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Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)By: AMIYA KUMAR GHOSHB.pharm,7th sem,4th yearRoll No. : 27701912002Registration No. : 122770210002 OF 2012-2013

NSHM Knowledge Campus-Kolkata, Group of Institutions


Chromatography is the physical separation of a mixture into its individual components. We can use chromatography to separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances.


Gas ChromatographyUsed to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below.

Paper ChromatographyCan be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances

Liquid ChromatographyUsed to identify unknown plant pigments & other compounds.

Thin-Layer ChromatographyUses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances.Examples of Chromatography

TLC:Thin layer chromatography (TLC) is an important technique for identification and separation of mixtures of organic compounds.

In TLC, components of the mixture are partitioned between an adsorbent (the stationary phase, usually silica gel, SiO2) and a solvent ( the mobile phase) which flows through the adsorbent.

Why use TLC:Identification of components of a mixture (using appropriate standards)following the course of a reaction,analyzing fractions collected during purification,analyzing the purity of a compound.


Easy to useCheapPossible multiple analysisPossible recovery of the productsNo sample preparation required2-dimensional analysisDrawbacks:

Slow (typically 30-60 minutes)Limited quality of the separationLimited reproducibilityEvaporation of the mobile phase (composition varies during the analysis)

Difference between TLC and HPTLC


As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate.Individual components move up at different rates, depending on intermolecular forces between the component and the silica gel stationary phase and the component and the mobile phase.The stationary phase is SiO2 and is very polar. It is capable of strong dipole-dipole and H-bond donating and accepting interactions with the analytes (the components being analyzed).

More polar analytes interact more strongly with the stationary phase in move very slowly up the TLC plate.

By comparison, the mobile phase is relatively nonpolar and is capable of interacting with analytes by stronger London forces, as well as by dipole-dipole and H-bonding.

More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase and move higher up the TLC plate.

STATIONARY PHASESilica is commonly used as stationary phaseThe separation of sample mixture will be depend on the polarity of sample.Some modified silica is also used in certain purposes.

Stationery phaseDescriptionApplicationSilica gel GSilica gel with average particle size 15m containing ca 13% calcium sulfate binding agent Used in wide range pharmacopoeial test

Silica gel GF254Silica gel G with fluorescence addedSame application with Silica gel G where visualization is to be carried out under UV light.

CelluloseCellulose powder of less than 30m particle sizeIdentification of tetracyclines

MOBILE PHASEThe ability of mobile phase to move up is depend on the polarity itselfVolatile organic solvents is preferably used as as mobile phase.

SOLVENTPOLARITY INDEXHexane0Butanol3.9Chloroform4.1Methanol5.1Ethanol5.1Acetonitrile5.8

Elution Strength of Mixed Solvents:

The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the components:eonet = eoA (mole % A) + eoB (mole % B)where:mole % A = (moles A) / (moles A + moles B)

Thus, to determine the eonet of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the eonet of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below:

eonet = eoEtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] + eohexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc]

thus: eonet = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]} {(1.0 mLEtOAc)(0.902g/mL)/88.11g/mole) + (9.0 mLhexane)(0.659g/mL)/86.18g/mole)}

andeonet = 0.067

Materials :TLC plateDeveloping container - chamber/ jar/ glass beakerPencilRulerCapillary pipeSolvents / mobile phase- organic solventsUV lamp

Procedure:1.Developing Container Preparation:

Solvent is transferred into the container with 0.5-1cm in dept from the bottom

2. TLC Plate Preparation

Commercialy obtained with 5cm x 20cm in sizePrepare your size when neccesaryLine 1 cm from the bottom with a pencil as a part should be spotted.

Image Notes1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder2. These were made with silica gel from dessicator packets, prepared in the same way as above but with less suspension to work with (hence the gaps near theedge)

3.Spotting TLC platesMake sure that your sample is liquified already. stick it using capillary pipe & spott onto the line

4.Develop the plateafter spotting, put the plate inside the chamber in the ascendant position Make sure that the depth of solvent doesnt touch the spotsLet it develop up to the 1cm from the top of plateAfter that, pull out the plate from the chamber and let the solvent be vaporized

5. Detection of spotsThe color samples are easy to be seen and no need to use UV lamp to detect them

Chromatogram of 10 essential oils,Stained with vanillin reagent.

Detection :Compound classDerivatizing agentGeneral Iodine vaporGeneral Sulphuric acid (50%)Acids Bromo cresol green Aldehyde and ketone 2,4 dinitro phenyl hydrazineAmines and amino acid ninhydrinAlkaloids Mercuric nitrate Barbiturate Diphenylcarbazone Lipids Bromo thymol blueSteroidsAntimony trichloridecarbohydrateAniline phthalate

Chromatogram of 10 essential oils,Stained with vanillin reagent.


The separation between two analytes on a chromatogram can be expressed as the resolution, Rs and can be determined using the following equation:

Rs = (distance between center of spots) (average diameter of spots)

In TLC, if the Rs value is greater than 1.0, the analytes are considered to be resolved.


Absorption of UV radiation is proportional to concentration

Quantification is possible

Rf value:

Rf of component A = dA dS

Rf of component B = dB dSThe Rf value is a decimal fraction, generally only reported to two decimal places


Rf values can be used to aid in the identification of a substance by comparison to standards.

The Rf value is not a physical constant, and comparison should be made only between spots on the same sheet, run at the same time.

Two substances that have the same Rf value may be identical; those with different Rf values are not identical.

Mobile Phase Mixture Composition Number% Ethyl Acetate% HexaneRfSalicylic AcidRfAcetylsalicylic Acid110000.5300.545290100.6200.600370200.2550.291450300.9450.291530500.3600.280620700.2700.250710900.1820.1648010000

Mobile Phase Mixture combinations for the separation of Salicylic Acid (1%) and Acetylsalicylic acid (1%)

Time (minutes)Salicylic acid RfAcetylsalicylic acid RfReaction Mixture Rf Lower SpotReaction Mixture Rf Upper Spot150.3850.3460.3850.481300.3200.3400.3600.500450.2640.3210.3210.491

Retardation factor (Rf) values for TLC of salicylic acid, acetylsalicylic acid, and reaction mixture in a 50/50 ethyl acetate/hexane solvent system over time.

Some problem:a. The spot shape is too broad- Diameter is supposed to be < 1-2mmb. The movement of solvent- should be straight up- unproportionality in stationary phase surface will inhibit the movement of solventc. streaking formation- caused by too concentrated sample

EXAMPLE :Mobile phase Stationary phaseHerbs and Herbal Products20 volumes of toluene+ 45 volumesof ethyl acetate,+20 volumes of glacial acetic acid + 5volumes of formic acidsilica gelAmalaki100 volumes of ethyl acetate+11 volumes of formic acid,+11 volumes of acetic acid + 25 volumes of water.silica gelAmraGlacial acetic acid.kieselguhr G.Arachis OilA mixture of 1 volume of hexane and 1 volumeof diethyl ether.silica gel GF254ArtemisiaToluenesilica gel GF254Clove OilA mixture of 90 volumes of toluene and10 volumes of ethyl acetatesilica gel GEucalyptus Oil

Derivatizing agent

anisaldehyde sulphuric acid reagentvanilin sulphuric acid reagentstarch solution1 volume of sulphuricacid and 0.5 volume of anisaldehydeanisaldehyde solution,anisaldehyde solution

Reference:R.A.Day, Jr. A.L.Undewood(1987). Analisis Kualititatif. Edisi ke Empat, ms: 474-529.David G. Watson(2005). Pharmaceutical analysis. Edisi ke-2, ms 315-331http// pharmacopeia, volume 3, 2007Kapp, Khail. Chapter 7, Thin Layer Chromatography, March 2, 2010 K