HPTLC by MAYUR009..

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PRINCIPLE & INSTRUMENTATION OF HPTLC:

PPT BY:-MAYURDHVAJSINH S.CHUDASAMA M.PHARM [Q.A] SEM:-1 ST SAMANVAY PHARMACY COLLEGE[S.I.P.E.R.] PRINCIPLE & INSTRUMENTATION OF HPTLC

CONTENTS :

CONTENTS Introduction Principle How TLC performed????? Characterization of separation Instrumentation Factors influencing HPTLC Application of HPTLC Reference

INTRODUCTION:

INTRODUCTION Chrometography ---> method used for separation of multi-component mixture. SEPARATE Analyze Identify Purify Quantify

INTRODUCTION:

INTRODUCTION H igh p erformance t hin l ayer c hromatography. Modified form of Planar Chromatography or flat bed Chromatography. Chromatography as here the Stationary phase is spread over a support material as a thin layer or flat bed.

PRINCIPLE:

PRINCIPLE Same as TLC Due to ADSORPTION or PARTITION or BOTH phenomenon depending upon nature of adsorbents used on plates & solvents system used on development.

SEPARATION PRINCIPLE:

SEPARATION PRINCIPLE During Adsorption the components dissolved in the mobile phase are adsorbed to the surface of silica / alumina . Partition results from the differences in the solubility of the substances in the mobile phases . Adsorption is suitable for separation of compounds differing in polarity while partitioning is for separation of substances differing in their solubility.

HOW TLC IS PERFORMED ??:

HOW TLC IS PERFORMED ?? Apply the sample at one end of the stationary phase Place the plate in the mobile phase in a closed chamber The components of the sample migrate at different rates as the mobile phase rises through the stationary phase Remove the plate from the mobile phase after it raises distance and quickly dry the plate Visualize the plate for compounds with naked eyes, under UV light and or by derivatization of the plate with suitable reagent.

CRITERIA FOR IDENTIFICATION OF ANALYTE BY TLC/HPTLC:

CRITERIA FOR IDENTIFICATION OF ANALYTE BY TLC/HPTLC Rf value of analyte should be within ±3 % compared to standard. Visual appearance of analyte and standard should be the same . For identification, additional co-chromatography in the TLC step is mandatory result only spot due to analyte should be intensified a new spot should not appear . Max of the analyte should be same as standard. The spectrum of analyte should not be visually different from that of standard .

CHARECTERIZATION OF SEPARATION:

CHARECTERIZATION OF SEPARATION Rf – Retardation Factor :In TLC compound identification is based on the Rf values compared to authentic standards. Rf value varies from 0 to1 , best between 0.1 to 0.8 Many factors influence the migration of a substance, which are very difficult to control at the same time. Hence Rf value is to be regarded as an approximate value. Distance moved by the solute Rf = -------------------------------------- Distance moved by the mobile phase front

WHY HPTLC ?????????????:

WHY HPTLC ????????????? Various Chromatographic techniques like TLC, GLC, HPLC etc are employed for qualitative & quantitative analysis of a drug. In case of GLC the sample to be analyzed or its derivative must be volatile and stable within possible temperature range. In case of HPLC requires solubility in appropriate solvent & sample must be free from all insoluble substances and dissolved gases i.e. degassing is required.

WHY HPTLC ?????????????:

WHY HPTLC ????????????? While TLC have least limitations of such type . Now a days TLC is employed in two ways: As a qualitative tool for separation of simple mixture where speed, low cost and simplicity are required. As a powerful tool for quantitative analysis with high sample throughput . The later one is now refered as HPTLC. In general both have similar approach but HPTLC uses technique in more optimized way.

DIFF. BETWEEN HPTLC AND TLC:

DIFF. BETWEEN HPTLC AND TLC PROPERTIES HPTLC TLC Layer of sorbent 100 µm 250 µm Particle size 4 to 8 µm [more uniform] 5 to 20 µm [less uniform] Separation 3 to s cm 10 to 15 cm Efficiency High [bcoz small partical size] Less Analyze time Less time for elution More time for elution Solid support Normal phase->silica gel, Reverse phase->c8,c18 Silica gel,Alumina & Kiesulguhr. Development chamber Less ammount of mobile phase More ammount of mobile phase

DIFF. BETWEEN HPTLC AND TLC:

DIFF. BETWEEN HPTLC AND TLC PROPERTIES HPTLC TLC Sample spotting Auto sampler Manual spotting Scanning Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram. Not possible. No. of HETP 4000 2000

INSTRUMENTATION:

INSTRUMENTATION

STEPS INVOLVING HPTLC:

STEPS INVOLVING HPTLC SAMPLE & STANDARD PREPARATION SELECTION OF CHROMATOGRAPHIC PLATES LAYER PRE-WASHING LAYER PRE-CONDITIONING APPLICATION OF SAMPLE CHROMATOGRAPHIC DEVELPOMENT DETECTION OF SPOT SCANNING & DOCUMANTATION

SAMPLE PREPARATION:

SAMPLE PREPARATION NORMAL PHASE--->NON POLAR SOLVANT REVERSE PHASE -->POLAR SOLVANT

SELECTION OF PLATES:

SELECTION OF PLATES It is important if the sample have tendency to react with the sorbent . Previously various hand made plates were used which are prepared by pouring & spreading the slurry of stationary phase over a solid support. various materials used are Cellulose, Cellulose with starch, Cellulose with fluorescent indicator silica gel etc . Now a days pre coated plates are available. In this the stationary phase is coated over a solid support like glass, polyester or aluminum. Generally the thickness of the sorbent layer is 100-250 μm is used. But for preparative work 1.0 and 2.0 mm are also available.

GLASS :

GLASS Advantages:- Resistant to heat & chemicals Easy to handle Superior flat and smooth surface Disadvantages:- Fragile High weight Additional packing material required High production cost so costly

POLYESTER SHEETS:

POLYESTER SHEETS Advantages : More economical Unbreakable Less packing material required Less shelf space for storage Easily cut out in desired size Spot can be cut and eluted Disadvantages : Charring at temperature more than 120°C as it is unstable beyond this temperature.

ALUMINUM SHEETS:

ALUMINUM SHEETS Advantages : More economical Unbreakable Less packing material required Less shelf space for storage Easily cut out in desired size Spot can be cut and eluted Increased temperature resistance compared to polyester Disadvantages : Elements containing high concentration of mineral acid or conc. ammonia may react with the support.

COMMERCIALLY PRECOATED PLATES:

COMMERCIALLY PRECOATED PLATES Silica gel 60F Aluminum oxide mainly for basic compound, alkaloids . High purity silica gel 60 Cellulose (microcrystalline) for amino acid, dipeptides Polyamide or micro polyamide Silica gel which is chemically modified by various functional groups like amino(-NH2), Cyno (CN), Pre screen TLC plates used to preview rapid and less expensive separation before HPLC

PLATE SIZE:

PLATE SIZE Generally precoated plates of size 20 X 20 cm are available with either of support. According to requirement the plates can be cut out in specific size. Though 20 X 20 cm size is most widely available plates, with different size are also available like pre screen plates are available in the size of 5 X 7.5 cm

PRE WASHING:

PRE WASHING To avoid any possible interference due to impurities It’s purification step Mainly menthol is used Essential for quantitative evaluation Stability testing Ascending, dipping and continuous are different modes of pre washing of plates.

PRE WASHING:

PRE WASHING After washing, the plates must be dried for a sufficient time to ensure complete removal of the washing liquids. Use of hot or cold air is avoided as laboratory air which is usually contaminated is blown over the layer and the purpose of cleaning the layer is defeated. The washed plates should be always stored in dust free atmosphere under ambient conditions. Preferably desiccators of suitable dimensions should be used for storage.

PowerPoint Presentation:

Advantages Signal to noise ratio is low Straight base line obtained Improves Limit of detection

SOLVENTS USED FOR PRE WASHING:

SOLVENTS USED FOR PRE WASHING Generally methanol is used for pre washing purpose but if the mobile phase is lipophilic then its cleaning power is not a good . In such cases the mixture of cleaning solution are used like Chloroform: Methanol(1:1), Chloroform-methanol-ammonia(90:10:1), Metylene chloride: methanol(1:1 ).----> BEST

ACTIVATION OF PRE COATED PLATES:

ACTIVATION OF PRE COATED PLATES Plates exposed to high humidity or kept on hand for longer time may have to be activated by placing in oven at 110-120°C for 30 minutes prior to sample spotting. This step removes water that has been physically adsorbed on the surface of the sorbent. The plates should always be dried in vertical position as in horizontal position drops of solvent may fall on the plate as a result of condensation. Activation at higher temperature should be avoided as it may lead to very active layers and there will be risk of sample being decomposed or artifact being formed .

APPLICATION OF SAMPLE:

APPLICATION OF SAMPLE Usual concentration range is 0.1 to µg/µl above this causes poor separation. Automatic applicator LINOMAT IV Hamilton syringes Camag nano applicator Clark contact spotter

SAMPLE APPLICATION:

SAMPLE APPLICATION Advantages Better separation because of rectangular area in which the compounds are present on the plate. Equal Rf value of ref. and sample Response of densitometer is high Spot broadening in the direction of development is smaller Large quantities of sample can be handled for application Densitometric scanning is less critical

PRE CONDITIONING:

PRE CONDITIONING Also called as CHAMBER SATURATION When the plate is introduced into an unsaturated chamber, during the course of development, the solvent evaporates from the plate mainly at the solvent front. Therefore larger quantity of the solvent shall be required for a given distance; hence resulting is increase in Rf value If the chamber is saturated (by lining of filter paper) prior to development, solvent vapours soon get uniformly distributed throughout the chamber. As soon as the plate is placed in such a saturated chamber, it soon gets pre loaded with solvent vapours, hence less solvent shall be required to travel a particular distance, resulting into lower Rf values.

CHAMBERS:

CHAMBERS Horizontal developing chamber Automatic developing chamber Automated multiple device chamber

CHROMATOGRAPHIC DEVELOPMENT & DRYING:

CHROMATOGRAPHIC DEVELOPMENT & DRYING After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere. Dry in vacuum desiccators - avoid hair drier - essential oil components may evaporate

DETECTION & VISUALIZATION:

DETECTION & 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) Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution . When individual component does not respond to UV - derivatization required for detection. Pre or post chromatographic derivatization is used.

PowerPoint Presentation:

In derivatization dipping or spraying technique is used . Hydrophilic dyes such as methylene blue,-wetted zone appears blue whereas non-wetted zone appears as pale . Flurorecents dye –rhodamin B is also used . Other commonly reagent are: antimony trichloride, anisaldehyde-sulphuric acid etc . These reagents yield sufficient stable colored spots for quantitative scanning .

QUANTITATION:

QUANTITATION DENSITOMETRY Densitometry is used for hptlc quantitation . In is a measurement of the UV absorbance / fluorescence characteristic of a solute directly on the plate. This is done by two methods Transmission mode Reflectance mode Transmission measurements are more sensitive than reflectance mode. Reflectance measurements are more sensitive to non uniformity of sample spot.

DENSITOMETER:

DENSITOMETER

INSTRUMENTATION:

For absorbance measurement: Single beam, double beam These includes: Light sources Monochromator Detector INSTRUMENTATION

INSTRUMENTATION:

LIGHT SOURCE For UV :- H2,Xenon For VISIBLE :- Tungsten lamp MONOCHROMATOR Filter or Prism DETECTOR Photo mutiplier tube[PMT] INSTRUMENTATION

INSTRUMENTATION:

Advantage: Both sample & reference beam scan the same part of the plate surface there by eliminating effects caused by variation in the plate. The transmission of the light in a translucent material as described by : Where, Io = Intensity of incident light, It = Transmitted intensity, Ir = Reflected intensity, Ix = Scattering intensity. INSTRUMENTATION Io = Ia +It + Ir +Ix

DOCUMENTATION:

DOCUMENTATION The type of TLC plate, chamber system ,composition of mobile phase, running time , detection method and all the relevant data should be recorded . E – Merck has recently introduced HPTLC pre-coated plates with imprinted identification code - supplier name. Item number, batch number and individual plate number – to Avoid manipulation of data at any stage - coding automatically get recorded during photo documentation.

FACTORS INFLUENCING HPTLC:

Type of stationary phase Type of pre-coated plates. For quantitative analysis, use of HPTLC pre-coated plate is essential . Layer thickness Mobile phase Solvent purity Size of the developing chamber Saturation of chamber Sample volume to be spotted Size of the initial spot Solvent level in chamber Temperature Flow rate of solvent Separation distance Mode of development FACTORS INFLUENCING HPTLC

APPLICATION OF HPTLC:

APPLICATION OF HPTLC Pharmaceutical industry Food analysis Clinical application Industrial application Forensic pharmacy

REFERENCE:

REFERENCE Pharmaceutical analysis by A.V.KASTURE www.pharmainfo.net Google images

PowerPoint Presentation:

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