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principle &instrumentation of hptlc:

principle &instrumentation of hptlc PRESENTED BY: Reeta vaishy m.Pharm ( qa ) Sem-1 st roll.no-13 GUIDED BY: Dr. n. j. shah (principal) m.Pharm , Ph.D DHARMAJ DEGREE PHARMACY college


CONTENTS Introduction How TLC are performed ? Separation principle Why HPTLC ? Instrumentation References


INTRODUCTION HPTLC - High Performance Thin Layer Chromatography . Thin Layer Chromatography was first established by Egon Stahl . Modified form of TLC or 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.

How TLC separations are performed :

How TLC separations are 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.

Separation principles : :

Separation principles : Two basic underlying principles of differences in affinity : adsorption &partition . 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 .

Criteria for identification of an analyte by TLC/HPTLC :

Criteria for identification of an analyte by TLC/HPTLC 1. Rf value of analyte should be within 3% compared to standard. 2. Visual appearance of analyte and standard should be the same. 3. 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. 4 . max of the analyte should be same as standard. 5. The spectrum of analyte should not be visually different from that of standard.

Characterization of Separation :

Characterization of Separation Rf – Retardation Factor : In TLC compound identification is based on the Rf values compared to authentic standards. Distance moved by the solute Rf = ------------------------------------------------- Distance moved by the mobile phase front Rf value varies from 0 to 1, 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.

Factors responsible for variation in Rf :

Factors responsible for variation in Rf Dimension and type of chamber Nature and size of layer Direction of mobile phase flow Volume and composition of mobile phase Equilibration conditions Humidity Sample preparation method preceding chromatography

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.

Slide 10:

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.

Slide 11:

HPTLC- is a sophisticated and automated form of TLC. Main Difference of HPTLC and TLC - Particle and Pore size of Sorbents. The other differences are: PROPERTIES HPTLC TLC Layer of Sorbent 100-200µm 200-250µm Efficiency High due to smaller particle size generated less

Slide 12:

PROPERTIES HPTLC TLC Particle size (µm) More uniform particle 4-8 (µm) . Less uniform particle size 5-20 (µm) Solid support silica gel for normal phase and C8 , C18 for reversed phase modes Silica gel , Alumina & Kiesulguhr Development chamber . require less amount of mobile phase More amount

Slide 13:

PROPERTIES HPTLC TLC Scanning Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram Not possible Analysis Time Less time for elution More time for elution Sample spotting Auto sampler Manual spotting No. of HETP 4000 2000

Steps involved in HPTLC are :

Steps involved in HPTLC are Selection of plates and sorbent Sample preparation including any clean up and pre chromatographic derivatization Sample application Development chamber Detection Quantitation Documentation

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.

Slide 17:

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

Slide 18:

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.

Slide 19:

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.

Slide 20:

Commercially available pre coated plates are :- Silica gel 60F Aluminium oxide mainly for basic compound, alkaloids . High purity silica gel 60 Cellulose (microcrystalline) for amino acid, dipetides Polyamide or micro polyamide Silica gel which is chemically modified by various functional groups like amino(-NH2), Cyano (CN), Diol 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 of pre coated plates: :

Pre washing of pre coated plates: To avoid any possible interference due to impurities with the chromatographic separation particularly in case of quantitative work, it is always recommended to clear the plate before actual chromatography. This process is called pre washing of plates . Ascending, dipping and continuous are different modes of pre washing of plates.

Slide 23:

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.

Slide 24:

The advantages of pre washing are:- 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 lipophillic 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.

Slide 27:

Application of samples: It is the most critical step for obtaining good resolution and quantification by HPTLC. Sometimes during application the layer may damaged resulting into unevenly spread spots. So generally use of automatic device are recommended for quantitative analysis. TLC HPTLC Volume of sample application (µl) 1-10 0.5-5 Size of the zones down to minimum (mm) 2-4 0.1-1

Some automatic sampler are: 1. Hamilton syringes 2. Camag nano applicator 3. Clark contact spotter :

Some automatic sampler are: 1. Hamilton syringes 2. Camag nano applicator 3. Clark contact spotter Automated sampler Linomet sample applicator

Slide 29:

Various advantages of sample application as band are :- 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 (Chamber saturation): :

Pre-conditioning (Chamber saturation): Chamber saturation has pronounced influence on the separation profile. 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.

Slide 31:

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.

Slide 32:

Horizontal developing chamber Automated multiple deve . chamber Automatic developing chamber

Slide 33:

Various factors influencing HPTLC separation and resolution of spots are listed below. 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

Slide 34:

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

Chromatographic development and drying :

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 desiccators - avoid hair drier - essential oil components may evaporate .

Detection and visualization :

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) 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 chromatogrphic derivatization is used.

Slide 37:

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 tricloride , phasphomolybdic acid, anisaldehyde-sulphuric acid etc. these reagents yield sufficient stable coloured spots for quantitative scanning.


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

Slide 39:

Desaga HPTLC densitometer CD 60

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.

Slide 41:

Various advantages of HPTLC over HPLC are as follows. Simultaneous processing of sample & standard under similar conditions leads to better analytical precision and accuracy. Extreme flexibility for various steps i.e. selection of Stationary phase, mobile phase, developing technique, detection with or without pre or post column derivatization. Co Chromatography or Co TLC is possible & often practised . Several analysts can simultaneously work on the same system.

Slide 42:

Technically simple to learn and operate. No skilled person is required. Low cost pre-coated HPTLC plates/ rolls are available. Entire spectrum can be seen at a glance. Negligible wear & tear so low maintenance cost . Sample preparation is simple. Choice of solvent for dissolving sample is not very critical as it is completely removed before developing chromatogram. Samples rarely require clean up. Turbid sample can also be applied.

Slide 43:

In situ derivatization is possible and routinely employed. Solvents need no prior treatment like filtration and degassing. Solvents of analytical grade are suitable. No need of extra purification. Mobile phase consumption per sample is extremely low , thus reducing the acquisition and disposal cost. Sample application is variable volumes an be applied. Spot or band. Manual or automatic

Slide 44:

There is no possibility of interference from previous analysis as fresh stationary phase and mobile phase are used for each analysis so no memory effect. No carry over hence no contamination. Mobile phase having pH more than 8 can be used which is not possible in HPLC as system being unstable in highly alkaline media. Detection Visual detection is possible Derivatization is simple and routinely used. Non UV absorbing compounds can also be detected by post chromatographic derivatization.


REFERENCES: 1. HPTLC- Quantitative analysis of pharmaceutical formulation by P.D.Sethi 2. Instrumental methods for analysis by Willard 3. A text book of pharmaceutical analysis vol,2 by g vidya sagar .



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