packed columns permit separation aithout centrifugal force; however, the separation bands on this type of column are not as sharp as those obtained with more densely packed oncs. Good separations are obtained in time 1)eriods ranging from 5 t o 30 minutes, depending upon the packing of the column and whether or not centrifugal force is employed. The bands were either eluted off the ends of the columns or the columns were extruded. The glass columns may he cut into shorter lengths before extrusion, as described by Williams (26). Separation of Amino Acids. Figure 5 illustrates the chromatography of arginine, glycine, valine, and histamine on ion exchange paper with buffer and centrifugal force. Figure 6 shows the separation of a mixture of 10 amino acids; seven zones separated, two of which contained more than one substance. Rechromatography of mixed zones in a second system is required for separation.
~
Figure 6. acids
The authors acknowledge the technical assistance of G. R. White, A. Cowperthwaite, K. Barthalamus, N. D. Tillett, M. B. Herndon, M. Montgomery, and L. Tarone. They are indebted to M. h y s e and Samuel hl. Greenherg of Smith, Kline & French; to Robert W. Percival and Robert Kunin of Rohm & Haas; t o J. E. Griffin of the University of Pennsylvania; and to Ralph Davis of Bendix Corp. for
Separation of ten amino
Conditions were same as for Figure 5 except that rotational speed was 550 r.p.m. for 40 minuter
’
~
their assistance and suggestions during the course of the experiments. LITERATURE CITED
( 1 ) Anderson, J. M., J . Chromatog. 4, 93 (1960). ( 2 ) Bersin, T., Muller, A., HeEu. Chim. Acta 35, 475 (1952). (3) Block, R. J., Durrum, E. L., Zweig,
G., “Paper Chromatography and Paper Electrophoresis,” 2nd ed., Academic Press, New York, 1958. (4) Boggs, L. A., ANAL.CHEM.24, 1673 (1952). 151 Caronna. G.. Chim. Znd. ( M i l a n ) 37. 113 (1955): ’ (6) Herndon, J. F., Touchstone, J. C., \
ACKNOWLEDGMENT
( 1 1 ) McDonald, H. J., Bermes, E. iV., Jr., Shepherd, H. G., Jr., Naturwasaenschuften 44, 9 (1957). (12) McDonald, H. J., McKendell, L. V., ’ Zbid., p. 616.’ 113) McDonald. H. J.. McKendell. L. V., Bermes, E. W., Fr:, J . Chromatog. 1, 259 (1958). (14) McDonald, H. J., Ribeiro, L. P., Banaseak, L. J., ANAL. CHEM.31, 825 (1959). (15) Reitsema, R. H., J . Am. Phurm. Assoc. 43, 414 (1954). (16) Roberts. H. R.. ANAL.CHEM.29, 1443 (1957). (17) Roberts, H. R., Bucek, W., Zbid., p. 1447. (18) Roberta, H. R., Kolor, M. G., Ibid., p. 1800. (19) Roberts, H. R., Kolor, h l . G., Nature 180, 384 (1957). (20) Roberta, H. R., Kolor, M. G.> Bucek, W., ANAL. CHEM. 30, 1626 (1958). (21) Tata, J . R., Hemmings, A. K., J. Chromatog. 3, 225 (1960). (22) Touchstone, J. C., Herndon, J. F.,
,
I
.
White, G., Cowperthwaite, A., Davis, C. N., “Horizontal Chromatography Accelerating Apparatus. 11. Separations of Mixtures of Amino Acids,” unpublished data. (7) Indovina, R., Ricotta, B. M., A n n . Chim. (Rome)45, 241 (1955). (8) Izmailov, N. A., Shraiber, M. S.,
Farmatsiya N r . 3, 1 (1938). (9) Kirchner, J. G., Miller, J. M., Keller, G. E., ANAL. CHEM.23, 420 (1951); Zbid., 25, 1107 (1953). (10) McDonald, H. J., Bermes, E. W., Jr., Shepherd, H. G., Jr., Chromatog. Methods 2, No. 1, 1 (1957).
White, G., Cowperthwaite, A., Davis, C. K., “Horizontal Chromatography Accelerating A paratus. IV. Separation of Indole icetic Acid and Phenolic Acids from Urine,” unpublished data. (23) Touchstone, J. C., Herndon, J. F., White, G., Davis, C. N.,“Horizontal Chromatography Accelerating A?; paratus. 111. Separation of Dyes, unpublished data. (24) Tuckerman, M. M., Osteryoung, R. A,, Nachod, F. C., Anal. Chim. Acta 19, 249 (1958). (25) Williams, T. I., “An Introduction to Chromatography,’’ pp. 25-6, Blackie & Son Ltd., London, 1946.
RECEIVED for review December 5, 1961. Accepted May 24, 1962. Division of Biological Chemistry, 140th Meeting, ACS, Chicago, Ill., September 1961. This is the first in a series of four articles on horizontal chromatography.
Separation of Isotopically Substituted Hydrocarbons by Partition Chromatography Thermodynamic Properties as Calculated from Retention Volumes W . E. FALCONER and R. J. CVETANoVlk Division o f Applied Chemistry, National Research Council, Ottawa, Canada
b A number of hydrocarbons have been effectively separated from their partially and fully deuterated isomers on a 300-foot nonpolar capillary column. Relative retentions are reduced by 0.72% at 25” C., 0.61% at 50” C., 0.52% at 80’ C., and 0.49% at 105” C., per substituent deuterium atom. Boiling points have been determined for a number of deutero species by the chromatographic method. From the relative retention volumes at several temperatures, differences in the enthalpy, entropy, and free energy of solution for several partially and fully deu-
1064
ANALYTICAL CHEMISTRY
terated isomers have been calculated, and the values are reported. In view of the small differences in these quantities from the values of the corresponding light isomers, the chromatographic method is particularly suitable for such determinations, as they would be difficult to make accurately by standard techniques.
T
developed for the separation of hydrogen isotopes on adsorption columns at low temperatures (8,9,19) have riot been adequate for the separation of isotopically substituted ECHNIQUES
hydrocarbons. Despite the observation by Wilzbsch and Riesz (13) in 1957 that extensively deuterated or tritiated organic compounds (cyclohexane and methylcyclohexane) could be separated from their light isomers on a conventional GLPC column, the potential of such separations has not been exploited during the past five years. More quantitative information on the separation of hydrocarbon isomers of varying deuterium content in particular appears necessary. I n the absence of a dipole moment, deutero hydrocarbons generally have somewhat higher vapor pressures (and
Table
I.
Retention Times, Boiling Points, and Calculated Thermodynamic Quantities for Deuterated Hydrocarbons from Data at Several Temperatures
AGD -
rR.L
Hydrocarbon 25" C. Isopentane-dlo 0.927 2,3-Dimethylbutane-d1~ 0.917 2,3-Dimethylbutane-ds 0,957 3,4-Dimethylhexane-d18 , . , 3,B-Dirnethy1he~ane-d~. . . 3-Methy1, 1,s hexadiene-dlz .. . PMethyl, l-hexenedi I ... b
50" C. 0.937 0.928 0.969 0.889 0.943
80" C. 105°C. 25" C. ... 0.0073 0; 936 . . . 0.0069 '
0:904 0:912 0.952 0.956
0,935 0.940 0.920 0.929
0.0072 ... ...
... ...
... ...
ASD
-
AG, ASH Ca1.l Entropy A H s , - Mole Units (1 - r H * L ) / n d AHs, at at 50°C. 80°C. 105°C. B.P.,. " C . 7al./Mole50° C. 50°C. 0.0063 ... 25.5(27.9) -83 42 -0.38 0.0060 0 .'dd53 . . . .55.6 (58.0) -78 48 -0.39 0.0052 56.9(58.0) -95 20 -0.36 0 0062 O.'dd53 O,'dd4g 114.4 (117.7) -113 76 -0.58 0.0063 0.0053 0.0049 116.0(117.7) -60 38 -0.30
0.0054 0.0050 0.0057 0.0051
.. . ...
78.2 (80.0b)
-40
42
-0.25
84.5 (86.7)
-73
55
-0.40
Boiling points of light isomers taken from (Ila)are given in parentheses. Boiling point from ( I l b ) .
Table II.
Retention Times and Boiling Points for Deuterated Hydrocarbons from Data at Single Temperature 1-
V
20
19
IS
17
16
15
c Lz a
AGD - AGH, B.P.," a C. Cal./Mole 25 0.927 0.0073 -3.0 (-0.5)b 45 (25") 50 0.923 0,0064 24.8(27.9) 51 50 0.959 0.0068 26.3(27.9) 26 50 0.943 0.0057 57.6(59.5) 38 50 0.919 0.0058 60.5(63.3) 55 50 0.929 0 0059 91.4(93.7)c 48 50 0.923 0,0064 91.2(93.7)c 52 n's-3,Methyl-lJ5heptadiene-& 50 0.908 0.0066 108.4 (111,O)c 63 ,. Boiling points of light isomers, taken from (lluj,are given in parentheses. Relat,ive retention for n-butane-& a t 50" C. was taken as 0.938, t o allow for observed decrease in isotope effect with increasing temperature. c Boiling points from ( I l b ) .
Hydrocarbon n-Butane-dlo Isopentane-d12 ISOpentane-ds l,5-Hexadiene-dlo 3-Methylpentane-dla trans-1,5-Heptadiene-d12 cis-1,5-Heptadiene-d12
TH.L
rH.L
nd
9
ELUTION TIME I m i n u t e t l
Figure 1 . Separation of 2,3-dimethylbutane and isopentane isomers on a 300-foot squalane capillary at
25'
C.
lower boiling points) than their protio isomers (3,6). On the other hand, the polarity and chemical reactivity of the trvo species are probably nearly identical. Good chromatographic resolution should therefore be obtained with a nonpolar liquid phase where the separation is determined primarily by boiling point differences. Porter, Deal, and Stross (10) have demonstrated the soundness of the values of partition coefficients and heats of solution calculated from GLPC measurements. Although careful control of the critical column parameters is a prerequisite for obtaining highly accurate absolute thermodynamic values from such measurements, relative values can be determined from relative measurements of retention volumes alone. Since the retention volumes do not differ widely for deutero-protio pairs, the partition coefficient, and hence the enthalpy, entropy, and free energy of solution of a deuterated species can be calculated with an accuracy approaching
that of the values known for the light compound. EXPERIMENTAL
Separationswere effected on a 300-foot stainless steel capillary, 0.010 inch i d . , coated with squalane (2,6,10,15,19,23hexamethyltetracosane), with about 130,000 theoretical plates (Perkin-Elmer U column: 154-0457). The chromatography apparatus was a PerkinElmer Vapor Fractometer fitted with a flame ionization detector, with a response of 2 pa. per mg. per second. Samples (0.01 to 1pmole) were flushed onto the column from a sampling trap connected to a Perkin-Elmer Gas Sampling Valve in a helium stream. A 64 to 1 by-pass system was inserted between the injection system and the column. The flow rate through the column was 1.6 cc. per minute. RESULTS
Figure 1 shows the separation of 2,3 - dimethylbutane - dlz, 2,3-dimethylbutane - de, and 2,3 - dimethylbutane on this column a t 25' C. The resolution of these compounds is good, and it appears likely that by proper choice of column length and temperature, isomers differing by still fewer and per-
haps eventually only by one deuterium atom may be separated. By comparison, although 2,3-dimethylbutane& separated completely from its light isomer on a 50-foot dimethyl sulfolane on firebrick column with about 4OOO theoretical plates a t 25' C. (Figure 2), separation of the mixture containing the c16 isomer was incomplete under these conditions. However, extensive peak broadening was observed. Table I shows retentions of deuterated hydrocarbons relative to their light isomers ( I H , L ) for compounds which have been investigated a t more than one temperature. [The nomenclature and symbols are those recommended in reference ( I ) . ] The change in relative retention per deuterium atom is also listed in each instance [(I - r H . L ) / nd]. The enthalpies, entropies, and free energies of solution have been calculated by the methods of Keulemans (7) and Porter, Deal, and Stross (20). Table I1 lists a number of relative retentions and changes in relative retentions determined a t one temperature only. The boiling points of the deuterated species, included in both tables, have VOL 34, NO. 9, AUGUST 1962
1065
been obtained from a plot of log t t B against boiling point prepared by Bednas (8) from the investigation of about 50 commercially available hydrocarbons. To increase the accuracy of the boiling points relative to the light isomer, the curve obtained by Bednas (9)was shifted for each compound so that it passed through the accepted value for the light isomer (If). The boiling points of deuterated compounds obtained in this way are probably reliable to better than ztO.3’ c. (relative to the light value), if departures from ideality are the same for both solutesi.e., yo^ = ~ O L ,where y o is the activity coefficient a t infinite dilution. The deuterated samples used in this investigation were obtained as follows. n-Butane-&, commercial sample from Merck, Sharpe, & Dohme, Ltd. 3,4-DimethylhexanedI8, combination of sec-butyldg radicals, produced by the mercury photosensitized decomposition of n-butanedlo. 3,4-Diniethylhexane-dg, cross-combination of sec-butyl-& radicals with light sec-butyl radicals, produced by the simultaneous mercury photosensitized decomposition of light and heavy nbutane. 2,3-Dimethylbutane-dl*, combination of sec-propylde radicals, produced (6) by the addition of hydrogen atoms to propylene-d6. 2,3-Dimethylbutane-d6, cross-combination of sec-pr0py1-d~radicals with light sec-propyl radicals, produced (6) by addition of hydrogen atoms to a mixture of light and heavy propylene. Isopentanedlo, cross-combination of ethyl-& and sec-propyl-& radicals. The mechanism of formation of isopentane in the reactions of hydrogen atoms with propylene will be discussed in a separate publication (6). Isopentane-d8, cross-combination of ethyl radicals with sec-propyl-& radicals. All other deuterated hydrocarbons given in the tables were major reaction products (4) in the mercury photosensitized reactions of butene-ld8. The corresponding light products used for reference were produced by parallel reactions using light reactants, and their identities were verified by mass or infrared spectra, or retention times on a number of different columns, or by a combination of these techniques. Commercial samples were used as standards for comparsion in most cases. DISCUSSION
Partially and fully deuterated hydrocarbon isomers in the Ch to C8 range have been effectively separated from each other and from their protio counterparts using a commercially available chromatographic apparatus and a high resolution nonpolar capillary column. The feasibility of a given separation can best be determined from a consideration of the decrease in relative retention per deuterium atom-viz., 0.72 ( =tO.Ol)yo a t 25” C., 0.61 (zt0.04)y0 a t 50’ C.,
1066
ANALYTICAL CHEMISTRY
c
E a
a $
I
&,&!,A
60
55
5C
45
55
50
45
IO
5
0
ELUTION TIME lminulesl
Figure 2. Separation of 2,3-dimethylbutane isomers on a 40-foot dimethyl sulfolane column at
25’ C.
0.52 (=kO.Ol)% a t 80” C., and 0.49% a t 105” C. (Bracketed figures are experimental mean deviations.) Isomers differing by only one deuterium atom exhibit, therefore, characteristic retention times, and their complete separation may eventually be possible by proper choice of column parameters. On the 300-foot squalane column, separation was increased roughly twofold by a 25” C. reduction in temperature. In view of its speed and potential accuracy, the chromatographic method of analysis of isotopic mixtures should prove advantageous. Thus, for example, the statistical factor of two in the cross combination of isopropyl radicals is well illustrated in Figure 1 (CeH8De). Although adequate separations of isotopic isomers can be made on conventional columns when the difference in degree of substitution is large (IS), separation of pairs differing only slightly in the number of substituted deuteriums would require packed columns of extraordinary efficiency. However, the application of packed columns to preparative separations should not be overlooked. Capillary columns are to be preferred to packed columns for determination of boiling point differences and thermodynamic quantities of the isotopic isomers by the gas chromatographic technique. Extremely small samples can be introduced, and conditions approach ideality as the solutions of solutes in the liquid phase approach infinite dilution. In the present work, approximately 10-8 mole of sample was sufficient to give full scale recorder deflection. In addition to the great rapidity of measurements, a further advantage of this technique is that samples of a high degree of purity are not required. Davis and Schiessler (6) found that the heats of vaporization of perdeutero benzene and perdeutero cyclohexane were not far removed from those for the corresponding light isomers, but they were unable to assign meaningful numerical values to the differences using calorimetric or vapor pressure measurements. Similar difficulties in expressing small differences in large quantities arise in determining differences in entropies and free energies of solution. The values for differences in heats of solution ( A H s ) listed in Table I can
probably be assigned to a close approximation to differences in heats of vaporization ( A H V ) . The slightly lower values found for the deuterated species agree in direction and magnitude with those observed by Davis and Schiessler
(6). Fully deuterated hydrocarbons boiled about 2” to 3” lower than their protio isomers; differences for partially deuterated species are proportionally less. The present differences of about 0.2” C. per deuterium atom agree well with values found by conventional methods (3, 6). ACKNOWLEDGMENT
The authors are grateful to M. E. Bednas for assistance with the analyses performed on the capillary column, and to L. C. Doyle for help with some of the preparatory work. LITERATURE UTED
(1) Ambrose, D., James, A. T., Keulemans, A. I. M., Kovkts, E., Rock, H., Rouit, C., Stross, F. H., “Gas Chromatography 1960,” R. P. W. Scott, ed., p. 423, Butterworths, London, 1960. (2) Bednas, M. E., National Research Council, Ottawa, Canada, unpublished data. (3) Craig, D., Regenaas, F. A., Fowler, R. B., J. Org. Chern. 24,240 (1959). (4) Cvetanovif, R. J., Doyle, L. C., J. Chem. Phys. in press. (5) Davis, R. T., Jr., Schiessler, R. W., J . Phys. Chem. 57,966 (1953). (6) Falconer W. E.,Rabinovitch, B. S., Cvetanovik, R. J., National Research Council, Ottawa, Canada, unpublished data. (7) Keulemans, A. I. M., “Gas Chromatography,” 2nd ed., Reinhold, New Ynrk ---I, 195Q (8) Kwan, T., J . Res. Imt. Catalysis, Hokkaido University 8, 18 (1960). (9) Lee, J. K., Rowland, F. S., J . Chem. Phys. 32, 1266 (1960); J . Phys. Chem. 64, 1950 (1960). (10)Porter, P. E.,Deal, C. H., Stross, F. H., J. Am. Chem. SOC.78, 2999 (1956). (11) (a) Rossini, F. D., “Selected Values of Physical and Thermodynamic Properties of Hydrocarbons and Related Compounds,” Carnegie Press, Pittaburgh, 1953; (b) Beilstein, F. K., “Bedsteins Handbuch der orgenischen (12) Smith, H. A,, Hunt, P. P., J . Phys. Chem. 64,383 (1960). (13) WiLbach, K. E.,Riesz, P., Science 126, 748 (1957). Chemie.” RECEIVEDfor review March 5, 1962. Accepted May 23, 1962.
