NILESH JAWALKAR

M.PHARM 1
ST

YEAR(PHARMACETICS)
HPTLC

HIGH PERFORMANCE THIN
LAYER CHROMATOGRAPHY


CHROMATOGRAPHY



Chromatography is a physical process of
separation in which the components to be
separated are distributed between 2 immiscible
phases a stationary phase which has a large
surface area and mobile phase which is in constant
motion through the stationary phase.


Introduction of HPTLC


HPTLC is the improved method of TLC which utilizes the
conventional technique of TLC in more optimized way.
HPTLC takes place in highspeed capillary flow range of
the mobile phase.
There are three main steps HPTLC procedure, they are

1] Sample to analyzed to chromatogram layer, volume precision
and exact position are achieved by use of suitable instrument.
2] Solvent (mobile phase) migrates the planned distance in layer
(stationary phase) by capillary action. In this process sample
separated into its components.
3] Separation tracks are scanned in densitometer with light
beams in visible or uv region




Steps Involving in HPTLC
Sample Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
5
Sample preparation
Normal phase chromatography: non polar solvent
Reversed phase chromatography: polar solvent

Selection of chromatography
layer
Depends on nature of material to be
separated
Commonly used(silica gel, alumina)
6
Pre-washing
It is purification step
Mainly methanol is used
Essential for quantitative evaluation
Stability testing

7
Pre-conditioning
Also called Chamber Saturation
Low polarity mob. Phase:- no need
High polar mob. Phase:- desirable
For reverse phase saturate chamber with
polar solvent
Sample application
1-5 l
Linomat IV Applicator
8
Linomat lV applicator
9
Fig. Hptlc instrumentation
Selection of HPTLC plates
Previously hand made plates is used in TLC for both
qualitative and quantitative work. Certain drawbacks with
that is nonuniform layer, formation of thick layer, paved for
advent of precoated plates.

Nowadays precoated plates are available in different format
and thickness by various manufactures. Precaoted plates can
be used for both qualitative and quantitative work in
HPTLC, they are

GLASS PLATES
POLY ESTER/POLYETHYLYNE
ALUMINIUM PLATES





Glass Plates:

Offers superior flat and smooth surface.
- fragile
- high weight
- higher production cost


Polyester/polyethylene plates:
Thickness of plate is 0.2mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packing material is required.
- Development of plate cannt be above temperature
120
0
c loses its shape.





Aluminium plates:
- Thickness of plate is 0.1mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packaging material is required.


SORBENTS USED IN HPTLC PLATES:

sorbents which are used in convential TLC are
also used in HPTLC with or without modification.
- silica gel 65F
- highly purified silicagel 60
- aluminium oxide
- cellulose microcrystalline
- silica gel
- reversed stationary phase


Layer thickness
The layer thickness in HPTLC is around 100-
200cm,in conventional it is 250mm.

Layer prewashing:
- Ascending method
- Dipping method
- Continuous method

ACTIVATION OF PRECOATED PLATES

The plates are activated by placing in an oven
at 110120
0
C for 30 min, this step will removes
water that has been physically absorbed on surface
at solvent layer.

Freshly opened box of HPTLC plates usually
does not require activation.

Activation at higher temp and for longer time is
avoided which leads to very active layer and there
is risk of sample being decomposed.


Solvents used in HPTLC

- Methanol (commonly used)
- Chloroform:methanol:ammonia(90:10:1)
- Chloroform:methanol(1:1)
- Methylene chloride:methanol(1:1)
- Ammonia(1%)solution

Application of sample and standard

Usual concentration range is 0.1-1g / l,above
this causes poor separation.

Linomat IV (automatic applicator) - nitrogen gas
sprays sample and standard from syringe on TLC
plates as bands.

Band wise application - better separation - high
response to densitometer.

Chromatographic development and drying

After development, remove the plate and
mobile phase is removed from the plate - to avoid
contamination of lab atmosphere.

Dry in vacuum desiccator - avoid hair drier
because essential oil components may evaporate.

Detection and visualization


Detection under UV light is first choice -
non destructive.

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 GF.


Non UV absorbing compounds like
ethambutol, dicylomine etc - dipping the plates in
0.1% iodine solution.

When individual component does not
respond to UV - derivatisation required for
detection .


HPTLC
100m
High due to smaller particle size
generated
3 - 5 cm
Shorter migration distance and
the analysis time is greatly
reduced
Wide choice of stationary
phases like silica gel for normal
phase and C8 , C18 for reversed
phase modes
New type that require less
amount of mobile phase
Auto sampler
Use of UV/ Visible/ Fluorescence
scanner scans the entire
chromatogram qualitatively and
quantitatively and the scanner is
an advanced type of
densitometer
TLC
250m
Less

10 - 15 cm

Slower

Silica gel , Alumina &
Kiesulguhr

More amount

Manual spotting


Not possible


APPLICATIONS

Pharmaceutical Researches
Biomedical Analysis
Clinical Analysis
Environmental Analysis
Food Industry
Therapeutic drug monitoring to determine concentration of drug
and its metabolite in blood, urine etc
Analysis of environmental pollutions levels
Quantitative determination of prostaglandins and thromboxanes
in plasma
Determination of mercury in water
Analysis of nitrosoamines in food and body fluids
Determination of sorbic acid in wine
Characterization of hazards in industrial waste

REFERENCES
1. Principles of instrumental analysis skoog, Holler,
Nieman
2. Instrumental methods of analysis Willard
,Merrit, Dean
3. Pharmaceutical analysis Munson
4. Sharma J.friedB.Handbook of TLC
5. Kasture A.V A text book of Pharmaceutical
Analysis (Instrumental methods), 14
th
edition, page
no.28-30
6. Elke Hahn Deinstrop Applied TLC, 2
nd
edition
7. Site www.Google.com (Google images)


THANK YOU