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HPTLC TechniqueHPTLC Techniquein in

Pharmaceutical ResearchPharmaceutical Research

Prof. (Dr.) A N Kalia(Former H.O.D, Pharm. Sci., MD Univ. Rohtak.

Director, Herbal Drug Research& H.O.D Pharmacognosy)

ISF College of Pharmacy, Moga

ChromatographyChromatographyChromatography

is a physical method of separation, in which

the components to be separatedare

distributed between two phases:

1. Stationary phase2. Mobile phase

Types of chromatographyTypes of chromatographyBased on the techniques being used, chromatography is broadly Based on the techniques being used, chromatography is broadly

classifiedclassified1. Adsorption Chromatography: 1. Adsorption Chromatography: the chemical mixture in question the chemical mixture in question

need to be passed over an adsorbent bedneed to be passed over an adsorbent beda. Adsorption column chromatographya. Adsorption column chromatographyb. Thin layer chromatography/ HPTLCb. Thin layer chromatography/ HPTLCc. Gasc. Gas--solid chromatographysolid chromatography

2. Partition Chromatography: 2. Partition Chromatography: a mixture is separated by making use a mixture is separated by making use of partition of a solute between two solventsof partition of a solute between two solvents

a. Partition column chromatographya. Partition column chromatographyb. Paper chromatographyb. Paper chromatographyc. Gasc. Gas--liquid chromatographyliquid chromatographyd. HPLCd. HPLC

3. 3. IonIon--exchange Chromatographyexchange Chromatography: : Affinity ChromatographyAffinity Chromatography4. Molecular sieve Chromatography: 4. Molecular sieve Chromatography: Gel Filtration Gel Filtration ChromatographyChromatography

Plates used in TLC & HPTLCa. Hand made platesa. Hand made plates

CELLULOSECELLULOSE (native(native))::

15 g of cellulose dissolved in 100ml of water15 g of cellulose dissolved in 100ml of waterPlate thicknessPlate thickness: : 250µm250µm

CELLULOSECELLULOSE WITHWITH STARCHSTARCH::

20 g acid washed cellulose, 0.4 gm starch in 60 ml of 20 g acid washed cellulose, 0.4 gm starch in 60 ml of waterwater

Plate Plate thickness: thickness: 200µm200µm CELLULOSECELLULOSE MICROCRYSTALLINEMICROCRYSTALLINE::

2525gg ofof cellulosecellulose inin 4040 mlml ofof CHCH33OHOH ++ 2020 mlml ofof waterwaterPlatePlate thicknessthickness:: 100100µmµm

Stationary phase Stationary phase ACETYLATED CELLULOSE + CaSO4 . ½ H2O:30 g of acetylated cellulose + 4.5 g of CaSO4 in 60 ml

of water +10 ml of CH3OH

Plate thickness: 130µm

SILICA GEL G:30 g of silica + 65 ml of waterPlate thickness : 250µm

SILICA GEL WITH STARCH:

30 gram of silica + 90 ml of boiled waterPlate thickness: 250µm

b. Precoated platesb. Precoated plates Types of Precoated Plates:

Glass support Polyster sheets

Aluminium sheets

Glass support

Thickness: 1.3 mm

Advantages: 1. Superior & smooth flat surface

2. Chemical & heat resistantDisadvantages: 1. Heavy weight

2. High production cost

PolyesterPolyester (plastic(plastic sheet)sheet) Thickness: 0. 2 mm

Advantages: 1. Unbreakable2. Less packing material

3. Less space (being in roll form)4. Spots can be cut & eluted

Disadvantages: 1. Charring reaction above 120O C

AluminiumAluminium sheetsheet Thickness: 0. 1 mm

Advantages: Same as polyester,

Heat resistant, Compatible withorganic solvent and acids

Disadvantages: 1. Mineral acids & conc. NH4OH can

attack

Commonly available precoated plates Commonly available precoated plates with their applicationswith their applications

SILICASILICA GELGEL 6060FF (( UnmodifiedUnmodified))::

usedused inin moremore thanthan 8080%% ofof analysisanalysis

ALUMINIUMALUMINIUM OXIDEOXIDE::

usedused forfor basicbasic substancessubstances ee..gg.. alkaloidsalkaloids && steroidssteroids

HIGHHIGH PURITYPURITY SILICASILICA GELGEL 6060::

usedused forfor aflotoxinsaflotoxins

CELLULOSECELLULOSE MICROLIMEMICROLIME::

used for used for amino acidsamino acids, , dipeptidesdipeptides , , sugarssugars, , antibioticsantibiotics & &

labile compounds labile compounds

cannot be chromatographed on active silica gel layer cannot be chromatographed on active silica gel layer

PEPEII IMPREGNATEDIMPREGNATED CELLULOSECELLULOSE::UsedUsed forfor

monomono andand oligonucleotides,cooligonucleotides,co--enzymesenzymes

sugarsugar phosphatesphosphates

POLYMIDE/MICROPOLYMIDEPOLYMIDE/MICROPOLYMIDE::UsedUsed forfor

dansyldansyl--amino acids, antibiotics, antioxidants, antipyretics, amino acids, antibiotics, antioxidants, antipyretics,

optical brighteners, optical brighteners, pesticides, dye stuffs, steroids, hormones, vitamins, pesticides, dye stuffs, steroids, hormones, vitamins,

sulfonamides & sugarssulfonamides & sugars

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Silica gel chemically modified plates:Silica gel chemically modified plates: NHNH22 ((Amino)Amino)

CarboxylicCarboxylic acids,acids, phenolsphenols,, nucleotidesnucleotides,, vitaminsvitamins

(B(B11,, BB22,, BB33),), uricuric acid,acid, xanthenesxanthenes derivativesderivatives CNCN ((cyanocyano))

PharmaceuticalsPharmaceuticals preservativespreservatives

CHIRCHIRResolutionResolution ofof enantiomericenantiomeric substancessubstances forfor opticaloptical

puritypurity suchsuch asas aminoamino acids,acids, dipeptidesdipeptides && lactoneslactones DIOLSDIOLS

HormonesHormones andand steroidssteroids

IMPREGNATEDIMPREGNATED PLATESPLATES

LiquidLiquid pparaffin,araffin, buffers,buffers, silversilver nitrate,nitrate, ionion exchangeexchange materials,materials,

acids,acids, basesbases oror detergentsdetergents

RPRP--22,, RPRP--88 ANDAND RPRP--1818

NonNon--polarpolar substancessubstances (lipids),(lipids), fattyfatty acids,acids, carotenoids,carotenoids,

steroids,steroids, cholesterol,cholesterol, andand itsits estersesters

PolarPolar compoundscompounds vitamins,vitamins, basicbasic andand acidicacidic drugsdrugs whichwhich areare

likelylikely toto bebe decomposedecompose onon activeactive layer,layer, antibiotics,antibiotics, alkaloidsalkaloids

(ephedrine,(ephedrine, atropine,atropine, scopolamine,scopolamine, codeine,codeine, morphine),morphine), barbiturates,barbiturates,

cardiaccardiac drugs,drugs, sweeteners,sweeteners, foodfood dyes,dyes, tranquilizerstranquilizers.. phenothiazinesphenothiazines,,

sulphonamidessulphonamides,, insecticides,insecticides, etcetc..

Preparatory platesPreparatory platesPlatePlate thicknessthickness::11--22 mmmm

ADVANTAGESADVANTAGES LargeLarge samplesample volumevolume appliedapplied asas streakstreak volumevolume

AvailableAvailable asas prepre--coatedcoated softsoft layerslayers EasyEasy toto recoverrecover thethe separatedseparated compoundscompounds

PreferredPreferred asas comparedcompared toto columncolumn chromatographychromatography (easy(easyinin recovery)recovery)DISADVANTAGESDISADVANTAGES::

GiveGive lessless samplesample resolutionresolution thanthan analyticalanalytical platesplates SampleSample volumevolume mustmust bebe moderatemoderate

Hybrid plates (RPHybrid plates (RP--18 WF 18 WF 254s254s))HPTLCHPTLC gradegrade silicasilica gelgel 6060 ((66 nmnm porepore sizesize))ADVANTAGESADVANTAGES ControlledControlled reactionreaction conditioncondition ensureensure thatthat certaincertain silanolsilanol

groupsgroups areare chemicallychemically bondedbonded withwith octadecyloctadecyl chainchain whilewhileremainingremaining silanolsilanol groupsgroups remainremain nonnon bondedbonded thusthus givinggiving

hybridhybrid (hydrophilic(hydrophilic andand hydrophobic)hydrophobic) characterscharacters toto platesplates SuitableSuitable forfor reversedreversed phasephase andand normalnormal phasesphases asas wellwell asasmixturesmixtures ofof aqueousaqueous-- organicorganic solventssolvents

DevelopmentDevelopment timetime isis significantlysignificantly reducedreduced

Prescreen TLC platesPrescreen TLC platesRPTLCRPTLC CC1818 PLATESPLATES

PlatePlate SizeSize:: 55××77..55cmcm

ADVANTAGESADVANTAGES ForFor inexpensiveinexpensive previewpreview ofof separationseparation traittrait priorprior toto

HPLCHPLC analysisanalysis HelpfulHelpful inin optimizationoptimization ofof samplesample preparationpreparation

parametersparameters forfor useuse inin HPLCHPLC

Dual phase TLC plateDual phase TLC plateDurasilDurasil 2525 UVUV 254254 andand NannoNanno--DurasilDurasil 2020 UVUV 254254

Base: Base: AluminiumAluminium or polyester baseor polyester base

Part of plate is coated with reversed phase and Part of plate is coated with reversed phase and

remaining with normal phase coating remaining with normal phase coating

Hard, water proof but still wetHard, water proof but still wet--able with hybrid able with hybrid

characters characters

Plate sizePlate sizePrePre--coatedcoated TLC/HPTLCTLC/HPTLC plateplate size=size=2020××2020cmcmCuttingCutting cancan bebe mademade withwith scissorscissor asas perper requirementrequirement

PRECAUTIONPRECAUTION ScissorsScissors mustmust havehave sharpsharp bladesblades andand inclinedinclined slightlyslightly toto

rightrightforfor cuttingcutting ofof platesplates TheThe loosenedloosened layerlayer shouldshould bebe removedremoved byby lightlylightly drawingdrawingthethe spatulaspatula overover thethe cutcut edgeedge toto obtainedobtained constantconstant RfRf

NoteNote-- It is important to note direction and the application It is important to note direction and the application of sorbentof sorbent

PrePre--washing of prewashing of pre--coated platescoated platesPrewashing is necessary for the removal of water Prewashing is necessary for the removal of water vapor, vapor,

volatile substances, volatile substances, elutableelutable components components & & binders binders

MethodMethod usedused forfor PrePre--washingwashing AscendingAscending (( superiorsuperior butbut longerlonger time,time, 1010--2020%% topcuttingtopcutting))

DippingDipping (( quickerquicker andand uniformuniform ,, 1010ml/chamber,ml/chamber, A,B,C)A,B,C) ContinuousContinuous modemode (Excellent(Excellent results)results)

MethanolMethanol isis commonlycommonly usedused

ADVANTAGESADVANTAGES ReductionReduction inin signalsignal toto noisenoise ratioratio

ImproveImprove thethe limitlimit ofof detectiondetection (LOD)(LOD)

Activation of Activation of precoatedprecoated platesplatesActivationActivation

Oven at 110Oven at 110--1201200 0 C for 30 C for 30 minsmins

AluminiumAluminium plates: plates: kept between two glass kept between two glass platesplates

Higher temperatureHigher temperature: Very active & risk of : Very active & risk of decomposition of sample applieddecomposition of sample applied

No activation is required for freshly opened boxNo activation is required for freshly opened box

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Sample preparationSample preparationSample is prepared for complete Sample is prepared for complete recovery of intact recovery of intact

compound(s) of interest and minimum matrix along with compound(s) of interest and minimum matrix along with

suitable concentration and suitable concentration and analytesanalytes

For For normal phase normal phase chromatographychromatography

SolventSolvent-- NonNon polarpolar andand volatilevolatile toto avoidavoid circularcircular

chromatographychromatography

ForFor ReverseReverse phasephase ChromatographyChromatography

SolventSolvent-- PolarPolar (sorbent(sorbent wetwet--ableable))-- layerlayer uniformityuniformity

StorageStorage-- RefrigeratorRefrigerator

ReferenceReference substancesubstance andand samplesample-- InIn samesame solventsolvent

It is most critical step for good resolution for

quantification HPTLC

1. Ensure for complete fill and empty of capillary

2. Apply through upper end of capillary hence reverse

capillary after filling

Band length- 2-4mm TLC, 0.5-1mm HPTLC

Concentration- 0.1-1µg/µml TLC/HPTLC

Application of sampleApplication of sample Mobile phaseMobile phaseAnalytical grade( AR) solvents are usedAnalytical grade( AR) solvents are used

SelectionSelection-- Trial Trial and error experienceand error experience

SolventSolvent-- v/vv/v

Composition sum total volume is 100 partsComposition sum total volume is 100 parts

Measure Measure the solvent the solvent separately and then placed in mixing separately and then placed in mixing vessel then introduced into developing chambervessel then introduced into developing chamber

Volatile Volatile solvent at room temperature should not be used solvent at room temperature should not be used for long timefor long time

Polar solvent portion of mobile phase may get adsorbed Polar solvent portion of mobile phase may get adsorbed during developmentduring development

Mobile phase should be simpleMobile phase should be simple

Preconditioning (chamber saturation)Preconditioning (chamber saturation)Chamber Chamber should be saturated (by lining with filter paper) prior to should be saturated (by lining with filter paper) prior to

developmentdevelopment

Saturation effects the Rf valueSaturation effects the Rf value

LOWLOW POLARITYPOLARITY SOLVENTSOLVENT ee..gg.. AliphaticAliphatic hydrocarbons,hydrocarbons, toluenetoluene andand

theirtheir mixturesmixtures,, prepre--saturationsaturation isis notnot recommendedrecommended

HIGHHIGH POLARITYPOLARITY SOLVENTSOLVENT ee..gg.. MethanolMethanol prepre equilibriumequilibrium isis

recommendedrecommended

PHASEPHASE SEPRATIONSEPRATION MIXTUREMIXTURE :: ParitialParitial saturationsaturation (( nn--butanol,butanol,

waterwater andand glacialglacial aceticacetic acid)acid)

RPRP--TLCTLC:: SaturationSaturation withwith methanolmethanol isis recommendedrecommended

ForFor humidityhumidity controlcontrol:: SSuitableuitable liquidsliquids isis placedplaced inin oneone ofof thethe

chamberchamber troughtrough

Drying of platesDrying of platesAfter development of plate, mobile After development of plate, mobile phase is removed phase is removed

completely completely & & quickly as possible preferably be quickly as possible preferably be

performed in fume cup performed in fume cup boardboard

HAIRHAIR DRYERSDRYERS NEEDNEED PRECAUTIONSPRECAUTIONS

EssentialEssential oiloil componentscomponents maymay evaporateevaporate

CompoundsCompounds sensitivesensitive toto oxygenoxygen maymay getget destroydestroy duedue toto

riserise inin temperaturetemperature whilewhile usingusing hothot hairhair dryerdryer

Detection and visualizationDetection and visualizationVisualizing reagent help in further confirmation of Visualizing reagent help in further confirmation of

findings findings Non destructive Non destructive

1. UV detection 1. UV detection 2. Iodine2. Iodine-- Universal detection reagent Universal detection reagent 3. Fluorescent Chemicals ( 3. Fluorescent Chemicals ( RhodamineRhodamine B, B,

phosphomolybdicphosphomolybdic acid and Antimony acid and Antimony trichloridetrichloride) ) DestructiveDestructive

1. Charring on heating after spraying with 1. Charring on heating after spraying with corrosive reagentcorrosive reagent

2. Dipping technique2. Dipping technique3. Embedding 103. Embedding 10--20% NH20% NH44SOSO44

4. Heating at 1504. Heating at 150°°C for 25C for 25--30 min30 min

InIn--situ Densitometry:situ Densitometry:Instrumental measurement of visible, UV Instrumental measurement of visible, UV

absorbance, fluorescence or fluorescence quenching directly absorbance, fluorescence or fluorescence quenching directly on the layer without resorting to scrapping or eluting sampleon the layer without resorting to scrapping or eluting sample

Quantitative evaluation is based on visual comparison of Quantitative evaluation is based on visual comparison of size & intensity size & intensity

of fluorescent or UV absorbing compoundof fluorescent or UV absorbing compound

Scanner converts the spot, splash band into chromatogram Scanner converts the spot, splash band into chromatogram consisting of peaks similar in appearance to HPLCconsisting of peaks similar in appearance to HPLC

The position of scanned peak is correlated to Rf value of the The position of scanned peak is correlated to Rf value of the spot/band and height/area is related to the concentration spot/band and height/area is related to the concentration

of the substance on the spotof the substance on the spot

Quantization (evaluation)Quantization (evaluation)

Figure shows separation of six compounds), with the relevant Densitogram, on a SiO2 plate & height/area is related to the

concentration of the substance on the spot

The signal which are measured represent the adsorption The signal which are measured represent the adsorption of transmitted or reflected light that passes through the of transmitted or reflected light that passes through the spot compared to blank portion and the sorbent layerspot compared to blank portion and the sorbent layer

CALIBRATIONCALIBRATION1. Single level calibration1. Single level calibration

Suitable for known target as stability testing, Suitable for known target as stability testing, dissolution profiledissolution profile

2. Multiple level calibration2. Multiple level calibrationThree level of calibration curve serve the purpose of for Three level of calibration curve serve the purpose of for

quantitative analysis & scanning is done at different quantitative analysis & scanning is done at different wavelengthswavelengths

IfIf adsorptionadsorption maximamaxima ofof individualindividual componentcomponent ofof thetheformulationformulation areare quitequite apartapart thenthen thethe chromatogramchromatogram mustmustbebe scanscan atat individualindividual adsorptionadsorption maximamaxima forfor obtainingobtainingmeaningfulmeaningful resultresult

Quantization (evaluation)Quantization (evaluation)

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Advantages of Advantages of HPTLCHPTLC

Advantages of HPTLCAdvantages of HPTLC TLC and especially HPTLC provides

Fingerprinting analysis for the complex components &

Quantitative determination of the marker compounds

No limitation to the composition of mobile phase to maximize the selectivity of the separation

Chamber requires very little time for equilibration

Choice of detection200 to 700nm, with or without derivitization of

plates

Cost and time efficacyMultiple samples can be analyzed on one plate – affects

the cost

Automation sample application takes 0.5 to 2.0 min per sample

Plate development requires 8 minutes

Equilibration time 10-15 min is sufficient

Drying normally do not exceed 10 minutes

Densitometric evaluation of a plate can be accomplished within 10 minutes

Advantages Advantages over TLCover TLC

Layer of Sorbent 100 µm (AmD),200 µm 250 µm

Efficiency High due to smaller particle size generated (5-6µm)

(10-12µm )Low

Separations 3 - 5 cm 10 - 15 cm

Analysis Time Shorter migration distance and the analysis time is greatly reduced Slower

Solid supportWide choice of stationary phases like silica gel for normal phase and C8, C18 for reversed phase

Silica gel , Alumina & Kiesulguhr

Development chamber

New type that require less amount of mobile phase More amount

Sample spotting Auto sampler Manual spotting

Scanning

1. Use of UV/ Visible/ Fluorescence

2. Scanner (densitometer) scans the entire chromatogram qualitatively & quantitatively

Not possible

HPTLCHPTLC TLCTLC

Advantages Advantages over over HPLCHPLC

HPTLCHPTLC HPLCHPLCPrecision & Accuracy

Simultaneous processing of sample & standard- leads better P & A

Simultaneous processing notpossible

Internal Standard

Less need Always needed

Flexibility Extreme flexibility in stationary phase, mobile phase, developing technique, detection

Limited flexibility

Co-chromatography

Co-TLC possible & often practiced

Co-HPLC usually notpracticed

Simple Technically simple to learn & operate

Skilled & well-trainedpersonnel are essential

Cost Less cost per analysis & low cost pre-coated HPTLC plates available

Columns are very expensive

HPTLCHPTLC HPLCHPLCOpen system Entire spectrum can be seen

at a glanceClosed system

Maintenance Negligible wear & tear, hence low cost

High maintenance cost

Sample preparation

Simple Most critical step-Laborious & time-consuming

Choice of solvent Not very critical & removedcompletely before developing chromatogram

Solvent must be compatible with HPLC column

Impuritiesinterference

Rarely require clean-up, turbid samples can be directly applied, solid particles do not interfere

Requires purification, exhaustive clean-up required

Derivatization In situ derivatizationpossible and employed

In situ derivatization not practical

Benefits of HPTLC compared to Benefits of HPTLC compared to HPLCHPLC

Enable concentration during application by up Enable concentration during application by up to a factor of 10,000to a factor of 10,000

Is capable of high throughput (300 or 1000 Is capable of high throughput (300 or 1000 runs per day at one workplace) with minimal runs per day at one workplace) with minimal costcost

Enables multiple detection (UV/VIS, Enables multiple detection (UV/VIS, FluoresenceFluoresence detector, MS, NMR etc).detector, MS, NMR etc).

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Using TLC with Using TLC with electroscopyelectroscopy ionization, the ionization, the mobile phase used for mobile phase used for seperationseperation can be can be chosen independently of MS consideration chosen independently of MS consideration because it is evaporated after chromatographic because it is evaporated after chromatographic separationseparation

a technique for the direct determination of a technique for the direct determination of TLC plates by matrixTLC plates by matrix--assisted laser desorption assisted laser desorption ionization mass spectrometry (MALDI MS). ionization mass spectrometry (MALDI MS).

The methodology have been developed, which The methodology have been developed, which enables the detection of TLC spots on the basis enables the detection of TLC spots on the basis of the relative molecular mass of the of the relative molecular mass of the compounds under analysis,compounds under analysis,

Mechanism of TLC/HPTLC Mechanism of TLC/HPTLC separationseparation

Three main types of separation mechanism Three main types of separation mechanism AdsorptionAdsorptionPartition Partition IonIon--exchangeexchange

Separation that occurs can’t be attributed to just one Separation that occurs can’t be attributed to just one mechanism may be result of two or more different mechanism may be result of two or more different

types of interaction including those above:types of interaction including those above:--ion pairing ion pairing charge transfer charge transfer π π --π interactionsπ interactions

Adsorption Separation Adsorption Separation (Silica Gel)(Silica Gel)

Adsorption is a surface phenomenon, sample Adsorption is a surface phenomenon, sample interaction with interaction with silanolsilanol groupgroup--OH. Dipole induced OH. Dipole induced interaction occur depending on the nature of the interaction occur depending on the nature of the solute. solute.

The weakly adsorbed The weakly adsorbed analytesanalytes move with move with the solvent front ( capillary action) leaving behind the the solvent front ( capillary action) leaving behind the

more strongly adsorbedmore strongly adsorbed

Hydrocarbons <ethers< nitro compounds < tertiary Hydrocarbons <ethers< nitro compounds < tertiary amines< esters< primary aminesamines< esters< primary amines

Two or more functional groups cause Two or more functional groups cause an additive effectsan additive effects

StearicStearic effect between functional effect between functional group e.g. formation of hydrogen bondinggroup e.g. formation of hydrogen bonding

Partition separationPartition separation Polar liquid (stationary phase) is held by a solid Polar liquid (stationary phase) is held by a solid

support (cellulose or a weak adsorbent)support (cellulose or a weak adsorbent)

Separation of sample is effected by virtue of Separation of sample is effected by virtue of difference in solubility's of its components in the difference in solubility's of its components in the developing solvents and stationary phase (water, developing solvents and stationary phase (water, formamideformamide, poly ethylene glycol etc), poly ethylene glycol etc)

Developing solventsDeveloping solvents Butanol:aceticButanol:acetic acid:wateracid:water (40:10:50)(40:10:50) Butanol:acetone:diethylamine:waterButanol:acetone:diethylamine:water (10:10:2:5)(10:10:2:5) 2 2 propanol:formicpropanol:formic acid:wateracid:water (42:10)(42:10)

Hence equilibration occur between the stationary and Hence equilibration occur between the stationary and mobile phase on TLC/HPTLC there are many mobile phase on TLC/HPTLC there are many equibiliriumequibilirium stages that are known as stages that are known as theoretical theoretical plates plates . .

Resolution obviously improve with the Resolution obviously improve with the number of theoretical plates number of theoretical plates

TLC > 600 Th. platesTLC > 600 Th. plates HPTLC typically >5000 Th. PlatesHPTLC typically >5000 Th. Plates

Ion exchange separation Ion exchange separation DependentDependent onon sorbentsorbent containingcontaining ionsions thatthat areare capablecapableofof exchangingexchanging withwith ionsions ofof likelike chargecharge inin thethe samplesampleoror mobilemobile phasephase

The degree of separation is dependent on The degree of separation is dependent on pH value of the developing solvent pH value of the developing solvent Ionic strength of the developing solventIonic strength of the developing solvent Adsorption property of ion exchange material.Adsorption property of ion exchange material.

Uses:Uses:Short chain carboxylic acids sugar phosphates of Short chain carboxylic acids sugar phosphates of

detergentsdetergents

Procedure of Procedure of HPTLCHPTLC

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Sample and standard Sample and standard Sample and standard Sample and standard preparationpreparation

Selection of chromatographic layer

Layer pre-washingLayer pre-washing

Layer preconditioningLayer preconditioning

Application of sample and standard

Chromatographic developmentChromatographic development

Detection of spots

Scanning and documentation of chromatoplate

HPTLC INSTRUMENTATION

Step 1-Method development An optimized TLC method is developed for thechromatograms to be run Method development depends upon solubility andchemical profile of the component (marker) to be detectedin the mixture

Method is developed in a view to standardize the drugmixture with respect to its marker component

Step 2 - Sample applicationBAND APPLICATION, NOT SPOTSACCURATE POSITIONING

ACCURATE VOLUMESSHARP START ZONES

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H P T L C H P T L C development chambersdevelopment chambers

Twin Trough Chambers

Automatic Development Chamber (ADC)

Development (Gradient Chamber) (AMD)

GradientGradient Up to 25 developments In same direction Polar Non-polar Each time front moves further Computer controlled All polarity covered Up to 40 components separated

POST CHROMATOGRAPHYPOST CHROMATOGRAPHYa. VISUALISATIONb. PHOTO DOCUMENTATIONc. DERIVATISATION

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Spectral MatchSpectral Match Validation of MethodValidation of Method“Validation is

establishing documented evidence that provides a high degree of assurance

that aspecific process will consistently produce a product meeting its pre-determined specifications and quality

attributes”

A properly designed system will provide a high degree of assurance that every step, process, and change has been properly evaluated before its implementation

FDA (1987), Guideline on general principles of Process Validation, US Food and Drug Administration, Maryland, USA

METHOD CYCLE :METHOD CYCLE :--

Validation

Development Optimization

Quality topicsQuality topics-- Method Method validation validation

Method is to be validated as per ICH guidelines using following parameters:

1. Linearity2. Specificity3. Precision 4. Limit of detection and quantification5. Robustness6. Accuracy 7. Ruggedness of Method

ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures, 1994

LINEARITYThe linearity of the analytical method

is its ability to elicit test results

that are directly proportional to the concentration of the analyte

in samples within a given range”Acceptance criteria: Linear regression rAcceptance criteria: Linear regression r22 > 0.95 (min 5 > 0.95 (min 5

concentration require )concentration require )Method

A stock solution of standard marker is prepared in solvent.Different volumes 1, 2, 4, 6, 8,10, 12µL, are spotted on the TLCplate to obtain different concentrations of e.g. 100, 200, 400,600, 800, 1000, 1200 ng spot−1 of standard, respectively.The data of peak areas are plotted against the correspondingconcentrations and treated by least-square regression analysis

LINEARITY

By Visual Inspection of plot of signals vs. analyte concentrationBy Appropriate statistical methods

Linear Regression (y = mx + c)Correlation Coefficient, y-intercept (c), slope (m)

PrecisionPrecision is a measure of “scatter or dispersion”

about the mean and among a set of identical measurements

It is usually expressed in terms of Standard deviation.

Precision can be divided into –a. Repeatabilityb. Intra-day and inter-day precisionC.ReproducibilityReproducibility (precision(precision betweenbetween laboratories)laboratories)

MethodRepeatability of the sample application and measurement of peak area can be carried out using six replicates of the same spot (e.g. 600 ng spot−1 of marker) and expressed in terms of percent relative standard deviation (%R.S.D.) and standard error (S.E.). The intra- and inter-day variation for the determination of marker should be carried at three different concentration levels of e.g. 400, 600 and 800 ng spot−1

The acceptance criteria is 10% (As per ICH guidelines)

Inaccurate & precise accurate & imprecise

Precision

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AccuracyThe percent recovery study of analytical procedure is

the ability to estimate the amount of the substance that has transverse the chromatographic plate after

development. It is thus

a measure of the accuracy of the analytical method

MethodThe pre-analyzed samples are spiked with extra 50, 100 and 150% or with 80, 100 and 120% of the standard marker and the mixtures are reanalyzed by the developed method. The experiment can be conducted several times to get maximum accuracy. This is done to check for the recovery of the marker at different levels in the formulations

Accuracy :Accuracy :--

Closeness of the test results obtained by the method to the true Closeness of the test results obtained by the method to the true value.value.

It Should be established across specified range of analytical It Should be established across specified range of analytical procedure.procedure.

It Should be assessed using a minimum of 3 concentration levels, each It Should be assessed using a minimum of 3 concentration levels, each in triplicate (total of 9 determinations)in triplicate (total of 9 determinations)

The acceptance criteria is mean value The acceptance criteria is mean value 10% deviation from true 10% deviation from true value. value.

LOD (Limit of detection)The limit of detection is a parameter of Limit test.

It is the “lowest concentration“

of the analyte in a sample that can be detected,

but not necessarily quantitative (less than 400 AU)

LOQ (Limit of quantification)It is the

lowest concentration of an analyte in a sample that can be determined with

acceptable precision and accuracy under the stated experimental conditions

ROBUSTNESSThe robustness of an analytical procedure

is a measure of its capacity to remain unaffected

by small, but deliberate variations in method parameters &

provides an indication of its reliability during normal usage

MethodBy introducing small changes in the mobile phase composition, mobile phase volume, duration of mobile phase saturation and activation of pre-washed TLC plates with methanol, the effects on the results are examined. Robustness of the method should be done in triplicate at a single concentration level e.g. 600 ng spot−1 and the %R.S.D and S.E. of peak areas are calculated

Ruggedness

It is a degree of reproducibility of test results

obtained by the analysis of same sample under a

variety of normal test conditions, such as

different laboratories, different analyst, at

different time intervals, etc.

Specificity/ SelectivityAn analytical method is specific,

if

the analytical response arises from the analysis from the analyzed of interest only &

does not arises from any other compound present in the sample

MethodThe specificity of the method can be ascertained by analyzing the standard drug and extract. The spot for marker in the sample is confirmed by comparing the Rf values and spectra of the spot with that of the standard. The peak purity of the marker can be assessed by comparing the spectra at three different levels, viz. peak start (S), peak apex (M) and peak end (E) positions of the spot

Methodology used in HPTLC Methodology used in HPTLC Analysis of a Marker in Extract Analysis of a Marker in Extract

and in Formulationand in Formulation

ExperimentalExperimentalMaterials

Standard (Marker), formulation and the drug used in the study were procured from authentic source

All chemicals and reagents used were of Analytical grade and were purchased from Ranbaxy Fine Chemicals, New Delhi, India.

Instrumentation and chromatographic conditions:-HPTLC software: Wincats Sample applicator: Linomat 5TLC chamber: Twin-trough glass

chamberDetection scanner: Camag scanner iiiWavelength: DesiredLamp: DeuteriumSlit dimension: 6 x 0.30 mm

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Chromatographic ConditionsStationary phase : Pre-coated silica gel 60F254 TLC Plate: (20x20 cm,0.2 mm thick)

Mobile phase : Developed by hit and trial

method

Saturation time : 20 min

HPTLC System : CAMAG, Switzerland.

Sample application Speed – 80 nl sec-1. (Depends upon type of solvent used)

Application position : 15 mm (Y axis)

Position of Solvent front: 75 mm

Calibration curve A stock solution of marker (100 µgmL−1) was prepared in methanol. Different volumes of stock solution 4, 6, 8,10, 12, 14µL, were spotted on the TLC plate to obtain concentrations of 400, 600, 800, 1000, 1200, 1400 ng spot−1 of marker, respectively. The data of peak areas plotted against the corresponding concentrations were treated by least-square regression analysis

Method validationLinearity :-

A representative calibration curve of marker is obtained by plotting peak area of marker against the

concentration of marker over a range and the correlation coefficient is determined

Chromatogram of standard vicine (Fig.1)

Calibration curveThe developed HPTLC method for estimation of marker showed a good

correlation coefficient (r2 = 0.9990±0.0002) in concentration range of 400–1400 ng spot−1 with respect to the peak area. Fig.2 displays three-dimensional image of the calibration samples and Fig.3 shows the calibration curve of marker at 278 nm

Linearity of Calibration curve of Marker Linearity of Calibration curve of Marker

Fig.3Fig.3Correlation coefficint rCorrelation coefficint r22 = 0.9990 Sdv = 1.85% Y= 285.8277+11.268*X= 0.9990 Sdv = 1.85% Y= 285.8277+11.268*X

PrecisionRepeatability of the sample application and measurementof peak area were carried out using six replicates of thesame spot (800 ng spot−1 of vicine) and was expressed interms of percent relative standard deviation (%R.S.D.)and standard error (S.E.).The intra-day and inter-day variation for thedetermination of marker was carried out at threedifferent concentration levels of 400, 600 and 800 ngspot−1

Ruggedness

A solution of concentration 1000 ng spot−1 was prepared and analyzed on day 0 and after 3, 6, 24, 48 and 72 h.

Data were treated for %R.S.D. to assess ruggedness of the method

PrecisionPrecision

Repeatability StudiesA spot of 800 ng was spotted in six replicates. The standard deviation (SD) and %RSD (Relative standard deviation) is found to be 1.08% and 0.012 %, respectively with standard error (SE) 0.4409

TrackTrack Amount fractionAmount fraction AreaArea

1.1. 800 ng800 ng 9020.299020.292.2. 800 ng800 ng 8903.698903.69

3.3. 800 800 ngng 8820.008820.004.4. 800 ng800 ng 8970.578970.575.5. 800 ng800 ng 9028.549028.546.6. 800 ng800 ng 9087.429087.42

SD = 1.08 %RSD = 0.012 SE = 0.4409

Intra-day precision Inter-day precision

Amount (ng spot-1)

Mean area

S.D. % R.S.D. S.E. Mean area

S.D. %R.S.D. S.E.

400 5226.39 4.30 0.082 1.75 5244.10 4.39 0.083 1.55

600 7454.28 2.08 0.028 0.85 7606.36 2.75 0.036 0.97

800 8914.04 1.26 0.014 0.51 8959.57 1.18 0.013 0.41

(b) Intra-day and inter-day precision The measurement of the peak area at three different concentration levels showed low values of S.E. and %R.S.D. (<1%) for inter- and intra-day variation, which suggested an excellent precision of the method.

n = 3

S.D. – Standard deviation%RSD – Percentage relative deviation

S.E. – Standard error

2. Ruggedness of the methodLow SD (3.11) and %R.S.D.(0.030) in the peak area with SE 0.89, proved the ruggedness of the method indicating that marker is stable during analysis.

Time Duration Track Area (AU)At 0 hrs. 1. 10159.81

2. 10312.59

After 3 hrs. 1. 10447.60

2. 10111.80

After 6 hrs. 1. 9980.80

2. 9931.60

After 24 hrs. 1. 10270.10

2. 10343.30

After 48 hrs. 1. 10478.70

2. 10329.40

After 72 hrs. 1. 10753.00

2. 11083.70

Mean Area= 10350.20 SD = 3.11 %RSD = 0.030 SE = 0.89

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3. Specificity 3. Specificity The specificity of the method is ascertained by analyzing the standard drug and extract. The spot for marker in the sample is confirmed by comparing the Rf value = 0.31 and spectra of the spot with that of the standard.

Spectra comparison between Standard and samples

4. Detection of related impuritiesThe spots other than the principal spot for marker from the standard solution were not more intense than the principal spot. The solution showed two additional spots at Rf = 0.04 and 0.72 having peak areas of 453.20 and 236.70 (Fig. 5), which were much less as compared to the standard

5. Robustness of the methodRobustness of the method was done in triplicate at a concentration level of 600 ngband−1 and the % R.S.D and S.E. of peak areas were calculated

Robustness of MethodBy introducing small changes in the mobile phase composition, mobile phase volume, duration of mobile phase saturation and different analyst; the effects on the results were examined.

Ta ble3. Robustness of the HPTLC method (n = 3, 600 ng band-1)

Parameter S.D. of Peak Area % RSD S.E.

(1) Mobile phase composition(Ethyl acetate:methanol:water:formic acid)

2.44 0.0297 0.8144

(2) Mobile phase volume (8, 10 and 12 mL) 1.12 0.0134 0.3749

(3) Duration of saturation ( 10, 20 and 30 min) 2.31 0.0282 0.7717

(4) Different analyst 1.39 0.0076 0.5674

6. Limit of detection and limit of quantificationLOD and LOQ were experimentally verified by diluting the known concentrations of vicine until the peak is detected and quantified

7. Recovery studiesThe pre-analyzed samples were spiked with extra 50, 100 and 150% of the standard vicine and the mixtures were reanalyzed by the proposed method. This was done to check for the recovery of the vicine at different levels in the formulation

Limit of Detection and Quantification Detection limit and limit of quantification was found to be 18.75 ng and

56.25 ng respectively, which indicate adequate sensitivity of method

Recovery Studies Results showed high extraction efficiency of vicine from formulation. The

proposed method afforded recovery in the range of 98.01–99.33 % as listed in Table 4. This confirms that the proposed method can be used for determination of vicine in herbal formulation containing M. charantia

Table 4. Recovery Studies (n=6)

Excess drug added to analyte (%)

Theoretical content (ng)

Amount found (ng)

Recovery (%)

%R.S.D.

S.E.

050100150

4006008001000

392.04590.74794.65982.61

98.0198.4599.3398.26

0.04510.02480.01110.0220

1.331.100.531.24

Estimation of Marker in herbal extractTo determine the content of marker in herbal extract, 1g of powdered drug was transferred into a 25mL volumetric flask containing 10mL methanol, sonicated for 30 min. The resulting solution was filtered and analyzed for the drug content. The filterate (40μL) was applied on the TLC plate followed by development and scanning as described. The analysis was repeated in triplicate. The possibility of interference from other components of the extract in the analysis was studied.

Estimation of Marker in formulationAn accurately weighed 1 g of powdered formulation was transferred into 25mL volumetric flask containing 10 mL methanol and sonicated for 30 min. The resultant solution was filtered and 40μL of the filtrate was applied on the TLC plate followed by development and scanning as described.

Estimation of Marker in Estimation of Marker in formulationformulation

A single spot at Rf = 0.31 was observed in the chromatogram of the extract of formulation along with other components.

The total content of marker was found to be 0.140% (w/w)

Substance Substance Amount Amount (average)(average)

CV%CV%

VicineVicine 563.06 ng563.06 ng 0.2530.253

n =2 Volume of sample =40 µl

Estimation of Marker in herbal extract

Chromatogram of herbal extract showed the presence of number of peaks with different Rf values along with a peak at Rf = 0.31 which is same for the marker when run in the same plate on different track. Spectra was found to be overlapped and the amount of marker present in sample extract has been evaluated. The total content of marker was found to be 0.097% (w/w).

SubstanceSubstance Amount Amount (average)(average)

CV%CV%

VicineVicine 390 ng390 ng 0.1900.190

Sample crude drug, Number of samples n = 2 Volume of sample = 40µl

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1:1 mixture of 5, 25-stigmastadien-3-ol glucoside and -sitosterol glucoside

(Pitipanapong et al, 2005)

Isolation and HPTLC finger printing of Charantin

Scheme for the isolation of Scheme for the isolation of CharantinCharantin from from Momordica balsaminaMomordica balsamina

Ground plant materialPetroleum ether

Petroleum ether extract Marc80% methanol

Marc ExtractConc. in vacuum

Suspend in 95% methanolpH 10 (by KOH)

After 48 hrs dilute suspension with waterExtract with diethyl ether

Aqueous phase Diethyl ether extractWashed withwater,5%HCl, with water again

Residue, recrystalized several times in methanol (CHARANTIN)

( Lolitkar et al, 1966)

2D-TLC of isolated charantin

HPTLCHPTLC

( I) HPTLC of isolated charantin was done in ( I) HPTLC of isolated charantin was done in order check its purity and absorbance pattern.order check its purity and absorbance pattern.

(II) HPTLC of methanolic extract and standard (II) HPTLC of methanolic extract and standard vicine was done to do the qualitative and vicine was done to do the qualitative and quantitative estimation of vicine in fruit pulp quantitative estimation of vicine in fruit pulp of of Momordica balsaminaMomordica balsamina. .

Sample and Standard Preparation Sample and Standard Preparation

The isolated charantin was dissolved in methanol and a clear The isolated charantin was dissolved in methanol and a clear solution was prepared .solution was prepared .

For qualitative and quantitative estimation of vicine , 1g of For qualitative and quantitative estimation of vicine , 1g of powdered fruit pulp was ultrasonicated with 10ml of methanol powdered fruit pulp was ultrasonicated with 10ml of methanol at room temperature for 30 min. The extract was filtered using at room temperature for 30 min. The extract was filtered using fine filter paper and two sample bands each of 40µl were fine filter paper and two sample bands each of 40µl were appliedapplied..

Stock solution of 100µg/ml of standard vicine was also Stock solution of 100µg/ml of standard vicine was also prepared in pure methanol by sonication for 30 minutes at prepared in pure methanol by sonication for 30 minutes at room temperature. For plotting the calibration curve of room temperature. For plotting the calibration curve of standard vicine different volumes of stock solution 2, 4, 6, 8, standard vicine different volumes of stock solution 2, 4, 6, 8, 10µl were applied on TLC plate simultaneously in form of 10µl were applied on TLC plate simultaneously in form of bands to obtain concentrations of 200, 400, 600, 800 and bands to obtain concentrations of 200, 400, 600, 800 and 1000ng per band respectively.1000ng per band respectively.

Chromatographic ConditionsChromatographic Conditions

Stationary phaseStationary phase : Pre: Pre--coated silica gel 60Fcoated silica gel 60F254254 TLC plate TLC plate

Mobile phase : Mobile phase :

1) Charantin1) Charantin-- Benzene:Methanol(80:20)Benzene:Methanol(80:20)

2)Vicine2)Vicine-- Ethyl Acetate: Methanol: Water: Formic Acid Ethyl Acetate: Methanol: Water: Formic Acid (7:5 : 3 : 1 : 0.1 v/v/v/v)(7:5 : 3 : 1 : 0.1 v/v/v/v)

Saturation time :Saturation time : 20 min20 min

HPTLC System : CAMAG, Switzerland.HPTLC System : CAMAG, Switzerland.

Sample application Speed Sample application Speed –– 80 nl sec80 nl sec--11

Application position : 15 mm Application position : 15 mm

Position of Solvent front :75 mmPosition of Solvent front :75 mm

Fig-3 Qualitative and quantitative Qualitative and quantitative estimation of vicine in fruit pulpestimation of vicine in fruit pulp

The presence of vicine in the methanolic extract was confirmed The presence of vicine in the methanolic extract was confirmed by Rf value and spectral comparison of sample with standard by Rf value and spectral comparison of sample with standard vicinevicine. Detection was done at 278nm. For quantitative estimation . Detection was done at 278nm. For quantitative estimation the calibration curve of standard the calibration curve of standard vicinevicine was plotted by applying was plotted by applying different volumes of stock solution (100µg/ml) to obtain different volumes of stock solution (100µg/ml) to obtain concentrations of 200, 400, 600, 800 and 1000ng per spot concentrations of 200, 400, 600, 800 and 1000ng per spot respectively. The sample (40µl) was applied along with standard respectively. The sample (40µl) was applied along with standard vicinevicine to determine the quantification of vicine in sample. The to determine the quantification of vicine in sample. The calibration curve of standard vicine was found to be linear calibration curve of standard vicine was found to be linear showing standard deviation 2.67%showing standard deviation 2.67%

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Spectral comparison of sample with the Spectral comparison of sample with the standard vicinestandard vicine

FigFig-- 5 Calibration curve of standard vicine5 Calibration curve of standard vicine ResultsResults One of the peak observed at Rf value 0.34 in One of the peak observed at Rf value 0.34 in

chromatogram in test sample matched with the chromatogram in test sample matched with the standard vicine and the peak purity was standard vicine and the peak purity was confirmed by spectral comparison at λmax confirmed by spectral comparison at λmax 278nm. This confirms the presence of vicine in 278nm. This confirms the presence of vicine in fruit pulp.fruit pulp.

The amount of vicine in fruit pulp of The amount of vicine in fruit pulp of Momordica Momordica balsaminabalsamina is found to be 0.0975% w/w.is found to be 0.0975% w/w.

Calibration curve of Calibration curve of gallicgallic acidacidDifferentDifferent volumesvolumes ofof stockstock solutionsolution ((500500 µg/µg/mLmL)) werewere appliedapplied onon thethe TLCTLCplateplate toto obtainobtain concentrationsconcentrations ofof 00..55,, 11,, 22,, 33 andand 66 µg/spotµg/spot ofof gallicgallic acid,acid,respectivelyrespectively.. TheThe datadata ofof peakpeak areasareas plottedplotted againstagainst concentrationsconcentrations andand wereweretreatedtreated byby leastleast--squaresquare regressionregression analysisanalysis.. RegressionRegression modemode waswas foundfound linearlinearrr2 2 =0.99853 with this equation, Y=2000.060 + 2.754*X & S.D was 4.24%=0.99853 with this equation, Y=2000.060 + 2.754*X & S.D was 4.24%

Calibration curve for standard Calibration curve for standard gallicgallic acidacid

Calibration curve of gallic acid

HPTLC chromatogram of peakof standard gallic acid

3D pattern for peaks of standard reference gallic acid

Quantity of gallic acid in samplesThe quantity of gallic acid found in Terminalia bellirica fruits, bahera

powder and gasex tablets herbal formulations were 1.107%, 0.740 % and

0.351% respectively.

Validation of HPTLC MethodValidation of HPTLC Method1. Linearity1. LinearityTheThe correlationcorrelation coefficientcoefficient forfor thethe usedused methodmethod waswas foundfound toto bebe 00..9985399853 andandthusthus exhibitsexhibits goodgood linearitylinearity betweenbetween concentrationconcentration andand areaarea..22.. PrecisionPrecision && repeatabilityrepeatabilityRepeatabilityRepeatability ofof thethe samplesample applicationapplication andand measurementmeasurement ofof peakpeak areaarea werewerecarriedcarried outout usingusing sixsix replicatesreplicates ofof thethe samesame bandband ((44µgµg ofof gallicgallic acid)acid) andand waswasexpressedexpressed inin termsterms ofof percentpercent relativerelative standardstandard deviationdeviation ((00..0202)) andand standardstandarderrorerror ((00..7777))

Sample (Sample (µg/mlµg/ml)) TracksTracks Area (AUC)Area (AUC)

44 TrackTrack--II 7518.537518.53

44 TrackTrack--IIII 7603.727603.72

44 TrackTrack--IIIIII 7496.127496.12

44 TrackTrack--IVIV 7464.827464.82

44 TrackTrack--VV 7328.937328.93

44 TrackTrack--VIVI 7148.317148.31

Table: Repeatability of gallic acid

Intra Day precisionIntra Day precision Inter Day precisionInter Day precision

Amount of Amount of

reference reference

standard standard (µg )(µg )

Mean areaMean area S.D.S.D. % R.S.D% R.S.D S.ES.E Mean areaMean area S.DS.D % R.S.D% R.S.D S.ES.E

22 3690.773690.77 3.383.38 0.090.09 1.381.38 3762.643762.64 3.113.11 0.080.08 1.091.09

33 6321.516321.51 3.913.91 0.060.06 0.020.02 6503.206503.20 3.853.85 0.060.06 1.361.36

44 7742.87742.8 2.062.06 0.020.02 0.840.84 7708.67708.6 2.212.21 0.020.02 0.780.78

Intra-day & inter-day precisionThe intra-day and inter-day precision for the determination of gallic acid was carried at three different concentration levels of 2, 3 and 4 µg/band.

Table: intra-day and inter-day precision for gallic acid

33.. RecoveryRecovery studiesstudiesTheThe prepre--analyzedanalyzed samplessamples werewere spikedspiked withwith extraextra 5050,, 100100 andand 150150%% ofof thethestandardstandard gallicgallic acidacid andand thethe mixturesmixtures werewere reanalyzedreanalyzed.. TheThe experimentexperiment waswas

conductedconducted sixsix timestimes.. ThisThis waswas donedone toto checkcheck forfor thethe recoveryrecovery ofof thethe gallicgallic acidacidatat differentdifferent levelslevels inin thethe marketmarket formulationsformulations..

Amt. of Amt. of G.AG.A

taken ‘A’ taken ‘A’ (µg) (µg)

Amt. of Amt. of G.AG.A

spiked ‘B’ spiked ‘B’ (µg)(µg)

Amt of Amt of A +B A +B (µg)(µg)

Theoretical Theoretical areaarea

(AUC)(AUC)‘C’ ‘C’

Practical Practical areaarea

(Mean (Mean Area)Area)

(µg) ‘D’(µg) ‘D’

Recovery Recovery D/CD/C××100100

(%)(%)

44 22 66 7911.017911.01 8277.708277.70 104.63104.63

44 44 88 9553.209553.20 10043.5210043.52 105.13105.13

44 66 1010 11310.23 11310.23 11088.20 11088.20 98.0398.03

Table: Recovery studies of gallic acid at different levels

The percentage recovery of gallic acid was found to be102.48% and Standard deviation (Avg.) = 1.3648Standard Error = 0.5571

4. Specificity4. SpecificityTheThe spectrumspectrum ofof samplesample gallicgallic acidacid waswas foundfound toto bebe similarsimilar andand overlapoverlapwithwith thatthat ofof thethe referencereference standardstandard spectrumspectrum andand goodgood correlationcorrelation((00..99829982)) overlainoverlain ofof spectraspectra waswas obtainedobtained betweenbetween samplesample andand standardstandard..

Fig: HPTLC fingerprint of T.bellirica fruit crude powder (1,2);Gasex tablets (3); Baherapowder formulation (4,5); Gallicacid (6,7)

Fig: Spectral comparison of standard and samples bands

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Terminalia bellirica Bahera powder

Gasex tablets

The Rf value of gallic acid in thesesamples were found be (0.33) whichare similar and specific.

5. Ruggedness5. RuggednessA solution of concentration 4 µg/band was prepared and analyzed at A solution of concentration 4 µg/band was prepared and analyzed at

Different levels 0,3,6,24,48 hrs. Data were treated for RSD to assess Different levels 0,3,6,24,48 hrs. Data were treated for RSD to assess ruggedness of the method. ruggedness of the method.

Time (Hrs.)Time (Hrs.) Peak Area (AUC)Peak Area (AUC)

00 7746.27746.2

33 7880.857880.85

66 7322.97322.9

2424 7656.27656.2

4848 7794.37794.3

Relative standard deviation (RSD %) = 0.03% and Standard error = 1.25

Table: Ruggedness of the HPTLC method

6. Limit of detection and limit of quantification (LOD & 6. Limit of detection and limit of quantification (LOD & LOQ)LOQ)

LimitLimit ofof detectiondetection waswas calculatedcalculated byby makingmaking thethe dilutionsdilutions ofof stockstocksolutionsolution andand determiningdetermining thethe minimumminimum amountamount whichwhich couldcould bebe detecteddetecteddensitometricallydensitometrically..

MinimumMinimum detectabledetectable limitlimit (LOD)(LOD) == 6060 ngngLimitLimit ofof quantificationquantification (LOQ)(LOQ) == 180180 ngng

77.. RobustnessRobustnessRobustnessRobustness ofof thethe methodmethod waswas donedone inin triplicatetriplicate atat aa concentrationconcentration levellevelofof 22 µg/µg/ bandband andand thethe %%RR..SS..DD andand SS..EE.. ofof peakpeak areasareas werewere calculatedcalculated..

ParametersParametersS.D. of S.D. of

Peak AreaPeak Area% RSD% RSD S.E.S.E.

Mobile Phase compositionMobile Phase composition 2.182.18 0.010.01 0.720.72

Mobile phase volumeMobile phase volume 2.672.67 0.010.01 1.081.08

Duration of saturationDuration of saturation 2.682.68 0.010.01 1.091.09