HPTLC Seminar

M.M.
U COLLEGE OF
M.M.U COLLEGE OF PHARMACY SEMINAR ON HPTLC PRESENTED BY AZIM ARSHI M.PHARMA 1ST ,
SUBMITTED TO MR. NIRMAL . T. HAVANAVAR PROFESSOR & HOD PHARMACEUTICAL CHEMISTRY
PHARMACY
RAMANAGARAM-571511
SEMINAR ON
High Performance Thin Layer Chromatography
PRESENTED BY SUBMIT TO
Mr. NIRMAL.T.HAVANAVAR
AZIM ARSHI
PROFESSOR & HOD
PHARMA.CHEMISTRY
1ST M.PHARMA
DEPARTMENT OF
Introduction
Chromatography is a physical process of separation in
which the components to be separated are distributed between
2 immiscible phasesa 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.
It is also known as planar chromatography or Flatbed

chromatography.
Principle
HPTLC takes place in highspeed capillary flow range of the
mobile phase. There are three main steps HPTLC procedure
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 it’s components.
3] Separation tracks are scanned in densitometer with light

beams in visible or uv region
Sample & slandered Preparation Selection Of Chromatographic lay
Layer Pre Washing
Layer Pre Conditioning
Application Of Sample & Standard
Chromatographic Development
Detection of spot
Scanning & documentation of chromatoplate
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.
qGLASS PLATES
qPOLY ESTER/POLYETHYLYNE
qALUMINIUM PLATES
qGLASS PLATES
Thickness of Plate 1.3 mm
vResistant to heat & Chemical
vEasy to handle
vOffers superior Flat & smooth surface
vFragile
vHigh weight
vHigher production cost
qGLASS PLATES
qPOLY ESTER/POLYETHYLYNE
vIt can be produced in Roll forms
vUnbreakable
vLess packing material required
vDevelopment of plate can not be above temp. 1200 losses of it shape
qALUMINIUM PLATES
vIt can be produced in Roll forms
vUnbreakable
vLess packing material required
vEluent with high Concentration mineral acid or ammonia chemical
attacks aluminum plate
SORBENTS USED IN HPTLC PALTES
Sorbent, which are used in conventional
TLC, are also used in HPTLC with or
without modification
üSilica gel 65F(modified)
üHighly purified Silica gel 60
üAluminium oxide
üCellulose Microcrystalline
üSlica gel PARTICAL SIZE OF
üReversed stationary phase SORBENTS
HPTLC 6 m, TLC 10 m.
üHybrid Plates
LAYER THICKNESS
The layer of thickness in HPTLC is around 100200 m,where as
250m in conventional TLC.
LAYER PRE WASHING
q Ascending method
q Dipping method
q Continuous method
Solvents used for pre washing
qMethanol (commonly used)
qChloroform: Methanol: Ammonia (90:10:1)
qChloroform: Methanol (1:1)
qMethylenechloride: Methanol (1:1)
qAmmonia solution (1%)
ACTIVATION OF PRECOATED PLATES
The plates are activated by placing in an oven at 110120 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 pleads to very active layer and there is risk of
sample being decomposed.
SAMPLE PREPATION
Proper sample prepation an in imp.prerequsite for success of TLC
separation.
Besides maximizing the yield of analyte in selected solvent, stability
of analyte during extraction and analysis must be considered.
Therefore choice of suitable solvent for given analysis is very imp.
Solvent for dissolving sample should be non polar and volatile as far
as possible, since polar solvents are likely to induces circular
chromatogram at the origin.
APPLICATION OF SAMPLE AND STANDERED SOLUTION
Sample application is one imp. And critical step for obtaining good
resolution for quantification by HPTLC. Sample / std. Is applied
as a sporty or band depending upon the analysis.
Spot application is done by using
1)Capillary tubes
2)Micro bulb pipettes
3)Micro syringes
4)Automatic sample applicator
compare sample / std. Application HPTLC from that of TLC
Parameter TLC HPTLC
Spotting vol 110l 0.12l
Spot diameter 36mm 12mm
Sample / std. 0.11g/ml 0.11g/ml

CAMAG LINOMAT
Camag Linomat with spray tech. Is

usually automated sample application
device. The sample is loaded in micro
syringe (Hamilton syring ) of 1.0 l
capacity. The sample is applied either as a
spot or band by programming instrument
parameters like spotting volume, band
length, no. of spot/ band, space between
bands etc.
The nozzle is placed at tip of syringe, air is
coming out at high pressure atomizes
sample solution into fine spray. It results
in concentration and spraying of sample as
a narrow band of suitable length.
CHROMATOGRAM DEVELOPMENT
After application of sample in HPTLC plate,
chromatogram is developed by dipping in
suitable solvent system taken in a
developing chamber. The solvent system is
rises over the layer by capillary action and
separation of sample in to different
components takes place.
qSelection of solvent system / mobile phase
qChamber saturation
qType of development and developing
device.
LINEAR & RADIAL DEVELOPMENT
In a close bed tec. Such as HPLC only Linear development
Is possible, But an open bed tec. Like HPTLC does not
suffer this limitation.
HPTLC can develop by
Ascending (linear )
Circular
Anti circular
Circular Chromatography
Anti Circular Chromatography
DETECTION OR VISULATION OF SPOTS / BANDS
There is no difficulty in

detecting the colored substances
or colorless substances
absorbing UVradiations or with
fluoresce (Riboflavin)
“Derivatisation”
Detection of spots / bands are

done by
1)Destruction / Nonreverse
2)Nondestructive / Reversible
3)Misc.methods
EVALUATION OF SPOTS / BANDS
After detection of spots / band, upon objective of expt.

Chromatogram is used for several purposes
qQualitative Evaluation
qQuantitative Evaluation
DOCUMENTATION OF CHROMATOGRAM
HPTLC plates that have been evaluated quantitatively and

qualitatively, should be documented as per guidelines of GMP,
GLP common methods of documentation are
ØPhoto documentation
ØVideo documentation
APPLICATIONS OF HPTLC
vPharmaceutical Researches
vBiomedical Analysis
vClinical Analysis
vEnvironmental Analysis
vFood Industry
vTherapeutic drug monitoring to determine concentration of drug
and it’s metabolite in blood, urine etc.
vAnalysis of environmental pollutions levels.
vQuantitative determination of prostaglandin’s and thromboxanes
in plasma.
vDetermination of mercury in water.
vAnalysis of nitrosoamines in food and body fluids.
vDetermination of sorbic acid in wine.
vCharacterization of hazards in industrial waste.
PARAMETERS TLC HPTLC
TYPE OF Handmade / PrecoatedPer coated

CHROMATOGRAP
HIC PLATE
ADSORBANT 200-250 m 100-200 m
LAYER
PARTICAL SIZE 5-20 m 4-8 m
RANGE
APPLICATION OF Manual / Semiautomatic /
SAMPLE Semiautomatic Automatic
SHAPE OF SAMPLE Spot Spot / Band
SPOT SIZE 3-6mm 1-2mm
SAMPLE VOLUME 1-10 l 0.1-2l

PARAMETERS TLC HPTLC
NO.OF SAMPLE 15-20 40-50

PER PLATE
OPTIMAL 10-15cm 5-7cm

DEVP.DISTANCE
DEVP. TIME Depends on Mobile 40% less than TLC
Phase
QUANTITATIONS Manual / Instrument Instrumental
REPRODUCIBILITY Difficult Reproducible

OF RESULTS
REFERENCES:
Principles of Instrumental Analysis,Skoog,Holler,Nieman.
Instrumental Methods of Analysis. Willard, Merrit,Dean.
Pharmaceutical Analysis. Munson.
Instrumental Methods of Chemical Analysis. Gurdeep R.
http://pharamcytimes.wordpress.com/category/instrumentalanalysisstudies/
Chatwal, ShyamK. Anand.
Sherma J, Fried B. Handbook of thin layer chromatography.
http://www.interchromforum.com/html/body_qnt_err_hptlc.html
3rd ed. New York: Marcel Dekker, Inc.; 2003. p. 34.
http://www.pharmainfo.net/reviews/validatedanalyticalmethodsdeterminati
Sethi PD. HPTLC: Quantitative analysis of pharmaceutical
http://www.selectscience.net/commNWDetails.aspx?mailID=1035
formulations. 1st ed. New Delhi: CBS Publisher; 1996. p. 4457.
Peter EW. Thin layer chromatography: A modern practical
approach. UK: The royal society of chemistry; 2005. p. 6154.
Tha n kyou

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M.M.

It is also known as planar chromatography or Flat­bed

1] Sample to analyzed to chromatogram layer, volume

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

3] Separation tracks are scanned in densitometer with light

Spot diameter 3­6mm 1­2mm

Sample / std. 0.1­1g/ml 0.1­1g/ml

Camag Linomat with spray tech. Is

There is no difficulty in

Detection of spots / bands are

After detection of spots / band, upon objective of expt.

HPTLC plates that have been evaluated quantitatively and

PARAMETERS TLC HPTLC

TYPE OF Handmade / PrecoatedPer coated

SPOT SIZE 3-6mm 1-2mm

SAMPLE VOLUME 1-10 l 0.1-2l

PARAMETERS TLC HPTLC

NO.OF SAMPLE 15-20 40-50

OPTIMAL 10-15cm 5-7cm

REPRODUCIBILITY Difficult Reproducible

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It is also known as planar chromatography or Flatbed

Spot diameter 36mm 12mm

Sample / std. 0.11g/ml 0.11g/ml