CHROMATOGRAPHY BASED CHEMOMETRIC FINGERPRINTING, ISOLATION & QUALITY CONTROL OF PHYTOCHEMICALS

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COLUMN CHROMATOGRAPHY [7,8,9]
Column chromatography efficiently separate and purify phytochemicals in laboratory as well as industrial scale without any elaborated instrumentation. Cold extraction of alkaloids, componential resolution of pigments such as chlorophyll & Xanthophyll, isolation and purification of amino acids & glycosides, determination of quinine and strychnine in elixirs are some of the phytochemical applications of this technique.

The Russian Botanist Mikhail Semyonovich Tsvet serendipitously discovered column chromatography while filtering petroleum ether extract of chlorophyll through a column packed with calcium carbonate. Principally, column chromatography is an analogues technique to that of thin layer chromatography, but differs from later in mode and direction of separation. In former technique phenomenon of separation takes place inside column towards the direction of gravity however same, but in a reverse manner is true for thin layer chromatography. Dimensions of chromatographic column, particle size of adsorbent, packing of chromatographic column, nature of solvent system employed for elution, temperature, solvent flow rate, run time of column, and quantity of sample are some of factors that affect overall resolution of this chromatographic procedure. In spite of expansive and tedious technique commercial utilization of column chromatography is none lesser than other techniques. Efficient handling of sample irrespective of its quantity and nature, availability of wide range of adsorbents, selection & recyclization of vast solvent system, sharp-end excellent purity of end product, and lesser space utilization are some of the edging points of this technique. On other hand consumption of greater quantity of mobile phase, complicated procedure, time consumption, need of technical hand, and higher cost of separated product are few tailing points of column chromatography.

                Adsorbent

Phytochemical screening

Calcium carbonate Napthaquinones and Xanthophylls
Calcium hydroxide Carotenoids
Calcium oxalate Anthraquinones
Calcium silicate Carbohydrates and their phenylosazones
Calcium sulphate Lipids and steroids
Dicalcium phosphate Carotenes
Magnesium silicate Esters, steroids, glycosides, lactones
Silica gel Fatty acids, sterols, glycerides, amino acids, sugars etc
Dextran gel Protein and nucleic acid
Charcoal & activated carbon Sugar amino acid
Sucrose Chlorophylls and xanthophylls
Polyethylene powder Esters and fatty acid
Polyamide Flavanoids
Aluminum silicate Sterols and glycosides
Aluminum oxide ore (Bauxite) Ergot

Table 02: Adsorbents and their role in phytochemical screening

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)[7,8,9]
Csaba Horvath in 1964 at Yale University introduced “High performance liquid chromatography” which is nothing but a highly advanced automated form of column chromatography. In terms of procedural aspect HPLC is somewhat analogous to column chromatography however distinguished from later in instrumentation and mode of solvent percolation. Isolation & analysis of various phyto-ingredients such as cardiac glycosides, bioactive alkaloids, anthocynides, xanthines, caffines, isofavones, and tannins can be done profoundly by reverse phase HPLC employing C-18 column. On other hand ion exchange HPLC aids up in separation and analysis of polar and non-polar vitamins. HPLC in general makes use of narrower bored optimally packed stainless steel columns through which mobile phase forces under high pressure (1000-300psi) enhancing multidimensional efficiency of the system. Rather than this, small sample size, overall automation of technique, computerized data handling, automated sample loading, better resolution, consuming least quantity of solvents, efficient handling of wide range multi-component mixtures, rapid in process, non-destructive methodology, flexibility in procedure, and instrumentation portability are some of the extraordinary features of this technique.

Packing material (stationary phase) used in high performance liquid chromatography is although same as that of column chromatography, but is of smaller size (3-10 micrometer) providing greater surface area for better resolution of sample into individual components. Reduction of particle size no doubt increases separation efficiency of this chromatographic system, but also impedes solvent flow through chromatographic column essentializing pressure pumps. Stability, chemical inertness, purity, uniformity in particle size, large surface area, pressure resistance, procedure adopted for fractionation, polarity of solvent system, and physiochemical characteristics of sample are some of the paramount features considered while seeking appropriate stationary phase for chromatographic fractionation. Likewise, an ideal solvent system for analytical as well as separation workout can be picked by trial and error method or by exhaustive literature survey. As discussed earlier, a preliminary solvent optimization for HPLC can also be done by employing thin layer chromatography.  

GAS CHROMATOGRAPHY [7,8,9]
Gas chromatography (GC) or gas liquid chromatography (GLC) is a sister chromatographic technique of high performance liquid chromatography, but distinguished from later in terms of sample preparation, its handling, physiochemical characteristics of sample, mode of sample injection, component detection, instrumentation, its cost, overall runtime, and detectors employed in measurement. HPLC still lacks universal detector in terms of measurement when compared with gas chromatography. The credit for inventing gas chromatography was goes to Martin & Synge (1941) however first true experimentation on this technique was done by Martin & James (1954). It is a well known fact that nature is enriched with numerous pharmacologically active volatile phytochemicals, gas chromatography aids up in their analysis and separation. Analysis of volatile components by this technique has number of advantages such as the technique provides perfect chemo-fingerprinting of volatile components which can be used to relate and identified various plant species and compare the concentration and quality of their volatile components. Secondly, the technique use for standardization & impurity profiling of herbal formulations by readily identifying volatile impurities present in them. Beside this, conjugation of gas chromatography with mass spectroscopy (GC-MS) enhances scope of this technique both qualitatively and quantitatively. The major applications of gas chromatography in phytoindustry includes qualitative and quantitative determination of volatile contents in herbal formulations, analysis of volatile oils, volatile impurity profiling in herbal medicines, analysis of herbal formulations containing belladonna, opium, and conium alkaloids, analysis of cardioactive glycosides, and amino acids.

Generally volatile, less polar, and thermally stable compounds are better candidate and injected directly into the chromatographic system however special consideration and efforts has to be made for high molecular weight, non-volatile, and highly polar samples since direct injection of same will leads to damage chromatographic column hence special techniques such as pyrolysis, photolysis, and sample derivatization are adopted for their efficient handling.  

SIZE EXCLUSION CHROMATOGRAPHY [10]
Size exclusion chromatography (SEC) also known as exclusion chromatography (EXC) is yet another important chromatographic technique employed in separating phytochemical mixture into individual components according to their size or molecular weight with an aid of highly crossed- linked polymer based stationary phase. When fractionating component of different sizes are passes through cross-linked macromolecular stationary phase, various components of fraction gets separated out owing to their tangling property with stationary phase. Large molecular weight component of mixture bypasses the pores of stationary phase; moves along with mobile phase hence get separated out. 

The most commonly used gels in SEC are hydrophilic in nature and swells-up when comes in contact with aqueous solvents. Xerogels of polyacrylamide, Sephadex (Dextran), and Agarose are some generally used stationary phase in size exclusion chromatography.  One of the most important key feature of this technique is separation of bioactive large molecular weight components without affecting their biological property. The technique of size exclusion chromatography efficiently employed in fractionation of large molecular weight biomolecules such as polysaccharides, polypeptides, proteins, and amino acids.

CONCLUSION
Phytochemicals including herbal products practically represents distinct class of chemical compounds necessitating utilization of modern analytical tool for their multidimensional analysis.  Since most of the marketed herbal products or crude phytoformulations are none the less complex mixture of chemical compounds, requiring chemo-specific-fingerprinting to elucidate their pharmacological activity. In recent years, different chromatographic techniques either alone or in combination idealistically implement isolation, characterization, and quality control of herbal products providing firm platform for their global authentication and commercialization.

REFERENCES
1. Ncube N.S., Afolayan A.J., Okoh A.I.; Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends; Afri. Jornl. Of Biotech; 2008; 7(2); 1792-1806.
2. Shikha S., Vijay K.L., Kamlesh K.P., Polyherbal formulation based Indian medicinal plants as antidiabetic phytopharmacol; 2012; 2(1); 1-15.
3. Omboon V.; Drug Discovery Research in Pharmacognosy; InTech; Shanghai; 2012.
4. Oluyemisi F., Omoregie H., Ochogu P; Standardization of Herbal Medicine- Review; Int. J. of Biodiv. & Conser; 2012; 4(3); 101-112.
5. Watson D.G., Pharmaceutical analysis; Churchill Livingstone, Edinburgh; 1999.
6. Helmut G, Alex W.; Basic Principles of Chromatography;  Wiley-VCH;  Weinheim; 2001; Edn 1, 173-197.
7. Beckett A.H., Stenlake J.B.; Practical Pharmaceutical Chemistry; CBS Publisher; New Delhi; 2005
8. Skoog D.A., Holler, F.J., Nieman T.A.; Principles of Instrumental Analysis; Harcourt Brace & Co.; 1998.
9. Jeffery G.H.,  Bassett J., Mendham J., Denny R.C.; Textbook of Quantitative Chemical Analysis; Longman Scientific & Technical; 1989.
10. Sharma, B.K.; Instrumental methods of chemical analysis-Introduction to analytical chemistry; Goel Publishing House; 2000.

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