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• TLC is a liquid-solid adsorption technique where the mobile phase ascends the thin layer of stationary phase coated on to a backing support such as glass by capillary action. • TLC is a simple, quick, and Inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture.

• • • • • • Selection of stationary phase Selection of mobile phase selection visualization technique develop TLC with initial conditions optimize the development validation .

. • Alumina and silica are the most common stationary phases used for TLC. preferably that has an identical media as the preparative column.Criteria for selection of stationary phase • Choose a scalable TLC plate. • Choose between normal and reverse phase based on sample polarity and solubility. • Several different types of stationary phases are listed according to polarity.

basic or neutral) Activated carbon charcoal.g.e. .C-18 Cellulose Starch Calcium sulphate Silica(silica gel) Magnesium silicate Magnesium oxide Alumina (alumina oxide .Chromatography Stationary Phase Polarities Polydimethyl siloxane Methyl/Phenyl siloxane Cyanopropyl siloxane Carbowax (polyethylene glycol) Reverse Phase(hydrocarbon-coated silica gel. acidic.

. • The first step in solvent selection is determination of the solubility of the sample. the mobile phase must have a greater solubility for the sample. • It is best to dissolve the sample in the mobile phase.Criteria for selection of mobile phase Solubility: • To elute a sample on TLC. • The desired mobile phase would provide the greatest solubility.

the sample will remain at the origin. .Solvent Solubility Screening Table Water Methanol Ethanol Acetone Diethyl Ether Ethyl Acetate Dichloromethane Toluene Chloroform Affinity: • To achieve a separation. the sample must have a relatively equal affinity for the solvent and packing material. • If the sample has a higher affinity for the stationary phase than the solvent.

solvent. a substitution in the polar solvent often results in a change in resolution. • Change in the less polar solvent results primarily in a change in Rf of the sample components. • A preparative mobile phase system contain less polar solvent. and support. • As a guide for method development. • Most TLC mobile phase systems contain a polar solvent. .Resolution • Resolution is improved by optimizing the affinity between sample.

• Review the result after visualization and adjust the Rf if necessary. • It is common to try 3-6 solvent systems for the first round of method development.Perform TLC under initial set of conditions • Look up the affinity for the type of compound as well as the solvent strengths to find a starting point for the method development. . • Increase the separation and evaluate visualization techniques to make sure you are seeing all necessary compounds.

5 Dinitro benzoic acid. Common techniques includes: • Non specific methods: Iodine chamber method. visualization techniques will need to determined.for cardiac glycosides .for Phenolic compounds & tannins Ninhydrin in acetone-for amino acids Dragendroff’s reagent – for alkaloids 3. Sulfuric acid spray reagent. • Specific methods: Ferric chloride.Selection of Visualizing agent Once a mobile phase is selected. UVchamber for flourescent compounds.

3-0. Rf = Distance travelled by solute Distance travelled by solvent front . • Rf value is specific and constant for every compound in a particular combination of stationary and mobile phase.8 .Optimizing TLC Separations • The optimum separation of compounds by TLC is usually achieved when Rf values are between 0.

Solvent front Distance travelled by the solvent Distance travelled by the spot Start line .

Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity. purity and potency of the drug substances and drug products. quality. 13 . strength.

• • • • • Specificity (Selectivity) Linearity Range Accuracy Precision    • • • • Repeatability Intermediate Precision Reproducibility (Ruggedness) Detection Limit Quantitation Limit Robustness System Suitability Testing 14 .

• Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. matrix. etc. degradants. 15 . Typically these might include impurities.

In this case a combination of two or more analytical procedures is recommended to achieve the necessary level of discrimination. 16 .• It is not always possible to demonstrate that an analytical procedure is specific for a particular analyte (complete discrimination).

17 .• The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

• The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision. 18 . accuracy and linearity.

• The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. 19 .

usually applied to standardization of methodology). Reproducibility expresses the precision between laboratories (collaborative studies. Repeatability is also termed intra-assay precision. Intermediate Precision expresses withinlaboratories variations: different days.• • • Repeatability expresses the precision under the same operating conditions over a short interval of time. 20 . etc. different analysts. different equipment.

• The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. 21 .

22 . and is used particularly for the determination of impurities and/or degradation products. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices.• The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

• The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small. 23 . but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

electronics.• System suitability testing is an integral part of many analytical procedures. analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. 24 . System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated. The tests are based on the concept that the equipment.

REFERENCES • C. VCH. Ng. Chromatography Today.: Validation of Chromatographic methods. 1991 • H. Jork . 1992 Lindal.7 to 28. Funk. page no. S. Wimmer. Thin layer Chromatography Reagents and Detection Methods. 2. Fischer. Amsterdam. H. Elsevier. Vol. 1994. W.F.K. Poole. Parameters for Validation of Chromatographic methods for drug substances And Drug product. • . Weinheim.Poole. W.