#HPTLC
#HPTLC #TLC # HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
High-performance thin-layer chromatography (HPTLC) is an enhanced form of thin-layer chromatography (TLC). A number of enhancements can be made to the basic method of thin-layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements.
Automation is useful to overcome the uncertainty in droplet size and position when the sample is applied to the TLC plate by hand. One recent approach to automation has been the use of piezoelectric devices and inkjet printers for applying the sample.[2]
The spot capacity (analogous to peak capacity in HPLC) can be increased by developing the plate with two different solvents, using two-dimensional chromatography.[3] The procedure begins with development of sample loaded plate with first solvent. After removing it, the plate is rotated 90° and developed with a second solvent.
Instrumentation
An example of an instrument for HPTLC is from CAMAG,[4] a Switzerland based company. It provides automated sample application (loading), plate development, detection and documentation.
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures.[1] Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.[2] The mobile phase has different properties from the stationary phase. For example, with silica gel, a very polar substance, non-polar mobile phases such as heptane are used. The mobile phase may be a mixture, allowing chemists to fine-tune the bulk properties of the mobile phase.
After the experiment, the spots are visualized. Often this can be done simply by projecting ultraviolet light onto the sheet; the sheets are treated with a phosphor, and dark spots appear on the sheet where compounds absorb the light impinging on a certain area. Chemical processes can also be used to visualize spots; anisaldehyde, for example, forms colored adducts with many compounds, and sulfuric acid will char most organic compounds, leaving a dark spot on the sheet.
To quantify the results, the distance traveled by the substance being considered is divided by the total distance traveled by the mobile phase. (The mobile phase must not be allowed to reach the end of the stationary phase.) This ratio is called the retardation factor (Rf). In general, a substance whose structure resembles the stationary phase will have low Rf, while one that has a similar structure to the mobile phase will have high retardation factor. Retardation factors are characteristic, but will change depending on the exact condition of the mobile and stationary phase. For this reason, chemists usually apply a sample of a known compound to the sheet before running the experiment.
Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents [3]
A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis. This method is referred to as HPTLC, or "high-performance TLC". HPTLC typically uses thinner layers of stationary phase and smaller sample volumes, thus reducing the loss of resolution due to diffusion.
TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the absorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC
he process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC