This article throws light upon the top four separation techniques used for the analysis of herbal medicines. The techniques are: 1. Thin Layer Chromatography 2. Gas Chromatography 3. High-Performance Liquid Chromatography 4. Capillary Electrophoresis.
Technique # 1. Thin Layer Chromatography (TLC):
TLC is an adsorption chromatography in which samples are separated based on the interaction between a thin layer of adsorbent and a selected solvent. A thin layer of an adsorbent attached on a plate is used as stationary phase, while the selected solvent is the mobile phase. TLC was the common method of choice for herbal analysis before instrumental chromatography methods such as GC and HPLC were established.
Even nowadays, TLC is still frequently used for the analysis of herbal medicines since various pharmacopoeias still use TLC to provide the first characteristic fingerprints of herbal extracts. TLC is mainly used as a simple method for initial screening, with semi quantitative evaluation together with other chromatographic techniques.
TLC has the advantage of many-fold possibilities of detection in analysing herbal medicines. In addition, TLC is rather simple and can be employed for multiple sample analysis. For each plate (20 x 20 cm), more than 30 spots of samples can be studied simultaneously.
Thus, the use of TLC to analyse herbal medicines is still popular. With the help of the CAMAG video store system (CA- MAG, Switzerland) and TLCQA-UV methods it is possible to get useful qualitative and quantitative information from the developed
TLC plate. Moreover, with the help of image analysis and digitized technique developed in computer science, the evaluation of similarity between different samples is also possible. TLC is a convenient method of determining the quality and possible adulteration of herbal products. For further details about TLC the readers could consult the book by Wagner et al. (1996).
Technique # 2. Gas Chromatography (GC):
GC is a method for separating components of mixtures of volatile compounds. In this technique, species distribute between a mobile gas and a stationary liquid phase. Thus, when sample molecules are in the liquid phase they are stationary. The rate of migration for a chemical species is determined by how much of it distributes into the gas phase.
The higher the percentage of material in the gas phase, the faster it will migrate. For example, a species that distributes itself 100% into the gas phase will migrate at the same rate as the flowing gas. On the other hand, a species that distributes 100% into the stationary phase will not migrate at all. Species that distribute themselves partly in both phases will migrate at an intermediate rate.
It is well-known that many pharmacologically active components in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by GC is very important in the analysis of herbal medicines. The GC analysis of the volatile oils has a number of advantages. Firstly, the GC of the volatile oil gives a reasonable “fingerprint” which can be used to identify the plant.
The composition and relative concentration of the compounds in the volatile oil are characteristic of the particular plant and the presence of impurities in the volatile oil can be readily detected. Secondly, the extraction of the volatile oil is relatively straight forward and can be standardised and the components can be readily identified using GC-M5 analysis.
The relative quantities of the components can be used to monitor or assess certain characteristics of the herbal medicines. Changes in composition of the volatile oil may also be used as indicators of oxidation, enzymatic changes or microbial fermentation.
The advantages of GC clearly lie in its high sensitivity of detection for almost all the volatile chemical compounds. This is especially true for the usual FID detection and GC-MS. Furthermore, the high selectivity of capillary columns enables separation of many volatile compounds simultaneously within very short times.
Thus, over the past decades, GC is a popular and useful analytical tool in the research field of herbal medicines.
With the use of GC-MS, reliable information on the identity of the compounds is available as well. However, the most serious disadvantage of GC is that it is not convenient for analysis of polar and non-volatile compounds. For this, it is necessary to use tedious sample work-up which may include derivatisation. Therefore, the HPLC become another necessary tool for the comprehensive analysis of the herbal medicines.
Technique # 3. High-Performance Liquid Chromatography (HPLC):
HPLC is a popular method for the analysis of herbal medicines because it is easy to learn and use, and is not limited by the volatility or stability of the samples. In a liquid chromatographic process, a liquid permeates through a porous solid stationary phase and elutes the solutes into a flow-through detector.
The stationary phase is usually in the form of small-diameter (5-10 mm) uniform particles, packed into a cylindrical column. The typical column is constructed from a rigid material (such as stainless steel or plastic) and generally 5-30 cm long and the internal diameter is in the range of 1 – 9 mm.
In general, HPLC can be used to analyse almost all the compounds in the herbal medicines, and it has therefore received the most extensive application in recent years in the analysis of herbal medicinal preparations.
Reversed-phase (RP) columns are probably the most popular columns used for the analysis of herbal medicines. It is noteworthy that the optimal separation conditions for the HPLC involves many factors; compositions and pH of the mobile phases, flow rate, mode of elution, sample preparation method, detection system, etc.
Thus, experimental suitable protocols should be designed for the optimal separation of each mixture of compounds.
In order to obtain better separation, some new techniques have been recently developed in the research field of liquid chromatography. These are micellar electro kinetic capillary chromatography, high-speed counter-current chromatography (HSCCC), reversed-phase ion-pairing HPLC (RP-IPC-HPLC), and strong anion-exchange HPLC.
These newer techniques will provide new opportunities for more effective separation for certain specific extracts of some herbal medicines. HPLC coupled to a photodiode array detector (HPLC-PDA) has been widely used for the analysis of crude plant extracts.
The UV spectra of natural products obtained on-line by HPLC-PDA give useful information on the type of constituents and in the case of certain classes of compound. HPLC-PDA has become a common technique in most analytical laboratories in the world now.
With the additional UV spectral information, the qualitative analysis of complex samples in herbal medicines turns out to be much easier than before. For instance, checking peak purity and comparing with the available standard spectrum of the known compound to the one in the investigated sample.
With the introduction of electrospray mass spectrometry, the coupling of liquid chromatography and mass spectrometry (LC-MS) has opened a new way for the wide and routine application of this technique to the analysis of herbal medicines. Several valuable review articles dealing with LC-MS and its applications have been published.
Technique # 4. Capillary Electrophoresis (CE):
CE was introduced in early 1980s as a powerful analytical and separation technique and has since been extensively developed. It allows an efficient way to acquire data on the purity and complexity of a sample, and can handle virtually every kind of charged sample component ranging from simple inorganic ions to DNA.
There has been an obvious increase in use of electrophoretic methods, especially capillary electrophoresis, in the analysis of herbal medicines in last decade.
CE is a promising technique for the separation and analysis of active ingredients in herbal medicines since it needs only small amounts of standards, and can analyse samples rapidly with very good separation ability. It is also a good tool for producing the chemical fingerprints of the herbal medicines, since it has similar technical characteristics of liquid chromatography.