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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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