ON RESEARCH     (ANKOSH R. WANOLE)

 HPTLC (High-Performance Thin Layer Chromatography)

INTRODUCTION

High-performance Thin Layer Chromatography (HPTLC) is a sophisticated and automated form of thin-layer chromatography (TLC) with better and more advanced separation efficiency and detection limits.
HPTLC is a well-known and versatile separation method, which shows many advantages compared to other separation techniques.
The basic difference between conventional TLC and HPTLC is only the sorbents’ particle and pore sizes. 

PRINCIPLE

HPTLC has a similar approach and employs the same physical principles of TLC (adsorption chromatography) i.e. the principle of separation is adsorption. The mobile phase solvent flows through because of capillary action. The components move according to their affinities towards the adsorbent. The component with more affinity towards the stationary phase travels slower. The components with the lesser affinity towards the stationary phase travel faster. Thus the components are separated on a chromatographic plate.

STEPS INVOLVED IN HPTLC 

"Steps involved in HPTLC" - HPTLC is a technique used in analytical chemistry to separate and identify the components of a mixture. The steps involved in HPTLC include sample application, development, detection, and quantification. The sample is applied as a small spot at the origin of a thin layer chromatography (TLC) plate. The plate is then placed in a developing chamber containing a solvent that carries the sample components up the plate. Once the components have separated, the plate is removed from the chamber and dried. The separated components are then visualized using detection methods like UV absorption, fluorescence, or by reacting with chemical reagents. Finally, the separated components are quantified by measuring the intensity of the signals or by comparing them with known standards."

SAMPLE PREPARATION AND APPLICATION 

A good solvent system moves all components of the mixture off the baseline but does not put anything on the solvent front. The peaks of interest should be resolved between Rf 0.15 and 0.85. The elution power of the mobile phase depends on a property called eluent strength which is related to the polarity of the mobile phase components

ACTIVATION OF PLATES 

A freshly opened box of HPTLC plate does not require activation. Plates that are exposed to high humidity or kept in hand for a long time require activation. Plates are placed in an oven at 110˚c-120˚c for 30 min before sample application. 

SAMPLE APPLICATION

Sample application can be done by using

 1] Capillary tubes 

2] Micro syringes 

3] Micro bulb pipettes 

4] Automatic sample applicator 

CHROMATOGRAPHIC SITE

1] Detection 

2] Photo Documentation

3] Densitometry Measurements

1] DETECTION - Detection under UV light is the first choice – nondestructive UV absorbing compounds like ethambutol, and dicyclomine - dipping the plates in 0.1% iodine solution. 

2] DENSITOMETRY MEASUREMENTS-Measure visible, UV absorbance, or Fluorescence. Convert the spot\ band into a chromatogram consisting of peaks

APPLICATIONS OF HPTLC 

Pharmaceutical-industry-Quality control, identity, purity test etc.

Food analysis – Quality control, additives, pesticides, stability testing, etc. 

Clinical applications – Metabolism studies, drug screening, stability testing, etc. 

Industrial applications - Process development and optimization etc. 

Forensic applications – Poisoning investigations 

CONCLUSION

Applications of HPTLC for phytochemical analysis, fingerprint analysis, and HPTLC future to combinatorial approach, HPTLC-MS, HPTLCC-FTIR, and HPTLC a power analytical tool in the field of analysis.