ANALYTICAL CHEMISTRY
420
toses, a solvent with ratio about 0.54 would be used. For sugars behaving like the oligosaccharides, assumed to be DP 7 and 8 in mixture A, a solvent ratio of about 0.8 would be preferable. The selection of a solvent combination is aided by the fact that nearly the same results are obtained over a moderate range of solvent ratio Rp maltose-RF glucose. This is of particular value when only one pair of sugars in a mixture have very similar RF values. The solvent chosen to separate this pair best will generally give very satisfactory separation of the other components of the mixture. The upe of the multiple development technique will depend on the approximate Rp value of the sugars to be resolved, as maxi1
mum resolution will be obtained in -
RF
- 1 developments.
Thus,
when chromatograming pentoselike sugars with solvents of RF maltose-RF glucose ratio about 0.54, no more than two developments will be profitable. With the oligosaccharides such as DP 7 or 8, on the other hand, multiple development was essential for separation into distinct spots, the positions and separations of which are indicated in Tables I1 and 111. ACKNOWLEDGMENT
The authors are grateful to Edna Montgomery and F. B. Weakley (18), who furnished the isomaltose [6-( a-D-gIucopyranosy1)-~-glucose], Ivan .4.Wolff and Paul R. Watson for supply-
ing purified gentiobiose, Marjorie J. Austin for the mixture of oligosaccharides, and the Research Department of Hiram Walker and Sons, Inc., for the crude fusel oil. LITERATURE CITED
(1) Atkinson, H. F., Nature, 162,858 (1948). (2) Bates-Smith, E. C., and Westall, R. G.. Biochm. Bwphys. Acta, 4,427-40 (1950). (3) Calkins, F. C., J . Chem. Education, 23,604 (1946). (4) Chargaff, E., Levine, C., and Green, C., J . Biol. Chem., 175, 67 (1948). ( 5 ) Clegg, D. L., A N A L . CHEU.,22,48 (1905). (6) Dimler, R. J., Bachmann, R. C., and Davis, H. -4., Cereal Chem., 27,488 (1950). (7) Edman, P.. Arkiv Kemi, Mineral. Geol., A22, No.3 (1946). (8) Hanes, C. S., and Isherwood, F. A, Nature, 164,1107 (1949). (9) Hough, L., Ibid., 165,400 (1950). (10) Jermyn, M. il., and Isherwood, F. A., Bkochen. J . , 44, 402 (1949). (11) Ma, R. M., and Fontaine, T. D., Science, 110, 232 (1949). (12) Montgomery, E. &I., Weakley, F. B., and Hilbert, G. E., J . A m . Chem. SOC.,71,1682 (1949). (13) Partridge, S. M.,Biochem. J . , 42,238 (1948). (14) Sumner, J. B., J . Biol. Chem., 62,287 (1925). (16) Trevelyan, W. E., Proctor, D. C., and Harrison, J. S., Nature, 166,444 (1950). (16) Williams, R. J., and Kirby, H. 51..S c h , 107,481 (1948). RECEIVED Septcinber 7, 1950
Separation and Identification of Some Terpenes by a New Chromatographic Technique ,J. C,. KIRCHNER, JOHN 31. MILLER, AND G. J . KELLER Bureau of .4gricultctral arid Industrial C h e m i s t r y , U . S . Depurtment of 4gricultrrre, Pasadena, Calif. 4 chromatographic method for separating terpenes was developed for the determination of the volatile flavoring constituents of citrus fruit. This new technique in organic chromatography has been introduced by using adsorbent-coated glass strips in a manner analogous to paper chromatography. After the mixture had been spotted near one end of the strip, the chromatogram was developed with the aid of capillary attraction by dipping in a suitable solvent. The sol*ent was then evaporated from the strip and the various zones were indicated by spraying with suitable reagents. On spraying with a
I
K T H E course of determining the volatile flavoring constitu-
ents of citrus fruit, the authors desired to develop a chromatographic method for the purification and identification of terpenes. The small amounts of oil obtainable from the fruit made this almost imperative, and in addition it was desirable, because of the nature of the terpenes, to eliminate any form of heat treab ment. Although a considerable number of papers (16, 17) have been published on the chromatography of triterpenes, very little has been done on the chromatography of the simpler terpenes, posFibly because of the lack of a suitable indicating reagent The work on the simpler terpenes, and most of the work on the sesquiterpenes, has been done by arbitrary separation of fractions of the eluting solvent. Winterstein and Stein (16),Carlsohn and Muller (9),and Spath and Kainrath ( I S ) separated a few of the simpler terpenes by chromatography. The advent of paper chromatography has seen a great increase
fluorescein solution and exposing to bromine vapor, compounds which absorb bromine faster than the fluorescein show up as yellow spots on a pink background. Very unreactive compounds can be located by spraying with a concentrated sulfuric-nitric acid mixture and heating to cause charring of the compounds. The technique can he applied to other types of compounds and is a rapid method of checking solvents and adsorbents for use on larger chromatographic columns. The term “chromatostrip” has heen suggested for application to these adsorbent-coated glass strips.
in the use of chromatographic techniques Applications of paper chromatography are limited and it was soon evident that ordinary filter paper was unsuitable for chromatographing terpenes. Impregnation of filter paper Kith various adsorbents has been used to increase the adsorhing strength of the paper both for organic chromatography ( I , 4, 5 , 8) and for inorganic analysis (6, 7), but is limited by the relativelv small number of adsorbents that can be used. In contrast to paper chromatography, column chromatography has a distinct disadvantage in work with colorless compounds, such as thr terpenes, becauqe of the difficulty in locating the zones. Fluorescent rompound8 can be located with ultraviolet light and the method of Sease ( I d ) can be used to advantage for many ultraviolet absorbing compounds. The authors desired to combine the advantages of paper and column chromatography in order to obtain a rapid chromatographic method to which zone-indicating developers could be
V O L U M E 23, NO. 3, M A R C H 1 9 5 1
421 he prepared without the addition of filter aid; this proved desirable because the strips containing Celite were weaker in sdsorptive power than the strips containing only adsorbent and binder. Therefore, the mixture contained 19 grams of adsorbent, 1 gram of Amioca starch, and (as fluorescingagent deaeribed later) 0.15 gram of zinc silicate and 0.15 gmm of ainc cadmium sulfide.
1
Preparation of suitahle strips involved careful attention to the details of preparation. The major difficulties were the cracking of the adsorbent and a surface that was too soft. To solve Clie difficulties, the followingprocedure was used: The specified amounts of matorid. thorouehlv blended while
2
beaker was rembved from the bath and 2 To 7 ml. of w&r 3 ~
~
~
.~ .... . -..
~~~~~
raphy has been employed by coating sheets of glass and devclo;ing in the name manner as filter paper.) It is necessary to ohtain a smooth surface both for writing and for ease of detection of spots; this can be accomplished by coating the glass while it is held between two glass mides 0.02 inch hieher than the elass strip. The strips were &en dried in a f o k d d r a f t oven a t 105°C. for 15minutes. This procedure resulted i n a stripapproximately 0.02 inch thick, with a minimum of cracks and a surface hard enoueh to write on with a blunt oencil. These &rim = - wonli not crumbio under mild handling.
.
4
5
Figure 1. Chromatographs Left. Mi*of fire tu-es as shown by fluaeeeein-bromine t e s t (From fop to bottom) 1. *Pinene 2. Limonens 3. Terpin71 acetate 4. .z-Te.pincol 5. Geraniol Right. Loostion of oinnsmaldchyde rith o-dianisidine resgent
easily applied. The method of Meinhard and Hall (9) on the radial surface chromatography of inorganic ions formed the basis of the present work. Their method was modified by coating the adsorbent, mixed with a hinder, on suitable glass strips; the strips were activated and then developed in a manner similar to paper strips in test tubes as used by Flood ( 6 ) and by Rockland and Dunn ( 1 1 ) . In order t o make the method as universally applicable as posaible, Sease's (19)idea of mixing two fluorescent inorganic materials-for example, zinc cadmium sulfide and zinc silicate-to the adsorbent was incorporated advantageously. Because not all of the terpenes absorb in the ultraviolet region and are thus not adaptable to the Sease technique, and because reagents were needed t o indicate certain specific functional groups, it was necessary t o develop a new series of tests for locab ing compounds on the chromatograms. For this new technique, the authors propose the name "chroma, tostrip." EXPERIMENTAL
which were heatebwith 18 ml. of distilled h e r until the'starch had coagulated and the mixture had formed a thick paste. The paste was then triturated with water to 8 consistency just thin enough to spread on the glass strips. Satisfactory strips could
~~
~
~~~~
Chromatostrips which were not dried prior to use in a uniform fashion exhibited a marked difference in Ra value (Ra = ratio of distance traveled by a spot to the distance traveled by the solvent). Limonene chromatographed with hexane on strips dried in a desiccator over phosphorus pentoxide a t 65 mm. of mercury for 0.5 hour had an Ra of 0.8, whereas this oil had an Ra of 0.4 when dried a t 3 mm. of mercury over phosphorus pentoxide for the same length of time. Exposure of strips t o atmospheric conditions for short periods of time also increased the Ra of some of the samples. Thus, i t was necessary to desiccate the strips in a standard manner and to limit the time in which they were exposed to the atmosphere before use. The strips were placed in a desiccator over powdered potae sium hydroxide and evacuated t o 3 mm. of mercury. (Reasons for this desiccant are explained later.) If placed in the desiccator while still warm from the oven, the strips reached equilibrium in 30 minutes. Before the strips were removed, i t was essential that the vacuum he broken only with dry air. For this reason, a tube packed with Ascarite was used to admit air t o the desiccator. The strips could not be used after exposure t o the atmosphere for periods longer than 10 minutes. Following these precautions, it was possible t o obtain reproducible Ra d u e s . For the spot test with a sulfuric-nitric acid mixture, it wm necessary t o eliminate the starch 8 8 a binder because of ita reaction with the hot acid mixture. Plaster of Paris (20%) was suhstituted 8s the binding agent. In preparing these strips, a small
Table I. Characteristics of Chromatostrips Made with Various Absorbents Adsorbent Coating Magnesium oxide Alumion Alumina silicic acid Calcium hydroxide Starch Dionleium phosphate Bentonite Calcium carbonate hlaenesium carbonate FiltFol Filtrol X202 Iiiltrol, Neutral E
+
A"eX
Florid Talc Siiioio soid
Physical Characteristics Of strip soft Excellent ElroeUent Soft, crumbly Good Fair Good Good Fair Good Good Good Fair Good Good Excellent
Resolution of Oila
ANALYTICAL CHEMISTRY
422 quantity of the dry mixture (enough for two strips) was mixed
1.5 ml. of fresh solvent. The strip was removed when the solvent reached the desired height (10 cm.), and the solvent was allowed ~ ~ ~ ~~ ~ . a$ k e ~~ ~ ' f~ ~ ~ ~~ ~ , i ~ ~ ~~~ ~ '~~ ~ $~~ > e ~ to evaporate from the strip before the qualitative tests were and were used as soon as they had cooled. applied. Method of Chromatography. The sample to be chromatoSolvents. With silicic acid-coated strips, a search was made graphed was placed as a small dot near the bottom of the strip for suitable solvents to be used in chromatographing terpenes. and pencil lines were made on the adsorbent to indicate the origiThe solvents tried were divided into four general classes. (Earlier nal position of the sample and the desired length of solvent experiments had shown that the strips could not be placed directly in a solvent containing water, because the adsorbent travel. The strip was then placed in a test tube which contained beneath the surface of the liquid tended to slide off the glass strip. Subsequently, this Table 11. Reactivity of Compounds with Various Zone-Indicating Tests difficultv was eliminated bv Sulfuricplacing the strip on a wad of UltraFluoresceinSulfurio Nitric Formula violet Compound Bromine (Concd.) (Concd.) cotton saturated with the solvent.) These four classes are + Brown described, together with the Limonene solvents falling in each class, as follows:
+
+
-
+
+ Brown
+
+
+
+ Yellow
+
Camphene
4-
+ Brown
+
Geraniol
+
+
+
a-Pinene
0
// Pulegone
,CHa
c,
C Ha-(II>=
HI
Purple
Carvone
+
+ Pink
+
p-Cymene
-
-
+
+
+ Green
+
+
+
Green
+
+ Green
+
a-Terpineol
OH
\-&-CHI
I
C Ha
Nopo
1,s-Cineol
Cinnamaldehyde
+
+
-
+
n-Capric acid
-
-
-
4-
Terpinyl aoetste
+
+ Brown
+
Camphor
-
-
'+
1. Those that carried all oils to the top of the column (ethyl alcohol, dioxane, diethyl ether, acetone, 1-nitropropane, pyridine, ethyl acetate, methanol) 2. Those that did not move the majority of the oils (hexane, petroleum ether, carbon tetrachloride, carbon disulfide) 3. Those that moved the oils a reasonable distance (chloroform, benzene) 4. Those that interfered with the fluorescein-bromine test (tetrahydrofuran, diacetone alcohol, amylene, ethyl triethoxy silane)
Various mixtures of group 1 with 2 and 3 were investigated, and 15% ethyl acetate in hexane was selected as one of the best solvents for this work. Adsorbents. Of the numerous adsorbents tested (Table I), silicic acid proved to be the best for terpenes. Merck's reagent grade, which had been sifted to pass a 100-mesh sieve, was used in the preparation of the strips for the terpene work. Color Tests. A wide variety of types of compounds are encountered in the examination of terpenes, so that it is necessary to have several qualitative tests to locate the spots formed on the chromatograms. It is also useful to have, in addition, a variety of tests for specific functional groups. The following tests were worked out and used in the task of locating compounds. Fluorescein-Bromine. The principle of adding bromine to unsaturated linkages was used in locating a large number of compounds with ethylenic-type double bonds. The completed chromatogram from which the solvent Lad evaporated was sprayed
VOLUME
23, NO. 3,
MARCH 1951
1
2
423
pounds could be detected as daxk spots when viewed under ultraviolet light (Figure 2). Aa suggested by Sease, the source of ultraviolet light was important in viewing these chromatograms. Unless ultraviolet light of short wave length w&s used, no spots were discernible. A Mineralight (Short wavc Model SL 2537) gave light of satisfactory wave length for this work. Table I1 indicates the types of compound8 detected by this means. o - D i a u i s i d i n e . Aldehydes can be detected as colored compounds with 0dianisidine (14). Cinnamaldehyde was detected by spraying with a solution of o-dianisidine in elacid acetic acid. Sulfuric Acid. Certain compounds resisted all means of detection because of the absence of reactive groups within the molecule. To detect these Compounds, concentrated sulfuric acid was sprayed ou the developed chromatogram. This reagent was used in locating l,&cineol. Table I1 indicates the color reactions and types of com3. p-Cymene, 3ss.f. Pulepone, 8.27, cinnsmsldehyde, 9.1 y pounds detected by this re4. Pclmene, 1.4 me.. Pulegone 32.87, cinnamntdehyda 3b4y Gent. Special equipment was needed to spray a reagent as corrosive as concentrated sulfuric acid. The spraying was done with an all-glass sprayer built in the laboratory (Figure 3). A small stainless steel booth for spraying was ConstNcted to protect the b a d , and the Sprayer was manipulated behind a glass window. Bromocresol Green. Fhmsey and Patterson (IO) have developed a method far the detection of acids on silica gel columns by incorporating bromocresol green in the adsorbent. Acids were detected on the strips by spraying with a solution of 0.3% bromocresol green in Soyo by volume methanol, to which had been added 8 drops of 30% sodium hydroxide per 100 ml. The acid appeared as yellow spots on LL green backgmund. Sulfuric-Nitric Acid Mixture. Camphor was so unreactive that i t was necessary to develop a special teat to indicate the location of this compound. This was accomplished by spraying with concentrated sulfuric acid to which 5% concentratad nitric acid had been added. The chromatograms were then heated on a strip of glass cloth, face down, on top of a hot plate which was turned to full heat and registered approximately 500' C. After the acid fumes had ceased coming off, the glass strip was carefully liited from the adsorbent and the latter was turned over by means of the glass cloth. The compound locations were observed as black spots on a white background. Chromatography of Terpenes. Fifty-eight samples of different commercial oils were chromatographed with five solvents on fluorescent silicic acid-costed strips. By this series of tests i t was hoped to test the applicability of the technique and to select I
(From top to bottom) 1. p-Cymene 2. Pulegone 3. Cinnemaldebydo
(Fmm left to right) Blank (spot due to traces of imputicy in solvent not m. moved by diefillation 2. p-cymene. 1431, pu1e.one. 3.31/, cinnnmsldchyde. 3.37 1.
avoided). The fluorescein rea&d with bromine to form the red dye,eosin. Wherever B material was present which could react with bromine, such as an ethylenic double hand, the fluorescein retained its normal yellow color. The location of these compounds was then readily apparent as yellow spots on a pink background (Figure 1). Amounts of some material aa small as 1 microgram were detected with certainty on chromatograms with fluoresceinbromine. Table I1 reveals the types of compounds which can be detected. Certain precautions must he observed in performing this test. Unless sufficient water is present, the pink color of eosin does not develop uniformly. For this reaaon, the dilute solution of Euorescein is necessary in order to build up the amount of water on the strip. If desired, a 0.1% solution of fluorescein may be used by first spraying with water. The presence of alkali enhances the red color, while acid prevents its formation. Strips desiccated wlth phosphorus pentoxide a p p m n t l y ahsorbed enough acid vapors to depress the formation of the red color; this difficulty was eliminated by using powdered potassium hydroxide as the desiccant. Fluorescence. Sease's ( I d ) method of fluorescent columns was incorporakd by adding aiuc cadmium sulfide and ainc silicate to the mixture of starch and adsorbent prior to adding the water. The dried strips gave a bright fluorescence; certain types of com-
424
ANALYTICAL CHEMISTRY
a variety of samples of good purity on which sensitivity and RF values could be determined. Of the 58 samples chromatographed, two were considered to be sufficiently pure; the other 56 oils gave chromatograms on which 2 to 9 spots were apparent when viewed under ultraviolet light and treated with fluorescein and bromine.
for comparing RF values. A minimum of five samples in each solvent was run to determine the RP value. Samples diluted with ethyl alcohol tend to have their position on the chromatngram distorted by the alcohol. This phenomenon was not observed when hexane was used as the diluent. On some chromatograms in which the concentration of the oil was high, the Rp wa.9 higher than on similar chromatograms with smaller amounts of oil. This was presumed to be caused by a dilution of the ascending solvent by the sample. When the concentration of the sample was high, this dilution was apparently enough to cause a change in the characteristics of the solvent. The results of the sensitivity determinations and the RF of the terpenes in the various solvents are given in Table 11.. DISCI'SSION
By using the technique described herein, a sample of oil can be rapidly chromatographed and many conclusions concerning purity and adulteration can be established a t once. Because a 125 ML. REACGVT
BOTTLE
SWLE
-
I
INCH
Figure 3. All-Glass Sprayer for Corrosive Liquids
Table 111. Preparation of Pure Compounds Limonene. B y fractionation from grapefruit peel oil B.p. a t 8 mm.: observed, 54' C.; literature, 53.35' C. a-Pinene. B y fractionation of a commercial sample B.p. a t 3 0 m m . : observed, 60.5' C.; literature, 80.5OC. Pulegone. B y fraction from oil of pennyroyal B.p. :it 7 mm.: observed, 86.5' t o 89' C.; literature 87.4' C . Camphene. Commercial sample. M.p.: observed, 45O to 4Q0 C.; literature 49' t o 52' C. Carvone. Purified by formine HzS addition product and then steam distilling. 5f.p.: observed, 219' to 224' C.: literature, 222. to 224'C. Geraniol. By forming CaCL addition product from commercial sample a n d then steam distilling p c y m e n e . Commercial sample (Paragon) Cineol. Fractionated from oil of Cajeput (b.p. a t 9.5 mm.: observed 56.4' C . ; literature 54.2' C.), and then further purified by recovery from resorcinol addition product a-Terpineol. Recrystallized commercial product from hexane, m.p. 34-34.5' C.; literature35' C. Nopol. Fractionated, b.p. 92.6' C. at 4 mm.; literature 71' C. a t 1 mm., 98O C. a t 5 mm. Terpinyl acetate. Prepared from a-terpineol and acetic anhydride by method of Boules (9) Cinnamaldehyde. Commercial sample (Paragon) by recovery from sodium bisulfite addition product n-Capric acid. Recrystallized from acetone. m.p. 33-34' C.; literature 31.3' C. Camphor. Sample used for molecular weight determinations, m.p. 178.8'C.; literature 178.8' C.
Fourteen compounds were then purified in this laboratory by various techniques. The method of purification and an indication of the purity of these samples are given in Table 111. All fractional distillations were made with a Podbielniak Hypercal column Many different types of compounds were selected in order to indicate the wide range of usefulnem of both the technique and the various spobindirating reagents. Each of the pure samples was chromatographed on fluorescentsilicic acid strips. Five solvents were used to characterize the oil: hexane (boiling point (35' to 69" C,), carbon tetrachloride, chloroform, benzene, and ethyl acetate in hexane (15% by volume). Sensitivity to the various color tests was determined by measuring the sample applied to the adsorbent, pure or in a suitable solvent, from a calibrated capillary pipet. (The ordinary mercury piston-type microburet could not be used because the'oils in contact with the mercury caused the mercury to fall out of the capillary. A calibrated Table IV. Chromatography of Pure Compounds capillary tube fitted with a ~ _ _ _ RF Values Limit syringe to control the column Carbon 15% ethyl Control, of HextetraChloroacetate limonene Sensiof liquid by air displacement ane chloride form Benzene in hexane in hexane tivity, 'I Compound Color Test was used.) The quantity of Limonene 0.41 0.37 0.93 0.66 ... 37 0.96 Fluorescein-bromine *-Pinene 0.83 0.89 0.95 0.96 0.83 Fluorescein-bromine 0.50 37 sample applied was reduced 0.01 0.01 Ultraviolet and fluoPulegone 0.09 0.07 0.49 0.51 4 in systematic fashion until the rescein-bromine 0.74 0 82 0.79 0.47 2000 Fluorescein-bromine Camphene 0.92 0.94 chromatogram failed to give Fluorescein-bromine Geraniol 0.00 0.00 0.05 0.05 0.21 0.46 1.5 Carvone the color test in question 0.00 0.01 0.07 0.04 0.45 0.47 0.4 Ultraviolet Fluorescein-bromine 5.U The last detectable concen100) p-Cymene 0.38 0.56 0.94 Ultraviolet 0.9j Fluorescein-bromine a-Terpineol 0.00 0.00 0.05 0.03 4.0 tration wm called the sensiFluorescein-bromine 0.00 0.11 0.06 1.o Sopol 0.00 Concd. sulfuric 0.6 1.8-Cineol 0.01 0.02 0.12 0.06 tivity limit. On each set of 0.3 o-Dianisidine Cinnamaldehyde 0.00 0.00 0.09 0.06 five chromatograms, a control Fluorescein-bromine 1.0 Terpinylacetate 0.00 0.00 0.26 0.25 Bromocresol green n-Capric acid 0.00 0.07 0.07 4.0 0.00 chromatogram of limonene Camphor Concd. sulfuric-nitria 0.00 0 00 0.28 0.22 0.8 chromatographed with hexane a Sensitivity of detection 1Sy with sulfuric-nitric acid. b Sensitivity of detection 30y with sulfuric-nitric acid. was run in order to make sure Compound run on strips with plaster of Paria instead of ntarch as a binder: bhua, RF value for limonene control the strips were dried properly has a different value. and to afford a reference ~~
C
V O L U M E 23, NO. 3, M A R C H 1 9 5 1 large number of terpene samples have been investigated and the great majority of them (56 out of 58) have been found impure by examination of chromatograms in five solvents, much of the tedious physical and chemical examination of the sample can he eliminated. The oils examined gave no sign of decomposing or isomerizing on silicic acid. Silicic acid is an excellent adsorbent for this work, because it resolves compounds of very similar t,ype, as illustrated in Table IV. The identification of terpenes should be facilitated by application of the principle of characteristic RF values to the chromatographed samples. In identifying constituents of natural products where the amounts of fractions concerned may be small, the volume of material necessary to secure identification can be substantially reduced. Amounts of oil as small as 0.5 microgram have been detected. Some of the color tests reported here have useful implications for indicating structure-for instance, from Table I1 it is evident that the fluorescein-bromine test reveals the possible presence of ethylenic-type double bonds. Other indications of structure are obtained from a study of Table 11, and many other tests can be applied which have specificity for certain types of structures. As a chromatographic method aside from the advantages to terpene chemistry, many features can be pointed out
425
\+ith Ivss (1r:tPtic revealing agents. These reagents have been used to inthrate traces of impurities in some terpenes which were thought to be pure by reason of physical constants and chromatography \vith other compound-indicating tests. The spray gun used for all of the work except for the sulfuric acid was an artist’s air brush. As shown by Meinhard and Hall (9),the completed chromatograms could be stripped off on Scotch tape and pasted on suitable cards for filing and reference purposes. This was not possible for strips sprayed with cold sulfuric acid, but the chromatograms which were heated after spraying with acid could be saved in the same manner. ACKNOWLEDGMENT
The authors are indebted to Richard Course of this laboratory for assistance in fractionating the oils. LITERATURE CITED
(1) (2) (3) (4) (5)
Boldingh. .J., Ezperientin, 4, 270 (1948). Boulea, V., Bull. soc. chirn. Belyes, 1, 117 (1907). Carlsohn, H., and Muller, G., Ber., 71, 858 (1938). D a t t a , P. E‘., and Overell, B. G., Biochm. .J., 44,XLIII 11949). D a t t a . Y. P.. Overell, €3. G., and Stack-Dnnne, MI.. .\ratwe, 164, ti73 11949). Flood, €I., Z.nn.ctl. Chem., 120, 327 (1940). Hopf. P. P., J . Chem. Soc., 1940, 785. Kirchner, J . Q., and Keller, G . J., J . Am. Chem. SOC., 72, 1867 (1950 j. Meinhard, J. E., and Hall, h’. E . , -4x.4~.GEM., 21, 186 (1949). Ramsey. L. L., and Patterson. W. L., J . Assoc. Ofic. A ~ T . Chemists,31,139 (1948). Rockland. 1,. E., and Dunn, 51. S.,Science, 109, 539 (1049). Sease, .J. JT., J . Am. Chem. Soc., 70, 3630 (1948). Spath, E., and K a i n r a t h , P., Be?., 70,2272 (1937). Wasicky. R., and Frehden, 0..Microchim. Acla, 1, 55 (1937). n’interstein. 4 . . and Stein, Q., 2. physiol. C’hem., 220, 247 (1933). Zechnieistri, L.. “Progress in Chromatography, 1938-1947,” Loiidon, (’hapman and Hall, 1949; New York. John Wiley & Sons. Zechnieister. L., and C:liolnoky, L.. “Principles and Practice of ( ’ h r o m a t o l 5 r a p h ~ , ”New York. John Wiley & S o ~ i s ,1941.
The chromtttostrip combines some of the advantages of paper and column chromatography. It adds the rapidity (a strip may be run in 0.5 hour) and the ease of spot development of paper chromatography to the wide range of adsorbents of column chrom:ttography . The method can be used for rapidly checking solvents and adsorbents for larger columns, because the results obtained are comparable. Although small conventional columns can be used for the same purpose, the proposed method is more rapid inasmuch as one man can easily run a set of 40 strips in an hour’s time. More drastic reagents can be applied to locate compounds than are permissible with paper strips. “Wet tails” (irregular flow of solvent up side of paper due t o touching of the glass wall) and similar annoyances of the flexible paper are avoided. I t is a microchromatographic method and is much more convenient. than a packed column for micro work.
(17)
The use of concentrated sulfuric acid and concentrated sulfuricnitric acid mixture for elucidating positions of organic materials on a chromatogram eliminates the uncertainty usually associated
KECEIVEU.June 26, 19,50. Presented hefore the Division of Analytical Chemistry a t the 118th hfeeting of the . i x E R r c a . u CAEYICAL S O C I E T Y , Chi. csgo, Ill.
(6) (7) (8) (9) (10) (11) (12) (13) (14)
(15) (16)
Automatic Fraction Collector for Chromatographic Separations DON4LD F. DUKSO, ELWYN I). SClI4LL,
AND
ROY I,. WHISTLER
Purdire University, Lxfayette, Znd.
A
METHOD has been developed recently for the chroniatographic separation of sugars on charcoal (1). For application of the method to separations on a larger wale, an automatic apparatus has been devised which feeds the mixture and the devtAlopers in succession to the top of the chromatographic column and collects each effluent in separate receivers. The device h:ts performed well in the fractionation of hydrolyzates of guaran, xylan, and starch, and may have general application for chromatographic separations. .4s shown in Figures 1 and 2, the apparatus consists of containers for the solutions to be fed to the column, receivers for the effluents, solenoid valves operating in pairs so that when energized one container and the corresponding receiver are connected to the column, an electronic control unit, a float chamber, a light source, a photoelectric unit, and a vacuum pump. The details
of the control circuit and a list of parts are given in Figure 3.
All the materials are readily available and easily assembled. unit can be built for approximately $200, exclusive of labor.
The
OPERATION OF CIRCUIT
The apparatus is readied for operation by filling the containera, connecting the receivers to the distributor, and inserting a previously packed and wetted column. The space in the tube above the adsorbent must be completely full of liquid to ensure a proper start. To initiate the operating cycle the main power switch, SW,, is placed in the “on” position, the reset button, SW,, is pressed, and the momentary contact switch, SWI, is closed. The operation of the apparatus is fully automatic from this point. After a delay of 70 seconds, the first solenoid valve in the upper bank is energized, allowing the solution in the first container to fill the float chamber and flow to the top of the column. At the same
