New Type of Chromatographic Column JOHN M. MILLER AND J. G. KIRCHNER Fruit and Vegetable Chemistry Laboratory, Pasadena 5, Calif. In the course of investigations on the volatile flavor-
the location of the resolved zone8 could be easily determined by spraying on the sides of the column a suitable reagent, which could he scraped off before recovery of the resolved compounds. A device is described for distributing solvent to the column for development. The operation of the column was demonstrated with some terpenes. This new ehmmatographic device has many advantages over conventional chromatographic columns, in addition to the convenience of locating resqlved zones.
ing constituents of citrus fruits, it became necessary to chromatograph certain fractions in quantities large enough to perform additional tests. To 80complish this a chromatographic column was developed which was self-supporting and was therefore not encumbered by a containing envelope. The column for which the name “ohmmatobar” has been suggested, was made fmm a desired adsorbent and plaster of Paris. After development,
I
so that approximately 6 inches of the rod extend from the head
N T H E course of work on the isolation and identification of the
of the mold and 0.125 inch from the base. According t o the adsorbent being used, various methods of drying or activating can he employed. With silicic acid columns, the bar is placed in a forced-draft oven a t 75” C. far a t least 8 hours. After drying, the column is oooled and stored in a desiocator over potassium hydroxide a t 3-mm. vacuum.
volatile Aavoring constituents of citrus juices, it became necessary to u8e chromatographic procedures for the sepsretion of certain terpenes. The use of the adsorbent-packed glass tubes common in chromatography on the colorless terpenes was difficult, because suitable methods for locating these compounds on a developed column were not available. Arbitrary cutting of the column, location by fluorescence, extrusion from the glass envelope and brushing on of an indicating reagent (4),and detection by means of strongly fluorescent columns according to the method of Sease (S) fell short of satisfying the need for a more convenient and accurate method. As part of the solution to this problem, the “chromatostrip” method of Kirchner et 02. ( f ) was developed. Although this method wm successful in the isolation and identification of colorless materials, a column of larger dimensions was needed so that larger quantities of material could be ohromattographed and recovered from the column for further confirmatory tests. Paper chromatographic methods have been applied to the isolation of larger quantities by Mitchell and Haekins (S). It appeared likely that the principles and techniques of the ohromattostrip could he applied to the making of a larger column, which would he free of a containing glass envelope and the con8equent disadvantages of columns paoked in glass tubes. This paper describes the preparation of a rigid, self-contained column, and ita application to the separation of some terpenes. The name “chromatobar” is proposed for this type of column.
Columns have been made of silicic acid, alumina, magnesium oxide, Anex, and Filtrol. The bars are rigid, maintain their shape, and can be handled without breaking. METHOD OF CHROMATOGRAPHY
These rigid columns me used for chromatography in much the same way as the chromatostrim, hut some mecautions and special techniques are necessary.
7
3
,RING
.
STAND
PBEPABATION OF COLUMN
The column
i8
made from a mixture of a suitable adsorbent
with calcium sulfate hemihydrate (plaster of Paris). For a column 1 inch aquare by 10 inches lona (2.5 X 25 cm.).
beaker, 135 &.6f water are added all a t once, ana this mixture is stirred vigorously and poured into 8 previously prepared mold. The amount of water to he added to a hatch of mixture must he determined empirically for esch different lot of an adsorbent and each different adsorbent. The final slurry should he just liquid enough to pour easily. The above-mentioned quantity of water is for a silicic acid adsorbent. The time between the addition of water and pouring of the slurry into the mold must he kept to a minimum (30 seconds), as the slurry sets very rapidly. After approximately 5 minutes, the bar can he removed from the mold. The mold consists of piece of metal formed into the shape of the column desired. A glass rod located in the center of the bar lengthwise must he used to give added strength to the column. For the preparation of 1 X 1 X 10 inch columns, a piece of metal 10 inches long m d 3 inches wide is bent to form a trough 1 inch wide, 1inch deep, and 10 inches long. The ends of the trough are made from stiff cardboard cut to a 1-inch square with a hole in the center to hold the glass rod. The trough is lined with waxed paper and the glass rad is inserted through the holes in the ends,
LOOSE PACK CA
HOOKS FOR -REMOVING DISTRIBUTOR
PLASTER PLUG
r
,
--7
Figure 1. Apparatus for Developing Chmmatohar 428
V O L U M E 23, NO. 3, M A R C H 1 9 5 1
429
end surfaces &resmooth'and ueruendioular to their sides. With a
solvent and the material to he chromatographed is placed on the end of the bar hy spreading evenly from a pipet, Dilute solutions of the materials to be chromatographed should he used to en8ure even distribution aero88 the surface of the bar. The bar with the material to be chromatographed is then ready for use.
The solvent is poured down the sides of the cylinder until a suitable amount is present, but is not allowed to overflow the sides of the distributor. The solvent rising through the solvent distributor and the column by capillary attraction advances up the column in an even manner. When the solvent has reached the desired level, the bar is removed and examined hy any of the techniques available for detecting the compounds chromatographed. If necessary, it can be returned to a new solvent for further development after the detecting reagent has been scraped off. Figure 1is 8, drawing of the assembly used far the ehromatography. SEPARATION OF SOME T E R P E N E S
The column can he used to separate materials in sufficient quantities to be examined by other means. The separations described were made on silicic acid (80 mesh or smaller) columns of 1square-inch cram section and 10 inches long. Because the analogy t o the chromatostrips is very close, numerous strips should he run to permit selection of the best adsorbent and solvent conditions for the desired separation.
on-a pihk background.
Figure 2. Chmma tograph of Crude Preparation of Isoeugenol on Fluorescent GIumn Photographed on "ltrariolef Light
1. s o 1 s e n r fmnt 2. lsoeugenol 3, 4, 5, 6. Unknown con-
Figure 3. Chromatog r a p h Showing Separation of (1) aPinene, (2) TeIpiny 1 Acetate, and (3) aTerpineol
Fixtire
4.
Se&a tion of (1) Limonene, (2)
TerDinvl A c e t a tk, and (3) aTerpineol Compounds located by
fluoreeoeinbromine test
C o m p o u n ds located by flll.3CeSO~ilL-
b m m i n e test
stituent*
The column is developed hy means of a solvent distributor. This distributor consists of a %inch section of glass tubing of suittLble size (2 inches for a I-inch bar), which is indented in three places at the bottom to allow free access of solvent and hns three glass hooks a t the top for convenience in removing it from the containing vessel. About half of the tube is filled with the same mixture of calcium sulfate hemihydrate and admrhent used in the bar to form a solid base and dried as in the preparation of the bar. Lame, easily packed material (suoh as calcium sulfate) is placed on the surfsee of this plaster plug and very lightly firmed with B wooden rod. The solvent distributor is placed in the bottom of a cylinder and the oolumn is gently hut firmly pressed into the cake of loose material an the distributor. The c o I u m is held firmly in place with slight pressure by means of a clamp on the glass rod in the center of the column. A small tamping rod is then used to press down the loose material around the bar and brush away any cxeess.
I mixture of limonene, a-terpineol, and terpinyl acetate natographed on a chromatostrip, it appeared that these npounds were separable with 15% ethyl acetate in However, when a mixture of 20 mg. of limonene, 20 mg. 01, and 20 mg. of terpinyl acetate was chromatograpbed on a column with 15% ethyl acetate in hexane, good separation of limonene and terpinyl acetate was not obtained. As experiments showed that a-terpineol and terpinyl acetate do not move when chromatographed with hexane whereas limonene is moved to an Rpvalue of 0.4, the above mixture of compounds was placed on a column m d developed with hexane until the solvent had traveled %/a of the column. The bar wa8 then transferred t o nnother solvent distributor and the development finished with 15% ethyl acetate in hexane. This resulted in a good separation of the three components, as shown in F i y n 4.
DISCUSSION
The rigid, envelopefree column was designed to overoome one of the disadvantages of the column packed in glass-i.e., the difficulty of locating colorless compounds without previous extrusion of the column. Large quantities of material can he isolated from, this column, and colorless compounds can he detected by the techniques of paper or chromatostrip chromatography. T h e square bars are very convenient for locating bands, because each of the four sides can be used for apmying a different reagent. In one experiment, citral was chromatogrnphed and sprayed with fluorescein-bromine on one side and with o-dianisidine on the other side. By this means the position of the aldehyde with ethylenic bonds was determined and it was thus differentiated from other aldehydes present as impurities. The surface of the bar can h e easily scraped free of the sprayed material and the product recovered without the presenoe of interfering substances. Use of the chromatobar eliminates the task of packing a satisfactory column in glass tubes-a particularly difficult task when columns of largo size are desired. The columns used in the illus-
ANALYTICAL CHEMISTRY
430 trations for this paper were 1 square inch in cross section; columns 2 and 4 inches square have been made without difficulty. When shaved with a knife, these columns show no evidence of nonuniformity in packing. By use of the capillary-ascent method of solvent travel combined with the rigid characteristics of the column, it is a matter of small concern to transfer the bar from one solvent to another. A preliminary examination of the position of the bands can be made by spraying an appropriate reagent on the column, scraping the reagent off the sides, and then returning the bar t o that solvent or a new solvent combination for further development. This is a distinct advantage over a packed chromatographic column, where further development is impossible once the column has been removed from the glass tube. Although conventional chromatographic borosilicate glass tubes can be used for fluorescent chromatography with the longer wave-length ultraviolet light, the shorter rays (230 to 290 mp) needed for excitation of the zinc silicate are absorbed by this glass. Therefore, this method is particularly adaptable to the use of the ability of compounds to absorb ultraviolet light, because columns made fluorescent with zinc cadmium sulfide and zinc silicate can be observed without being removed from the solvent and without interference due to absorption of the short wave-length ultraviolet light by a glass envelope. A number of developed columns have been examined for uniformity of the bands through the interior of the column. Successive small layers have been removed from the surface and the new surface examined by spraying or by observation in ultraviolet light. The zones have been found t o be uniform throughout the cross section of the column. The zones have been more uniform
than have the zones of columns packed in glass, because the solvent in these columns tends to travel more rapidly along the glass surfaces than through the adsorbent. Occasionally a column will crack in a direction perpendicular to the supporting glass rod during the drying period. This defect can be remedied by gently pressing the bar together; columns with such cracks have been used without any effect on the movement of solvent or compounds. Round bars have been made with thin plastic casings as molds; however, the square bars are more convenient for applying colordeveloping reagents by means of sprays. Solvents containing water have been used to develop columns; although they impart fragility to the column, such columns can be successfully handled. These columns can he accurately cut with a coping saw and areas of interest removed and extracted with suitable solvents. ACKNOWLEDGMENT
The authors are particularly grateful to George J. Keller of this laboratory for the preparation of the drawing of Figure 1. LITERATURE CITED (1)
Kirchner, J. G.. Miller, J. M., and Keller, G. J., . 4 ~ . 4 ~Cmmf., . 23, 420 (1951).
(2) Mitchell, H. K., and Haskins, F. A., Science, 110, 278 (1949). (3)
Sease, J. W., J . Am. Chem. Soc., 70,3630 (1948).
(4) Zechmeister, L., Cholnoky. L., and Ujhelyi, E., Bull. soc. chim. hiol., 18, 1886 (1936). RECEIVED July 7, 1950. Presented before the Division of Analytical Cheniistry a t the 119th Meeting of t h e AMERICAXCHEMICAL SOCIETY, Chicago, Ill. Report of a study made under the Research and Marketing Act of 1946.
Compound Types in Gasoline by Mass Spectrometer R . A . BROWN, The Atlantic Refining Co., Philadelphia, P a .
A
NUMBER of laboratories throughout this country use commercial model mass spectrometers to obtain high precision analyses on a wide variety of organic compound mixtures (6, 7 , 10, 12, 14). Notable success has been achieved in the field of hydrocarbon analysis, where very small samples containing up to thirty components can be analyzed in 1 or 2 man-hours (2, 3, 16). In view of the speed, accuracy, and small sample requirements of these methods it appeared highly desirable to extend the scope of the mass spectrometer to compound-type analysis of complex liquid hydrocarbon mixtures such as gasolines. A study of this possibility has resulted in a quick and accurate spectrometric method, which appears to be a worth-whjle addition to numerous other procedures previously described (6, 8, 9, 11, 15). This method has been applied .to some 500 samples, and the resulting data have been used to evaluate laboratory and pilot plant products, commercial grade gasolines, and solvents over a period of 2 years. Analytical data are obtained on total paraffins, total cycloparaffins and/or mono-olehs, aromatics, and the group designated as the “coda” type-namely, cyclomono-olefins, diolefins, and acetylenes. Calculation of aromatics with unsaturated side chains has not been evaluated here, although their concentration is easily estimated. Pl’ormally such compounds are absent but, if present, appear in relatively small and constant amounts. In the case of eome catalytically cracked gasolines, for instance, such compounds constitute 6% of all aromatics. This method reports the calculation of total aromatics only, although average sensitivity data and 10 minutes’ additional cal-
culation time are all that is needed to resolve aromatics according to molecular weight. Mono-olefins are differentiated from cycloparaffins by an auxiliary procedure, such as bromine number or nitrosation (1). Accuracy achieved is found to be *l% for aromatics and *2% for “coda” compounds in all types of mixtures. Other components are determined within *2oJ, in wide boiling mixtures but only within *4y0 in mixtures of 15” C. or less range. Calibration data presented here can probably be used directly by other
Table I. ATO.
of
Carbon .4toms 4 5 6 7 8 9 10 11 12 13 14 15 16
Total
Numerical Summary of Compounds Studied
Aliphatic paraffins 2 3 5 9
18 10 1 0 1 1 1 0 2 53
Kumher of Isomers Studied CycloAliphatic olefine, Cyclomonodienes, Aroparaffins olefins acetylenes matics .. 3 2 .. 5 3 .. 1 2 1 2 11 2 1 7 3 5 2 4 21 2 .. 8 4 14 1 4 0 .. 1 0 .. 1 0 2 0 0 1 1 .. .. .. 0 .. .. .. 1 34 11 34 40 Total = 172 . I
