Thin layer chromatography(TLC).
Thin-layer chromatography is a technique used to separate complex mixtures of substances into their individual components. Separation essentially takes place as a result of the individual components being distributed between the mobile and stationary phases.
A fully automatic sampler is used to apply the mixture to be separated to the so-called stationary phase. The stationary phase may take the form of a thin layer of silica gel, which has been applied to a carrier sheet made of glass or aluminium. Other support media include aluminium oxide and silylated silica gel, which is used for reversed phase chromatography. Stationary phases with homogeneously distributed minute particles of material are used in high-performance thin-layer chromatography (HPTLC).
The actual separating process takes place in a TLC chamber that has been filled with the mobile solvent required for the separation. The mobile solvent (also referred to as the mobile phase) is usually a mixture of different solvents. The composition of the mobile solvent must be adapted to suit the substances being separated. The prepared TLC plate is placed vertically in the TLC chamber, which is saturated with mobile solvent vapour, in such a way that part of the plate is immersed in the solvent.
The mobile phase then moves up the plate through the stationary phase by capillary action. When the mobile solvent reaches the applied sample mixture, the mixture dissolves and the actual separation process begins.
The dissolved particles interact with the mobile and stationary phases to a greater or lesser extent according to their respective properties. A particle therefore remains closer to the starting point or moves further up the plate according to the ratio of forces. The compounds in the sample mixture react with the stationary and mobile phases to a varying degree and they therefore travel up the plate at different rates, separating the mixture into its individual components. Molecules with properties similar to those of the mobile phase travel faster than those that react more strongly with the stationary phase. The results of the separation can often be detected by simply looking at the plate. In some cases, however, it may be necessary to chemically transform (derivatise) the substances on the plate using certain reagents in order to make them detectable.
Apart from optimised facilities for sample preparation, sample application, derivatisation and detection, PhytoLab also uses state-of-the-art equipment for documentation of the results obtained. Digital documentation systems offer a means of filing and archiving the results of analyses and making them available to customers on request at any time. Apart from identification and purity testing, thin-layer chromatography can also be used for the quantitative determination of individual constituents. The interesting zones on the TLC plate undergo densitometric analysis for this, using special TLC scanners. Evaluation then takes the form of comparing the values measured for the sample with those obtained for a standard, which has also been applied.
Each plant contains a characteristic spectrum of constituent substances (e.g. flavonoids, alkaloids, anthraquinone), which can be used for identification purposes in a similar way as a fingerprint. Following preparation and analysis according to one of these constituent substances, the TLC fingerprint of a thin-layer chromatographic separation frequently offers a means of identifying the examined material and/or testing for contamination or adulteration. When identifying plant material, PhytoLab's analysts apply authentic reference material, qualified reference substances and commonly mistaken or adulterated plants to the TLC plate alongside the actual sample. PhytoLab is in possession of a comprehensive collection of comparison drugs, which are used for this. They include an extensive range of Chinese drugs that appear in the Chinese Pharmacopoeia (Ph. Chin.). The authenticity of these comparison drugs is certified by the NICPBP (National Institute for the Control of Pharmaceutical and Biological Products), a Chinese drug surveillance authority. These comparison drugs also put PhytoLab in a position to be able to reliably identify herbal drugs used in the field of Traditional Chinese Medicine (TCM) and to check their purity by use of reference subtances. The test for aristolochic acid by tlc or more specific by hplc ms is of high interest in last years.Reference substances are qualified at PhytoLab and are available for purchase.
PhytoLab has established all of the thin-layer chromatographic methods listed in the Ph. Eur. as routine procedures. It goes without saying that methods laid down in other pharmacopoeias (German Homeopathic Pharmacopoeia (HAB), Chinese Pharmacopoeia, British Pharmacopoeia, United States Pharmacopoeia, Swiss Pharmacopoeia, Ph. Franc. X etc.) and monograph collections are performed as a matter of routine as well.
The TLC specialists at PhytoLab are continuously developing new methods and procedures in addition to those described in these works of reference. Special attention is focused on the development of TLC methods in the herbal drug (phytopharmaceutical) sector, where they are used to identify individual herbal components in mixtures. These procedures are then carried out at regular intervals within the framework of stability testing for registration or market authorisation projects. The ideal facilities at the PhytoLab laboratory produce optimum, reproducible results.
Participating in the work carried out by the European Pharmacopoeia Commission's Expert Group 13 B (Phytochemistry B), PhytoLab is intensively involved in the establishment of new methods of testing and analysing herbal active substances and drugs.
