Principle

TLC is based on the classic chromatography principle where mixture components are separated between a fixed stationary phase and a liquid mobile phase by differential affinities between the two phases. During development of the chromatogram, the mixture of substances is first transported by the mobile phase, then resides on the stationary phase for a while, and is carried along again. The process repeats many times, and each substance is slowed down at different rates relative to the velocity of the mobile phase. The more a substance preferentially resides on the stationary phase, the slower its progress will be. Even substances with similar affinities for the two phases demonstrate differences in their chromatographic run, and can be separated.

The process steps of HPTLC are identical to classical TLC. The main difference between them is in the characteristics of the separation plate. HPTLC plates are based on optimized silica gel 60 with a significantly smaller particle size than used for classical TLC. This allows a higher packing density and a smoother surface. Hence, sample diffusion is reduced, resulting in compact bands or spots. Furthermore, the smaller particle size and thinner layer significantly increase detection sensitivity and analysis speed.

Applications

Biosciences

Procedure

1. Sample Preparation
2. Pre-Rinsing the Layer
3. Choice of Stationary and Mobile Phases in TLC Process: The TLC stationary phase consists of a TLC plate coated with a layer of sorbent material.
4. Pre-Conditioning the Layer: Unless special precautions are taken during sample application, humidity in the laboratory can diminish the activity of the TLC layer within minutes, as equilibrium is reached between the lab atmosphere and the sorbent. Pre-conditioning the TLC plate helps to avoid its deterioration.
5. Sample Application: Samples can be applied as spots or bands. In general, broadening of spots in the direction of development is less common with band application than with point application. Solvent polarity is another factor to consider during sample application.
6. Manual/Automatic/Semi-Automatic Application

Materials

• Sample: Urine; Bodily fluids; Blood; Saliva; Serum; Plasma; Drugs; Organic Compound; Liquids; Pesticides; Steroids; Alkaloids; Nucleotides; Glycosides; Carbohydrates, Fatty Acids; Environmental Pollutants; Water; Food & More
• Equipment: HPTLC RP-18 F254s Plates; Chamber; Watch Glass; Capillary; UV-light; Silica Gel
• (Optional): Solvent

Notes

When can you use Thin-Layer Chromatography? It may be used if:
1. Substances to be analyzed are soluble in a solvent or mixture of solvents.
2. The substances are non-volatile or of low volatility.
3. The substances are strongly polar, of medium polarity, nonpolar, or ionic.
4. A large number of samples must be analyzed simultaneously, cost-effective, and within a limited period of time.
5. All sample components are individually detectable (remain at the start or migrate with the front) after separation, or can be subjected to various detection methods (e.g. in drug screening).
6. The substances cannot be detected by liquid chromatography (LC) or gas chromatography (GC), or only with great difficulty.
7. Samples to be analyzed would damage LC or GC columns.
8. The solvents used would attack the sorbents in LC columns.