MAHARSHI ARVIND INSTUTE OF PHARMACY MANSAROVAR, JAIPUR.

GUIDED BY . Prepared by Mr. Kapil sharma. Jasmin .H. modi. M pharma (Pharmaceutics)

I semester

Contents.Introduction.Introduction.Introduction to H.P.T.L.C. Principle.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:Introduction: chromatography is a physical process of

seperation in which the component is to be separated are disturbeted between two immiscible a stationary phase with has large surface area and a mobile phase which is in constant motion through the stationary phase.

Introduction to H.P.T.L.CH.P.T.L.C is the improved method of T.L.C which

utilize the conventional technique of TLC in more optimize way .

It is also known as a planer chromatography or Flat- bad chromatography.

Principle Principle HPTLC take place in high speed capillary flow rang

of the mobile phase,There are three main step in HPTLC

1] sample to analyzed to chromatogram layer volume precision and suitable position are achieved by use of suitable instrument.

2]solvent (mobile phase) migrates the planned distance in layer(stationary) by capillary action in this process sample separated in its components.

3] separation tracks are scanned in densitometer with light beam in visible or uv region

Selection of HPTLC plates.Previously hand made plate is used in TLC for both qualitative and quantitative work, certain draw back with that is non uniformly layer , formation of thick layer paved for advant pre coated plates.Now a days pre coated plates are available in different formet and thickness by different manufactures. these plates are used for both qualitative and quantitative purpose in HPTLC.

glass plates .Polyester /polyethylene.Aluminium plates.

GLASS PLATES Resistance to heat Easy to handle Thickness 1.3mm Offer superior plane and smooth surface.

Fragile High weight High production cost

POLY ESTER 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. 1200losses of its shape.

Aluminium plates.Thickness of plate 0.1mm It can be produce in Roll form. Unbreakable. Less packing material required. Development of plate is not above temp. 1200losses of its

shape.

Sorbents used in HPTLC Plates.Sorbent used in conventional TLC can be used in HPTLC with or with out modification.Silica gel 65F(modified)Highly purified silica gel60.Aluminium oxide.Microcrystalline.Silica gel G particle size of sorbent Reversed stationary phase. HPTLC 6 m Hybrid plates. TLC 10 m

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

Layer prewashig . Ascending method . continuous method. Deeping method. solvent used for washing methanol chloroform: methanol:amonia(90:10:1) Chloroform: methanol(1:1) Ammonia solution (1%)

Activation of pre coated plates. The plates are activated by placing 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 PREPARATION: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 during extraction and analysis must consider. there for choice of suitable solvent for given analysis is very important .Solvent for dissolving the sample should be non polar and non volatile as far as possible since polar solvent are likely to induce circular chromatogram at the origin.

Application of sample and standard solution.Sample application is one imp and critical step for obtaining the good resolution for quantification by HPTLC. sample/std are applied as sport or 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. compare sample/ std applications

FIG:Automatic HPTLC sampler

CAMANG LINOMAT Camang inomat with a spray tech is Automated sample application device.The sample is loaded in micro syring(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 APPLICATOR

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

CHROMATOGRAM DEVELOPMENT.After application of sample in HPTLC plate,

chromatogram is developed by dipping 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 :DEVELOPMENT CHAMBER

LINEAR AND RADIAL DEVELOPMENT. In close bad tech like HPLC linear

development is possible but an open bad technique does not suffer this limitation.

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

FIG: HPTLC of Ginseng.

Detection or visulation of spot/ band.There is no diffucult in detecting the coloured

substance.or colour les substance absorbing the uv radiationor with fluoresce(Riboflavin)

Detection of spots/band are done by 1) destruction/Non reverse.2) Nondestructive/reversible.3) Misc. method.

FIG:HPTLC VISULIZER

Evaluation of spot or band. After detection of spot /band upon objective of

experiment chromatogram is used for several purpose.

Quality Evaluation . Quantitative Evaluation .

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 prostaglandin s and

thromboxanes in plasma.Determination of mercury in water.Characterization of hazard in industrial waste.

PARAMETER TLC HPTLC

TYPES OF CHROMATOGRAPHIC PLATES

HANDMADE/PRECOATED

PRECOATED

ABSORBENT LAYER 200-250 m 100-150m

PARTICLE SIZE RANGE 5-20 m 4-8 m

APPLICATION OF SAMPLE MANUAL/SEMIAUTOMATIC

MANUAL/SEMIAUTOMATIC

SHAPE OF SAMPLE SPOT SPOT/BAND

SPOT SIZE 3-6mm 1-2mm

SAMPLE VOLUME 1-10 0.1-2

DIFFERENCE BETWEEN TLC AND HPTLC.

PARAMETER TLC HPTLC

NO OF SAMPLE OER PLATE 15-20 40-45

OPTIMAL DEVELOPMENT DISTANCE

10-15cm. 5-7cm

DEVELOPMENT TIME DEPEND UPON MOBILE PHASE

40 % LESS THAN TLC

QUANTITATION MANUAL MANUAL /INSTRUMENTION.

REPRODUBILITY OF RESULT DIFFUCULT REPRODUCIBLE.

References. Principle of Instrumental analytical ,skoog, Holler,

Nieman. Instrumental method of analysis, willard,

merriet ,dean. Pharmaceutical analysis,munson. Instrumental method of chemical analysis

gurudeep chatwal, shyam k anand