High-Performance Thin Layer Chromatography

1. HPTLC
Dr. Ashwani K. Dhingra
Professor
Guru Gobind Singh College of Pharmacy

2. WHAT IS PLANAR /THIN LAYER
CHROMATOGRAPHY
HPTLC is the improved method of TLC which utilizes
the conventional technique of TLC in more optimized
way with enhancements intended to increase the
resolution of the compounds to be separated and to
allow quantitative analysis of the compounds. e.g use
of high quality TLC plates and with finer particle sizes
in the stationary phase
• It is also known as planar chromatography or Flat-
bed chromatography.

3. WHY
HPTLC
• Some of the official HPLC methods which
used for the analysis of some drugs suffered
from some drawbacks.
• Many of these drawbacks are related
primarily to the use of the conventional
columns which have low resolution power
and consume long run time (e.g. the run time
was 100 min. in HPLC official method for
Roxithromycin (ROX).
• The use of the gradient elution is the other
reason behind the disadvantages of some
official HPLC analytical methods because of
its own disadvantages as the long time
needed for column (re-) equilibration, limited
choice of detectors, base-line drift on varying
the eluent, lower signal-to-noise and signal-
to-background ratios, spur peaks (impurities
in weak eluent) and increased instrument
complexity

4. WHY HPTLC
• Furthermore, some of official HPLC methods are using
sample solvent different than that which used as mobile phase
which can affects the analysis results of some drugs.
• In addition to unsuitable column temperature which is used
in some official HPLC method (e.g. 15 Cº and 60 Cº for analysis
of ROX and Doxycyclin (DOX) respectively).
• Also, there are some drawbacks of the official TLC analysis
of some drugs, as using a large developing chamber which
requires large TLC pates and consume large quantity of the
mobile phase as well as developing time. Complex mobile phase
for some TLC purity test and type of sample solvent (e.g. it
contains high quantity of water) are also important
disadvantages.

5. Conventional
TLC
• A small aliquot of a sample solution is
applied in either a spot or band to a thin
sorbent layer supported by a substrate
(glass, plastic, aluminum foil) near one end
of the TLC plate.
• After the sample has dried, the TLC plate
is placed into a chamber where solvent is
introduced to the end of plate where the
sample was applied and capillary action
wicks the solvent to the other side of the
plate.
• Components of the sample mixture are
separated-based on their different
migration rates in the particular stationary
and mobile phase combination.
• Detection is often performed by visually
observing the separated compounds, using
either white or ultraviolet light, using
necessary visualization agents to impart
color or fluorescence to the compounds by
using fluorogenic derivatizing agents.

6. TLC to
HPTLC
• Instrumentation was developed
to permit more precise spotting of
the sample onto the plates and
the quantitative evaluation of the
separated spots.
• Improvements in the technique
itself resulted in higher
separation power and faster
analysis.
• Just as the name change of LC
to (HPLC) character-ties, this
improved TLC was also named
HPTLC

7. TLC to HPTLC
• The main difference between conventional TLC and HPTLC
was in the particle size and range of the adsorbent. The
original "silica gel for TLC had a fairly broad particle size
range (10-60 μm), with an average of about 20 μm, but the
material for HPTLC had a narrower range and an average
particle size of only about 5 μm.
• The plates were also smaller, 10 x 10 cm against the
conventional 20 x 20 cm, and the sample volume was reduced
by an order of magnitude. The method of sample application
was also improved with the design of mechanical applicators
(dosimeters) permitting a reduction in the diameter of the
starting spots.
• These improvements significantly reduced the time needed
for an analysis, with a simultaneous increase of the separation
efficiency.

8. Difference
between
TLC and
HPTLC
TLC HPTLC
Plate particle size:
10 - 25 µm 5 - 7 µm
Separation distance: 100 - 150 mm 60 mm
Development time: 30 - 200 min 3 - 20 min
Application: manual automated/semi-
automated
Development: manual automated
Derivatization: spraying dipping
Analysis data: no documentation fully documented
Quantitative analysis: no yes
Environment: no control no problems
Resolution: often poor very good
Procedure: flexible fully standardized
Reproducibility: impossible highly attainable
cGMP Compliant: usually not YES!!

9. THE PRINCIPLE
HPTLC takes place in high-speed capillary flow range of the mobile
phase. There are three main steps HPTLC procedure:
• SAMPLE APPLICATION
Sample to analyzed to chromatogram layer, volume precision and
exact position are achieved by use of suitable instrument.
• CHROMATOGRAM DEVELOPMENT
Solvent (mobile phase) migrates the planned distance in layer
(stationary phase) by capillary action. In this process sample
separated into it’s components.
• CHROMATOGRAM EVALUATION
Separation tracks are scanned in densitometer with light beams in
visible or UV region

10. Pre-
treatment
of HPTLC
Plate
Although for most qualitative
analysis TLC plates can be used
without any pretreatment.
However, the impurities on the
plate accumulate not only from the
laboratory atmosphere but also
from packing material such as
shrink- wrapping foil. Therefore,
it is important to consider a
standardized cleaning procedure if
the analytical method has to be
validated and reproducible results
are required as the developed
method aim was for stability test.

11. Pre-treatment of HPTLC Plate
• Washing-Methanol as washing agent
• Activation- Drying in oven at 1200C for one hr. to
maximize activity. At this temp. Adsorbed water is
completely removed from surface (Rf value will be less
than the un-activated plates)
• Humidity prevention: during transport and sample
application, the stationary phase is again in contact with
relative humidity of the environment. It is useful to
equilibrate the active plate with the humidity of the
surrounding by cooling it down to room temperature in a
dust and fume free environment in desiccators.

12. TLC Plate
HPTLC Plate
TLC
vs
HPTLC

14. STEPS OF THE HPTLC PROCEDURE

16. Selection of HPTLC plates
Previously hand made plate is used in TLC for both
qualitative and quantitative work, certain draw back
with that is non uniformly layer, formation of thick layer
paved for advent precoated plates. Now a days pre coated
plates are available in different format and thickness by
different manufactures. These plates are used for both
qualitative and quantitative purpose in HPTLC.
• glass plates.
• Polyester /polyethylene.
• Aluminum plates

17. Application
of sample
and
standard
Sample application is one of
the important and critical step
for obtaining the good
resolution for quantification by
HPTLC.

18. LINOMAT 1V

19. AUTOMATIC
TLC
SAMPLER

20. DEVELOPMENT
CHAMBER
Chromatogram development:
After application of sample in
HPTLC plate, chromatogram is
developed by dipping in suitable
solvent system taken in developing
chamber. The solvent system rises
over the layer by capillary action
and separation of sample in
different components take place.

21. LIGHT WEIGHT
TWIN TROUGH
CHAMBER

22. Automatic
Developing
Chamber
ADC

23. DENSITOMETRIC
CHROMATOGRAM EVALUATION
Detection or visulation
of spot/ band: There is
no difficult in
detecting the colored
substance, or color les
substance absorbing
the UV radiation or
with fluoresce
(Riboflavin).

24. Photo & Video –
Documentation /
Video
Densitometry
Reprostar.

26. Fig. 2: TLC of CH2Cl2 and MeOH extracts (Hexane/EtOAc 8:2)
UV λmax = 254 UV λmax = 366
After spraying with different reagents

27. HPTLC fingerprints of successive
extracts of M. Longifolia:
• Qualitative Analysis:

28. HPTLC
fingerprint
profiles of
different
extracts of M.
longifolia:
S.N Extracts
Solvent
system
Wavelength (nm)
No. of peaks (Rf
values)
1 Hexane
Hexane:
ethyl
acetate
(8:2)
254
6 (0.21, 0.32, 0.49,
0.56, 0.67, 0.73)
366
4 (0.12, 0.20, 0.54,
0.74)
550
10 (0.08, 0.18, 0.20,
0.27, 0.32, 0.42,
0.49, 0.56, 0.61,
0.67)

29. HPTLC
fingerprint
profiles of
different
extracts of
M.
longifolia:
2 Chloroform
Hexane: ethyl
acetate
(8:2)
254
7 (0.15, 0.20, 0.24, 0.33, 0.49,
0.65, 0.68)
366 3 (0.20, 0.65, 0.74)
550
10 (0.08, 0.16, 0.20, 0.27, 0.32,
0.38, 0.42, 0.49, 0.60, 0.77)
3 Methanol
Hexane: ethyl
acetate
(8:2)
254 4 (0.20, 0.33, 0.46, 0.68)
366 3 (0.12, 0.20, 0.74)
550
8 (0.10, 0.20, 0.28, 0.32, 0.42,
0.50, 0.61, 0.76)

30. Thin Layer chromatography (TLC)
Detection of the analytes
Coloured analytes
Derivatisation procedures
Densitometry with UV scanner
UV light
beam
Reflected
beam
Detector
Pseudo-chromatogram

31. Thin Layer chromatography (TLC)
Detection of the analytes
Absorption of UV radiation is proportional to concentration
Quantification is possible

32. Densitometry
Densitometer measures the difference in
absorbance or Fluorescence Signal between
a TLC ZONE (in the form of a peak) and
the empty plate background (Baseline) and
relate the measured signal from a series of
standards to those of unknown samples
through a calibration plot.
The Kubelka-Munk equation is usually used
to relate signal intensity and zone
concentration (weight per zone) for
reflectance mode of densitometry.
(1-R2)/2R=2.303e (C/S)
Where R- Light reflected from infinity thick
opaque layer
e-molar absorption coefficient of the analyte,
C is the zone weight and S is the scattering
coefficient

33. Densitometry
The plate is mounted on a stage or platform that can be moved in x- or y direction
controlled by a stepping motor drive to allow each chromatogram track to be
scanned in or opposite to the direction of mobile phase development.
A tungsten –Halogen lamp is used for scanning colored spots in visible region
Deuterium lamp is used for UV region scanning
Monochromator used is Grating
Detector is a PHOTOMULTIPLIER TUBE
For normal Fluorescence scanning, a high intensity Xenon or Mercury vapor line
is used.
Densitometer can be performed in absorbance or fluorescence mode
Compounds are quantified by measuring the decrease in reflectance as a result of
absorbance of radiation.

34. Quantitative
Analysis:
• Biomarker compound/ Standard
compound
• Pure compound/Single
compound
• Large amount
• Therapeutic activity

35. Quantification of pulegone in
methanolic extract of M. longifolia
• HPTLC
chromatogram of
standard
Pulegone

36. HPTLC chromatogram of standard Pulegone
HPTLC chromatogram of methanolic extract of M. longofolia

37. UV Spectra of
standard
pulegone and
different
extract of M.
longofolia

38. TLC plate of pulegone standard and different extract of M. longofolia at UV
λmax = 254
TLC plate of pulegone standard and different extract of M. longofolia after
sraying with anisaldehyde suphuric acid

39. Determination of the amounts of Caffeine
in Coffee seed subjected to different
treatments
Experimental:
Sample preparations:
The samples were purchased from the local market at Al-
Kharj city. The seeds were powdered and 5 gm from each
sample were extracted separate by boiling with water for
two minutes. The resulted decoctions were filtered and
filtrates were transferred to 100 ml volumetric flask.
Mixture of EtOH and H2O were used to complete the
volume with final ration of 1:1 EtOH and H2O.

40. Standard Solution:
Standard solution was prepared by dissolving 10 mg of caffeine in 100
ml of 1:1 EtOAc/H2O mixture. A volume of 1, 2, 3, 4, 5, 6, 7, 8 mL were
applied on silica gel plates to obtain the calibration curve.

41. Chromatographic Conditions:
The TLC system composed of EtOAc/MeOH 85:15 was used as mobile
phase. It resulted in a symmetric nice resolved spots corresponding to
caffeine at Rf value = 0.38.
Chromatogram of standard Caffeine

42. Chromatogram of standard and samples of Caffeine extracted
from different coffee samples.

43. Fig. UV absorption spectrum of caffeine.

44. TLC plates of standard and caffeine extracted from different coffee samples.

45. • Pharmaceutical research.
• Biomedical Analysis.
• Clinical Analysis.
• Environment Analysis.
• Food industry.
• Therapeutic drug monitoring to
determine its concentration and
metabolites in blood urine etc.
• Analysis of environment pollution
level.
• Quantitative determination of
prostaglandin s and thromboxanes in
plasma.
• Determination of mercury in water.
Applications
of HPTLC