and i were mixtures of two and three fluorwent compounds, respectively, all in small amounts. None of the unit lentified compounds is umbelliprenin, imperatorin, or xanthotoxin, all known to o w i r in this species (9,11). Phenolic Coumarins. Phenolic couniarins are normally removed during standard isolation procedures, together with other phenols, by extraction of an ethereal solution with dilute aqueous alkali. The problem of separating them chromatographically from neutral coumarins does not arise. Fewer of them are apparently found in nature than neutral coumarins, and it is rare t o find a species in which more than two have been reported. -45these coumarins tend to vaporize only with difficulty, excessively high temperatures were found necessary to elute them from even relatively short columns in reasonable times. The peaks usually tailed badly. A much more satisfactory approach involved acetylation of the free phenolic groups with acetic anhydride and sodium acetate, a reaction which is conveniently conclucted and gives excellent yields of the esters. The relative retention times of the five phenolic coumarin acetates studied are given in Table I, and a chromatogram of a synthetic mixture is reproduced in Figure 4. The separations were satisfactory except for daphnetin and scopoletin, which emerged together as a single peak. As no species appears to be known which contains both of these (9, I I ) , their failure to separate is not a practical drawback. High vacuum sublimation, over a 100’ to 220’ C. range, of phenolic reactions from Heracleum and Angelica
yielded in each case an oily material, which was acetylated and chromatographed on the silicone grease column. The Heracleum extract showed 10 minor peaks, but paper chromatography together with available authentic compounds in no case yielded any clue to identity. Several of the compounds were fluorescent, and could have been acetates of phenolic coumarins. The Angelica mixture also contained a number of minor peaks, and a major component with a relative retention time of 3.7 (umbelliferone = 1). When chromatographed on paper and then sprayed with 2N sodium hydroxide, it fluoresced a brilliant greenish-blue, and it may also be the acetate of a phenolic coumarin. At least two phenolic coumarins of which no samples were available occur in Angelica ( I O ) . Umbelliferone, which was expected in both sublimates on the basis of earlier reports (2, l o ) , was not detected, and paper chromatography of the solution before acetylation confirmed its absence from both mixtures. The techniques described in this paper, in conjunction with existing preparative and analytical methods, promise to be of value in investigations of naturally occurring coumarins. Isolation techniques used heretofore, even those employing column chromatography, often required kilogram quantities of dried plant. The use of gas liquid chromatography, together with paper chromatography, should permit a survey of coumarin patterns on less than 100 grams of dried plant or its equivalent of fresh material. I n most cases it should be possible to obtain fractions sufficiently pure for positive identification by mixed melt-
ing point, and for radioactivity determinations in biosynthetic studies. ACKNOWLEDGMENT
’The authors are indebted to the following for providing authentic samples of various coumarins used: A. Baerheim Svendsen, Oslo; Gerhard Billek, Vienna; Asima Chatterjee, Calcutta; Giovanni Rodighiero, Padova; G. H. S. Towers, Montreal. A number of helpful suggestions from B. M. Craig and E. von Rudloff during the course of this investigation are also gratefully acknowledged. The gas chromatograph employed was constructed by T. M. Mallard of the National Research Council laboratory at Saskatoon. LITERATURE CITED
(1) Baerheim Svendsen, A., “Zur Chemie Sorwegischer Umbelliferen,” p. 24,
Johan Grundt Forlag, Oslo, 1954. (2) Baerheim Svendsen, A., Otteatad, E., Pharm. Acta Helv. 32, 457 (1957). (3) -Brown, S. A., National Research
Council, Saskatoon, Sask., Canada, unpublished data, 1962. (4)Craig, B. M., Chem. & Ind. (London) 1960,i442. (5) Craig, B. M., Murty, N. L., J . Am. Oil C’hemistsSOC.36, 549 (1959). 16) Cropper, F. R., Heywood, A., Nature 172, 1101 (1953). ( 7 ) Zbid., 174, 1063 (1954). (8) Dean, F. M., Progr. Chem. Org. Nut. Prod. 9, 225 (1952).
(9) Karrer, W., “Konstitution und Vor-
kommen der organischer Pflanzenstoffe.” DD. 535. 554-5. Birkhauser Verlag, ~ t i t t g a r,’t1958. ’ (10) Xarasimhachari, N., Rudloff, E. M. von, Can. J . Chem. 40, 1123 (1962). (11) Spath, E., Ber. 70A, 83 (1937). (12) Stanley, W. L., Vannier, S. H., J . Am. Chem. Soc. 79, 3488 (1957). RECEIVED for review April 4, 1962. Accepted May 31, 1962.
Horizontal Chromatography Accelerating Apparatus Description of Apparatus and Applications J. F. HERNDON, H. E. APPERT, J. C. TOUCHSTONE, and C. N. DAVIS The Malvern Institute, Malvern, Pa.
An apparatus is described which permits horizontal chromatography, or ion exchange separations, to be carried out on strips of media, or circular disks. The apparatus permits these techniques to be accelerated at choice by the introduction of rapid solvent flow rate, heat, and centrifugal force. This apparatus has found use in accelerating a wide variety of chromatographic separations.
C
has been employed to accelerate horizontal paper chromatographic separation of compounds in mixtures ( I , 6, 7, 10-14). Recently, Tata and Hemmings (21) described centrifugally accelerated paper strip chromatography using an instrument of their own design. Roberts et al. (16-20)used elevated temperatures t o accelerate horizontal chromatograms. Williams (26) has speeded separation ENTRIFUGAL FORCE
in adsorption columns by centrifugation. Izmailov and Shraiber (8) originally described thin layer, open column chromatography-more recently improved by Kirchner, Miller, and Keller (9) and Reitsema (16). Tandem chromatography described by Tuckerman, Osteryoung, and Nachod (24) has introduced a novel means for accomplishing two-dimensional chromatography with strips. VOL. 34, NO. 9, AUGUST 1962
1061
All of these techniques have been used with a high degree of reproducibility with a new apparatus as described. This apparatus may be used for conventional or centrifugated horizontal chromatography with paper strips, columns, impregnated papers, or thin layers of adsorbents. Disks or circular chromatograms may also be developed. The chromatograms resemble those seen with conventional techniques but are very often superior. Five means are employed, together or alternately, to accelerate the chromatograms. They are: increased rate of solvent delivery; fast media; application of sample to the media in thin bands; centrifugal force; and elevated temperatures. Where strips of media are used, separation patterns can be examined by any suitable scanning device or means applicable to routine chromatography. Because of the design of the solvent delivery system and the multiple grooves in the plate, different kinds of media may be used in a single run, or both paper and adsorption columns may be developed simultaneously.
a
R/
9
Figure 1 . Cutaway drawing of horizontal chromatography accelerating apparatus with solvent delivery system a.
b. C.
d. EXPERIMENTAL
Apparatus. The apparatus (Figure 1) consists of four principal parts: A . A vapor proof chromatographic housing. B . A Teflon coated aluminum plate 31.5 em. in diameter, which has a central depressed area and eight grooves inch wide for holding strips of media or columns. This Teflon coated aluminum plate can be reversed for wedges, wide strips, or circular paper chromatograms. The plate is mounted on a supporting post which is connected to a variably controlled motor. C. A solvent delivery housing located on the center of the Teflon coated plate distributes developing solvent uniformly and evenly. When strips are being developed, the solvent flows down the individual strips within the radial grooves; or where disks or segments are used, the solvent flows out from the center toward the periphery in an ever increasing elliptical pattern. D. A device for delivering the solvent to the reservoir. The device for delivery of solvent consists of a modified separatory funnel equipped with an air inlet to provide for a constant head of pressure. A glass tube is fitted inside the stem of the funnel for observation of the drop rate. A hypodermic needle is attached to the stem and the solvent dripped into the solvent reservoir. Recently a device has been developed which employs a closed buret with siphon side arm enclosing a solenoid valve that can be automatically operated to give a predetermined intermittent or continuous solvent delivery. An instrument with the fully automatic solvent delivery system was used. 1062
ANALYTICAL CHEMISTRY
e. f. g.
h. I.
i. k.
m. n.
P.
9.
Stopper and constant pressure tube Solvent reservoir Stopcock Glass dripping outlet for observing drop delivery Glass funnel 18-gage stainless steel hypodermic needle and polyethylene stopper Polyethylene stopper Solvent delivery housing Grooved plate Gloss cover plate Variably controlled motor Drive shaft Variable control which permits speeds between 100 to 1000 r.p.m. Rotation speeds were measured by strobe Strip holding grooves. Solvent delivery housing (h) and strip holding plate (i) are mounted on motor shaft (m). To carry out circular chromotography on disks, the strip holding plate is inverted Stainless steel unit which houses '/B-hp. motor ( k )
Heating was accomplished by a thermostatically controlled heating bulb located within the housing. Temperatures could be raised to 38-40' C. and maintained during the experiment. Paper Strip Chromatograms. A fiber glass or filter paper washer, 4.32 cm. in diameter, is placed in t h e center depression of the grooved plate. Filter paper or ion exchange paper strips, 11 mm. by 14 cm., are then placed in the grooves.' The strips must overlay the washer a t least 5 mm. The strips should be cut with the fibers linear to the machine direction of the paper. A second fiber glass or filter paper washer is placed over the strip ends. These washers are predampened with the developing solvent before the solvent housing is set in place. The central ends of the paper are held in place by the weight of the solvent reservoir. The peripheral ends are
held in place by the ledges located a t the longitudinal edge of each groove. The mixtures for separation are applied in a thin band equidistant from the central reservoir housing. Reference samples are banded on opposite strips. The strips can be developed in the conventional way or rotated a t speeds up to 900 r.p.m. with or without heating (38-40' C.). McDonald, Ribeiro, and Banaszak (14) and Tata and Hemniings (21) have described an apparatus with similar features. Circular Paper Chromatograms. The nongrooved side of the plate was used to support media for circular chromatograms. The circle or segments are held in place by the solvent delivery housing. The materials to be separated are banded equidistant (1 cm.) from the solvent housing. Conventional or centrifugated separations can be effected with or without heating. Open Column Chromatograms. Various adsorbents, such as silicic acid, starch, and alumina can be applied on paper (Whatman No. 17 or Schleicher and Schuell No. 470) and developed as described for the paper strip chromatograms. Closed Column Chromatograms. Adsorbents are packed in glass tubes (3,5, or 6 mm. diameter by 13 cm. long) or rectangular plastic tubes (2 X 8 mm. and 4 X 8 mm. by 12 em. in length). A triangular wick of Whatman No. 17 paper is placed in the head of the column with the trailing end lying between the disks located in the solvent housing in the same manner as for strips. The columns are held in place by adhesive tapes. Development is carried out with centrifugation. The technique resembles that described by Williams ($6). RESULTS
The factors that influence horizontal chromatography as carried out with this apparatus are: rate of solvent delivery which affected distance traveled by the solvent front, nature of the medium, degree of centrifugation, and type of solvent. Temperature varixtions from day to day may cause changes in the results. A reduction of delivery rate increased separation time and an increase of delivery rate reduced it; however, the maximum that a separation can be speeded is limited by the absorbency of the medium. Fast media such as Whatman No. 17 and 31 and Schleicher and Schuell No. 470 permitted a very rapid rate of solvent delivery. The solvent delivery could be easily controlled and made highly reproducible when drop rate was manually controlled by use of the stopcock and stopwatch. The data presented herein were obtained using a commercially available automatic feed system which delivered a constant volume of solvent. The use of a separatory funnel device for dripping solvent as described by Bersin
n Lit
not further reduce separation time. As illustrated in Figure 3 slow solvent delivery with higher speeds of centrifugation may result in a decrease of migration of the solvent front. With organic solvents higher rotational speeds may result in a decrease of front movement due to evaporation of solvent from the paper. The use of centrifugal chromatography thus will require selection of the optimum conditions of solvent delivery dependent upon the media used and the nature of the solvent itself and the compounds to be separated. As illustrated in Figure 4, a t a given solvent delivery rate there is a linear relationship between the migration of the solvent front and time. Effect of Temperature on Separations. Heat greatly facilitated the separation of carbohydrates on strip chromatograms. The separation of arabinose, glucose, galactose, raffinose, and maltose occurred a t 38" C. This separation could not be obtained with the same conditions at 20" C. either with or without centrifugation. This finding concerning the beneficial value of heat is in agreement with those described by Roberts et d. (16-20). The effect of temperature on evaporation of solvent from the strips during development must be controlled by presaturation of the chamber, even during conventional operation without heat. Comparison of R, Values Obtained with and without Centrifugal Force. The reproducibility of a separation with a given set of experimental conditions depended upon the centrifugation used with a specific solvent delivery. The standard error of an Rj of 0.36 with the conditions described in Figure 3 was =k0.008when 700 r.p.m. was used with a solvent delivery of 0.05 ml. per minute; it was =k0.002 when the solvent delivery was 0.07 ml. per minute. In the caae of Rj values as determined at the optimum conditions for separation of methyl red using the experiments illustrated in Figure 2, the Rf values with centrifugation tended to be lower than those obtained without centrifugation. The greatest changes in R f values occurred when highly volatile solvents were used with centrifugation. The R, values although constant were decreased with increase in centrifugal force. Mixed Media Chromatography. By attaching chromatographic strips in tandem with other chromatographic strips, ion exchange strips, or cellulose derivative paper strips, a wide variety of separating media are obtained. This technique is related to that described by Boggs in 1952 (4) and Tuckerman, Osteryoung, and Nachod (84). This tandem technique was employed to effect the comparable
" 1
50 ? 4 0
I $30 2 0
i:rB 0 0
3 0 0R P Y
500
700
Figure 2. Relationship between centrifugal farce and movement of volatile solvent
Time required for 5-cm. movement of solvent front of NH4OH-n-propanol, 80:20, a n Whatman No. 4 paper, at different rotational speeds i s given. Each point represents average of 2 4 replicates. With a 20-gage needle, one drop/ 7 seconds gave 0.12 ml./min.; one drop/l3 seconds gave 0.09 ml./min.; and one drop/l7 seconds gave 0.07 ml./min. Chamber (20.0' C.) was presaturated with 3 ml. of solvent
and Muller (2) has proved useful. For optimal sharpness of separation as well as duplication of results, a balance between solvent delivery and movement of solvent on the strips must be obtained. Delivery rate depends on size of outlet and solvent used. Temperature control and presaturation of the chamber are necessary. Figures 2 and 3 illustrate correlation between movement of solvent front, time, and centrifugal force. At rotational speeds below 300 r.p.m., puddling of the solvent occurred with fast solvent delivery. Using centrifugal force and Whatman No. 1, 3, and 4 papers, separation times were greatly reduced. On faster papers, Whatman No. 17 and 31 and Schleicher and Schuell No. 470, centrifugal force did -
n
1
t
.-I
I
1
I
i
.I
O I
0
300
500
100
RPY
Figure 3. Relationship between centrifugal force and movement of aqueous solvent Time required for 5-cm. movement of solvent front of 296 NaZC03 on Whatman No. 1 paper, at different rotational speeds, is given. Each point represents average of 2 4 replicates. With a 22-gage needle, one drop/5 seconds gave 0.09 ml./min.; one drap/7 seconds gave 0.07 ml./min.; and one drop/9 seconds gave 0.05 ml./min. Chamber (24' C.) was presaturated with 3 ml. of solvent
i0
" 3
2
6 YlNUTES
4
8
$2
10
Figure 4. Relationship between time and movement of solvent front and speed of migration
Movement of solvent front from origin on Whatman No. 1 paper was measured at speed of 500 r.p.m. employing 2% Na2COb Each point represents average of 2 4 replicates. With a 22-gage needle, one drop15 seconds gave 0.09 ml./min.; one drop/7 seconds gave 0.06 ml./min. Chamber (24' C.) was presoturated with 3 ml. of solvent
separations of two-dimensional chromatography. Bands of separations were cut from previous runs, dried, and attached in tandem to new media to effect further separations. This was particularly effective with amino acids (6),dyes (23), and indole and phenolic acetic acid derivatives (29). Circular Chromatography. Using the apparatus as a chamber for horizontal chromatography, good separations on filter paper disks, with filter papers of various grades, were obtained, both with and without centrifugal force. Under these circumstances, the best separations are obtained with Whatman No. 1, 4, and 3 MM papers. As was true in the case of strip chromatography, the technique of dripping the solvent into the center housing permits ease of effecting separations and good reproducibility. Closed Column Chromatography. Centrifugal force permitted uniform packing of adsorbents in closed columns, and the resulting chromatograms were very discrete. Rotational speeds ranging from 500 to 800 r.p.m. are necessary to move developing solvents through this type of packed column. Loosely
f
i I
,
,
,
I
0
I
7.
3
4
,
,
,
?
5 6 7 8 CENTIMETERS
S
I
'
l
9
10
11
12
Figure 5. Separation of arginine, glycine, valine, and histamine H. Reeve Angel Amberlite cation exchanger p a p e r w e a k ocid type; containing amberlite IRC-50 resin; hydrogen form was used. Solvent was 0.025M sodium acetate, at rate of 0.04 ml./min. (drop/l3 seconds-22 gage needle). Rotational speed was 550 r.p.m. for 2 0 minutes. Chamber (27' C.) was prdsaturated with 1 0 ml. of solvent. Developed with ninhydrin (3) VOL 34,
NO. 9, AUGUST 1962
1063
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
