🔬 HPTLC – High Performance Thin Layer Chromatography | Principle, Instrumentation & Applications This presentation provides a clear, concise, and exam-oriented explanation of High Performance Thin Layer Chromatography (HPTLC), an advanced and widely used analytical technique in pharmaceutical analysis, quality control, and research laboratories. 📌 Topics covered in this PPT include: • Definition and principle of HPTLC • Difference between TLC and HPTLC • Instrumentation and components of HPTLC • Sample application and development techniques • Detection, derivatization, and densitometric scanning • Chromatographic parameters (Rf value, resolution, sensitivity) • Method development and validation • Applications of HPTLC in pharmaceuticals, herbal drugs, food analysis, and forensic science • Advantages and limitations of HPTLC 🎯 This PPT is highly useful for B.Pharm, M.Pharm, D.Pharm, MSc students, researchers, and professionals preparing for university exams, GPAT, NIPER, interviews, and industrial QA/QC roles. 📈 Why this presentation? ✔ Easy-to-understand language ✔ Diagram-based explanation ✔ Exam-oriented content ✔ Industry-relevant applications 🔑 Keywords / Tags: HPTLC, High Performance Thin Layer Chromatography, Pharmaceutical Analysis, Chromatography Techniques, TLC vs HPTLC, Method Validation, QA QC, Herbal Drug Analysis, GPAT, Pharmacy Notes 📥 Download, share, and save this PPT for quick revision and conceptual clarity.

1. HIGH PERFORMANCE
THIN LAYER
CHROMATOGRAPHY
(HPTLC)
Under the guidance of :
Dr. Anil. P. Dewani
( M. Pharm , PhD )
Presented By :
Mr. Prajval V. Tidke
( M. Pharm 1st
year )

2. 01
02
03
04
05
06
CONTENT
INTRODUCTION
PRINCIPLE
OF HPTLC
DIFFERENCE
BETWEEN TLC
AND HPTLC
STEPS
INVOLVED IN
HPTLC
APPLICATION OF
HPTLC
FACTOR
AFFECTING
HPTLC

3.  Chromatography is physical method of separation in which
the components to be separated are distributed between two
phase, one of which is stationary phase while the other mobile
phase moves in a definite direction
INTRODUCTION
 HPTLC – Sophisticated form of thin layer chromatography it
involves the same theoretical principle of thin layer
chromatography.
 Traditional thin layer chromatography & its modern
instrumental quantitative analysis version HPTLC are very
popular for many reason such as
Visual chromatogram
Simplicity
Multiple sample handling

4. PRINCIPLE
 Separation may result due to adsorption or partition
or by both phenomenon depending upon the nature of
adsorbents used on plates and solvents system used
for development
 The mobile phase flows through the plate because of
capillary action the components move according to
their affinities toward the adsorbent.
 The component with more affinity towards stationary
phase travels slower the component lesser affinity
towards stationary phase travel faster

5. DIFFERENCE BETWEEN
TLC & HPTLC
FEATURES OF HPTLC
 Low analysis time
 Less cost per analysis
 Low maintenance cost
 Simple sample preparation
 Low mobile phase consumption
per sample
 Visual detection possible
 Accuracy
 No contamination
Parameters HPTLC TLC
100 micrometer 250 micrometer
High Less
3-5 cm 10-15 cm
Faster Slower
Wide choice ( Silica,
C8,C18)
Limited ( silica,alumina,
kieselguhr)
Automatic Manual
UV/ Visible/
Fluorescence
Not Possible
Separations

6. STEPS INVOLVED IN
HPTLC
1. Selection
chromatographic of
Plates
2. Layer pre-
washing
3. Activation of
pre-coated plates
4. Sample
preparation
and
application
5. Selection of
mobile phase
6. Pre-
conditioning
7. Chromatographic
Development and
drying
8. Detection &
visualization
9. Documentation

7. 1. Selection chromatographic of Plates
 Hand-made plates ( cellulose based) are rarely
used nowadays because ready-made plates are
easily available
 Pre-coated plates are widely used for both
qualitative and quantitative analysis.
 Support materials used for plates:
• Glass
• Polyester/ polyethylene
• Aluminium
 Sorbents used in plates:
• Silica gel 60F
• Aluminium oxide
• Cellulose
• Smaller silica particles give better resolution
& higher sensitivity
2. Layer pre-washing
 It is a cleaning step for HPTLC plates
 Removes moisture and impurities from air.
 Silica gel has iron impurity, removed by
Methanol : water (9:1)
 Methods: Ascending, Dipping, Continuous
 Solvents: Methanol; Chloroform : Methanol;
Chloroform : Methanol : Ammonia

8. 3. Activation of pre-coated plates
 Fresh HPTLC plates do not need activation
 Activation is needed if plates are moist or
handled for long time.
 Plates are heated at 110-120C for 30 min to
remove moisture.
 Overheating is avoided to prevent sample
decomposition
4. Sample preparation and application
 Proper sample gives better separation
 Sample and standard are dissolve in the same
solovent
 Use concentrated solution; apply small
amount
 Dry plate and store dust-free
 Solvents: Methanol; Chloroform-Methanol;
Ethyl acetate-Methanol; Chloroform-
Methanol-Ammonia.

9. 5. Selection of mobile phase
 Choose based on sample and
sorbent properties
 Measure and mix solvents
separately
 Requires less solvent than TLC
 Do not resue mixed mobile phase
due to evaporation and adsorption
6. Pre-conditioning
 Unsaturated chamber – high Rf
 Saturation gives uniform solvent vapours
and lower Rf
 Needed for polar mobile phase, not for
non-polar
 Affects separation quality

10. 7. Chromatographic Development and drying
 Spot the sample, air dry and place the plate in the
chamber
 Develop using methods like ascending or descending
 Remove the plate and let the solvent evaporate
 Dry the plate in a vaccum desiccator away from heat
and light
8. Detection & visualization
 Detection under UV light is the first
choice
 Fluorescent compounds can be seen
at 254nm or 366nm
 Non- fluorescent compounds can be
seen using fluorescent plates like
silica gel
 Non- UV absorbing compounds are
detected by dipping the plate in
0.1% iodine solution
9. Documentation
• The scanner converts each spot into peak
• Peak height or peak area shows the amount of
substance present
• These values are measured by the instrument and
recorder

11. Factor affecting HPTLC
 Types of stationary phase
 Mobile phase
 Layer thickness
 Temperature
 Mode of development
 Amount of sample
 Dipping zone

12. Applications of HPTLC
 Pharmaceutical industry
 Food analysis
 Clinical applications
 Industrial applications
 Forensic
 Biomedical analysis
 Environmental analysis
 Cosmetics
 Finger print

13. Thank
You