HPTLC PPT

SEMINAR ON HPTLC MAHARSHI ARVIND INSTUTE OF PHARMACY MANSAROVAR, JAIPUR.

GUIDED BY .

Mr. Kap Kapil il sh sharm arma. a. modi.

Prepared by

Jasmi Jas min n .H. M pharma

(Pharmaceutics) I semester

Contents. 

Introduction.



Introduction to H.P.T.L.C.



Principle.



Selection of HPTLC plates.



Activation of pre coated plates.



SAMPLE PREPARATION:



Evaluation of spot or band.



Application of HPTLC.



DIFFRENCE between TLC andHPTLC.

Introduction: chromatography  is a phys physical pr ocess of  se sep pera rattion in which the com omp ponent is to be se separa parated ted ar e distu sturb rbeted eted bet etw ween two immiscibl ble e a stationary phase with has has larg large e sur  su r f fa   ce ar ea and and a mobile phase which is in const onsta ant motion thr ough ough the st sta ation onary ary phase. phase. Introduction Introd uction to H.P H.P.T .T.L.C  .L.C  oved met meth hod of  T.L.C which utilize the is the impr oved onal al technique of  of T T C in mo mor  r e optimize way . convention H.P.T.L.C

It is al also so kno now wn as a plane planer  r chr oma omato tography graphy or FlatFlat- ba bad d omato tography graphy.. chr oma

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Principle

HPT

C tak ake e place in high speed capillary f low ran rang of  the

mob mo bile pha phase se,Th The er e ar e thr ee ee main ste step p in

HPT

C

1] sampl ple e to analyzed to chr oma omato togra gram m lay laye er  r vo vollume pr ecision and suitabl able e position ar e achieved by use of  suitabl able e inst nstr  r ument. ument. 2]solvent (mo 2]sol mob bile pha phase) se) migra grates tes the pla planned nned dist sta ance in laye lay er( r(st sta ation onary ary)) by capillary action in this pr ocess sampl ple e separa se parated ted in its com omp ponents. 3] se separa parattion tracks ar e scanned in densitomete tometerr with ligh ghtt beam in visible or uv r egion

Selection

of HPTLC plates.

Previously hand hand made plate is used in TLC for both qualitative and quantitative work, certain draw back with that is non uniformly layer , formatio form ation n of thick thick layer layer paved paved for advan advant t pre coated plates. Now a days pre coated plates are available in differen dif ferent t formet formet and thickn thickness ess by differen different t manufactures. manufa ctures. these plates are used for both qualitative and quantitative purpose in HPTLC. 

glass

plates .



Polyester /polyethylene.



Aluminium plates.

GLA LASS SS PLATE PLATES S   

Resistance to heat Easy to handle

Thickness 1.3mm

Offer superior plane and smooth surface.

Fragile High weight High production cost

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POLY

T

POLYETHYLENE.

Thickness of plate 0.2mm It can be produce in Roll form. Unbreakable. Less packing material required.

Development of plate is not above temp. 120 0losses of its shape.

 Aluminium pla plates. tes. Thickness of plate 0.1mm ´

It can be produce in Roll form. Unbreakable. Less packing material required. Development of plate is not above temp. 120 0losses of its shape.

Sorb rbents ents used in HPTLC Pla lates. tes. Sorbent used in conventional

TLC can be used in HPTLC with or with out modification. 

Silica

gel



Highly purified silica



Aluminium oxide.



Microcrystalline.



Silica

gel

65F(modified) gel60.

G

particle size

of sorbent 

Reversed stationary phase.



Hybrid plates.

HPTLC TLC

Layer thickness in HPTLC 100200 m in TLC 250 m

6 m 10 m



Lay aye er pr ewa was shig . Ascendin g method .



continuous method.



Deeping method.

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solvent used for washin g methanol chloroform: methanol:amonia(90:10:1) Chloroform: methanol(1:1) Ammonia solution (1%)

 Activation

of  pr e coated pla plates tes..

The plates are activated by placin g in oven at 110 1200c for 30 minutes,this step will remove the water that has been physical absorbed on the surface at solvent layer. Freshly open box of HPTLC plates usually not requird activation. Activation at higher temperature and long time is avoided which may tends to vary active layer and sample decomposition .

SAMPLE PREP A SAMPLE  AR R ATION: Proper sample preparation is pre requsite for the success HPTLC separation.

Beside maximizing the yield of analyte in the selected solvent ,stability of the analyte durin g extraction and analysis must consider. there for choice of suitable solvent for given analysis is very important . Solvent for dissolvin g the sample should be non polar and non volatile as far as possible since polar solvent are likely to induce circular chromato gram at the origin.

 Application of  of s sampl ple e and st sta and ndar  ar d so sollution. Sample application is one imp and critical step for obtaining the good resolution for quantification by HPTL HP TLC. C. samp sample/ le/std std ar are e appli applied ed as as spor sport t or band band depending upon the analysis spot application is done by using 1)

Capillary tubes.

2)

Micro bulb pipetts.

3)

Micro syring.

4)

Automatic sample applicator. compar com pare e sample/ sample/ std std app applica licatio tions ns

C AMA  AMANG NG LINO NOMA MAT Camang inomat with a spray tech is Automated sample application device. The sample is loaded in micro syrin g (Hamilton syring )of 1.0 capacity. The sample is applied as a spot or band By programming instrument parameter Like spotting volume ,band length, No of spot/band , space between spot/ band.

Fig

CAMANG LINOMAT APPLICATO APPLICATOR R

The nozzle is placed at tip of syring ,air is coming out at high pressure atomize the sample solution in to fine spray

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CHRO HROMA MATOGR AM DEVELOP EVELOPM MENT.

After application of sample in HPTLC plate, chromatogram is developed by dippin g in suitable solvent system Taken in developing chamber. The solvent system rises over the layer by capillary action and separation of sample in different components take place. selection of solvent system Chamber saturation Type of development and developing device. Fig :DEVELOP EVELOPM MENT

 AMBER ER CH AMB

DEVELOP EVELOPM MENT. ´ In close bad tech like HPLC linear development is possible but an open bad technique does not suffer this limitation. ´

LINE NE A  AR R  A  AN ND R ADI A  AL L

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

FI : HPTL

f

in

n .

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Detection or  r v visu sula lattion of s of  spot/ ba band. nd.

There is no diffucul There diffucult t in detec detectin ting the coloured substance. subst ance.or or colour les les substance substance absor absorbin bing the uv radiati rad iationor onor with fluoresce fluoresce(Rib (Ribofla oflavin) vin) Detection of spots/band are done by 1)

destruction/Non reverse.

2)

Nondestructive/reversible.

3)

Misc. method.

FIG:HPTLC VISULIZER

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Eval alu uation

of s of  spot or band. band.

After detection of spot /band upon objective of experiment chromatogram is used for several purpose. Quality Evaluation . Quantitative Evaluation .

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 Application

of  HPTLC.

Pharmaceutical research. Biomedical Analysis. Clinical Analysis. Environment Analysis. Food industry. Therapeutic drug monitoring to determine its concentration and metabolites in blood urine etc. Analysis of environment pollution level. Quantitative determination of prosta glandin s and thromb thr omboxa oxanes nes in plasm plasma. a. Determination of mercury in water. Characterization of hazard in industrial waste.

DIFFERENCE HPTLC.

BETWEEN

TLC  AN  AND

PARAMETER

T LC

HPTLC

YPES OF TYPES HROMATOGR APH  APHIC CHROMA PL ATES

H AN  ANDMADE/P

PRECO ATED

RECO ATED

 ABSOR  ABSORB BENT L AYER  AYER

200-250 m

100-150m

P AR  ARTIC  ANGE TICLE SIZE R ANGE

5-20 m

4 -8 m

 APPL  APPLIC ATION OF

MANU MA NU A  AL L/SEMI

MANU MA NU A  AL L/SEMI AU  AUTO

SAMPLE SAMPLE

 AU  AUTOMATIC TIC

MATIC TIC

SH APE  APE OF SAMPLE SAMPLE

SPOT

SPOT/BAN BAND

SPOT SIZE

3-6mm

1-2mm

SAMPLE SAMPLE VOLUM VOLUME

1-10

0.1-2

PARAMETER

TL C

HPTLC

NO OF SAMPLE SAMPLE OER PL ATE

15-20

40-45

OPTIMA MAL L

10-15cm.

5-7cm

EVELOPMENT TIME DEVELOPM

DEPEND UPON MOBILE PH ASE  ASE

LESS TH AN  AN TLC 40 % LESS

 ANTIT ATION QU AN

MANU MA NU AL  AL

MANU MA NU AL  AL

EVELOPMENT DEVELOPM

 ANCE DIST AN

RUMENTION. /INSTRUM REPRODUBILITY OF RES RESULT

DIFFUCULT

REPRODUCIBLE.

´ ´

Refe efer  r en ences.

Principle of  Inst nstr  r umental umental an analy alyttical ,s ,sk koo oog g, Holllle er , ema an. Niem

´

Inst nstr  r umental umental met meth hod of  analy alys sis, willar d, d, me merr  rr iet ,de ,dea an.

´

Phar maceutical

´

analy an alys sis,munson.

Inst nstr  r umental umental met meth hod of  chemical an analy alys sis gur udeep udeep hattwal wal,, shya hyam m k an anand cha