HPTLC – Principle, Instrumentation and Application – Pharmacognosy and Phytochemistry II B. Pharma 5th semester PDF Notes

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

1. visual chromatogram
2. simplicity
3. multiple sample handling
4. 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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