MAHARSHI ARVIND INSTUTE OF PHARMACY

MANSAROVAR, JAIPUR.

GUIDED BY . Prepared by
Mr. Kapil sharma. Jasmin .H. modi.
M pharma (Pharmaceutics)
I semester
 Contents.
Introduction.

Introduction to H.P.T.L.C.
 Principle.
Selection of HPTLC plates.

Activation of pre coated plates.

 SAMPLE PREPARATION:

Evaluation of spot or band.

Application of HPTLC.

DIFFRENCE between TLC andHPTLC.

 Introduction:
chromatography is a physical process of seperation in which the
component is to be separated are disturbeted between two
immiscible a stationary phase with has large surface area and a
mobile phase which is in constant motion through the stationary
phase.

Introduction to H.P.T.L.C
H.P.T.L.C is the improved method of T.L.C which utilize the
conventional technique of TLC in more optimize way .
It is also known as a planer chromatography or Flat- bad
chromatography.
 Principle
HPTLC take place in high speed capillary flow rang of the mobile
phase,There are three main step in HPTLC

1] sample to analyzed to chromatogram layer volume precision and

suitable position are achieved by use of suitable instrument.

2]solvent (mobile phase) migrates the planned distance in

layer(stationary) by capillary action in this process sample
separated in its components.

3] separation tracks are scanned in densitometer with light beam in

visible or uv region
 Selection of HPTLC plates.
Previously hand made plate is used in TLC for both
qualitative and quantitative work, certain draw back with
that is non uniformly layer , formation of thick layer paved
for advant pre coated plates.
Now a days pre coated plates are available in different formet
and thickness by different manufactures. these plates are
used for both qualitative and quantitative purpose in HPTLC.

 glass plates .
Polyester /polyethylene.

Aluminium plates.
 GLASS PLATES
 Resistance to heat

Easy to handle Thickness 1.3mm

 Offer superior plane and smooth surface.

Fragile
High weight
High production cost
 POLY ESTER POLYETHYLENE.
Thickness of plate 0.2mm
It can be produce in Roll form.
Unbreakable.
Less packing material required.

Development of plate is not above temp. 1200losses of its shape.

 Aluminium plates.
Thickness of plate 0.1mm
It can be produce in Roll form.
Unbreakable.
Less packing material required.

Development of plate is not above temp. 1200losses of its shape.

 Sorbents used in HPTLC Plates.
Sorbent used in conventional
TLC can be used in HPTLC with or
with out modification.
Silica gel 65F(modified)
Highly purified silica gel60.

Aluminium oxide.

Microcrystalline.

Silica gel G particle size of sorbent

Reversed stationary phase. HPTLC 6 m
Hybrid plates. TLC 10 m
Layer thickness in HPTLC -100-200 m
in TLC -250 m
 Layer prewashig .
 Ascending method .
 continuous method.
 Deeping method.

solvent used for washing

methanol
chloroform: methanol:amonia(90:10:1)
Chloroform: methanol(1:1)
Ammonia solution (1%)
 Activation of pre coated plates.

The plates are activated by placing in oven at 110-120 0c for 30

minutes,this step will remove the water that has been physical
absorbed on the surface at solvent layer.

Freshly open box of HPTLC plates usually not requird activation.

Activation at higher temperature and long time is avoided which
may tends to vary active layer and sample decomposition .
 SAMPLE PREPARATION:
Proper sample preparation is pre requsite for the success HPTLC
separation.
Beside maximizing the yield of analyte in the selected solvent
,stability of the analyte during extraction and analysis must
consider. there for choice of suitable solvent for given analysis is
very important .
Solvent for dissolving the sample should be non polar and non
volatile as far as possible since polar solvent are likely to induce
circular chromatogram at the origin.
Application of sample and standard solution.
Sample application is one imp and critical step for obtaining the
good resolution for quantification by HPTLC. sample/std are
applied as sport or band depending upon the analysis spot
application is done by using
1)Capillary tubes.

2)Micro bulb pipetts.

3)Micro syring.

4)Automatic sample applicator.

compare sample/ std applications

FIG:Automatic HPTLC sampler

 CAMANG LINOMAT
Camang inomat with a spray tech is
Automated sample application device.
The sample is loaded in micro syring
(Hamilton syring )of 1.0 capacity.
The sample is applied as a spot or band
By programming instrument parameter
Like spotting volume ,band length,
No of spot/band , space between spot/
band. Fig CAMANG LINOMAT
APPLICATOR

The nozzle is placed at tip of syring ,air is coming out at high

pressure atomize the sample solution in to fine spray
 CHROMATOGRAM DEVELOPMENT.
After application of sample in HPTLC plate, chromatogram is
developed by dipping in suitable solvent system
Taken in developing chamber.
The solvent system rises over the layer by
capillary action and separation of
sample in different components
take place.

selection of solvent system

Chamber saturation
Type of development and developing device.
Fig :DEVELOPMENT
CHAMBER
 LINEAR AND RADIAL DEVELOPMENT.
 In close bad tech like HPLC linear development is

possible but an open bad technique does not suffer

this limitation.
HPTLC can develop by
Ascending .
Descending .
Circular.
Anti circular.

FIG: HPTLC of Ginseng.

 Detection or visulation of spot/ band.
There is no diffucult in detecting the coloured substance.or colour
les substance absorbing the uv radiationor with
fluoresce(Riboflavin)

Detection of spots/band are done by

1) destruction/Non reverse.

2) Nondestructive/reversible.

3) Misc. method.

FIG:HPTLC VISULIZER
 Evaluation of spot or band.

After detection of spot /band upon objective of experiment

chromatogram is used for several purpose.
Quality Evaluation .
Quantitative Evaluation .
 Application of HPTLC.

Pharmaceutical research.
Biomedical Analysis.
Clinical Analysis.
Environment Analysis.
Food industry.

Therapeutic drug monitoring to determine its concentration and

metabolites in blood urine etc.
Analysis of environment pollution level.
Quantitative determination of prostaglandin s and thromboxanes in
plasma.
Determination of mercury in water.
Characterization of hazard in industrial waste.
DIFFERENCE BETWEEN TLC AND
HPTLC.
PARAMETER TLC HPTLC
TYPES OF HANDMADE/P PRECOATED
CHROMATOGRAPHIC RECOATED
PLATES
ABSORBENT LAYER 200-250 m 100-150m
PARTICLE SIZE RANGE 5-20 m 4-8 m
APPLICATION OF SAMPLE MANUAL/SEMI MANUAL/SEMIAUTO
AUTOMATIC MATIC
SHAPE OF SAMPLE SPOT SPOT/BAND
SPOT SIZE 3-6mm 1-2mm
SAMPLE VOLUME 1-10 0.1-2
PARAMETER TLC HPTLC
NO OF SAMPLE OER PLATE 15-20 40-45
OPTIMAL DEVELOPMENT 10-15cm. 5-7cm
DISTANCE
DEVELOPMENT TIME DEPEND UPON 40 % LESS THAN TLC
MOBILE PHASE
QUANTITATION MANUAL MANUAL
/INSTRUMENTION.
REPRODUBILITY OF RESULT DIFFUCULT REPRODUCIBLE.
 References.
 Principle of Instrumental analytical ,skoog, Holler, Nieman.
 Instrumental method of analysis, willard, merriet ,dean.
 Pharmaceutical analysis,munson.
 Instrumental method of chemical analysis gurudeep chatwal,
shyam k anand