1. High Performance Thin
Layer Chromatography
(HPTLC)
Anas Ejaz Shaikh
13FET1006
Analysis of Foods-2 (Instrumental)

2. Contents of the Presentation
Introduction
Principle
Advantages of hptlc over other techniques
Differences between tlc and hptlc
Steps involved in hptlc
Derivatization
Instrumentation
Applications

3. Introduction
Chromatography may be regarded as a analytical technique
employed for the separation, identification and purification of
the constituents of a mixture.
The term chromatography is derived from two Greek words.
 khromatos :colour
 graphos : written
High performance thin layer chromatography is an improved,
sophisticated and an automated form of thin layer
chromatography.
It is also called as flat bed chromatography.
By using HPTLC the samples are precisely and accurately
assayed using high performance grade of adsorbents.

4. Principle
HPTLC have similar approach and employ the same physical principles
of TLC (adsorption chromatography) i.e. the principle of separation is
adsorption.
 The mobile phase solvent flows through because of capillary action.
The components move according to their affinities towards the
adsorbent.
The component with more affinity towards the stationary phase travels
slower.
The component with lesser affinity towards the stationary phase travels
faster.
Thus the components are separated on a chromatographic plate.

5. Advantages of HPTLC Over Other
Chromatographic Methods:
1.HPTLC permits simultaneous analysis of sample and standard
solution there by provides better analytical accuracy & precision.
2.Lower analysis time & less cost per analysis.
3.HPTLC is very simple to operate and is less time consuming.
4.In HPTLC, the sample preparation is simple.
5. Solvent used in HPTLC needs no prior treatment like filtration &
degassing.
6. In HPTLC, the Mobile phase consumption for sample is extremely
low.
7. More number of spots can be applied on a single plate which aids in
identification of large number of compounds in single run.
8.Migration distance with HPTLC(3-5 cm) is less when compared to
TLC (10-15) which shortness the development time.

6. Differences between tlc and hptlc

7. Steps involved in hptlc
1.Selection of chromatographic plates(HPTLC plates)
2.Selection of chromatographic layer
3.Pre-washing of the plates
4.Activation of the HPTLC plates
5.Selection of mobile phase(solvent system)
6.Preparation of Sample and standard
7.Pre-conditioning (chamber saturation)
8.Application of sample.
9.Chromatographic development.
10.Detection of spots.
11.Scanning & documentation.

8. Schematic Flow Chart of hptlc steps

9. 1. Selection of chromatographic plates:
Most commonly ,standard plates of sizes
20 x 20cm or 5 x 7.5cm,5×5cm,10×20 are used in
HPTLC,
For qualitative and quantitative analysis.
a) Handmade plates:
b) pre coated plates:

10. a) Handmade plates:
Adsorbents like silica gel(most commonly
used),cellulose, microcrystalline cellulose with or
without a suitable binder are used for the
preparation of handmade plates.
b) Pre coated plates:
Commercially pre coated HPTLC plates of different
thickness are available.
Pre coated plates are being preferred over handmade
plates as they offer uniform and dense adsorbent
layers.

11. 2. Selection of Chromatographic layer:
The selection of the chromatographic layer in HPTLC
depends on the nature of material to be separated.
Commonly used adsorbents like Silica gel 60F, Alumina,
Cellulose, polyethylene impregnated cellulose etc.
The particle size of adsorbents employed for HPTLC
plates is smaller (4-8µm) than the TLC (5-20µm) plates.

12. 3. Pre-washing of the plates:
Prior to the actual chromatography, the plates are
pre washed to remove any adhering impurities.
This is done by developing them in solvent like
methanol, chloroform, ammonia by ascending or
descending method.
This helps to avoid the appearance of dirty zones or
spots.

13. 4. Activation of HPTLC 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.

14. 5. Selection of mobile phase:
Selection of mobile phase in HPTLC is done by trial
and error method by considering the chemical
properties of the solute and solvent, solubility of
the analyte, thickness of the adsorbent layer etc.

15. 6. Preparation of sample and standard:
This is done by dissolving the dosage form in an
appropriate solvents which helps in complete recovery
of the desired constituents.
Generally, same solvents used for dissolving both the
sample and standard materials.
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 .

16. 7. PRE-CONDITIONING (Chamber
saturation):
Pre conditioning of HPTLC chamber is required for
efficient separations.
It is not required when non polar solvent system is
used for development ,but is highly recommended
in case of highly polar solvent system.

17. 8. Application of sample and
standard:
To obtain a better resolution , both standard and
sample solutions are to be applied in volumes of 1.0 -
5µl in the form of bands.
Application carried out by Linomat applicator on the
plates which give uniform, safe & standard results.

18. •Semi automatic
sample applicator
Automatic sample
applicator

19. 9. Chromatographic development:
Ascending, descending, horizontal, continuous,
gradient, multidimensional…
HPTLC – migration distance of 5-6mm is sufficient,
after development, plates removed & dried.

20. TwinTrough Chambers:

21. Vario chamber development
VARIO CHROMATOGRAM
21

22. Horizontal Development
22
•HPTLC plate is developed from
both opposing sides towards the
middle.
•Plate sizes 10x10cm and 20x10cm

23. Automatic Multiple
Development(AMD)
23

24. Automatic Developing Chamber
(ADC):
In the ADC this step is fully
automatic and independent of
environmental effects
The activity and pre
conditioning of the layer,
chamber saturation,
developing distance and final
drying can be pre set and
automatically monitored by
ADC

25. Common problems encountered
during Development are as follows…
1.Tailing: due to the presence of traces of
impurities, this can be reduced by buffering the
mobile phase.
2.Diffusions: This is seen as zones on
chromatographic plates. This may arise due to
non-uniformity of mobile phase.

26. 10. Detection and visualization:
Detection under UV light is first choice - non
destructive.
Spots of fluorescent compounds can be seen at 254
nm (short wave length) or at 366 nm (long wave
length).
Non UV absorbing compounds like ethambutol,
dicylomine etc - dipping the plates in 0.1% iodine
solution.

27. Visualizer

28. 11. Scanning & Documentation:
HPTLC plates are scanned
at selected UV regions by
densitometer & the
detected spots are seen on
computer in the form of
peaks.
The scanner converts band
into peaks & peak height or
area is related to the
concentration of the
substance on the spot.

29. Chromatogram

30. Derivatisation techniques:
Some times when the compound can not be detected,
a spray reagent is used which react with the
separated compound and can be easily detected by
densitometric scanner.
The purpose of derivatisation is as follows:
a) To make non detectable substances to detectable
ones.
b) To increase detection limits.
c) To convert non fluorescent compounds to
fluorescent compounds.

31. Instrumentation of HPTLC consists of
following:
1) Lamp selector
2) Entrance lens slit
3) Monochromator entry slit
4) Grating
5) Mirror
6) Slit aperture disc
7) Mirror
8) Beam splitter
9) Reference photo multiplier
10) Measuring photo multiplier
11) Photo diode for transmission measurements.

32. Instrumentation

33. • Theory: According to the theory, the transmission of light in
a translucent material can be described by:
I0 = Ia + It + Ir + Ix
Where,
I0 = Intensity of incident light.
Ia = Intensity of absorbed light.
It = Intensity of transmitted light.
Ir = Intensity of reflected light.
Ix = Intensity of light lost due to
scattering.

34. • The Densitometer work by
2 modes:
1. Transmission mode
2. Reflectance mode

35.  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 and
converted in to absorbance values
 According to Goodman & Goodall transmission
measurements are more sensitive than reflectance
measurements

36. Fluorescence measurement in densitometry:
1) Measurement of direct fluorescence
2) Measurement of fluorescent quenching.
1. Direct fluorescent measurement:
• This method is followed if the spot exhibit fluorescence
when exposed to UV light.
• In this two monochromators are used for selection of
excitation & emission wavelength. The fluorescence is
measured in reflectance mode.

37. 2) Fluorescence quenching measurement:
• As the name indicates, it utilizes the ability of analyte to
absorb & quench fluorescence light.
• In this technique fluorescent background is incorporated
into the layer. When excited by short wavelength radiation
the plate fluorescence's uniformly.
• If UV absorbing substance is present in the plate, a portion
of the fluoresced light is absorbed & consequently
quenched.
• This fluorescence diminution is measured as a function of
amount of analyte in the spot.
.

38. Advantages of densitometer scanner:
The purpose of scanner is to convert the spot/band on the layer into
densitogram consisting of peaks similar in appearance to HPLC.
The position of the scanned peaks on the recorder chart is related to Rf
values.
Quantitation is faster, reliable accurate & reproducible

39. Applications in Food Industry
Analysis for :
1) Pesticide residues
2) Flavours (Vanillin, Ethyl vanillin, Coumarin)
3) Organic acids (Sorbic acid, ascorbic acid, benzoic acid)
4) Mycotoxins (Aflatoxins in groundnut, mustard oil, ghee, etc)
5) Antibiotics
6) Additives (sweeteners like Aspartame, Saccharin, anti-oxidants like BHA, BHT,
TBHQ)
7) Food colours (Natural, Coaltar Food colours)
8) Carbohydrates
9) Proteins (Quality determination)
10) Pigments and dyes
11) Different adulterants in food (Kesari dal in edible dal, karanja oil in other oils)

40. References
1) HPTLC - Quantitative Analysis of Pharmaceutical
Formulations by Dr.P.D.Sethi, Page No.3 – 72.
2) Pharmaceutical Analysis vol-II by Dr. A. V. Kasture,
Dr. K. R. Mahadik Nirali Publishers page no 28-30.
3) Puri, A., Ahmad, A. and Panda, B. P. (2010),
Development of an HPTLC-based diagnostic method
for invasive aspergillosis. Biomed. Chromatogr., 24:
887–892. doi: 10.1002/bmc.1382
4) www.pharmainfo.com
5) www.camagusa.com