High performance thin layer chromatography

1. HPTLCHPTLC
((High performance thin High performance thin
layer chromatography)layer chromatography)
VAISHNAVI U.S.D.N
M.Pharmacy(Pharmaceutical Chemistry)
170213884006
UNDER THE GUIDENCE:
CEEMA MATHEW,M.pharmacy
Asst.Professor

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INDEXINDEX

INTRODUCTIONINTRODUCTION

ADVANTAGES OF HPTLC OVER HPLC & ADVANTAGES OF HPTLC OVER HPLC &
TLCTLC

FEATURES OF HPTLCFEATURES OF HPTLC

STEPS INVOLVED IN HPTLCSTEPS INVOLVED IN HPTLC

DIFFERENCES BETWEEN TLC AND DIFFERENCES BETWEEN TLC AND
HPTLCHPTLC

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INTRODUCTIONINTRODUCTION

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CHROMATOGRAPHY CHROMATOGRAPHY is a broad is a broad
range of physical methods used range of physical methods used
to separate and or to analyze to separate and or to analyze
complex mixtures. The complex mixtures. The
components to be separated are components to be separated are
distributed between two phases: a distributed between two phases: a
stationary phasestationary phase bed and a bed and a
mobile phasemobile phase which percolates which percolates
through the stationary bed. through the stationary bed.
  

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High performance thin layer High performance thin layer
chromatographychromatography ( (HPTLCHPTLC) is an ) is an
enhanced form of enhanced form of
thin layer chromatographythin layer chromatography (TLC). (TLC).
A number of enhancements can be A number of enhancements can be
made to the basic method of thin made to the basic method of thin
layer chromatography to automate layer chromatography to automate
the different steps, to increase the the different steps, to increase the
resolution achieved and to allow resolution achieved and to allow
more accurate quantitative more accurate quantitative
measurements.measurements.

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
Automation is useful to overcome Automation is useful to overcome
the uncertainty in droplet size and the uncertainty in droplet size and
position when the sample is position when the sample is
applied to the TLC plate by hand. applied to the TLC plate by hand.
Principle of separation is ADSO
RPTIO
N

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Advantages of HPTLC over HPLCAdvantages of HPTLC over HPLC

Sample and standard, both can be Sample and standard, both can be
used at a time.used at a time.

Evaluation time is less.Evaluation time is less.

Use of internal standard is not Use of internal standard is not
necessary.necessary.

Simultaneously 2 or 3 persons can Simultaneously 2 or 3 persons can
work but not in case of HPLC.work but not in case of HPLC.

Solvent degasing and removal of Solvent degasing and removal of
impurities is not necessary. impurities is not necessary.

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Advantages of HPTLC over TLCAdvantages of HPTLC over TLC

Visual chromatogramVisual chromatogram

simplicitysimplicity

Multiple sample handlingMultiple sample handling

Low running and maintenance costs Low running and maintenance costs
and disposable layer etcand disposable layer etc

Detection by TLC scannerDetection by TLC scanner

Spotting by applicatorSpotting by applicator

Highly precisedHighly precised

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FEATURES OF FEATURES OF
HPTLCHPTLC

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
Simultaneous processing of sample and standard Simultaneous processing of sample and standard
- better analytical precision and accuracy less - better analytical precision and accuracy less
need for Internal Standard need for Internal Standard

Several analysts work simultaneously Several analysts work simultaneously

Lower analysis time and less cost per analysis Lower analysis time and less cost per analysis

Low maintenance cost Low maintenance cost

Simple sample preparation - handle samples of Simple sample preparation - handle samples of
divergent nature divergent nature

No prior treatment for solvents like filtration and No prior treatment for solvents like filtration and
degassing degassing

Low mobile phase consumption per sample Low mobile phase consumption per sample

No interference from previous analysis - fresh No interference from previous analysis - fresh
stationary and mobile phases for each analysis - stationary and mobile phases for each analysis -
no contamination no contamination

Visual detection possible - open system Visual detection possible - open system

Non UV absorbing compounds detected by post-Non UV absorbing compounds detected by post-
chromatographic derivatizationchromatographic derivatization

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STEPS STEPS
INVOLVED IN INVOLVED IN
HPTLCHPTLC

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Selection of chromatographic layerSelection of chromatographic layer
· · Precoated plates - different support Precoated plates - different support
materials - different Sorbents available materials - different Sorbents available
· 80% of analysis - silica gel GF · 80% of analysis - silica gel GF
· Basic substances, alkaloids and steroids - · Basic substances, alkaloids and steroids -
Aluminum oxideAluminum oxide
· Amino acids, dipeptides, sugars and · Amino acids, dipeptides, sugars and
alkaloids - cellulose alkaloids - cellulose
. Non-polar substances, fatty acids, . Non-polar substances, fatty acids,
carotenoids, cholesterol - RP2, RP8 and carotenoids, cholesterol - RP2, RP8 and
RP18RP18
· Preservatives, barbiturates, analgesic and · Preservatives, barbiturates, analgesic and
phenothiazines- Hybrid plates-RPWF254s phenothiazines- Hybrid plates-RPWF254s

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Sample and Standard PreparationSample and Standard Preparation
To avoid interference from impurities and To avoid interference from impurities and
watervapours watervapours
Solvents used areSolvents used are
Methanol, Chloroform: Methanol (1:1)Methanol, Chloroform: Methanol (1:1)
Ethyl acetate: Methanol (1:1)Ethyl acetate: Methanol (1:1)
Chloroform: Methanol: Ammonia Chloroform: Methanol: Ammonia
(90:10:1)(90:10:1)
Methylene chloride : Methanol (1:1)Methylene chloride : Methanol (1:1)
1% Ammonia or 1% Acetic acid 1% Ammonia or 1% Acetic acid
Dry the plates and store in dust free Dry the plates and store in dust free
atmosphere atmosphere

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Layer pre-washing & pre-conditioningLayer pre-washing & pre-conditioning

If any impurities are present, those If any impurities are present, those
are need to be removed hence pre are need to be removed hence pre
washing is done.washing is done.

Conc.HCl : Methanol(1:9) Conc.HCl : Methanol(1:9)
Plate is dipped in the soln, allowed for the
solvent to pass throughout the plate. Hence
impurities are washed out

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Activation of pre-coated platesActivation of pre-coated plates
Freshly open box of plates do not Freshly open box of plates do not
require activation require activation
Plates exposed to high humidity or Plates exposed to high humidity or
kept on hand for long time to be kept on hand for long time to be
activated activated
By placing in an oven at 110-120ºc By placing in an oven at 110-120ºc
for 30’ prior to spotting (pre for 30’ prior to spotting (pre
conditioning)conditioning)
Aluminum sheets should be kept in Aluminum sheets should be kept in
between two glass plates and between two glass plates and
placing in oven at 110-120ºc for 15 placing in oven at 110-120ºc for 15
minutes. minutes.

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Application of sample and standardApplication of sample and standard
· · Usual concentration range is 0.1-1µg / Usual concentration range is 0.1-1µg /
µl µl
· Above this causes poor separation· Above this causes poor separation
· Linomat IV (automatic applicator) - · Linomat IV (automatic applicator) -
nitrogen gas sprays sample and nitrogen gas sprays sample and
standard from syringe on TLC plates standard from syringe on TLC plates
as bandsas bands

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Selection of Mobile phaseSelection of Mobile phase
Can be explained by STAHL’S TRIANGLECan be explained by STAHL’S TRIANGLE
E
M S
Hydrophilic
Lipophilic
Active
Inactive
Polar
Non-Polar
E- ELUENT
S- SORBANT
M- MIXTURE
EG:If
E- hydrophilic
then
M- polar &
S- inactive

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
Trial and error Trial and error
- one’s own experience and Literature - one’s own experience and Literature

Normal phase
- Stationary phase is polar
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
Reversed phase
- Stationary phase is non polar
- Mobile phase is polar
- Polar compounds eluted first because of lower affinity with stationary phase
- Non-Polar compounds retained because of higher affinity with the stationary phase

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Chromatographic development and dryingChromatographic development and drying

After development, remove the plate and After development, remove the plate and
mobile phase is removed from the plate to mobile phase is removed from the plate to
avoid contamination of lab atmosphereavoid contamination of lab atmosphere


Dry in vacuum desiccator Dry in vacuum desiccator

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Chromatographic DevelpmentChromatographic Develpment
1. TWIN TROUGH CHAMBER1. TWIN TROUGH CHAMBER

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Detection and visualizationDetection and visualization
· · Detection under UV light is first choice - non Detection under UV light is first choice - non
destructive destructive
· Spots of fluorescent compounds can be seen at 254 · Spots of fluorescent compounds can be seen at 254
nm (short wave length) or at 366 nm (long wave nm (short wave length) or at 366 nm (long wave
length) length)
· Spots of non fluorescent compounds can be seen - · Spots of non fluorescent compounds can be seen -
fluorescent stationary phase is used - silica gel GFfluorescent stationary phase is used - silica gel GF
· Non UV absorbing compounds like ethambutol, · Non UV absorbing compounds like ethambutol,
dicylomine etc - dipping the plates in 0.1% iodine dicylomine etc - dipping the plates in 0.1% iodine
solutionsolution
· When individual component does not respond to UV · When individual component does not respond to UV
- derivatisation required for detection - derivatisation required for detection

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APPLICATIONSAPPLICATIONS

Pharmaceutical researchPharmaceutical research

Bio medical analysisBio medical analysis

Clinical analysisClinical analysis

Environment analysisEnvironment analysis

food industryfood industry

Quantification of herbal extractsQuantification of herbal extracts

Therapeutic drug monitoring to determine its Therapeutic drug monitoring to determine its
concentration and metabolite in blood urineconcentration and metabolite in blood urine

Analysis of environment pollution levelAnalysis of environment pollution level

Characterization of hazards in industrial wasteCharacterization of hazards in industrial waste

Quantitative determination of prostaglandins & Quantitative determination of prostaglandins &
thromboxanes in plasma.thromboxanes in plasma.

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DIFFERENCES DIFFERENCES
BETWEEN BETWEEN
TLC AND HPTLCTLC AND HPTLC

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PARAMETERPARAMETER TLCTLC HPTLCHPTLC
Type of Type of
chromatographic chromatographic
platesplates
Hand made/pre Hand made/pre
coatedcoated
Pre coatedPre coated
Absorbant layerAbsorbant layer200-250mm200-250mm 100-150mm100-150mm
Particle sizeParticle size5-20 micrometer5-20 micrometer4-8 micrometer4-8 micrometer
Application of Application of
samplesample
Manual/semi Manual/semi
automaticautomatic
Semi automaticSemi automatic
Shape of sampleShape of samplespotspot bandband

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Spot sizeSpot size 3-6mm3-6mm 1-2mm1-2mm
Sample volumeSample volume1-10 microlit1-10 microlit0.1-2microlit0.1-2microlit
No of sample per No of sample per
plateplate
15-2015-20 40-4540-45
Optimal Optimal
development development
distancedistance
10-15cm10-15cm 5-7cm5-7cm
Development Development
timetime
Depend upon Depend upon
mobile phasemobile phase
40% less than 40% less than
TLCTLC
QuantizationQuantizationManualManual Manual/ Manual/
instrumentationinstrumentation
Reproducibility Reproducibility
of resultof result
difficultdifficult ReproducibleReproducible

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REFERENCESREFERENCES

Ajaz Ahmad, M Mujeeb, Bibhu Prasad Panda (2010) An HPTLC Method Ajaz Ahmad, M Mujeeb, Bibhu Prasad Panda (2010) An HPTLC Method
for the Simultaneous Analysis of Compactin and Citrinin in Penicillium for the Simultaneous Analysis of Compactin and Citrinin in Penicillium
citrinum Fermentation Broth. Journal of Planar Chromatography-modern citrinum Fermentation Broth. Journal of Planar Chromatography-modern
TLC 23 (4), 282–285TLC 23 (4), 282–285

1994 Rebecca Carrier and Julie Bordonaro - Intro to Biochemical 1994 Rebecca Carrier and Julie Bordonaro - Intro to Biochemical
Engineering Term Project, merged with the 1997 project by Kevin YipEngineering Term Project, merged with the 1997 project by Kevin Yip


Puri, A., Ahmad, A. and Panda, B. P. (2010), Development of an HPTLC-Puri, A., Ahmad, A. and Panda, B. P. (2010), Development of an HPTLC-
based diagnostic method for invasive aspergillosis. Biomed. Chromatogr., based diagnostic method for invasive aspergillosis. Biomed. Chromatogr.,
24: 887–892. doi: 10.1002/bmc.1382 24: 887–892. doi: 10.1002/bmc.1382

WikipediaWikipedia


Pharmainfo.dotPharmainfo.dot

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