Hptlc Nilesh

8/11/2019 Hptlc Nilesh

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NILESH JAWALKAR

M.PHARM 1STYEAR(PHARMACETICS)

HPTLC


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HIGH PERFORMANCE THIN

LAYER CHROMATOGRAPHY


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CHROMATOGRAPHY

Chromatography is a physical process of

separation in which the components to be

separated are distributed between 2 immiscible

phases a stationary phase which has a largesurface area and mobile phase which is in constant

motion through the stationary phase.


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Introduction of HPTLC

HPTLC is the improved method of TLC which utilizes theconventional technique of TLC in more optimized way.

HPTLC takes place in highspeed capillary flow range ofthe mobile phase.

There are three main steps HPTLC procedure, they are

1] Sample to analyzed to chromatogram layer, volume precisionand exact position are achieved by use of suitable instrument.

2] Solvent (mobile phase) migrates the planned distance in layer(stationary phase) by capillary action. In this process sampleseparated into its components.

3] Separation tracks are scanned in densitometer with lightbeams in visible or uv region


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Steps Involving in HPTLC

Sample Preparation

Selection of

chromatography layer

Pre-washing

Pre-conditioningApplication of sample

Chromatography development

Detection of spots

Scanning & documentation

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Sample preparation

Normal phase chromatography: non polar solve

Reversed phase chromatography: polar solvent

Selection of chromatographylayer

Depends on nature of material to beseparatedCommonly used(silica gel, alumina)

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Pre-washing

It is purification step

Mainly methanol is usedEssential for quantitative evaluation

Stability testing

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Pre-conditioning

Also called Chamber SaturationLow polarity mob. Phase:- no need

High polar mob. Phase:- desirable

For reverse phase saturate chamber withpolar solvent

Sample application1-5l

Linomat IV Applicator8


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Linomat lV applicator

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Fig. Hptlc instrumentation


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Selection of HPTLC plates

Previously hand made plates is used in TLC for bothqualitative and quantitative work. Certain drawbacks with

that is nonuniform layer, formation of thick layer, paved foradvent of precoated plates.

Nowadays precoated plates are available in different formatand thickness by various manufactures. Precaoted plates can

be used for both qualitative and quantitative work in

HPTLC, they are

GLASS PLATES POLY ESTER/POLYETHYLYNE

ALUMINIUM PLATES


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Glass Plates:

Offers superior flat and smooth surface.

- fragile

- high weight- higher production cost

Polyester/polyethylene plates:Thickness of plate is 0.2mm.

- It can be produced in roll forms.

- Unbreakable.

- Less packing material is required.

- Development of plate canntbe above temperature

1200c loses its shape.


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Aluminium plates:- Thickness of plate is 0.1mm.

- It can be produced in roll forms.

- Unbreakable.

- Less packaging material is required.


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SORBENTS USED IN HPTLC PLATES:

sorbents which are used in convential TLC are

also used in HPTLC with or without modification.

- silica gel 65F

- highly purified silicagel 60- aluminium oxide

- cellulose microcrystalline

- silica gel

- reversed stationary phase


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Layer thickness

The layer thickness in HPTLC is around 100-

200cm,in conventional it is 250mm.

Layer prewashing:

- Ascending method

- Dipping method

- Continuous method


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ACTIVATION OF PRECOATED PLATES

The plates are activated by placing in an oven

at 1101200 C for 30 min, this step will removeswater that has been physically absorbed on surfaceat solvent layer.

Freshly opened box of HPTLC plates usuallydoes not require activation.

Activation at higher temp and for longer time isavoided which leads to very active layer and there

is risk of sample being decomposed.


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Solvents used in HPTLC

- Methanol (commonly used)- Chloroform:methanol:ammonia(90:10:1)

- Chloroform:methanol(1:1)

- Methylene chloride:methanol(1:1)

- Ammonia(1%)solution


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Application of sample and standard

Usual concentration range is 0.1-1g / l,above

this causes poor separation.

Linomat IV (automatic applicator) - nitrogen gas

sprays sample and standard from syringe on TLC

plates as bands.

Band wise application - better separation - high

response to densitometer.


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Chromatographic development and drying

After development, remove the plate and

mobile phase is removed from the plate - to avoid

contamination of lab atmosphere.

Dry in vacuum desiccator - avoid hair drier

because essential oil components may evaporate.


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Detection and visualization

Detection under UV light is first choice -non destructive.

Spots of fluorescent compounds can be

seen at 254 nm (short wave length) or at 366 nm(long wave length).

Spots of non fluorescent compounds can be

seen - fluorescent stationary phase is used - silicagel GF.


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Non UV absorbing compounds like

ethambutol, dicylomine etc - dipping the plates in

0.1% iodine solution.

When individual component does not

respond to UV - derivatisation required for

detection .


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HPTLC

100m

High due to smaller particle sizegenerated

3 - 5 cm Shorter migration distance and

the analysis time is greatlyreduced

Wide choice of stationaryphases like silica gel for normalphase and C8 , C18 for reversedphase modes

New type that require lessamount of mobile phase

Auto sampler

Use of UV/ Visible/ Fluorescencescanner scans the entirechromatogram qualitatively andquantitatively and the scanner isan advanced type of

densitometer

TLC

250m

Less

10 - 15 cm

Slower

Silica gel , Alumina &Kiesulguhr

More amount

Manual spotting

Not possible


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APPLICATIONS

Pharmaceutical Researches Biomedical Analysis Clinical Analysis Environmental Analysis Food Industry Therapeutic drug monitoring to determine concentration of drug

and its metabolite in blood, urine etc Analysis of environmental pollutions levels Quantitative determination of prostaglandins and thromboxanes

in plasma

Determination of mercury in water Analysis of nitrosoamines in food and body fluids Determination of sorbic acid in wine Characterization of hazards in industrial waste


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REFERENCES1. Principles of instrumental analysis skoog, Holler,

Nieman

2. Instrumental methods of analysis Willard

,Merrit, Dean

3. Pharmaceutical analysis Munson

4. Sharma J.friedB.Handbook of TLC

5. KastureA.V A text book of PharmaceuticalAnalysis (Instrumental methods), 14thedition, page

no.28-306. Elke Hahn Deinstrop Applied TLC, 2ndedition

7. Sitewww.Google.com (Google images)


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THANK YOU


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Hptlc Nilesh

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