Thin-layer chromatography:- [T.L.C]
In 1985 Stahl demonstrated the wide applicability of TLC. In outline the method consists of preparing, on a suitable glass plate, a thin layer of material, the solvent which may be either an adsorbent as used in column adsorption chromatography. The mixtures to be resolved are dissolved in a suitable solvent and placed as spots on the film towards one end of the plate; this end is then dipped in a suitable solvent mixture and the whole enclosed in a airtight container. The solvent front travels up the film and after a suitable time the plate is removed. The solvent front is marked. The solvent is allowed to evaporate and the positions of the separated compounds are determined by suitable methods.

1. Tradionations can effect more rapidly with smaller quantities of the mixture.
2. The separated spots are usually more clearly demarcated from one another and the nature of the film is such that drastic agents, such as conc. H2SO4 which would destroy a paper chromatogram can be used.
3. With adsorption TLC various substances exhibit different adsorptive capacities and any one material may vary in its activity according to the pretreatment.
4. Method of detection does not place any restriction on choice of eluent easily inspected.
5. Neutral, basic, acidic or purely aqueous eluents can be employed. The whole chromatographic system is flexible.
6. TLC can be exploited in the investigation and cultivation of medicinal plants it is possible to run many samples of extracts from different chemical races simultaneously with authentic standards and high performance individuals can be recognized, selected and bred.

High performance Thin layer chromatography {HPTLC}
HPTLC is a major advancement of TLC principle requiring shorter time and better resolution. The basic difference between conventional TLC and HPTLC is only in particle and pore size of the sorbents. The use of smaller particle size helps in greater resolution and sensitivity. About 3-6 cm solvent front migration is sufficient to effect proper separation. Whaman HPTLC plates are produced from 4-5m. silica gel with an inert binder to form a 200m layer. About 7cm development distance is achieved in about 4 minutes.

Sample preparation in HPTLC needs a high concentrated solution, as very less amount of sample need be applied. The size of the sample must not exceed 1mm in diameter. There are different techniques for the spotting of samples. One of them is self loading capillaries in which small volume of samples may be applied to the HPTLC plate surface using platinum iridium tubing fused into the end of a length of glass tubing. Contact spotting, pre adsorbent, or concentration zones. Chemicals focusing and programmed multiple developments are the other methods of spotting the samples.

The analytical profiles for cardenolides, tropane alkaloids flavanoids, steroidal compounds, antracene glycosides, lipids etc. have been developed using the technique HPTLC.
TLC/ HPTLC techniques are conveniently applied for completion of profiles pertaining to varied range of bioconstituents such as berberine, quinine and related alkaloids, opium alkaloids, coclchicine, chelidonine, sanguinarine, serpentine, raubasine, asarone, elemicin, eugenol, thymol, coumarin, pulegon sarsapogenin lanatosides digitoxigenin, purpurea glycoside A, flavones, diosgenin, silymarin, silybin, glycyrrhizin, robinin aesallitin, polygallic acid, ginsenosides, catechin, antraqunone derivatives, valtrate, rotenone, aristolchic acids, gebberlins antibiotics and number of other compounds of natural origin.

Gas Liquid Chromatography:- [GLC]
It was introduced by James and Martin in 1952, based on the suggestion of Martin and Synge Gas Chromatography separates volatile substances by percolation a gas stream over a stationary phase. The basis of separation in GLC is partitioning of the sample in and out of the film of liquid spread over an inert solid. There exists a wide range of liquid phases usable up to 450 degrees Celsius. In pharmaceutical analysis, GLC is applied to 3 types of problems.

1. Assay of the starting materials and drug substances.
2. Quantification of drug in formulations.
3. Assay of impurities and or solvents in raw materials or in durg substances.
Applications of GLC:-
1. Examination of many volatile oils, plant acids, alkaloids of opium, tobacco, conium and belladonna resins of cannaba steroidal compounds. Cardioactive glycosides and aglycons; sugars and aminoacids etc.
2. The technique has strong separation power and even complex mixture can be resolved into constituents.
3. The sensitivity of the method is quite high. It is a micro method only a few mg of the sample is sufficient of analysis.
4. It gives good lprecision and accuracy.
5. The analysis is complited in a short time.

Principle of gas chromatography separations:-
When a gas or vapour comes in contact with an adsorbent certain amount of it gets adsorbed on the solid surface. The phenomenon takes place according to the well known laws of Freaundlich i.e. x/m=K1C+K2C
Where x= mass of the gas or vapour sorbed in mass m or sorbent
C= Vapour concentration in the gas phase
K,K2,K1= Constants

Instrumentation:-
The gas chromatographic separation is carried out in a tubular column made of glass metal or Teflon. In this column a sorbent is filled as the stationary phase. the adsorbent re packed in the form of fine size graded powder where as the liquids are coated as fine film on the column wall of first coated over an inert size graded porous support such as fire brick powder followed by packing in the column. A gas serving as mobile phase flows continuously through the column. It is known as the carrier gas and serves to transport sample components in the column. The sample is introduced in the vapor form at the carrier gas entrance end of the column. Different components of the sample are sorbed on the stationary phase to different extents depending upon their distribution coefficients. The portion of each component in the gas phase is swept further immediately by the carrier gas. As result a fraction of the sorbed amount also desorbs out to maintain the K-value. At the same time out of swept amount some amount will go into the sorbent at the next point in the column again to maintain the K-value. This goes on successively and continuously and as a whole the band for each component moves further in the column and having the shape of more or less Gaussian distribution.

It should be noted that the gas is the driving force for the movement of zones through the column and the solid which provides the selective retarding force. The detector can be regarded as the brain of the chromatograph and the column as it heart.,

High performance Liquid Chromatography:- [HPLC]
Here in this method the separations take place with a packed column. The material is the stationary phase and it may be a solid or a liquid coated over an inert material. A liquid mobile phase is used as eluent in HPTLC. The mobile phase is forced through the column under high pressure either with autocratic elution or gradient elution.

It has become the most versatile safest, dependable, fastest and sensitive chromatographic technique for the quality control of drug components. The durgs like morphine, papaverine, codeine, emetine, antibiotics, steroids, ergot alkaloids, cardiac glycosides, sennosides, capsaicin, vitamins, rhubarb constituents etc. are analyzed by HPTLC, Digoxin and gitoxin are analysed using RP column 65% methanol. The reverse phase packing material (spherisorb- ODS) is produced by the bonding of ocladecylsilyl groups (C18H37Si) to silica gel.


Column chromatography:-
Basically it is a liquid chromatography in which mobile phase in the form of liquid passes through (over) the stationary phase packed in a column. Column chromatography has been derivetised into the other forms like gel permeation, ion exchange and column partition. (Affinity)
In the column chromatography, fairly large number of adsorbents are used like starch CaCO3, magnesia lime, silica gel, alumina, charcoal etc. To optimize the resolution various mobile phase used wither singly or in combination are petroleum ether, cyclohexane, chloroform, acetone, water, pyridine and organic acids for the better separation it is essential to consider the polarity of the sample, adsorbent and the mobile phase.

Gel permeation chromatography - (Gel filtration, molecular sieves)
It is also called as size exclusion chromatography. Te separation occurs not on the basis of adsorption of partition but on the effective size of solutes present in the solution. For the separation purpose, the stationary phase used are cross linked polymers which given an open network with a large number of pores of fairly uniform size. During the flow of mobile phase through such stationary phase. The very large sized molecules cannot enter the pores and hence get "excluded" and continue to travel further along with mobile phase but he molecules with low molecular size enter freely into different pores and hence find a longer path through the column. During the elution the largest molecules in the mobile phase elute first followed by the molecules with decreasing order in sizes. This phenomena is observed because the extent to which the molecules are retained depends on the size of included molecules relative to size of pores.
The various type of gels used as stationary phases are either soft or semi rigid gels or rigid gels.

Soft gels e.g. Agarose(sepharose) Dextran (sephadex) and polyacrylamide (bio-gel)
The semi rigid or rigid gels are alkylated dextran, polystyrene, glass beads.
Soft gels are used with primarily aqueous mobile phase and process is called gel filtration. If the other mentioned gels are used along with non aqueous m.p. like acetone, pyridine, chloroform, etc process is called gel permeation. Gel filtration is employed for separation of large biomolecules like proteins and polypeptides. Determination of molecular size, studies of plasma binding of drugs etc.

About Author / Additional Info:
i am M.Pharma ic cognosy and M.sc in chemistry.