Normal phase is the terminology used when the stationary phase is polar ; for example silica gel, and the mobile phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase.
Reversed phase is the terminology used when the stationary phase is a silica bonded with an organic substrate such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary phase.
2. GLASS PLATE Specific dimensions- 20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm Microscopic slides can also be used Plates should be of good quality & withstand high temperatures 3. PREPARATION & ACTIVATION OF TLC PLATES ♦ Pouring ( simplest methods ) ♦ Dipping (used for small plates ) ♦ Spraying ( difficult to get uniform layers ) ♦ Spreading ( best technique ) TLC Spreader
Activation of Plates ○ After spreading -> Air dry (5 to 10 minutes) ○ Activated by heating at about 100 ˚C for 30 min. Then plates may be kept in desiccators 4. APPLICATION OF SAMPLE » Using capillary tube or micropipette » Spotting area should not be immersed in the mobile phase 5. DEVELOPMENT TANK ▫ Better to develop in glass beakers, jars to avoid more wastage of solvents ▫ When standard method is used, use twin trough tanks ▫ Do chamber saturation to avoid “edge effect”
6. MOBILE PHASE M.P used depends upon various factors ► Nature of the substance ► Nature of the S.P ► Mode of Chromatography ► Separation to be achieved, Analytical/Preparative e.g. -> pyridine, pet. ether, carbon tetrachloride, acetone, water, glycerol, ethanol, benzene….
Spray reagents or Detecting agents or Visualizing agents
Same as P.C
Rx value – The ratio of distance traveled by the sample & the distance traveled by the standard.
R f value -
> Direct & Indirect method
APPLICATIONS OF TLC »Purity of sample »Examination of reaction »Identification of compounds »Biochemical analysis »In pharmaceutical industry »Separation of multicomponent pharmaceutical formulations »In food and cosmetic industry