HPTLC, or high-performance thin-layer chromatography, is an advanced form of thin-layer chromatography that offers better resolution and sensitivity due to smaller particle and pore sizes. It is a powerful analytical method suitable for both qualitative and quantitative analysis. HPTLC utilizes pre-coated plates, sample application, development in twin trough chambers, detection under UV or visible light, and densitometric scanning to produce chromatograms for analysis. The high efficiency and resolution of HPTLC allows for detection in the nanogram to picogram range, reduced analysis times, and quantitative analysis through measurement of peak areas in densitograms.

1. HPTLC
Dr.Prasanth B
Associate Professor
Nirmala College of Pharmacy
Muvattupuzha

2. • HPTLC : Advanced and sophisticated form of TLC.
• Offer better resolution and sensitivity.
• Principle is Adsorption.
• It is a powerful analytical method equally suitable for
qualitative and quantitative analytical tasks.
• It is also known as planer or flat bed chromatography.
• HPTLC is very popular for many reasons such as-
• Visual chromatogram
• Multiple sample handling.
Detection limit in nanogram range with UV- absorption
detection and in picogram range with fluorimetric
detection.
• Large no of theoretical plates in minimum area of plates .
• Analysis time is greatly redused in HPTLC due to shorter
migration distance.
• Higher efficiency/resolution due to smaller particle size
(5 μm).

3. • Main difference between TLC is the particle and pore size
of adsorbents.
• TLC: 40-50 micrometer.
• HPTLC: 5-10 micrometer.
• Reduction in particle size results in increased surface area
and increase in no of theoretical plates and thereby better
resolution.
• 1. 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.
• Supports: • Glass • polyester sheets • Aluminium sheets
Some of the sorbents used in HPTLC:
• Silica gel 60F (Unmodified )
• Alluminium oxide
• Cellulose (microcrystalline )
• Silica gel chemically modified

4. • 2. 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.
• 3. 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.
• 4.Application of samples
• Samples and standards are sprayed to precoated plates
in the form of concentrated solutions using a
pressurised nitrogen system.
• (Eg: Linomat sample applicator)

7. • Twin trough chambers are
used instead of conventional
TLC chamber .
• These offer
1. Lesser developmental
time.
2. Rapid saturation.
3. Vapour phase control of
pH and humidity.
4. Pre-conditioning or
Saturation of chamber
5. Developing the plate

8. • 6.Detection and visualization-
• • Detection under UV light is first choice.
• • Non destructive and spots of fluorescent
compounds can be seen at 254 nm (short wave
length) or at 366 nm (long wave length).
• • Spots of non fluorescent compounds can be
seen fluorescent stationary phase is used - silica
gel G.
• • Non UV absorbing compounds like ethambutol,
dicylomine dipping the plates in 0.1% iodine
solution

9. • Densitometric scanning and Photo
documentation
• An important aspect of HPTLC is the
densitometric scanning of plates under UV light.
• In Densitometry , UV light moves as thin curved
lines over the surface of the HPTLC plate and
optical density in reflectance mode is measured.
• The difference in optical density of light sensitive
materials are measured in Densitometry. The
variation in optical signal reflected by normal
stationary phase and place of stationery phase
where compound is adsorbed is measured and
used to sketch a densitogram(HPTLC
chromatogram).

10. HPTLC CHROMATOGRAM OR DENSITOGRAM
(X-axis: Rf value , Y axis AU:Area under the peak)
The Rf value of marker compounds identified.
The position of marker peaks in the test chromatogram is
identified and the area under the peak of marker peak in test
gives concentration of the same.
HPTLC chromatogram of Vasavaleha
Marker compounds: Vasicine and Piperine

13. • Thank You