Chromatography is a separation technique wherein the different components of a mixture are separated. The mixture to be separated is run through the stationary phase with the help of a mobile phase which separates the different components of a mixture on the basis of partition or adsorption.
The history of this technique dates back to 19th century only but it’s true use has attributed to a Russian Botanist named “Mikhail Tsvet” who used this technique to separate the plant pigments which are known by the name of Xanthophylls, Carotenes, Chlorophyll.
The use of this technique was limited till the mid 20th century when the researchers Martin and Synge developed Partition chromatography to separate chemical substances with minimum separating coefficients.
Paper Chromatography and Thin-layer Chromatography are the two among many chromatographic techniques that were developed with time and have been used continuously in different departments for separation of components.
Definition of Paper & Thin-Layer Chromatography
Paper Chromatography is a Solid-Liquid partition based technique for the component separation based on their polarity towards both the phases. The stationary phase in this technique is the Cellulose based filter paper while the mobile phase is the liquid.
TLC is a Solid-Liquid Adsorption based technique in which the components of a mixture are separated based on their polarity towards both phases. The stationary phase in this technique is glass-coated with the silica gel layer while the mobile phase is the liquid.
Difference between Paper and Thin Layer Chromatography
| PROPERTIES | PAPER CHROMATOGRAPHY | Thin-Layer Chromatography |
|---|---|---|
| Principle | Partition | Adsorption |
| Stationary Phase | Cellulose paper with water present in its pores | Glass coated with silica gel or Aluminium plate coated with silica gel |
| Mobile Phase | Pure solvents or Buffer solutions or a mixture of solvents. Hydrophilic Mobile phase-> Methanol:Water Hydrophobic Mobile Phase-> Kerosene:Isopropanol | Pyridine, Acetone, Carbon Tetrachloride, Glycerol, Ethanol, Benzene, Ether, etc. |
| Time Preparation | Very less | Comparatively more |
| Use of Silica Gel | No Silica gel required in this process | Silica gel required for preparing Stationary phase |
| Separation Efficiency | Efficient for Polar compounds | Efficient for less polar compounds |
| Use of Corrosive reagents | Cannot use | Can use |
| Chromatogram evaluation | Can’t be evaluated under UV | Can be evaluated under UV |
| Separation Time | Takes more time (1-2 hrs) | Takes less time (20-45 min.) |
| Cost | Cheap | Comparatively costly |
| Development Techniques | Ascending, Descending, Radial | One dimensional, Two-dimensional |
| Visualizing agents | Iodine Chamber method, Ninhydrin in acetone, Dragendroff’s reagent, Ferric Chloride, 3,5-dinitro Benzoic acid, etc. | Same as PC |
| Quantitative & Qualitative analysis | Suitable for only Qualitative analysis | Suitable for both |
| Applications | Identification of drugs and impurities, Separation of carbohydrates or vitamins or proteins or drugs, Analysis of metabolites in blood or Urine, etc. | Purification of samples, Isolation of compounds, Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics, separating and identifying colors, sweetening agents, preservatives, etc. |
| Heat | Cannot apply heat | Doesn’t gets affected by heat |
| Sensitivity | Less sensitive | Highly sensitive |
| Sample requirement | Less sample required | Requires comparatively more sample |
In short, TLC has many more advantages over Paper chromatography in terms of separation of mixtures.
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