Improved Method for Preparative Layer Chromatography Martin H. Stutz, William D. Ludemann, and Samuel Sass Chemical Research Laboratory, Research Laboratories, Edgewood Arsenal, Edgewood Arsenal, M d . 21010
WITHTHE STANDARDIZATION of the thin-layer chromatography (TLC) technique by Stahl (I) and the subsequent outgrowth of preparative layer chromatography (PLC), a method became available whereby reasonable quantities of pure compounds can be obtained with relative ease, PLC generally involves the mechanical steps of TLC with minor modifications. These modifications consist of increasing the thickness of the adsorbent layer, and the application of a more concentrated sample as spots, or as narrow bands. Reviews of this technique are given by Honegger (2) and Bobbitt (3), among others. Disadvantages of most of the published PLC techniques are the limited yields of material and the elution procedure after separation. The majority of the workers simply scrape the adsorbent containing the desired component(s) from the glass plate and elute the material with an appropriate solvent. This step is time-consuming, as it is usually found necessary to perform the additional step of filtration or centrifugation to eliminate adsorbent fines that are carried over in the eluting solvent. Examples of this technique are given by Gilmore ( 4 ) and Seikel (5). These methods usually involved the evaporation of relatively large quantities of eluant to obtain small quantities of sample. The purpose of this paper is to describe a recently developed procedure that eliminates the necessity for scraping or vacuuming the adsorbent from the glass plates, eliminates the carry-over of fines with the eluting solvent, reduces the volume of eluant, and significantly reduces the time necessary t o preparatively chromatograph a compound while increasing the yield of purified material. For demonstrating the practicality of the technique, the PLC of a dye mixture is described, although the system has been found applicable to a wide variety of compounds. EXPERIMENTAL
Materials and Procedure. The commercially available, reagent-grade solvents utilized in this study were used without further purification. The chromatographic apparatus and adsorbent were obtained from Brinkmann Instruments Inc., Westbury, N. Y . Thin-layer plates, 200 x 200 mm, are coated to a thickness of 750 ~.rusing silica gel G (Merck). After air-drying and activation for 30 minutes at 105-110” C, the plates are stored over silica gel until used. Two 200- x 200-mm plates are prepared simultaneously. Each plate is scored across the center, which essentially divides the plate into two 200- X 100-mm chromatograms. A sample of test dye mixture (Gelman Instrument Co., Ann Arbor, Mich.) is spotted, parallel to the center scored-line and 1 cm from the 200-mm edge o n both chromatograms of each plate. Filter paper wicks are placed on both of the 200mm edges of this plate such that their edges are just below the row of spotted samples. The second spotted plate is (1) E. Stahl, G. Schroter, G. Kraft, and R. Renz, Pharmazie, 11, 633 (1956). (2) C. G. Honegger, Helc. Chim. Acra., 46,1772(1963). ( 3 ) J. M. Bobbitt, “Thin-Layer Chromatography,” Reinhold, New York, 1963. (4) D. R. Gilmore and A. Cortes, J. Chromatog., 21 (l), 148 (1966). ( 5 ) M. K. Seikel, M. A. Millet, and J. F. Saeman, J. Chromatog., 15, 115 (1964). 258
ANALYTICAL CHEMISTRY
placed on top of the first plate (adsorbent layers facing), to make a sandwich with the adsorbent layers of both plates in contact with the wicks. For handling convenience, the plates are fastened together with pinch clamps (See Figure 1). The developing chamber consists of a tray, 18 x 12 x 21/2 inches, with a cover sheet of plate glass having slightly larger dimensions. Two solvent troughs (10 X 2 X 11i2inches) to contain the developing solvent, are placed in the tray approximately 180 mm apart. The plate sandwich is then placed on the troughs so that the exposed portion of the wicks extends downward into the troughs. Fifty milliliters of developing solvent (chloroform :dichloromethane, l :l ) are then added t o each trough. The chamber is covered, and the plates are allowed to develop until the solvent front reaches the scored center line of both plates (see Figure 1). After development, the plate sandwich is separated and the plates are allowed to air-dry. This results in four developed chromatograms on the two plates (see Figure 2). After allowing the plates to air-dry, each component in the four chromatograms is isolated from the others by scoring the adsorbent layer between them. The scored areas are carefully tapered along one edge of both plates since elution is made from this edge (Figure 2). Bleeder wicks are cut from glass fiber paper (type GF/A, H. Reeve Angel & Co. Inc., Clifton, N. J.) to approximately the same width as the scored and tapered areas. The length of the bleeder wicks is approximately 2.5 inches. The glass fiber wicks are placed on one of the plates such that their ends extend approximately 5 mm onto the adsorbent layer of the elution (tapered adsorbent) side of the plate. Wicks are also placed over those areas from which no eluant is to be collected, in order to allow run-off for the solvent flowing through these areas. On the opposite edge of the plate, a filter paper (feeder) wick, 50 X 200 mrn, is placed so that it extends 10 mm onto the adsorbent layer. The sandwich is then reassembled with each scored area in alignment with its counterpart on the opposite plate. The sandwich is again fastened together with pinch clamps (see Figure 3). The same chamber as previously described for initial separation is also used for the elution step. The tray is suspended over a flat, constant temperature water bath in order to maintain an internal temperature of 26-28“ C, which was found effective, in the described system, for complete elution. In a temperature-controlled room, this bath would not be necessary. One developing trough is used to contain the elution solvent (methanol), while the second one is used as a plate support and t o aid in the saturation of the chamber. The trough which will contain the elution solvent is placed approximately 10 mm higher than the supporting trough. When finally assembled, the result is a slight downward angle to the plate sandwich in the direction of solvent flow. The plate sandwich is then placed on the troughs. The wicks extending from the areas that are to be eluted and collected are bent and placed in 5-ml beakers so that their tips lie on, or touch the bottom of, these collecting beakers. The remaining wicks are bent so that their tips lie on the floor of the chamber, Methanol is poured into the supporting and elution troughs and into two small beakers (or petri dishes) placed a t the end of the chamber opposite the elution trough. The glass cover plate is then placed in position and the edges are sealed (Figure 3). The elution is continued until the components are completely transferrea into the beakers from both plates and wicks. Once the chamber is sealed, it requires no further attention. Under the above conditions, the total elution time for the dyes is approximately 16 hours.
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Figure 2. Isolation of same components by scoring after initial development Glass plates Component 1 (at origin) C. Component 2 D. Component 3 E. Component 4 F. Center scored line Ends tapered to facilitate alignment A. B.
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Figure 1. Assembled apparatus for initial development A. B. C.
Developing chamber Plate glass cover Solvent trough D. Assembled glass plates E. Filter paper wicks F. Pinch clamps
RESULTS AND DISCUSSION
The improved PLC procedure was found applicable to any type or thickness of adsorbent (cellulose, silica gel, alumina, etc.) that could be maintained during handling. In this case, a 750-p layer was the maximum thickness that could be handled. Poor adhesion of the layer to the plate was observed during tests with 1-mm layers, especially when the plate was inverted after scoring. Recently a new adsorbent has become available which the manufacturer (6)claims can be used in layers up to 5-mm thickness. The solvent chosen for the elution should be considerably more polar than the development solvent, preferably one that carries the components of the sample along with the solvent front in a normal development. The physical configuration of the wick was immaterial, provided one end was no wider than the scored area from which the component was to be eluted and the other end was in contact with the bottom of the collection beaker. Filter paper and other similar materials produced the desired elution, although glass microfiber wicks gave the best results of those tested. The tapering of the elution edge of the plate not only aids in alignment of the sandwich, but also prevents overflow of solvent from one scored area to the next. A single sandwich produced 1 to 2 ml of eluate per 5-ml beaker in approximately 16 hours. The dye mixture was utilized as an aid in the observation of progress during the various stages of development of the method. A colorless, but spectrofluorescent sample of other material was also investigated as a check on the method, and its progress followed with the aid of an ultraviolet lamp. The results in this case were also very good. If desired, an inorganic fluorescent indicator (such as the lead-manganese activated calcium silicate phosphor supplied by Kensington Scientific Corp.) can be incorporated in the (6) Bulletin No. BR 153, Brinkmann Instruments Inc., Westbury, N. Y . , p. 35.
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B
A I
Figure 3. Assembled apparatus for elution development Developing chamber Plate glass cover Solvent trough D. Support trough E . Assembled glass plates F. Collection beakers G . Pinch clamps H . Beakers or Petri dishes A. B. C.
adsorbent layer to permit observation of the progress of development. With a relatively clean starting sample (90%), 2 mg of material could be applied on each spot on the 750-p adsorbent layer. With a maximum of 17 spots per half plate, a total of 136 mg of sample for each plate sandwich could be applied. A theoretical yield of 122 mg of chromatographically pure material, per sandwich, is possible. The number of plate sandwiches that could be run at any given time was limited only by the height (depth) of the developing chamber. The time for a complete elution varied from 6 to 18 hours depending on how rapidly a particular component was eluted. The adsorbent fines usually carried over in the eluting solvent were eliminated, for all practical purposes, by the passage of the solvent through the wick. Methanol fractions from blank, adsorbent plates showed no spectrofluorescence or ultraviolet adsorption in the range of 230 to 350 mp. RECEIVED for review July 25, 1967. Accepted September 22, 1967. VOL 40, NO. 1, JANUARY 1968
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