HPTLC

Chinmay Kurambhatti 12 FET 1019

INTRODUCTION
HPTLC is an advanced form of
thin layer chromatography.
It provides superior separation
power using optimized coating
material, novel procedures for
mobile-phase feeding, layer
conditioning, and improved
sample application.
It promotes for higher
separation efficiencies, shorter
analysis time, lower amounts
of mobile phase, and efficient
data acquisition and

Basic Principle

FUNDAMENTALS OF HPTLC
Distribution factor (Ka):
A solute with large Ka has a great afnity for

stationary phase and will travel slowly through the

layer.

Retention/Retardation factor (Rf): The position of

any solute spot is characterised by its Rf.
Rf = Distance travelled by analyte/Distance
travelled by the solvent front.
Rf values range from 1.0 for analyte migrating to the
solvent front to 0.0 for an analyte strongly retained
at the point of application

FUNDAMENTALS OF HPTLC
Capacity factor (k): the ratio of

retention time in the stationary phase(ts)

to that in the mobile phase(tm).
k = ts/tm
measurement of capacity factor will help
to determine whether retention shifts are
due to the stationary phase (capacity
factor is changing with retention time
changes) or due to the mobile phase
(capacity factor remains constant with
retention time changes)
k can also be described by the ratio of
total number of moles of analytes in each
phase.

Spot capacity : The separation

number or spot capacity is dened

as the maximum number of
substances, which are completely
separated between Rf =0 and Rf =
1, provided that the separation
conditions are isocratic.
HPTLC has a separation number
of 1020

IMPLEMENTATION OF HPTLC
Pre-Coated Layers
Precoated plates that use small quantities of very high molecular weight

polymer as binder.
Abrasion resistant, very uniform in layer thickness, reproducible, preactivated, and ready to use.
Available with glass (high quality results)or aluminium (cheap) or polyester
support.
Layers containing a fluorescent indicator F 254 are used which enables the
visualization of samples in a UV cabinet in a non-destructive manner.

IMPLEMENTATION OF HPTLC
Hand operated plate coater
Plates are pushed through by hand one
after the other and lifted off the other side.

Automatic plate coater

The glass plates to be coated are
conveyed underneath a hopper lled with
adsorbent solution are moved by a
motorized conveying system at a uniform
feeding rate of 10cm/sec.

IMPLEMENTATION OF HPTLC
SAMPLE PREPARATION
Sample needs a highly concentrated solution

as very little amount of sample needs to be

applied.
Usual concentration of applied samples: 0.1 to
1 g/ l for qualitative analysis.
Normal phase chromatography: silica gel precoated plates, solvent: non polar(diethyl
ether, methylene chloride, and chloroform)
Reverse phase chromatography: Lipophilic C18, C-8, C-2; phenyl chemically-modied silica
gel phases polar solvents(methanolwater or
dioxanewater) used to dissolve samples.

SELECTION OF
CHROMATOGRAPHIC LAYERS
Silica gel is the most widely used for

pre coating the HPTLC plates, available

in very ne particle sizes.
PLATES: Similar to conventional TLC
plates
Fine particle size (4 to 8 mm)of silica
gel (adsorbent) accounts for higher
resolution and sensitivity
4 to 5 mm silica gel layer with an inert
binder to form a 200 mm layer & 10 by
10 cm or 20 by 10 cm plates are used

IMPLEMENTATION OF HPTLC
PLATE PREPARATION

SAMPLE APPLICATION
Spots: 0.5 to 5 L sample volume

Pre washing
Plates need to be washed to remove
water vapour or volatile impurities.
The plates are cleaned by methanol.
Conditioning
The pre washed plates are kept in
the oven at 120 C for 15 to 20 mins.

HPTLC takes upto 1 L. The size of the

spot applied should not exceed 1 mm
in diameter.
Spraying-on samples as narrow bands

allows the application of signicantly

larger volumes
There are automatic, semi-automatic &

manual sample applicators.

The classication is also done on the

type of sample to be analysed

SAMPLE APPLICATION

SAMPLE APPLICATORS

Automatic

Manual

Semi-Automatic

IMPLEMENTATION OF HPTLC
PRE-CONDITIONING
For low polarity mobile phase, no

need of saturation but is needed

for high polarity mobile phase.
Chamber saturation is done by
lining with lter paper 30 min
prior to development which leads
to uniform distribution of solvent
vapours.

MOBILE PHASE

The solution of proper mobile

phase is made by trial and error

method in which chemical
properties of solute and solvent
solubility of analytic adsorbent
layer are considered.

PROCESSES IN DEVELOPING CHAMBER & EFFECTS

Processes.
Saturation due solvent vapours. (Pre-Saturation reduces secondary front)
Absorption of gas phase polar molecules on stationary phase.
Transfer of non-polar components from mobile phase to gas phase.
Components of the mobile phase can be separated by the stationary phase

Effects
During chromatography, components of the developing solvent, which have been

loaded onto the dry layer via the gas phase (2), are pushed ahead of the true but
invisible solvent front called secondary front.
Mobile phase concentration decreases as solvent trough moves ahead.
Two plates are placed or unsaturated chambers then secondary fronts are prominent.
Pre-saturation & pre-conditioned layers secondary front is not observed.

Chromatic Development

IMPLEMENTATION OF HPTLC
AUTOMATIC DEVELOPMENT CHAMBER
Fully automatic, reproducible,

independent of environmental effects.

Gradient & Isocratic modes.
Automatically monitors:
1. Activity and pre conditioning of layer
2. Chamber saturation
3. Developing distance
4. Final drying

IMPLEMENTATION OF HPTLC
Immediately after the development is complete, the plates are

removed from the chamber and dried to remove mobile phase.

Detection is done under UV light-spots of fluorescent compounds can
be seen at 254 nm or 366nm
Spots of non-fluorescent compounds can be seen using fluorescent
stationary phase-silica gel GF
For non-UV absorbing compounds, the plates are dipped in 0.1% iodine
solution.
When individual compound does not respond to UV, derivatization is
required.

IMPLEMENTATION OF HPTLC
DERIVATIZATION
Substances without

chromophores or colour
can be visualized or made
detectable through
derivatization.
This can be done by :
Chromatogram immersion
device
HPTLC sprayer

IMPLEMENTATION OF HPTLC
SCANNING AND DOCUMENTATION
The development of HPTLC plates is scanned at

the selected UV regions (254nm, 366nm) or

visible light & the spots are seen on the
computer in the form of peaks.
The peak height or area is related to the

concentration of the substance on the spot.

Scanning densitometer:
Has 3 light sources: a tungsten Lamp, a high

pressure Hg lamp, A dueterium lamp

Scanning speed: 1 to 100mm/sec
Quantitative evaluation is performed with the

TLC Scanner using WINCATS software

DIFFERENCE BETWEEN
TLC & HPTLC

ESTIMATION OF TRANS-RESVERATROL IN GRAPE

BERRY SKIN EXTRACT BY HPTLC
Introductio
n

20g of grinded grape dried skin (35 C for 6 days)

macerated with 99.9% methanol acidied with 0.1% HCl
for 24hrs at 30 C. and stored in the dark at temperature
range
of -0.7 to -1.5 C.
Sample
Preparatio
n

Trans-Resveratrol
(3,5,4'-trihydroxy-transstilbene)

Nutraceutical.
Anti-cancer, anti-ageing ,
Cardiovascular health.
Analysed previously by
HPLC
Detected at
=308nm(trans) &

HPTLC ANALYSIS
SAMPLE APPLICATION,
DEVELOPMENT & ANALYSIS

Stock solution preparation

Stock solution of resveratrol :10.8mg

of resveratrol with 25ml of methanol.

The stock solution was stored in the
dark at -0.7 to -1.5 C.
Validation : By dilution, solutions of

100%, 80%, 70%, 50%, 25% prepared

and each solution was applied on a
single 10cmx20cm HPTLC glass plate
and developed with chloroform-ethyl
acetate-acetic acid (2.5:1.0:0.1) after
a saturation time of 20 minutes.

Test and standard samples (5l each) applied onto the HPTLC plates as 10mm x15mm wide apart
from middle of brands by spray on technique along with air for simultaneous drying of Camag
linomate V sample applicator lled with a 100ul syringe.
The plates developed to a distance of 70mm with 30ml chloroform-ethyl acetate-glacial acetic acid
(2.5:1:0.1) for resveratrol as the mobile phase. The chamber saturated for 20mins at RT(30C).

Wavelength for detection was evaluated from complete UV spectrum of resveratrol &
Densitometric scan performed with a Camag TLC scanner at = 313nm. The Rf of resveratrol was
obtained at 0.34.

RESULTS & CONCLUSIONS

Area under the curve(AU) against Rf
value of grape extract samples
(i) Blank solvent;
(ii) Resveratrol (std);
(iii) Fresh green grape skin;
(iv) Fresh green grape flesh;
(v)Fresh red grape flesh;
(vi) Fresh red grape skin;
(vii) Dried red grape skin

RESULTS & CONCLUSIONS

Trans-resveratrol standard.
Rf of 0.34 area of 19 774.6 was of transresveratrol

Fresh green grape skin extract

Rf of 0.34 area of 242.2 => conc. of 0.005185mg/ml

RESULTS & CONCLUSIONS

Fresh green grape flesh extract

No peak at Rf of 0.34 => conc. of 0mg/ml

Fresh red grape flesh extract

No peak at Rf of 0.34 => conc. of 0mg/ml

RESULTS & CONCLUSIONS

Fresh red grape skin extract

No peak at Rf of 0.34 => conc. of 0mg/ml

Dried red grape skin extract

Rf of 0.34 area of 374.7 => conc. of 0.00802mg/ml.

RESULTS & CONCLUSIONS

.

Despite the observed small amount in grape skin, the metabolites of trans-

resveratrol, the isomer of trans-resveratol or other compounds

predominating in all the samples.
since the trans- isomer is affected much by UV and heat, the compound
might have deteriorated during the sell or storage of the grapes in the
local market. However, a relatively good concentration might have been
presentinitially.