Appendix XI W. High-Performance Thin-Layer Chromatography of Herbal Drugs and Herbal Drug Preparations

(Ph. Eur. method 2.8.25)

High-performance thin-layer chromatography (HPTLC) is used for qualitative analysis of herbal drugs and herbal drug preparations. It is a thin-layer chromatographic technique (2.2.27) that, unless otherwise stated in an individual monograph, uses a glass plate coated with a uniform, porous layer (average pore size 6 nm), typically 200 µm thick, of irregular particles of silica gel between 2 µm and 10 µm in size and with an average size of 5 µm, a polymeric binder and a fluorescence indicator (F254). The results are qualified using a system-specific suitability test.

EQUIPMENT

The equipment used for qualitative HPTLC typically consists of:

— glass plates, as described above, usually 20 × 10 cm in size;
— devices suitable for the application of specified volumes of solutions as bands and allowing control of the dimensions and position of application;
— a device suitable for conditioning the stationary phase at the prescribed relative humidity;
— a suitable chromatographic tank (for example, a twin trough chamber);
— a device suitable for the reproducible drying of the developed plate;
— devices suitable for the application of reagents to, and heating of, the plate as part of the derivatisation procedure;
— a system suitable for the electronic documentation of chromatograms under 254 nm UV, 366 nm UV and white light.

NOTE: Normal thin-layer chromatographic methods using glass plates or sheets coated with particles of 5-40 µm or HPTLC aluminium-backed sheets may be used, provided that the results obtained fulfil the general system suitability criteria that the bands develop perpendicular to the lower edge of the plate and the solvent front is parallel to the upper edge of the plate, and satisfy the system-specific suitability test stated in the individual monograph.

METHOD

Preparation of test solution

Unless otherwise stated in the individual monograph, the test solution is usually prepared as follows.

For dry herbal drugs or dry herbal extracts, mix 0.5 g of the powdered herbal drug or 0.1 g of the dry herbal extract with 5 mL of methanol R and sonicate for 15 min; filter or centrifuge and use the filtrate or supernatant as the test solution.

For essential oils, dissolve 50 µL of the essential oil in 1 mL of toluene R and use this solution as the test solution.

Preparation of reference solutions

Unless otherwise stated in the individual monograph, reference solutions are usually prepared as follows. Prepare a 1 mg/mL solution of suitable reagent(s) or reference standard(s) in methanol R or, for essential oils, in toluene R. Prepare a second reference solution (diluted reference solution) by mixing 1 volume of this solution and 3 volumes of the same solvent. Both solutions are used as intensity references.

Intensity marker

Use one or more of the substances in the reference solution and in the diluted reference solution as intensity marker(s) for the evaluation of the chromatogram.

Preparation of system-specific suitability solution

Prepare the solution as stated in the individual monograph.

Sample application and plate layout

Unless otherwise stated in the individual monograph, samples are applied as narrow bands of 8 mm in length at a distance of 8 mm from the lower edge of the plate. The centre of the first track, which is used for the system-specific suitability solution, is positioned 20 mm from the left edge of the plate. The minimum distance between tracks (centre to centre) is 11 mm. A maximum of 15 tracks are applied onto a standard plate. If no electronic solvent front detection device is used, the development distance is marked with a pencil close to the right or left edge of the plate.

Conditioning of the plate

Following sample application and unless otherwise stated in the individual monograph, expose the plate to air with a suitable relative humidity obtained using a saturated solution of magnesium chloride R (for example, by allowing the plate to stand in a closed chamber containing such a solution for 1 h or by using preconditioned air).

Preparation of the tank and development of the plate

Unless otherwise stated in the individual monograph, the chromatographic separation is performed in a saturated tank. Where a twin trough chamber is used, place a piece of filter paper in the rear trough. Load the tank with a sufficient quantity of mobile phase to wet the filter paper completely and achieve a level of 5 mm in both troughs. With the lid closed, leave the tank for 20 min for saturation. Introduce the plate in a vertical position into the front trough of the tank so that the coating layer faces the filter paper. When the mobile phase has reached 70 mm, remove the plate from the tank and dry in a vertical position in a stream of air at room temperature. Other tank configurations and developing distances may be specified in an individual monograph.

NOTE: Other tanks may be employed if the results obtained fulfil all of the system suitability criteria.

Visualisation

Chromatograms on the plate are visualised as stated in the individual monograph. Where derivatisation reagents are used, typically 3.5 mL of reagent solution is homogenously sprayed onto a plate of size 20 × 10 cm, or the plate is immersed into the reagent solution, typically at a speed of 5 mm/s for a dwell time of 1 s. Observation may be performed under 254 nm UV, 366 nm UV or white light prior to and/or after derivatisation. When pictures are digitally recorded, exposure time should be adjusted based on the track with the system-specific suitability solution.

System-specific suitability test

This test is based on the separation of 2 substances that have similar retardation factors (RF values) but that are barely separable under the specified chromatographic conditions (for example, chlorogenic acid and hyperoside in chromatographic systems used for flavonoids). The results for the test and reference solutions are only valid when the system-specific suitability solution satisfies the separation requirement stated in the individual monograph.

Visual evaluation

The chromatograms obtained with the test and reference solutions are compared against the description, under the results section in the individual monograph, with respect to zone position and colour, as well as intensity for the test solution. Zones of the test solution described in the results table without a descriptor have intensities similar to the zone of the intensity marker in the reference solution. Zones described as ‘intense’ are visually more intense than the zone of the intensity marker in the reference solution; zones described as ‘faint’ are visually less intense than the zone of the intensity marker in the reference solution, but equal to or more intense than the zone of the intensity marker in the diluted reference solution; zones described as ‘very faint’ are visually less intense than the zone of the intensity marker in the diluted reference solution.