HPTLC
Objective
At the end of the lecture, student will be able to
- Explain the principle of HPTLC
- Discuss the instrumentation of HPTLC
- Discuss the application of HPTLC in various fields
- Isolate and estimate phytoconstituents from natural source using HPTLC
Introduction
- Sophisticated form – thin layer chromatography
- Same theoretical principle – thin layer chromatography
- Traditional Thin Layer Chromatography & its modern instrumental quantitative analysis version HPTLC are very popular for many reasons such as
- visual chromatogram
- simplicity
- multiple sample handling
- low running and maintenance costs, disposable layer etc
Principle of Separation
- Separation may result due to adsorption depending upon the nature of adsorbents used on plates and solvents system used for development
Features of HPTLC
- Simultaneous processing of sample and standard – better analytical precision and accuracy, less need for Internal Standard
- Several analysts work simultaneously
- Lower analysis time and less cost per analysis
- Low maintenance cost
- Simple sample preparation – handle samples of divergent nature
- No prior treatment for solvents like filtration and degassing
- Low mobile phase consumption per sample
- No interference from previous analysis – fresh stationary and mobile phases for each analysis – no contamination
- Visual detection possible – open system
- Non UV absorbing compounds detected by post-chromatographic derivatisation
Steps involved in HPTLC
Selection of chromatographic layer
Sample and standard preparation
Layer pre-washing
Layer pre-conditioning
Application of sample and standard
Chromatographic development
Detection of spots
Scanning
Documentation of chromatic plate
Stationary phase – Precoated plates
- 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
Prewashing
- 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 Precoated Plates
- Freshly open box – plates – do not require activation
- Plates exposed to high humidity or kept on hand for long time to be activated
- Oven – 110-120˚c – 30min 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
- 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 – 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 – 10 -15 ml – mobile phase
- Components of mobile phase – mixed – introduced – twin – trough chamber
Chamber saturation – Preconditioning
- 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
CAMAG development Chamber
Drying
- After development – remove the plate and mobile phase is removed from the plate – to avoid contamination of lab atmosphere
- Dry in vacuum desiccators – avoid hair drier – essential oil components may evaporate
Visualizer
Detection
- Detection under UV light is first choice – non destructive
- Spots of fluorescent compounds – 254 nm (short wave length) or at 366 nm (long wave length)
- Spots of non-fluorescent compounds – 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
Applications
- Simultaneous determination of benazepril hydrochloride and hydrochlorothiazide
- Analysis of semi-permanent hair dyes
- Application of HPTLC for the determination of active ingredients in herbal and pharmaceutical formulations.
- Cosmetic and environmental analysis
- Metallurgy, electroplating
- Toxicology, forensic analysis
Summary
- Principle of HPTLC is adsorption
- HPTLC is automated and sophisticated instrument
- Usage of precoated plates provides better separation of compounds
- Sample application – Micro syringe – CAMAG applicator – nitrogen gas
- Development – Twin trough chamber – reduce the time of development
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