Sample preparation is at the beginning of the HPTLC process and can significantly influence the result. For practical reasons, it should be kept as simple as possible. Three main steps are included:
- The sample homogenization, comprising drying, grinding, freezing, lyophilizing and/or milling step(s);
- The choice of solvents;
- The extraction method.
Choosing a suitable extraction solvent involves several considerations:
- It should be non-hazardous (health, explosion, flammability), stable, and not reactive with the sample;
- It should be inexpensive and easily removable from the plate.
Polar solvents are water, ethanol, methanol;
Medium polarity solvents are ethyl acetate, dichloromethane, ethers;
Non-polar solvents are toluene, cyclohexane;
Some extraction methods are:
- Maceration and percolation;
- Reflux/Soxhlet;
- Steam distillation (for essential oils / terpenes);
- Supercritical fluid extraction;
- Shaking;
- Extraction in the ultrasonic bath (sonication);
- Warm water-bath (boiling);
- Liquid-liquid extraction;
- Microwave-assisted extraction;
- Accelerated solvent extraction.
For samples with unknown properties, or as a convenient starting point, CAMAG has proposed a simple extraction scheme covering different solvent polarities: 0.5 g of the powdered sample (amount can be adjusted) are extracted by sonication for 10 minutes with 5.0 mL of the following solvents:
| Neat solvents and their mixtures | Acidic mixtures | Basic mixtures |
| Heptane/Toluene tert. Butyl methyl ether Dichloromethane Ethyl acetate Acetone Acetonitrile Ethanol Methanol Water Ethanol – water 7:3 (v/v) Methanol – water 8:2 (v/v) | Methanol – acetic acid 9:1 (v/v) Water – acetic acid 9:1(v/v) | Methanol – ammonia 25% 8:2 (v/v) Water – ammonia 25% 8:2 (v/v) |
The 15 test solutions can be analyzed in parallel with a mobile phase from the literature or a universal solvent system(s).
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