T hin-Laye r Chromat ography Recent Developments in Equipment and Applications E. G. WOLLISH, MORTON SCHMALL, and MARY HAWRYLYSHYN Analyticof Research Laborotory, Hoffmonn-LaRocheInc., Nutfey, N. 1.
b Thin loyer chromatography has rapidly attained popularity in Europe, where standardized adsorbent and equipment have become available. The procedure has recently also found enthusiastic supporters in the United States. This paper describes several pieces of improved equipment. The current literature is reviewed ond a p plications in different fields, a s well a s examples of separations that can b e achieved, a r e cited. The unique advantages of this method, for the separation not only of lipides and essential oils, but also of a great variety of other organic compounds, a r e pointed out.
T
HIN LAYER chromatography has
recently found wide recognition, particularly in Europe, where Stahl (564Z)popularized this method, standardizing adsorbents and equipment. Although over 30 years ago Ismailov and Schraiber ($0)had separated mixtures of organic compounds on layers of adsorbent powder, examining the rings produced under ultraviolet light, it was not until 1949 that Meinhard and Hall (38) prepared microscope slides with a layer of alumina, held together hy starch, and developed circular chromatographic zones on them. Considerable progress was achieved by Kirchner et ai. @2-#6, 89-41), who perfected Chromatostrips by coating narrow strips of glass as well as plates for two-dimensional technique with various adsorbents held on the plate with starch or plaster of Paris. The above investigators used them for the separation of terpenes, essential oils, and other compounds by ascending technique. Reitsema (50,51)coined the term "chromatoplates" for his procedure, using larger glass plates mated with silicic acid with starch as binder, and successfully partitioned essential oils on them. Several publications d a l t with the adsorption mechanism of silicic acid (9, 30, 3%56, 62), while Wren (68) reviewed the chromatography of lipides on silicic acid. Miller and Kirchner (41) were the first to describe an apparatus for coating glass strips with an adsorbent mixture of
1138
ANALYTICAL CHEMISTRY
Figure 1. Applicetor silicic acid and starch paste. These authors specified silicic acid to pass a 100-mesh screen (25). Stahl (56, 57, 60, 6$) also pointed out the necessity for the standardization of silicic acid with respect to particle size and likewise added about 15% plaster of Paris (calcined calcium sulfate) to the silica gel a8 a binder. Such a silica gel can now he obtained under the name Silica Gel G (from Merck, Darmstadt. Importers: Terra Chemicals Inc., 500 Fifth Ave.. New York 36. N. Y.: Brinkman Instruments, I&, 115' Cutter Mill Road, Great Neck. L. I.. N. Y.). This investigator also developed several useful pieces of apparatus for the preparation and storage of coated glass plates, which equipment is now commercially available (manufactured by DESAGA, Heidelberg, Germany. Importer: Brinkman Instruments, Great Neck, L. I.).
9'/* inch coated plates. We utilized this t,ool also for applying the adsorbent to several na.rroy glass strips simultaneously and haye constructed a similitr larger applicator for the preparation of two-dimensional plates.
The apparatus, as slightly modified by us (Figure I), consists of a ridged base plate ( A , Figure 2) of aluminum, a/, inch thick. on which the elass d a t e ;&-be moved smoothly wzhout' adhering. A guide strip '/*inch high and 13/a inFhes wide prevents the plate from sliding sideways on its movement through the applicator. The chamber serving as reservoir for the adsorbent slurry consists of tu-o vertical side plates with '/s-incli-deep slots at a distance of 1s/8 inches from each other. The stainless steel gate plates are l'h inches high, 3 3 / 1 ~inches wide, and '/a inch thick. The front gate rests on top of the glass plate, while the rear gate has a sharpened edge. Two 2-inch thumbscrews are soldered to the inner side of the rear gate plate in our modRECENT DEVELOPMENTS IN APPARATUS DESIGN ification, while in Mutter .and Hof~ ~ ~ Recently, f i Mutter ~ ~and ~ stetter's ~ ~applicator . they are drilled through the aluminum gate blade and Hofstetter (45) have a serve to regulate the thickness of the particularly practical applicator for the uniform coating of glass plates with an adsorptive layer (manufactured by The drawing of our modification of CAMAG, Muttenz, Homhurgerstrasse the applicator, including measurements 24, Switzerland. This company also for both sizes (one-dimensional and furnishes other accessories and adtwo-dimensional plates) is shown in sorbents, such as aluminum oxide Fluka Figure 2. and silica gel Fluka.). This apparatus Chromatographic Chamber. Standpermits the regulation of the thickness ard rectangular borosilicate glass jars of the adsorbent (about 250 microns with eround edees (Cornine 6944) of desirable) regardless of the thickness I- or %gallon clpacpaciti for ii/2 x 'Q,/$ of the glass. Thus, inexpensive, good and 8 X 8 inch plates, respectively, quality window glass (single thickness, pan be used. Measurements of I-gallon about '/e inch) may be used instead of jar: 5J/s X 63/8 X lo*/* inches; plate glass for the preparation of 3'/2 X 3-gallon jar: 9% X 7'/8 X W/,inches. ~~~~~
Figure 3. chamber Figure 2.
Chromatographic
Details of applicator
The complete setup is visible in Figure
3. Plate Holder. Our plate holder (drawing of both sizes shown in Figure 4 ) consists of a 8/&ch-wide strip of stainless steel */&inch thick, which is held by four springs against the walls of the jar. It is supported by four 6/rsinch-diameter round glass rods 9'/~ inches long, which are inserted into the rings at each corner of the s u p port. Three chromatographic plates can he held in vertical position by clips, which grip their upper edge and at the same time fasten the plate to the stainless steel support. The glass rods are rimmed a'/* inches from one end. The jar is covered with a heavy glass plate, 6'/* X 7'/* inches for the small chamber, or SI/%X 101/* inches for the twodimensional one. TNOglass strips, about 3/4 inch wide and '/,. inch thick and slightly shorter than the length of the chamher, may be placed on the bottom of the jar to support the chromatographic plates during equilibration. Storage Cabinet. The storage cahinet (Figures 5, 6, and 7) is made of stainless steel sheet in the form of a cube with the top open, 12-inch side measurement. The inner edge of the cover is lined wit.h a neoprene rubber gasket to provide a tight seal with the top rim of the cube. The prepared chromatographic plates are held in a rack, which is removable and is constructed from 11-gage stainless steel yire ('/*-inch diameter). The plates rest at a slant in grooves on the rack. A desiccant, such as magnesium perchlorate or granular porous barium oxide (Barium and Chemicals, Inc., Willoughhy, Ohio), is placed on the bottom of the storage cabinet. Glass plates, from single-thickness window glass, are cut in three sizes: 6/a X 10 inches (for exploratory chromatography); 3'/* X 9'/2 inches (for onedimensional chromatography) ; and 8 X 8 inches (for two-dimensional chromatography).
Preparation of Chromatographic Plates. The plates must be thoroughly cleaned, preferably with sulfuric-chromic acid, followed by water, and dried. A slurry is prepared by mixing 15 grams of Silica Gel G with 50 ml. of water and shaking for about 1'/2 minutes, and is used promptly. A plate is placed under the trough of the applicator, with the leading edge protruding about 1 inch beyond the gate. About 15 ml. of the slurry are poured into the trough to prepare singledimensional plates, or about 35 ml. for two-dimensional plates. Another glass plate is immediately pushed under the gates of the trough in a Continuous movement. The coated plates are immediately placed on a level surface for superficial drying. After about hour they are transferred to an oven of 50' C., where the plates are dried overnight. If desired, the plates can also be activated at 100' C. by drying for 1 hour. They are then transferred to the storage cabinet, containing the desiccant.
In preparing the narrow strips for exploratory work, six strips laid side by side are pushed simultaneously through the applicator with a clear single-dimensional glass plate. The strips are inserted in individual 100-ml. glass cylinders without pouring spout and can he used for exnerimental work after closing the cylinher with a cork stopper. PROCEDURE
Application of Sample. The sample is applied from micropipet a t a line ahout inch from the end of the plate. A plastic template may be used for the correct spacing of various samples along the starting line, After evaporation of the solvent in which the sample was applied, the plate is inserted into the chromatographic chamber, containing sufficient of the desired solvent to cover the bottom, hut without reaching the surface of the glass strips, on which the plate rests. To obtain rapid equilibration, i t is advantageous to have one side of the jar I 2 .,1
~
1 FLLT
Figure 4. Battery jar plate holder VOL. 33, NO. 9, AUGUST 1961
1139
chromatography. While R, values will always show the proper position in relation to another Compound, a standard should always be run alongside the sample for identification purposes. The wide range of usefulness of thinlayer chromatography is apparent from Table I, which shows typical examples of applications described in the literature.
lined with a sheet of filter paper which extends into the solvent at the bottom. The jar is covered and allowed to saturate with the solvent vapors. Then additional solvent is carefully poured down one side of the jar until about I / h inch of the chromatographic plate is immersed, thus starting the development. After the solvent front has reached a height of about 5 in.ches, or a/, of the length of the adsorbent, the plate is removed, dried, examined under short-wave and long-wave ultraviolet light for fluorescent or absorbent spots which may he conveniently markede.g., by dotting with a needle-and then sprayed -.ith the reagent. Example.
Separation of Sulfonamides
I x 1 x 1,sJ . S A N C L E LEGS t 4 1 REOD
Figure 6.
Box end cover
Welded conrtruction, 16-gage 316 sloinlerr Iteel All dimensions in inche,
Solvents. The selection of a solvent
(Figure 8 )
or solvent mixture can follow the
Bolvent Chloroform-heptaneetbanol system 1 1 1 Adsorbent Silica Gel G Sample 10 mg. dissolved in 100 acetone. 0.01 mi. applied to plate Spray O.l%p-dimethylaminobenzaIdehyde in ethanol, acidified 1%with coned. HC1
known principles of adsorption chromatography. Trappe (04) proposed an "eluotropic solvent series," presented in Stahl's (60) modified sequence in which the eluting power increases approximately with rising polarity: hexane
(petroleum ether)
0.9
0.7 0.8 0.4
0.02
RJ values based on the ratio of the distance traveled by the spot to the distance traveled by the solvent front are not as reproducible as in paper 1140
ANALYTICAL
CHEMISTRY
carbon disulfide
