3. Definition:
Chromatography is a physical process of
be
separation in which the components to
separated are distributed between 2 immiscible
phases-a stationary phase which has a large
surface area and mobile phase which is in
constant motion through the stationary phase.
4. Introduction:
HPTLC is the improved method of TLC
•
which utilizes the conventional
TLC in more optimized way.
technique of
• It is also known as planar chromatography
or Flat-bed chromatography.
5. 5
Advantages of HPTLC over HPLC
• Sample and standard, both can be
used at a time.
• Evaluation time is less.
• Use of internal standard is not
necessary.
• Simultaneously 2 or 3 persons can
work but not in case of HPLC.
• Solvent degasing and removal of
6. 6
Advantages of HPTLC over TLC
• Visual chromatogram
• simplicity
• Multiple sample handling
• Low running and maintenance costs
and disposable layer etc
• Detection by TLC scanner
• Spotting by applicator
• Highly precised
7. Differences
Parameter
between TLC
TLC
and HPTLC:
HPTLC
Chromatographic plate Hand made /pre-coated Pre-coated
Sorbent layer thickness 250 mm 100-200mm
Particle size range 5-20 μm 4-8 μm
Pre-washing of the plate Not followed Must
Application of sample Manual/Semi automatic Semi auto/Automatic
Shape Spot Spot/Band
8. Spot size 2-4mm 0.5-1mm
Sample volume 1-10 μl 0.2-5 μl
Application
volume
of larger Spotting which leads to Can be applied as bands
over loading
No.ofsamples/plate
(20X20)
15-20 40-50
Optimum
distance
development 10-15 cm 5-7 cm
Development time Depends on mobile phase 40% Less than TLC
Reproducibility of results Difficult Reproducible
9. SCANNING AND DOCUMENTATION OF CHROMOPLATE USING
PC CATS SOFTW
ARE
DETECTION OF SPOTS
CHROMATOGRAPIC DEVELOPMENT
APPLICATION OF SAMPLE
LAYER PRE-CONDITIONING
LAYER PRE-W
ASHING
SELECTION OF CHROMATOGRAPHIC PLATES
STEPS INVOLVED IN SAMPLEAND STANDARD PREPARATION
10. Selection of HPTLC plates
• Hand plates were available which are made up
of cellulose and other materials which are not
used much now-a –days.
11. • Pre coated plates:
The plates with different support materials
and sorbent layers with different format and
thickness are used.
Plates with sorbent thickness of 100-250μm are
used for qualitative and quantitative analysis.
12. Supports
Advantage
Materials
Glass
Disadvantage
1. Fragility
2.Relatively High wt
3.Costs more for additional
packaging
1.Ressistant
chemicals
to heat and
2.Easy to
superior
work
1.More
handle and offers
flat surface for
Polyester
thick)
sheets (0.2 mm economical as 1.Charring reactions if
temperature exceeds 120oc
as the plates are
dimensionally unstable
beyond this temperature
produced even in roll forms
2.Unbreakable
3.Less packing material
4.Spots can be cut and
eluted thus eliminates dust
from scrapping
Aluminum Sheets(0.1mm) 1.Increasesed
resistance
temperature 1.Eluents containing high
concentration of mineral
acids or ammonia can attack
chemically on aluminum
13. Some of the sorbents used in HPTLC:
Applications
80% of analysis is done on this layer.
SN
1.
Examples
Silica gel 60F
(Unmodified )
Aluminium oxide
2. Basic substances ,alkaloids and steroids
3. Cellulose
(microcrystalline )
Amino acids ,peptides ,sugars and other
liable compounds which cannot be
of
chromatographed on the active
silica gel.
layers
4. Silica gel chemically
modified COOH ,Phenols ,Nucleotides
Pharmaceutical preservations.
a) Amino group
( NH2)
b ) CN
14. Some of the binders used:
• Gypsum (G)
• Starch (S)
• Layer containing fluorescent indicator (F)
16. Pre washing of pre coated plates
The main purpose of the pre-washing is to
remove impurities which include water vapours and
other volatile substances from the atmosphere when
they get exposed in the lab environment.
Silica gel 60F is most widely used sorbent. The
major disadvantage of this sorbent is that it contain
iron as impurity. This iron is removed by using
Methanol : water in the ratio of 9:1.This is the
major advantage of the step of pre-washing.
17. Some common methods
:involved in pre-
washing
Ascending method:
• In this
are run
sample
technique the chromatographic plates
blank (i.e. before application of the
with suitable solvent / mobile phase.
The solvent/mobile phase carries the
impurities to the top of the plate.
It takes longer time but cleaning effect is
superior.
The disadvantage of this technique is active
dirt gets accumulated at the solvent front.
•
•
18. Dipping method:
In this technique, the chromatographic plate is
dipped in a suitable solvent for specified
period of time ,removed from the chamber
and finally dried.
Dipping method is quicker and yields uniform
layer but cleaning effect is often not as good
as Ascending method.
•
•
19. •
•
Continuous method:
In this technique, the plate to be washed is placed
in chamber having an entrance and exit slits.
The solvent is made to flow continuously through
the chamber that carries the impurities from the
plate.
The wanted plates should always be stored in a
dust free atmosphere, under ambient conditions.
Usually desiccators of suitable size are used for
storage of plates.
•
•
•
21. Activation of plates:
Freshly opened box of HPTLC plates doesn’t
need activation.
Plates exposed to high humidity or kept in hand
for long time require activation.
Plates are placed in oven at 110o-120oc for 30 min
prior to the sample application.
Activation at higher temperature for longer period
is avoided as it may lead to very active layers and
risk of the samples being decomposed.
•
•
•
•
22. Sample Preparation:
• Proper sample preparation is an important pre-
requisite for success of TLC separation.
For normal chromatography: Solvent should be
non-polar and volatile.
For reversed chromatography: Polar solvent is
used for dissolving the sample
Sample and reference substances should be
dissolved in the same solvent to ensure
comparable distribution at starting zones.
•
•
•
23. Application of sample:
The selection of sample application
technique and device to be used depends
primarily on:
Sample volume
No. of samples to be applied
Required precision and degree of
automation.
•
•
•
24. 1.It is the most critical step for obtaining good
resolution for quantification by HPTLC.
2.Some applicators used for spotting are:
a)
b)
c)
Capillary tubes
Micro bulb pipettes
Micro syringes,
d)Automatic sample applicator.
– The major criteria is that they shouldn’t
damage the surface while applying sample.
25. • The sample should be completely transferred to the
layer.
Micro syringes are preferred if automatic
application devices are not available.
•
• Volume recommended for HPTLC-0.5-5μl to keep
the starting zone down to minimum of 0.5-1 mm in
concentration range of 0.1-μg/ml
Sample spotting should not be excess or not low.
Problem from overloading can be overcome by
applying the sample as band.
•
•
26. Advantages of application of
sample as band are
Better separation because of rectangular area.
Response of densiometer is higher in case of band
that observed from an equal amount/equal volume
sample applied as a spot.
Large quantities of the sample can be handled for
•
• than
of
•
application, thus reducing the need of concentrating
which may be damaging in case of liable substances.
Position of plate for densitometric scanning is less
critical as composition of the compounds is uniform in
the entire area of band.
•
27.
28. Automatic applicators used:
1) CAMAG Nanomat: Samples applied in
the form of spots. The volume is controlled
by disposable platinum iridium of glass
capillary which has volume of 0.1-0.2μl.
29.
30. 2) CAMAG Linomat
Automated sample application device. Sample is loaded
in micro syringe (Hamilton Syringe) 1μl capacity. Sample
can apply either as spot or band by programming the
instrument with parameters like spotting volume ,band
length etc.
33. 3) CAMAG automatic TLC sampler III :
Applies sample as spot or bands
automatically from the rack of sample vials.
34.
35. Heated nozzle of the ATS4 (option)
Raising the temperature to 60 °C reduces the time for application of an aqueous sample to
about one halve. Especially in trace analysis the use of an ATS4 with heated nozzle is
advantageous because large volumes usually have to be applied in order to increase
detection sensitivity
36. Mobile phase
• Mobile phase should be of high graded.
• Chemical properties , analytes and sorbent layer factors
should be considered while selection of mobile phase.
• Use of mobile phase containing more than three or four
components should normally be avoided as it is often
difficult to get reproducible ratios of different components
37. • Mobile phase optimization is necessary
while
performing HPTLC.
• Various components of MP should be measured
separately and then placed in mixing vessel. This
prevents
contamination of solvents and also error arising from
volumes expansion or contraction on mixing.
• Trough chambers are used in which smaller
volumes
of MP usually 10-15 ml is required.
38. • Different components of MP are mixed first in mixing vessel
and then transferred to developing chambers.
• Chambers containing multi component MP are not generally
used for re-use for any future development , due to differential
evaporation and adsorption by layer and also once the chamber is
opened , solvents evaporate disproportionally depending on their
volatilities.
42. Pre-conditioning : (Chamber Saturation)
• Chamber saturation has
separation profile.
a pronounced influence on the
• Time required for the saturation depends on the
phase.
mobile
• If plates are introduced into the unsaturated chamber
,during the course of development , the solvent
evaporates from the plate mainly at the solvent front and
it results in increased Rf values.
43. • If tank is saturate
d
prior to the developme
nt
,
solvent
vapor
s
soo
n
get uniformly distributed
through
out the
chamber
.
chambe
r
A
s
,it
soon as the plate is kept in such a
saturated
soo
n
get
s
is
pre-
loaded
with solven
t
vapor
s
thus les
s
solven
t
required to travel a particular
distance
,resulting
in lower Rf
values.
• But in some
cases
depending on their
polarity
saturatio
n
and non-saturation of chambers are
required:
44. • Eg: Pre-equilibrium is often recommended in
case of
solvent with high polarity.
• Developme
nt
in a non-
saturation
or partially
saturated atmosphere is recommended in solvents
used
in a composition leading to phase separation
such
mixture of n-butanol , water and glacial acetic
acid.
a
s
• Preloading of dry layer with solvent vapors
should
avoided for low polar molecules.
be
45. Development and Drying:
The different methods used for development of chambers are like-
•
Ascending , descending .2-dimentional, horizontal , multiple
overrun , gradient ,radial ,anti-radial ,multimodal ,forced flow
planar chromatography.
Plates are spotted with sample and air dried and placed in the
developing chambers.
After the development plate is removed from chamber and mobile
phase is removed under fume cup-board to avoid contamination of
laboratory atmosphere.
•
•
• The plates should be always laid horizontally because when
mobile phase evaporates the separated components will migrate
evenly to the surface where it can be easily detected
46. Simulation chamber
The simultan developing chamber is a
thick walled clear glass tank with
vertical
grooves and a heavy ground-glass lid.
Round chamber
These cylindrical chambers are ideal
for
use with narrower width plates.
Nano chamber
The nano chamber is suitable for the
development of 10x10cm TLC plates
and
features a heavy glass lid for
gas-tight seal and optimum vapour
saturation.
HPTLC chamber
47. Drying :
Drying of chromatogram should be done in vacuum desiccators
with protection from heat and light.
•
• If hand dryer is used there may be chances of getting contamination
of plates ,evaporation of essential volatile oils if any present in the
spot or compounds sensitive to oxygen may get destroyed due to the
rise in temperature.
48. Factors influencing separation and
resolution
Type of stationary phase
of spots:
•
•
•
•
•
•
•
•
•
•
Type of pre-coated
Layer thickness
Binder in layer
Mobile phase
Solvent purity
Size of developing
plates
chamber
Sample volume to be spotted
Size of initial spot
Solvent level in chamber
49. • Gradien
t
• Relative
humidity
• Temperature Flow rate
in
solven
t
• Separation
distance
• Mode of
Derivatization
Greater the
difference
betwee
n
two spots and smaller the
initial
spot diameter of sample and better will be the
resolution
50. Detection and visualization
One of the characteristic feature of HPTLC is the
derivatization
possibility to
utilize post-chromatographic off line
Detection are of two types:
• Qualitative
• Quantitative
51. • Qualitative detection;
HPTLC is routinely used for qualitative analysis of raw
materials
, finished products ,plant extracts etc. It involves the
identification of
unknown
sample
components
with
mixture by
comparing
the standards.
theRfvalue
s
of thesampl
e
• Quantitation Evaluation:
Quantitative of the
chromatogram
direct and indirect methods;
by HPTLC basically
involves
52. Indirect method; It involves removal of analyte from the
plate followed by quantitation. Eg; Scrapping and elution which is
followed by analysis of eluant by convenient methods like
1)
2)
3)
Spectrophotometry
Flourimetry
Colourimetry
Collection of samples from scrapping will results in the
loss of sample, so
used.
vaccum devices and elution chamber are
54. • It is a instrumenta
l
measurement of
visible
, UV absorbanc
e
,
fluorescence quenching directly on the layer without resorting
to
scrapping and elution
• It involve
s
resolvin
g
of a compound
s
on thin layer plate
,
visualizing the spots and measuring the optical density of each
spot /
band directly on the plate.
• Th
e
amount of material /
compound
in the unknown are
measured by comparing them to a standard curve from
reference
standard chromatographed with the same condition.
• Chromatographi
c
zones
remit
a lower light intensity
than
the
environmen
t
around it. Absorption
spectra
can
be
directly
determined on the plate by comparison with substance free
area of
sorbent layer .
55. • Measurements are usually made by reflection from the
plate
using single beam ,double
beam
operation of scanning
instrument.
or single-beam dual
wavelength
• the scanner present
converts
chromatogram consisting
peaks
the spot / band on the Layer
into
• Position of scanned peaks on recorder chart are related
to Rf
values of the spots on the layer and peak height or area is
related
the concentration of the substance on spot.
to
• Signals
which
are measure
represent
the adsorptio
n
of
to
transmitted or reflected light passes through the spot
compared
57. It
•
basically consists of :
Light source
– Visible Radiation(800-400nm)
– UV Radiation(400-200nm)
• Wave length selection device
• Condensing and focusing system
• Photo-sensing detectors
58. Theory
The transmission of light in a translucent material can be described by:
Io= Ia +It + Ir + Ix
Where,
Io=Intensity of incident light
Ia=Intensity of absorbed light
Ix=Intensity of transmitted light
Ir=Intensity of reflected light
60. • In transmission mode the ratio of It/Io is
measured
and converted in to absorbance values
• In reflectance mode the ratio Ir/Io is
measured
converted in to absorbance values
and
• But it was found that transmission
measurements
are more sensitive than reflectance
measurements.
61. Advantages of densitometer /Scanner
• The purpose of scanner is to convert the spot /band on the layer in
to
to densitogarm consisting of peaks similar in appearance
HPLC.
• The position of
Rf values.
the scanned peaks on recorder chart is related to
• Peak height/area is related to the concentration of the
the spot.
substance in
• Quantitation is faster , reliable ,accurate and reproducible
• Photodocumentation of HPTLC plates is possible.
62. Documentation:
1. Documentation is important because labeling every single chromatogram
can avoid mistake in respect of order of application.
2. Type of plate, chamber system, composition of mobile phase, running
time and detection method should be recorded.
3. To assist the analysts and researchers E .merck has introduced HPTLC
pre-coated plates with an imprinted identification codes.
4. Suppliers name, item number, batch no. , individual plate no. are
imprinted near upper edge of pre-coated plates. This will not only help in
traceability of analytical data, but will also avoid manipulation of data at any
stage as coding will automatically get recorded using photo-documentation.
63. Applications of HPTLC:
• Pharmaceutical industry: Quality control, content
uniformity, uniformity test, identity/purity check.
• Food Analysis: Quality control , additives , pesticides
,stability testing ,analysis of sub-micron levels of
aflotoxins, etc.
• Clinical Applications: Metabolism studies , drug
screening ,stability testing etc
• Industrial Applications; Process development and
optimization, In-process check ,validation etc.
Forensic : Poisoning investigations
Detection of herbal drugs
•
•
