Chromatography is the science which studies the separation of molecules based on differences in their structure and/or composition. In general it involves moving (in a mobile phase) preparation of materials to be separated over a stationary support. Based on their differential affinity between the mobile and stationary phase, molecules in the preparation get separated between the phases. Chromatographic separations can be achieved on a variety of supports. Immobilized silica on glass plates in TLC, highly subdivided silica on aluminium sheets in HPTLC, volatile gases in GC, paper in PC and liquids with incorporated hydrophilic, insoluble molecules in HPLC are some of the supports effecting separations.

Chromatographic techniques are invaluable in the analysis of herbal drugs as they offer very powerful separation ability such that even complex chemical components in herbal drugs can be separated into relatively simple sub-fractions. Despite advances in chromatography with sophisticated instrumentation, basic techniques like column chromatography and TLC are still invaluable aids for the separation of phytoconstituents in quantity and identification of phytoconstituent profiles respectively.

1. Thin layer chromatography
   TLC was the common method of choice for herbal drug analysis long before instrumental chromatographic methods like GC and HPLC were established and in use even now. Its ease of usage, versatility, sensitivity, simplicity of sample preparation and simultaneous applicability to multiple samples make it a convenient tool for the detection of quality and adulteration in herbal drugs. TLC is a powerful and relatively rapid technique to distinguish between chemical classes, where macroscopy and microscopy may fail. Chromatograms of essential oils are widely published in scientific literature and can be of invaluable help in herbal drug identification.
2. High performance thin layer chromatography
   It is a sophisticated, advanced and an automated version of TLC, which combines the simplicity and precision of TLC with the speed and efficient quantitation which modern instrumentation provides. It employs high performance precoated silica gel plates which give more efficient and reproducible separation than conventional grades of silica. Very small accurately measured volumes may be applied at predetermined rate on exact locations at measured distances from neighbouring spots if any. Development time is much smaller and the developed chromatogram may be evaluated by comparison with reference standards. It may also be quantified with greater sensitivity and precision than is possible with conventional TLC.Forced-flow planar chromatography (FFPC), rotation planar chromatography (RPC) and over pressured layer chromatography (OPLC) are some of the updated techniques in TLC being employed in herbal drug analysis.
3. Gas chromatography
   Separation and analysis of volatile oil components such as essential oils and fatty oils are best undertaken on GC. A GC chromatogram gives a reasonable fingerprint of the volatile oil that is used to identify the plant. The composition and relative concentration of the organic compound in the volatile oil are characteristic of the particular plant and presence of impurities may be readily detected. GC enables straightforward extraction of volatile oil and it can be standardized by component identification using GC-MS analysis. GC-MS is the first online combination of chromatography and spectroscopy and is widely used in the analysis of essential oils. Monitoring relative quantities of components will assess changes in volatile oil composition, indicating oxidative and other changes. GC with flame ionization detector (FID) gives sensitivity of detection of all volatile compounds in a mixture. However GC is not convenient for polar compounds and non-volatile compounds.
4. High performance liquid chromatography
   HPLC is the most preferred method for quantitative analysis of more complex mixtures. It is easy to learn and use and is not limited by the volatility or stability of the compound. HPLC in general can be used to analyse almost all compounds in herbal drugs. Extensively applied for herbal drug analysis, reversed phase columns are most used for analytical separation of phytocompounds. It is simplest to use once conditions are optimized that require skill, expertise and experience.Micellar electrokinetic capillary chromatography (MECC), high-speed counter current chromatography (HSCCC), low-pressure size exclusion chromatography (SEC), reversed phase ion-pairing HPLC (RP-IPC-HPLC), strong anion-exchange HPLC (SAX-HPLC) are some of the newer methods that provide better opportunities for good separation of specific extracts of some herbal drugs. Their versatility is an advantage since most phytoconstituents are non-chromophoric. HPLC coupled with evaporative light scattering detector (ELSD) is an excellent detector of non-chromophoric compounds. It provides direct analysis of many pharmacologically active components in herbal drugs. The response of ELSD depends on size, shape and number of eluate particles rather than on the structure and/or chromatophore of analytes as does a UV detector.HPLC-MS is useful for direct structure elucidation of phytoconstituents in herbal drugs. It provides the advantage of both chromatography as separation method and MS as identification method.
5. Chromatographic fingerprint analysis
   Herbal drugs, singularly and in combinations, contain a myriad of compounds in complex matrices in which no single active constituent is responsible for the overall efficacy. This creates a challenge in establishing quality control standards for raw materials and standardization of finished herbal drugs. Botanical extracts made directly from crude plant material show substantial variation in composition, quality and therapeutic effects. Standardization involves adjusting it to a defined constituent or group of constituents with known therapeutic activity. When active principals are unknown, marker constituents are to be established among the known constituents of the extract for standardization purposes.

Markers are chemically defined constituents of an herbal drug that are important for the quality of the finished product. Ideally, the chosen marker should also be responsible for the therapeutic effect of the herbal drug. When the marker is a definite constituent or group of constituents associated with therapeutic activity, it is true standardization.

Ginkgo with its 26% ginkgo flavones and 6% terpenes is a classic example. Such extracts are highly standardized and no longer represent the whole herb and are now considered as phytopharmaceuticals.

Markers are basically of the following three types:

• Active principals – One or a few constituents specific to the particular herbal drug responsible for the claimed activity. E.g. curcumin for turmeric, E- and Z- guggulsterones for guggulipid, ginsenosides for ginseng, andrographolide for kalmegh, silymarin for milk thistle, sennosides for senna, piperine for pepper.
• Chemical markers – Compounds reported from the respective drugs but not specific to them. Activity specific to the marker may or may not be proven. E.g. Vasicine from Adhatoda vasaca is its major alkaloid, active principal and an important biomarker. However it cannot be considered as marker specific for Vasaka because it is also present in other species of Adhatoda, Sida sps, Paganum harmala etc. Likewise hyoscyamine is present in several Solanaceous species. On the other hand constituents such as rutin, quercetin or lapachol are found in a number of related plants and therapeutic activity of the drugs may not be attributed to these compounds.
• General markers – Compounds widely present in many plants such as gallic acid, lupeol, stigmasterol, β-sitosterol for which activity may or may not be reported.

Thus traditionally only a few markers of pharmacologically active constituents were employed to assess the quality and authenticity of complex herbal medicines. However, the therapeutic effects of herbal medicines are based on the complex interaction of numerous ingredients in combinations which are totally different from those of chemical drugs. Thus many kinds of chemical fingerprint analysis methods to control the quality of herbal drugs have gradually come into being. Development of fingerprint profiles of plant extracts is one of the most salient requirements in herbal drug standardization.

Chromatographic fingerprint of a plant extract is its chromatographic pattern developed under a standard, repeatable set of experimental conditions.

Developing such reproducible and highly specific chromatographic patterns for herbal drug extracts has been a significant milestone in herbal drug standardization. Thus these extract-specific chromatographic patterns are considered the ‘fingerprint’ of the analysed extract.

TLC fingerprinting is of key importance for herbal drugs made up of essential oils, resins and gums. In TLC fingerprinting, the data that can be recorded using a HPTLC scanner includes the chromatogram, retardation factor (R_f) values, the colour of the separated bands, their absorption spectra, λ_max, and shoulder inflections of all the resolved bands. Information generated from a HPTLC chromatogram has potential application in the identification of an authentic drug by excluding adulterants and also in maintaining the quality and consistency of the drug. HPLC fingerprinting includes recording of the chromatograms, retention time of individual peaks and the absorption spectra with different mobile phases. GC is used for generating the fingerprint profiles of volatile oils and fixed oils.

Such fingerprint profiles are usually distinctive and form a benchmark for the drug when either the active principals are not known or when chemical markers are not available. Also markers, either active principals or chemical markers, can be quantified by HPLC, GC and HPTLC methods from the developed chromatograms. E.g. phyllanthin, hypophyllanthin, gallic acid, ellagic acid content in Phyllanthus amarus whole plant; eugenol, gallic acid, ursolic acid, oleanolic acid content in Ocimum sanctum leaf. Several methods for quantification of phytochemicals using HPLC are widely reported in literature.

Fingerprint analysis of herbal drugs therefore represents a comprehensive qualitative approach for the purpose of species authentication, evaluation of quality and ensuring the consistency and stability of herbal drugs and their related products. The entire pattern of compounds can then be evaluated to determine not only the presence or absence of desired markers or active constituents but the complete set of ratios of all detectable analytes.

According to the concept of photoequivalence the chromatographic fingerprint of an herbal product is to be identical or similar to a similar profile of a clinically proven reference product.

Apart from serving the purpose of standardization and quality control, the fingerprint profiles, especially of TLC, aid in the experiments for bioassay-guided fractionation leading to the isolation of active compounds.

The chemical fingerprints obtained by chromatographic and electrophoretic techniques, especially by hyphenated chromatographies, are strongly recommended for the purpose of quality control of herbal medicines, since they might represent appropriately the ‘chemical integrities’ of herbal medicines and therefore be used for authentication and identification of the herbal products.

To summarize, TLC, HPLC and GC, quantitative TLC (QTLC) and HPTLC can determine the homogeneity of a plant extract. OPLC, infrared and UV-visible spectrometry, MS, GC, liquid chromatography (LC) used alone, or in combinations such as GC-MS, LC-MS and nuclear magnetic resonance (NMR) spectroscopy and electrophoretic techniques especially by hyphenated chromatographic techniques are powerful tools, often used for standardization and in quality control of both the raw material and finished herbal drugs including polyherbals. The results from these sophisticated techniques provide a chemical fingerprint as to the nature of chemicals or impurities present in the plant or extract. Methods based on information theory, similarity estimation, chemical pattern recognition, spectral correlative chromatograms (SCC), multivariate resolution, the combination of chromatographic fingerprints and chemometric evaluation for evaluating fingerprints are all powerful tools for quality control of herbal products.