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
CONTENTS
• Introduction
• Principle of HPTLC
• Difference between TLC & HPTLC
• Steps involved in HPTLC
• Material used for plates
• Mobile phase
• Sample application
• HPTLC Plate development
• Applications of 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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