high performance thin layer chromatography
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HPTLCHPTLC((High performance thin High performance thin layer chromatography)layer chromatography)
VAISHNAVI U.S.D.NM.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: distributed between two phases: a 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 more resolution achieved and to allow more accurate quantitative measurements.accurate quantitative measurements.
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Automation is useful to Automation is useful to overcome the uncertainty in overcome the uncertainty in droplet size and position droplet size and position when the sample is applied to when the sample is applied to the TLC plate by hand. the TLC plate by hand.
Principle of separation is
ADSORPTION
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Advantages of HPTLC over HPLCAdvantages of HPTLC over HPLC
Sample and standard, both can be used Sample and standard, both can be used at a time.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 work Simultaneously 2 or 3 persons can work
but not in case of HPLC.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 Low running and maintenance
costs and disposable layer etccosts and 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 need better analytical precision and accuracy less need for Internal Standard 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 - no stationary and mobile phases for each analysis - no contamination 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 (90:10:1)Chloroform: Methanol: Ammonia (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, If any impurities are present, those are need to be removed those are need to be removed hence pre washing is done.hence pre 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-Usual concentration range is 0.1-1µg / µl 1µg / 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 standard from syringe on TLC plates as bandsplates as bands
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Selection of Mobile phaseSelection of Mobile phaseCan be explained by STAHL’S TRIANGLECan be explained by STAHL’S TRIANGLE
E
M S
Hydrophilic
Lipophilic
Active Inactive
Polar
Non-Polar
E- ELUENTS- SORBANTM- MIXTURE
EG:If E- hydrophilic thenM- 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 After development, remove the plate and mobile phase is removed from the and mobile phase is removed from the plate to avoid contamination of lab plate to avoid contamination of lab atmosphereatmosphere
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 · Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long 254 nm (short wave length) or at 366 nm (long wave length) wave 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 · When individual component does not respond to UV - derivatisation required for detection UV - 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 layer 200-250mm200-250mm 100-150mm100-150mm
Particle sizeParticle size 5-20 micrometer5-20 micrometer 4-8 micrometer4-8 micrometer
Application of Application of samplesample
Manual/semi Manual/semi automaticautomatic
Semi automaticSemi automatic
Shape of sampleShape of sample spotspot bandband
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Spot sizeSpot size 3-6mm3-6mm 1-2mm1-2mm
Sample volumeSample volume 1-10 microlit1-10 microlit 0.1-2microlit0.1-2microlit
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
QuantizationQuantization ManualManual 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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