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



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

• Isolate and estimate phytoconstituents from natural source
using HPTLC


• 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


• 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


multiple sample handling

low running and maintenance costs, disposable
layer etc

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

• 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

• No prior treatment for solvents like filtration and

• 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

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


Documentation of chromatic plate

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


• 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

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

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

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

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

development Chamber


• 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



• 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


• 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


• 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


• Simultaneous determination of benazepril hydrochloride and

• 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


• Principle of HPTLC is adsorption

• HPTLC is automated and sophisticated instrument

• Usage of precoated plates provides better separation of

• Sample application – Micro syringe – CAMAG applicator –
nitrogen gas

• Development – Twin trough chamber – reduce the time of


 For Detailed PDF Notes Click on Download Button 

Leave a comment