Basic Principles of HPTLC

HPTLC- High Performance Thin Layer Chromatography is a sophisticated and automated form of TLC.

Main Difference of HPTLC and TLC - Particle and Pore size of Sorbents.

The other differences are

Selection of chromatographic layer

· Precoated plates - different support materials - different Sorbents available
· 80% of analysis - silica gel GF · Basic substances, alkaloids and steroids - Aluminum oxide
· Amino acids, dipeptides, sugars and alkaloids - cellulose
· Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18
· Preservatives, barbiturates, analgesic and phenothiazines- Hybrid plates-RPWF254s

Sample and Standard Preparation

To avoid interference from impurities and water vapours
Low signal to noise ratio - Straight base line- Improvement of LOD
Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform: Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid
Dry the plates and store in dust free atmosphere

Activation of pre-coated plates

Freshly open box of plates do not require activation
Plates exposed to high humidity or kept o­n hand for long time to be activated
By placing in an oven at 110-120ºc for 30’ prior to spotting
Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes.

Application of sample and standard

· Usual concentration range is 0.1-1µg / µl
· Above this causes poor separation
· Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe o­n TLC plates as bands
· Band wise application - better separation - high response to densitometer

Selection of mobile phase

- Trial and error
- one’s own experience and Literature
- Normal phase
- Stationary phase is polar
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
· Reversed phase
- Stationary phase is non polar
- Mobile phase is polar
- Polar compounds eluted first because of lower affinity with stationary phase
- Non-Polar compounds retained because of higher affinity with the stationary phase
- 3 - 4 component mobile phase should be avoided
- Multi component mobile phase o­nce used not recommended for further use and solvent composition is expressed by volumes (v/v) and sum of volumes is usually 100
- Twin trough chambers are used o­nly 10 -15 ml of mobile phase is required
-· Components of mobile phase should be mixed introduced into the twin - trough chamber

Pre- conditioning (Chamber saturation)

· Un- saturated chamber causes high Rf values
· Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform distribution of solvent vapours - less solvent for the sample to travel - lower Rf values.

Chromatographic development and drying

· After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere
· Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate

Detection and visualization

· Detection under UV light is first choice - non destructive
· Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length)
· Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF
· Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution
· When individual component does not respond to UV - derivatisation required for detection

Quantification

· Sample and standard should be chromatographed o­n same plate - after development chromatogram is scanned
· Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode - scanning speed is selectable up to 100 mm/s - spectra recording is fast - 36 tracks with up to 100 peak windows can be evaluated
· Calibration of single and multiple levels with linear or non-linear regressions are possible · When target values are to be verified such as stability testing and dissolution profile single level calibration is suitable
· Statistics such as RSD or CI report automatically
· Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors

Documentation

E - Merck introduced plates with imprinted identification code - supplier name. Item number, batch number and individual plate number - Avoid manipulation of data at any stage - coding automatically get recorded during photo documentation

Validation of analytical method

All validation parameters such as precision, accuracy, LOD, LOQ, Ruggedness, Robustness can be performed