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
V O L U M E 28, NO. 9, S E P T E M B E R 1 9 5 6
1367 These results m-ere evaluated by variance analysis to determine the significant factors. The statistical methods for calculation of polynomial coefficients were then employed to determine the form of the relationship between effect and variable. For molar acid content the significance of linear, quadratic, and cubic functions was tested while only the first t-ivo were fitted to the acetate-water ratio variable. A discussion of statistical methods is beyond the scope of this report and the reader is referred to any of the basic tests in this field.
IS
RESULTS AND DISCUSSION
The equations derived for expressing rate and degree of separation in terms of the variables are as follows. Glucose rate, r = 7.2 0
10
20
30 MOLE
40
PER C E N T
50
ACETIC
60
70
(1)
Galactose RglU = 0.94
I
0'
+ O.llA - 3.38B
80
ACID
5.29
- 0.03A
Mannose RglU.=
Figure 1. Development rate us. solvent composition
(2)
+ 0.0002A2
(3)
4
Arabinose R,,,, = 1.50 - 0.007A
+ 0.22R
(4)
Xylose R,I, = 1.69 - 0.01A - 0.24R
26
(5)
where r = glucose movement, millimeters per hour RglU,= ratio of sugar displacement to that of glucose R = molar ratio, ethyl acetate to water A = molar content of acetic acid in per cent
2.4
2.2
w
2
By arbitrary choice of A and R values, these equations lead to families of curves showing the effect of each independent variable. Plots for Equations 1 and 5 are shown in Figures 1 and 2, respectively.
2.0
=-,i I I Y
3
1.6
x >
A
1.4
1.2
1.0
o
Figure 2.
to
20
30 MOLE
40
PER CENT
OD ACETIC
60 ACID
70
no
Xylose separation us. solvent composition
studied. Acetic acid content was chosen as one independent variable. Because the other two factors, ethyl acetate and water contents, would then be dependent, they would not fill the requirements of the statistical experiment. The requirement was choice of factors of such nature that values for either might be chosen independently of values assigned t o the other. Therefore, the other independent variable selected was the molar ratio of ethyl acetate to water. The levels used for molar ratio of acetate to water were 2 to 1, 7 t o 6, and 2 to 3. The compositions of the 12 mixtures used in this work are presented in Table I. The 12 solvents cover more than half of the area above the two-phase region of this system. Any other ordered arrangement of compositions would have been equally suitable if the major portion of the area were experimentally investigated. Rhatman No. 1 filter paper was spotted with 2.5-pl. aliquot8 of a mixture of equal parts of glucose, galactose, mannose, arabinose, and xylose a t a total concentration of 5 y per p1. Solvent mixtures were made up from reagent grade acetic acid and ethyl acetate, and distilled water. The paper was cut into strips 7 X 23 inches with machine direction in the 7-inch dimension. This was done to reduce the length of the spots. For each solvent duplicate papers were irrigated in standard descending chromatographic equipment consisting of sealed glass jars 12 inches in diameter, 24 inches high, and containing stainless steel solvent assemblies. Runs were made a t room temperature without attempt to maintain constant conditions, since one purpose of this work was to ensure reproducibility of results but a t the same time to eliminate operator attention from elaborate schemes. ;\fter irrigation the papers were dried overnight in a hood and sprayed with 10% ammoniacal silver nitrate to develop the spots. The position of maximum density for each spot was located with a Photovolt densitometer, Model 501A. The results were tabulated as glucose movement rate in millimeters per hour and degree of separation for each of the other sugars as the ratio of sugar displacement to glucose displacement.
Figure 3.
Glucose rate and mannose composition
Rglu. us.
solvent
B,.l,M. D.
Solvent corn ositions of others ( I , 4-6). Volume ratio of ?to 3 to 2, ethyl acetate-acetic acid-water.
Rate of development as typified by glucose movement is directly related to the amounts of acetic acid and water, and inversely related to ethyl acetate content. Choosing ethyl acetate-water ratio and acid content values producing the same rate of movement leads to the curves shown in Figure 3. Here the relationship of result to each of the components is presented graphically. Degree of separation of the sugars is affected in a manner opposite to rate of movement by the same solvent composition changes. The typical curves obtained for Equations 4 and 5 by arbitrary substitution of A and R values are shown in Figure 2. Selection of values for an equal degree of separation then leads to the plots shown in Figures 4 and 5 .
ANALYTICAL CHEMISTRY
1368
Figure 4.
Arabinose R,-lu. vs. solvent composition
Table 11.
Characteristics of Solvent Mixture
(6 t o 3 to 2 parts ethyl acetate-acetic acid-water) Glucose rate 8 mm./hour
R~Iu.
Galactose Mannose Arabinose Xylose
0.94 1 16 1.45 1 55
Glucose, arabinose, and xylose values are linearly related to changes in solvent composition, while the mannose relationship requires a quadratic equation in acetic acid content only. The ratio of galactose to glucose movement is fixed for this solvent system a t 0.94. The mannose values, shown as M values on Figure 3, decrease as acid content increases but are fixed for any given acid content regardless of acetate-water ratio. The points labeled B , J , and JI on Figure 3 refer to the solvent compositions used by Boggs, Jeannes and others and Jermyn and Isherwood, and McCready, respectively ( I , 4-6). The general applicability of the findings reported here is attested to by the fact that the results of these other workers, in terms of rate and degree of separation. can be accurately predicted from Figures 3, 4, and 5.
Figure 5 .
Xylose Rglu. cs. solvent composition
round spots. This, of course, is necessary in order to permit use of the densitometer for quantitative work. Through choice of aliquot size and time of development, the spots are completely separated. This finding applies to all of the experimental points y i t h the exception of those mixtures containing 75% acetic acid. I n these, some tailing up to the starting point was observed. An example of the application of Figures 3 , 4 , and 5 is provided as follows. A mixture of xylose and arabinose will not separate if one uses a solvent composition defined by a glucose movement rate of 8 mm. per hour and a ratio of 1.06 for mannose to glucose displacement. However, the 6 to 3 to 2 mixture cited above, while moving the sugars a t the same rate, separates xylose and arabinose in a satisfactory manner. Other aspects of the system become apparent when one prepares transparencies of these figures and compares by superimposing them. Rather than recommend this system or the selected mixture as a panacea for all chromatographic problems, the authors wish t o emphasize the method of evaluating solvents. With a small number of ordered experiments, one can develop desired compositions in less time than trial-and-error work in which variations by volume ratio may appear large but in molar composition are actually insignificant. It is hoped that, by stimulating others t o study their favorite mixture and present their results in a similar fashion, some progress may be made in reducing chromatography to a science.
CONCLUSIONS
ACKNOWLEDGMENT
This is a generally applicable method for study of chromatography solvent systems. For selection of any particular solvent, Figures 3, 4, and 5 are superimposed. The point where curves of selected rate and degree of separation cross is the mixture desired. For work in this laboratory, the mixture chosen consists of 27.3, 23.3, and 49.4 niolar quantities of ethyl acetate, acetic acid, and water. In volumes the ratios are 6 t o 3 to 2 (point D,Figure 3). I n a period of 20 t o 24 hours, this solvent produces separations sufficient for quantitative work without interference from temperatuie variations as great as f l O o from a normal temperature of 25' C. The rate and degree of separation obtained are summarized in Table 11. This solvent mixture has been used t o separate mixtures ranging in composition from 99 parts of glucose, 0.5 part of mannose, and 0.5 part of xylose to those containing essentially equal parts of the five sugars studied. Because the papers are irrigated crosswise to machine direction, the spots are only slightly elliptical-that is, they are essentially homogeneous
The authors wish to express their appreciation to 1. F. Johnson for his aid in the statistical aspects of this work. LITERATURE CITED ( 1 ) Boggs, L. A . , ANAL.CHEM.24, 1673 (1952).
(2) Hardy, T. L., (1955).
Holland, D. O., Kagler, J. H. C., Ihid., 27, 971
(3) Hirst, E. L., Hough, L., Jones, J . K. L-., J . Chem. SOC.1949, 928. Wise, C. S., Dimler, R. J., ANAL.CHEX.23, 4 1 5 (4) Jeannes, -L, (1961). (5) Jermyn, IT.A., Isherwood, F. A , , Biochem. J . 44, 4 0 2 (1949). (6) McCready, R. M.,NcCornb, E. .1.,. ~ X A L . CHEM. 26, 1645 (1954).
(7) hliiller, R. H., Clegg, D. L., Ihid.. 23, 408 (1951). (8) Partridge, S. hI., Westall, R. G., Biochem. J . 42, 238 (1948). RECEIVEDfor review September 26, 1955. Accepted M a y 17. 19.56. Divibions of Carbohydrate Chemistry and -4nalytical Chemistry, Symposium on Carbohydrate Chromatography, 128tb meeting, ACS, Minneapolis. Minn., September 1955.
