HPTLC
- 2. 1.High Performance Thin Layer Chromatography (HPTLC). 2.Recent development led to the improvements in various operations involved. 3.Difference b/w TLC & HPTLC – practical technique than of the physical phenomenon (partition, adsorption) on which separation is
- 3. Difference TLC Stationary phase – thin layer of sorbent (cellulose powder / silica gel) Coating on an inert, rigid glass plate foil – separation on flat with two dimensiona l surface HPTLC Improved separation technique, high resolutions & improved quantitative analysis
- 4. • Similar approach & same physical properties of TLC. • Principle of separation – adsorption. • Mobile phase flows through b/c of capillary action. • Components move according to their affinities towards adsorbent. • Components – separated on chromatographic plate.
- 5. Components - Greater affinity towards stationary phase travels slower Components - Lesser affinity towards stationary phase travels faster
- 8. High concentrated solution, as less amount of sample – applied. Normal phase – silica gel is pre - coated Reversed phase – polar solvents are used for dissolving the sample. Plates solvents – non polar of volatile type.
- 9. Layer – pre- coated silica gel : adsorbent. Plates – similar as TLC. Particle size – very fine particles. Plates : 4-5mm silica gel to form 200mm layer Fine particles – greater resolution & sensitivity. TLC : 5 – 20mm HPTLC : 4 – 8mm (particle size)
- 10. Plates Pre - washing Remove water Volatile impurities Cleaned by methanol conditioning Pre – washed plates placed in oven at 120c for 15 – 20 minutes (conditioning)
- 11. Size not >1mm diameter Self – loading capillary for small volume of samples Surface using platinum – iridium tubing fused into end of a length of glass tubing
- 12. Low polarity of mobile phase – no need for saturation. High polarity of mobile phase – saturation is needed. Solution by trial & error where chemical properties of solute & solvent solubility of analytic adsorbent layer are considered.
- 13. Linear development method. plate – vertically. placed in solvent system in a suitable container. solvent – fed by capillary action & chromatogram – from both the sides. Chromatographic development - Methods Circular development Anti – circular device Multiple development
- 14. Immediately after the development is completed, the plates are removed from the chamber and dried to remove the frees of mobile phase. Detection - known by iodine vapor in iodine chamber.
- 15. HPTLC – supplies with computer & data recording & storing devices. Development – scanned at selected UV wavelength by instruments & detected spots - peaks Scanner – converts bond into peak & peak heights / area related to concentration of substance on the spot. Peak heights & area under spot – measured by instrument & recorded as % on the printer.
- 16. FEATUR ES Quality of adsorbed layer Methods of sample application Sample into adsorbent layer Scanning densitometer s
- 17. • Pure silica gel with narrow particle size. • Diameter : 3-5µm. • Chemically bonded layers as reverse phase plates. • Layers – improved adsorbents of 5000 plates. • Difficult separation in shorter time than TLC 1. Quality of adsorbed layer • Lower sample capacity & so the layer is reduced. • Sample volume : 100 – 200 cm gives 1mm diameter. • After plate development – 10 times better detection limits. • Advantage – compact starting spots – increased no. of samples. 2. Methods of sample application
- 18. • Critical process. • Capillary volume : 100/200 nL – sealed into glass support capillary – convenient spotting device in quantitative measurements. • Capillary tip – polished for smooth planar surface of small area (0.05mm) when used as mechanical applicator minimizes damage to surface of plate. 3. Sample into adsorbent layer • Scan individual spot by reflectance / absorption of a light beam. • Transmitted / reflected radiation – photograph on chromatogram revealing dark / light zones for separated compounds. 4. Scanning densitometers
- 19. Clinical purpose (analysis of drugs in blood) Environmental analysis Separation of compounds Quantitative analysis of volatile compounds Separation & quantitative analysis of thermally stable compounds