Some Improvements in Chromatographic Techniques for Terpenes JOHN M. hIILLER AND J. G . KIRCHXEH Fruit and Vegetable Chemistry Laboratory, C’. S , Department of Agriculture. Pasadena, Cali$.
IQUID chromatograms have been used a t various times in separating mixtures of colorless compounds. To locate the zones as they are washed into the filtrate, various procedures have been employed: evaporation of aliquots of the percolate and subsequent determination of melting points and equivalent weights of acids of the residue (Z), changes in pH (3,G), changes in conductivity ( d l ), ultraviolet absorption and ultraviolet absorbency ratios ( I ) , polarimetry (Zd), paper chromatography (IG), and changes in refractive index (4, 7,8,10,12,17-d0). I n working with liquid chromatogram of the colorless, volatile flavoring constituents of citrus fruits, it became desirable to have a method which would not only indicate when a new fraction appeared in the filtrate, but \\-auld also indicate the purity of that fraction. The “chromatostrip” method of Kirchner, Miller, and Keller (13) offered possibilities in this respect and was investigated with this object in mind. .Ipplication of the chromatostrip to liquid chromatograms showed that the manner of packing the columns is very important in obtaining uniform zones whose boundaries are perpendicular to the direction of travel without coning, and without the w a v ~ boundary which is sometimes a feature in the ordinary chromatogram. Packing the dry adsorbent with a tamper yields a column which often exhibits the familiar coniiig (24). The other alternative, packing by means of a slurry, has been used to some extent ( 3 , 6, 9, 14, 16, ZS), but the authors found that certain details of slurry packing must bc adhered to in order t o obtain strictly uniform packing. ZOh-E DETECTION BY CHROMATOSTRIPS
material. At least one batch of silicic acid contained watersoluble acids which interfered with the ultraviolet and fluorescein tests on the chromatostrips. For these reasons the adsorbent is washed with acetone and then with water. The washed adsorbent is mixed with 5% of its dry weight of Amioca starch and 200% of its dry w,ei ht of water. This slurq. is then hedted in a water bath a t 85 until it thickens. The paste is spread to a thickness of 0.25 inch on glass plates and dried for 2 hours a t 80’ C. in a forced draft oven. During the drying period it is protected from atmospheric contamination by sealing the oven tightly and introducing air that is filtered through a silicic acid pad. (With the commercial forced draft oven available, it was necessary to place a packing gland around the fan shaft.) The dried adsorbent is powdered in a ball mill .for 30 minutes and stored in a vacuum desiccator at 3 mm. of mercury over ascarite. The adsorbent, treated as just described, is slurried with four to five timee its weight of the solvent to be used for development. The well-stirred slurry is then poured into the glass column arid vigorously stirred again. Any adsorbent on the sides of the,glass is washed down FTith solvent, because if allowed to remain, it dries and “cakes” there. To eliminate this dificulty, the slurry may be poured into the column through a funnel designed to minimize stirring of the packed portion of the column. After the slurry is added, pressure is applied to the column by means of nitrogen to pack down the material and to remove excem solvent. ( I n using the volatile solvents which appear necessary for separation of terpenes, it is inadvisable to use too great a pressure on the column; otherwise when the pressure is released t o add the solution or to add more solvent, the dissolved gases in the solvent expand rapidly and break the column of adsorbent. With volatile solvents, pressures of over 5 pounds per square inch should be avoided. Readily soluble gases such as carbon dioxide are undesirable RS pressure sources for the same reasons.) Grritle
8.
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In a systematic separation by means of a liquid chromatogram, the effluent is collected in suitable fractions, the size of the fractions depending on the proportionate amounts of the components and on the proximit’y of the fractions to one another. These factors, and a determination of the most satisfactory solvent, are established by means of chromatostrips prior to running the column. The course of the chromatographic separation is followed by checking every fifth fraction (or other convenieiit, nuniber) with a chromatostrip by spotting a small amount of eluate a t the origin of the strip and developing it in the same solvent as that used for the column. On completion of develop ment, the zones will then determine the location of these materials in the eluate portions. JVhenever a fraction is located which contains more than one compound, additional fractions on both sides are checked to find the starting points of the pure fractioIic. The eluate fractions can then be combined as indicated by the chromatostrip tests.
ESSENTIAL
c-
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BEST SOLVENT FOR THE SEPARATION 15 SELECTED. 0Y CHROMATOGRAPHY ON CHROMATOSTRIPS’ STARCH TREATED SILICIC ACID ADSORBENT
PREPARATION OF ADSORBEhT AIiD PACKING OF COLUMh5
It has been found in this laboratory that the silicic acid obtained for chromatography frequently contains a yellow oily matelial abich is eluted from the column by some organic solvents such as ethyl acetate or acetone. Blank runs made with thit silicic acid resulted in the collection of 50 to 100 mg. of this oily material from 100 grams of adsorbent. Solvent-extracted silicic acid dried in the atmosphere or in the ordinary mechanical convwtion oven readily picked up additional quantities of oily
UFigure 1. Use of Chromatostrips for Detection of Zones and Zone Purity 1480
V O L U M E 2 4 , NO. 9, S E P T E M B E R 1 9 5 2
1481
tapping of the Rides of the column aidids in uniform settling. By selecting the proper amount of eluate to be placed OD the Successive portions of the slurry me added to the column its strip for chromatographing and using this Same amount lor testa each section is Dncked dawn. (The actual auiEnt,it,v ~~~"in eneh .~~~~ of succeeding portions, the location of zone boundaries is esportion Seem8 td he immaterial, 'and if reservoir is provided pedited, since the front edge of a fraction contains larger quantiat the top, the slurry may be added all a t one time.) Each ties of material than the trailing edge. Because of this, larger, more positive zones appear on the chromatostrip prepared from a fraction on the leading edge than from the trailing edge. \There removed by pressure to the level of the a d n o z e h , t,he solution is then forced into the adsorbent by pressure, and fresh solvent two compounds have very similar R, values, the appearance of an is carefully poured on the top. intense spot followed by a fading out. and then the reappearance of a strong test indicates the emergence of 8 new fraction. BeBefore treatment, a 32 X 300 mm. column of silicic acid a i t h C&UE of the rapidit,], with which large numbers of chromatostrips hexane as solvent passes about 1 ml. of solvent per 3 minutes may be run, the checking of zones by this method does not add under 1-pound pressure; after treatment, a 32 X 300 mm. column appreciably to the time required to run a column. will pass about 3 ml. of solvent per minute. This enhancement A B of solvent flow is apparently accomplished without increase in particle size. Before treatment, 80% of the adsorbent passes a 200-mesh screen in contrast to 90% aft,ertreatment. The volume occupied by the adsorbent in a packed column is not changed by the treatment with starch. The moisture content, determined with Karl Fischer reagent, of thesilicic acid which has been dried at 80" C. averages about 5.5%. This degree of dryness of the adsorbent has been found to give good separations of the terpenes without undesirable reactions such as polymerization or isomerization.
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a
~~~
SEPARATION OF SOME TERPENES
T o illustrate the use of these techniques the following examples were selected: Separation of the reaction products from the reduction of carvane to carveal by aluminum isopropoxide: On a 32 X 300 mm. silicic acid column, 13.6 grams of oil, obtained by ether extraction of the reaction mixture, were adsorbed. Development was a;ocomplished with 15% ethyl acetate in hexane. The eluted fractions were collected in test tubes in approximately 2-ml. volumes. Tubes 1 to 21 contained 4.95 grams of unreduced carvone, tubes 22 to 36,0.6 gram of a mixture of carvone and carveol, tubes 37 to 75, 1.62 grams of carveol. Purification of linalool monoxide: On a 32 X 300 mm. column of silicic mid, 0.8 gram of product, obtained from the reaction of perbenzoic acid with linslo81, was adsorbed. The flowing chromatogram W B I ~developed with 10% ethyl acetate in chloroform. Tubes 1 t o 3 contained traces of a mixture of perbeneoio acid and unreacted linalool. Tubes 7 t o 50 contained 418.9 mg. of linalool monoxide. Tubes 51 ti59 contained solvent only, and tubes 60 to 93 contained 58.2 mg. of an unidentified crystalline compound. Since the object of this experiment was to isolate the Durified linrtlotll monoxide. the remainder of the column was disca'rded. Purification of N-methyl methyl anthranilate: On a 32 X 300 mm. column of silicic acid, 2 grams of a commercial preparation of N-methyl methyl anthranilate were adsorbed and developed with 15% ethyl acetate in hexane. Tubes 2 to 22 contained 1.88 grams of N-methyl methyl anthranilate, tubes 23 to 30, 0.03 gram of a mixture of methyl anthranilate and the dimethyl anthranilate, tubes 30 to 40, 0.06 gram of r-LL-' . . ' I . . . : ' ' RESULTS AND DISCUSS,ION
The Dnethods ordinarily used for the deter:tion of zones emerging .,a no+.n m r i o r l "l*e*Ee. into the eluate in liquid chromatograms ha.ful in the chromatography of terpenes or terpene derivatives. Usually the zones follow so closely upon one another and are so similar in physical characteristics t h a t physical measuremenbs do not give a sharp indication of zone boundaries. When two zones are separated by only inch on the column, a small to amount of effluent may contain material from bath zones, because of mixing after leaving the adsorbent, Where the ultimate aim is separation and purification of the oomponents of a misture, it is desirable to be able to isolate theso small amounts of efRuent mixtures which are obtained between pure fractions. The use of chromatostrips for following the progress of a liquid chromatogram has proved very satisfactory for locating zones in the resolution of the components of essential oil materials. Figure 1 is a schematic representation of a typical experiment.
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111~"11
Figure 2. Axial Sections of Columns to Indicate Differences in Zones of Slurry-Packed and Dry-Packed ro1unIns Y
A. SI"my- poked B . Dil'?-palckod
The method of slurry filling of the column results in a great improvement in the evenness of the zones on the column. Figure 2 compares two methods of column packing: Column A was packed by the slurry method, while colunm B was packed by the conventional dry-pack metbad. Both columns were placed in Lucite tubes and sectioned lengthrvise on completion of develop ment. The relative thinness of the adsorbent-solvent slurry influences the evennem of packing, a thick slurry preventing even settling of the ad sorbent with resulting distortion of the zones I n z similar manner the stirring of each portion of slurry after it has been added to the column cannot be omitred xvithout obtaining uneven zones. Extreme care must be exercised in introducing both the solution to he chromatographed and the developing solvent, because the slighted disturbance of the surface of the adsorbent will result in uneven zones. I f such irregularities do occur in starting the chromatogram, they may he evened out by quickly releasing the pressure and stirring the adsorhent uniformly and evenly
1482
ANALYTICAL CHEMISTRY
down to the level to which the mixture has run. T o eliminate disturbances of the surface when adding solution to the column, a special device used. This consisted of a funnel with the stem end cut off at right angles to the stem and with a flat piece of glass, inch in diameter, fused across t'he end by means of three g l a s bead?. This provided a baffle t o divert the descending fluid into horizontal flou-. In determining the proper conditions for packing columns and for adding mat,erial and solvent, a visible check was provided by using a dye, Calco Oil Blue N.A., with 2.5% isopropyl ether in hexane as the developing solvent (Figure 2). -432 X 300 nun. column contains 125 grams of adsorbent and requires approximately 1 hour to pack by the slurry method. Although parking bj- this method is slower than by tamping the dry adsorl)ent, the greatly improved evenness of zones justifies the additional time in packing.
Hagdahl, L., Acta Chem. Scand., 2 , 574 (1948). Holmes, H . N . , Cassidy, H., AManly,R. S., and Hartzler, E. R., J . Am. Chern. Soc., 57, 1993 (1935).
Hurd, C. D., Thomas, G. R., and Frost, A. A , , I b i d . , 72, 3733 (1950).
James, A. T., Martin, A. J. P., and Randall, S.S..Biochem. J . , 49, 293 (1951).
Jeffrey, R. K., ASAL. CHEX, 23, 936 (1951). Kirchner, J. G., Mller, .I. M.,and Kelier, G. J., Ibid., 23, 490 (1951).
Levy, A. L., Chemistry & Industry, 1945, 380. Partridge, S. XI,,Biochem. J . , 44, 521 (1949). Schwab, G. hl., and Jockers, K., d n y e w . Chem., 50, 546 (193i). Tiselius, A . , A r k . Kerni iMineral. Geol., 14B, No. 22, 1 (1940). Ibid., No. 32, 1 (1940). Tiselius, A., . l ' a t u r ~ i s s e n s c h ~ n37, , 25 (1950).
Tiselius, A , , "The Svedburg 1884-1944," p. 370, Uppsala, illmqvist and Wiksells, 1944. Tiselim, A., and Claesson, S., A r k . Kenii Mineral. GeoZ., 15B, h-0. 18, 1 (1942).
Whistler, R. L., and Durso, D. F., J . A m . Chem. SOC.,72, 677 REFERENCES
(1950).
Rei,oza, >I., ASAL. CHEM.,22, 1507 (1950). Cassidy, H. G., J . Am. Chem. Soc., 63, 2735 (1941). Castle, D. C., Gillam, A. E., Heilbron, I. M., and Thompson, H. XT-., Biocheni. J . , 28, 1702 (1934). Claesson, S., A r k . Kemi Mineral. Geol., 23A, N o . 1, 1 (1946). Cleaver, C. S., Hardy, R. 9., Jr., and Cassidy, H. G., J . Am. C'hmn. Soc., 67, 1343 (1945). Euler, H., and Schlenk, F., 2. physiol. Chem., 246, 64 (1937). Glcnn, R. .I.,Wolfatth, J. S., and DeU'alt, C. W., Jr., ANAL. ('HEM., 24, 1138 (1952).
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Zechmeister, L., and Cholnoky, L., "Principles and Practice of Chromatography." p. 67, New York, John Riley & Sons, 1941. RECEIVED for review iMarch 4. 1952. Accepted April 2 6 , 1952. Report of a study made under the Research and Marketing Act of 1946. T h e mention of special instruments or materials throughout this paper does not imply t h a t they are endorsed or recommended by the Department of Agriculture over others of a similar nature not mentioned.
Radiocarbon Combustion and Mounting Techniques ERSEL A . EVANS AND J. L. HUSTON Oregon State College, Corvallis, Ore.
THE course of H-ork on exchange reactions in the solvents 1Sacetic acid and acetic anhydride ( 4 ) , and on an isotope effect
on the rate of combustion of sodium acetate by chromic acid (3). useful modifications were developed of published procedures for performing wet combustions t o give barium carbonate, and for mounting barium carbonate in very small amount for radioassay. In preliminary work on wet combustions, much difficulty was encountered with blanks due to carbon dioxide from the air and t o sulfur trioxide evolved from the Van Slyke-Folch oxidizing mixture, the latter difficulty being especially troublesome because sodium acetate was difficult to oxidize and required considerable heating of the oxidizing fluid. Figure 1 showe the apparatus used t,o eliminat'e these blanks. The design of this apparatus was based largely on that described by Lindenbaum et al. ( 6 ) , but differed from theirs in permitting more convenient introduction of fikered barium hydroxide solution and in dispensing with a bubbling tube, carbon dioxide being absorbed in vacuo. It !vas also designed to permit positive elimination of barium sulfate blanks by redistillation of carbon dioxide. The authors believe the use of barium hydroxide solution is more convenient than the use of carbonat'e-free sodium hydroxide, but the use of the latter reagent m-ould be preferable if it were desired to determine the amount of carbon dioxide evolved as well as t o make a sample for radioassay ( 2 ) . Combustion took place in the lo\yer flask, C, the stopcock, S3, and ground joint of which were lubricated with a mixture of phosphorus pentoxide and phosphoric acid; all other ground surfaces were lubricated xvith stopcock grease. The sample (usually about 50 mg.) was placed in this flask, the apparatus was assembled and evacuated t,hrough stopcock S2 by means of an aspirator, barium hydroxide solution was admitted from one of t h r upper cups, J , through its sintered-glass filter, and 82 was closed. The c,ombustion fluid was then admit,ted to the lower flask by means of S3. S o potassium iodate was used ( 2 ) . The large stopcock, F , could be used to allow the combustion flask to communicate with either side; the apparatus was designed t o permit taking two barium carbonate samples when
the isotope rate effect was being studied, and could be simplified when used for simple routine combustions alone. The combustion flask was heated with a small flame for 10 to 15 minutes (a much shorter period would be required if a substance more readily oxidizable than sodium acetate were being used), and was allowed to stand another 15 minutes, the barium hydroxide solution being stirred all the while. The stirring was done by means of a small glass-enclosed bar magnet, actuated by a Precision Scientific magnetic stirrer. Barium carbonate free of barium sulfate was now prepared as follows. P was turned to close off the barium hydroxide from the combustion flask, air was admitted, and this flask was removed and replaced by another flask which was exactly alike except that the side cup contained a sintered-glass filter. After re-evacuation, barium hydroxide solution was filtered into this flask, hydrochloric acid was run in from the upper cup, J , and the evolved carbon dioxide was absorbed as before. When the absorption was complete the lower flask wvns removed and its contents were filtered quickly. Rather than follow the normal procedure of preparing Van Slyke oxidizing fluid by heating a mixture of fuming sulfuric acid, sirupy phosphoric acid, and chroniium trioxide ( 7 ) to 150" C. to accomplish solution of the chromium trioxide, oxiFigure 1. Wet Combustion dize organic mateApparatus
