Separation Techniques and Herbal Medicine

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.