This document discusses High Performance Thin Layer Chromatography (HPTLC). HPTLC is an automated, sophisticated form of Thin Layer Chromatography (TLC) that allows for multiple sample handling and enables complex separations. It has detection limits in the nanogram and pictogram range for UV absorption and fluorimetric detection, respectively. The document compares HPTLC to TLC and HPLC, noting advantages like greater efficiency, resolution, and sensitivity for HPTLC due to smaller particle sizes and automated instrumentation. HPTLC is commonly used for purity testing in pharmaceuticals, pesticides, steroids, food dyes, and more.

1. NIBHA INSTITUTE OF PHARMACEUTICAL SCIENCES
NAME – ANUGRAH KUMAR
B.PHARM(3rd SEMESTER)
Section- 2020-2024
PHARAMACEUTICAL

2. Introduction
– HPTLC (High performance thin layer chromatography) is the
automated, sophisticated form and improved method of TLC.
It is also known as planer or flat bed chromatography
– HPTLC is very popular for many reasons such as-
– Visual chromatogram,
– Multiple sample handling,

3. – Enables the most complicated separation
Detection limit in nanogram range with
Uvabsorption detection and in pictogram range with
fluorimetric
detection.
• Large no of theoretical plates in minimum area of
plates .

4.  HPLC and HPTLC methods were validated for the
simultaneous determination of the three
drugs. For HPLC, column, while for HPTLC method only
10 μL was spotted on TLC plates.
 HPLC using an RP-Select B (5 gm) column and a
post-column reaction procedure. The
positive results were confirmed by high-performance
thinlayer chromatography(HPTLC).
 Due to retained components inside
the column from previous applications.
However, there are several disadvantages
of HPTLC methods comparing to HPLC.
Electrocyclic Reactions column

5. Selection of chromatographic plates
– Hand made plates which are made up of cellulose
and other materials which are
not used much now a day.
– Pre-coated plates- The plates with different support
materials and sorbent
layers with different format and thickness are used for
qualitative and
quantitative analysis.

6. Support materials used in plates-Glass
Polyester/polyethylyne Aluminium
Sorbents used in plates- Silica gel 60F,
Aluminium oxide, Cellulose, silica gel
chemically modified –
a)amino group(NH2)
b) CN group
Note: Smaller particle size of silica helps in
greater resolution and sensitivity

7. Layer pre-washing-
1. It is purification step.
2. 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. In case of silica 60F( most widely used
sorbent) the major disadvantage
of this sorbent is that it contain iron as impurity.

8. STEPS IN HPTLC
Selection of chrographic layer
Layer pre-wash
Sample and standard preparation
Application of sample and standard
Chromatographic development
Detection of spot
Scanning and documentation of choromatoplate

9. Advantages/Disadvantages of
HPTLC over TLC
Most recently HPTLC is used as alternative to
classical
TLC and it is a valuable tool for reliable
identification. It is
instrument controlled by software. HPTLC,
which is used for
impurity determination, relies more on plain
but the most
potent silica gel hydrophilic phase meeting the
criteria of
most of the pharmacopoeias.

10. HPTLC Development
Vertical Development.
Vario method development.
Horizontal development.
Automatic Multiple Development (AMD)

11. HPTLC Instrumentation
Hptlc is one type of planar chromatography
and Most advanced from of from of instrumental TLC.
HPTLC is more useful than TLC and HPTLC. Because
HPTLC is independent of sample application ,
chromatogram development , detection.etc.

12. PARAMETER TLC HPTLC
Technique manual instrumental
Efficience less High
Layer Lap made/pre
coated
Pre-coated
Mean partical
size
10-12 um 5-6 um
Layer thickness 250um 100 um
Plate hight
30 um 12 um
Soild support
Silics gel,
alumina
Silicagel-normal
phase c8 and c18
reverse phase
Sample spotting Manual spotting
capillary
Auto sample

13. HPTLC is used for purity control of
chemicals, pesticides, steroids, and water
analysis.
HPTLC is also widely used for analysis of
vitamins, water-soluble food dyes, pesticides
in fruits, vegetables, and other food stuffs.
HPTLC USE