30 21 11.7 51 39 27 15
