Preparative thin-layer chromatography
Preparative TLC is used mainly to purify drugs or to isolate drug metabolites and impurities in amounts of about 1–100 mg for subsequent use as reference materials, structural elucidation, biological activity evaluation and other purposes. Scale-up from analytical TLC is achieved by increasing the thickness of the layer (loading capacity increases with the square root of the layer thickness) and by increasing the plate length used for sample application. Precoated TLC plates for preparative chromatography vary in size from 20 cm X 20 cm to 20 X cm 40 cm and are coated with 0.5–10 mm thick layers, with the most popular thickness being 1.0–2.0 mm. As the average particle size (~25 μm) and size distribution (5–40 μm) are larger for preparative layers, and as sample overload conditions are used commonly in preparative chromatography, invariably inferior separations in a longer time (~1–2 h) are obtained compared with analytical separations.
Resolution can be increased significantly by using wedge-shaped, gradient-thickness layers. These layers have a uniform increase in thickness from 0.3 mm at the bottom to 1.7 mm at the top. Sample bands are focused during migration by the negative mobile-phase velocity gradient created by the layer geometry.
Sample application is a critical step in preparative TLC, and if performed improperly can destroy all or part of the separation. The sample, usually as a 5–10% (w/v) solution in a volatile solvent, is applied as a band along one edge of the layer to give a maximum sample load of about 5 mg/cm for each millimetre of layer thickness. Sample loads are usually lower for difficult separations and for cellulose and chemically bonded layers. Any of the automated band applicators for analytical TLC are suitable for sample application in preparative TLC. Manual sample application by syringe or glass pipette must be performed carefully to avoid damaging the layer and producing irregularly shaped migrating zones. A short predevelopment, of about 1 cm with a strong solvent, is often useful to refocus manually applied bands. Preparative layers with a preadsorbent zone are useful for manual sample application, since the focusing mechanism can be used to correct for poor sample-application technique. In all cases, it is important that the sample solvent is evaporated fully from the layer prior to the start of the separation to avoid the formation of distorted separation zones. It is usual to leave a blank margin of 2–3 cm at each vertical edge of the plate to avoid uneven development as the solvent tends to move differentially at the edges of the plate.
Most of the changes in preparative TLC over the past decade have occurred in the method of development. Conventionally, ascending devel opment in large-volume tanks that hold a number of preparative layers in a rack is commonly used. In laboratories that perform preparative TLC on a regular basis, higher resolution and shorter development times are achieved by using forced-flow development or rotation planar chromatography (accelerated development using centrifugal force). These methods allow conventional development and elution with on-line detection and automated fraction collection to be used.
After development, physical methods of zone detection are used to identify the sample bands of interest. Layers that contain a UV indicator for fluorescence quenching or the adsorption of iodine vapours are useful for this purpose because they are nondestructive. If a reactive spray reagent is used for visualisation, it should be sprayed on a small strip of the chromatogram only, so as not to contaminate the remainder of the material. Some form of masking arrange ment can be employed to protect the area of the plate where the spray reagent is not to be applied. Once the bands of interest are located (by examining the area of the plate which has been sprayed), the corresponding zones in the non-sprayed area are scraped off the plate carefully with a spatula or similar tool. A number of devices based on the vacuum-suction principle for removing the marked zones from the plate are also available. Soxhlet extraction, liquid extraction or solvent elution with a polar solvent is used to recover drugs from the sorbent. For solvent extraction, water is often added to dampen the silica gel prior to extraction with a water-immiscible organic solvent. Chloroform and ethanol (methanol is less suitable because of its higher silica solubility) are widely used for solvent elution. Colloidal silica can be removed by membrane filtration prior to vacuum stripping of the solvent.
