this presentation presents introduction about high performance thin layer chromatography, its features, principle and instrumentation along with its applications. it also gives comparison between TLC and HPTLC. instrumentation is given in a sequence for easier understanding of instrument.

2. 1.High Performance Thin
Layer Chromatography
(HPTLC).
2.Recent development led
to the improvements in
various operations
involved.
3.Difference b/w TLC &
HPTLC – practical
technique than of the
physical phenomenon
(partition, adsorption)
on which separation is

3. Difference
TLC
Stationary
phase –
thin layer
of sorbent
(cellulose
powder /
silica gel)
Coating on
an inert,
rigid glass
plate foil –
separation
on flat with
two
dimensiona
l surface
HPTLC
Improved
separation
technique,
high
resolutions
& improved
quantitative
analysis

4. • Similar approach & same physical
properties of TLC.
• Principle of separation – adsorption.
• Mobile phase flows through b/c of
capillary action.
• Components move according to their
affinities towards adsorbent.
• Components – separated on
chromatographic plate.

5. Components - Greater
affinity towards
stationary phase travels
slower
Components - Lesser
affinity towards
stationary phase travels
faster

8. High concentrated
solution, as less
amount of sample
– applied.
Normal phase –
silica gel is pre -
coated
Reversed phase –
polar solvents are
used for dissolving
the sample.
Plates solvents –
non polar of
volatile type.

9. Layer – pre-
coated silica gel :
adsorbent.
Plates – similar as
TLC. Particle size
– very fine
particles.
Plates : 4-5mm
silica gel to form
200mm layer
Fine particles –
greater resolution
& sensitivity.
TLC : 5 – 20mm
HPTLC : 4 – 8mm
(particle size)

10. Plates
Pre - washing
Remove water
Volatile impurities
Cleaned by methanol
conditioning
Pre – washed
plates placed in
oven at 120c for 15
– 20 minutes
(conditioning)

11. Size not >1mm diameter
Self – loading capillary for
small volume of samples
Surface using platinum –
iridium tubing fused into end
of a length of glass tubing

12.  Low polarity of mobile phase – no need for
saturation.
 High polarity of mobile phase – saturation is
needed.
 Solution by trial & error where chemical
properties of solute & solvent solubility of analytic
adsorbent layer are considered.

13.  Linear development method.
 plate – vertically. placed in solvent system in a
suitable container.
 solvent – fed by capillary action &
chromatogram – from both the sides.
Chromatographic
development -
Methods
Circular
development
Anti – circular
device
Multiple
development

14.  Immediately after the
development is
completed, the plates
are removed from the
chamber and dried to
remove the frees of
mobile phase.
 Detection - known by
iodine vapor in iodine
chamber.

15. HPTLC – supplies with computer &
data recording & storing devices.
Development – scanned at selected UV
wavelength by instruments & detected
spots - peaks
Scanner – converts bond into peak &
peak heights / area related to
concentration of substance on the
spot.
Peak heights & area under spot –
measured by instrument & recorded as
% on the printer.

16. FEATUR
ES
Quality of
adsorbed
layer
Methods of
sample
application
Sample into
adsorbent
layer
Scanning
densitometer
s

17. • Pure silica gel with
narrow particle size.
• Diameter : 3-5µm.
• Chemically bonded
layers as reverse phase
plates.
• Layers – improved
adsorbents of 5000
plates.
• Difficult separation in
shorter time than TLC
1. Quality of
adsorbed
layer
• Lower sample capacity
& so the layer is
reduced.
• Sample volume : 100 –
200 cm gives 1mm
diameter.
• After plate
development – 10
times better detection
limits.
• Advantage – compact
starting spots –
increased no. of
samples.
2. Methods of
sample
application

18. • Critical process.
• Capillary volume :
100/200 nL – sealed into
glass support capillary –
convenient spotting
device in quantitative
measurements.
• Capillary tip – polished
for smooth planar surface
of small area (0.05mm)
when used as mechanical
applicator minimizes
damage to surface of
plate.
3. Sample into
adsorbent layer
• Scan individual spot by
reflectance / absorption
of a light beam.
• Transmitted / reflected
radiation – photograph
on chromatogram
revealing dark / light
zones for separated
compounds.
4. Scanning
densitometers

19. Clinical purpose (analysis of
drugs in blood)
Environmental analysis
Separation of compounds
Quantitative analysis of volatile
compounds
Separation & quantitative analysis
of thermally stable compounds