THIN LAYER CHROMATOGRAPHYTHIN LAYER CHROMATOGRAPHY

Presented By:Presented By:Rakesh AhirwarRakesh Ahirwar

(M.Sc. Geo-Informatics)(M.Sc. Geo-Informatics) Ph.D. Research ScholarPh.D. Research Scholar

Chemical Sciences August 2015Chemical Sciences August 2015

Academy of Scientific and Innovative Research( वैजािनक तथा नवीकृत अनुस ंधान अकादमी)

CSIR-Advanced Materials and Processes Research Institute, Bhopal( पगत पदाथ र एव ं पकम अनुस ंधान ससंथान, भोपाल)

GUIDED BY:

Dr. Sorna GowriDr. Sorna GowriSenior Principal ScientistSenior Principal ScientistCSIR AMPRI BHOPALCSIR AMPRI BHOPAL

ContentContent

1.1. DefinitionDefinition2.2. Phases usedPhases used3.3. Materials & MethodsMaterials & Methods4.4. Application Application 5.5. Advantages & DisadvantagesAdvantages & Disadvantages

Thin Layer ChromatographyThin Layer Chromatography

One of analysis method that is used to One of analysis method that is used to identify the unknown compounds and to identify the unknown compounds and to determine the purity of mixture.determine the purity of mixture.

This method is simple, rapid and cheapThis method is simple, rapid and cheapWidely used in pharmaceutical & food Widely used in pharmaceutical & food stuff industry.stuff industry.

IntroductionIntroduction- A plate of TLC can be made from aluminum - A plate of TLC can be made from aluminum or glass which is coated by a solid matter as or glass which is coated by a solid matter as a stationary phase.a stationary phase.

- The coated material has 0.1-0.3mm in - The coated material has 0.1-0.3mm in thicknessthickness

-some of them has been added by -some of them has been added by fluorescent indicator that will make it fluorescent indicator that will make it florescence during the UV light exposure.florescence during the UV light exposure.

STATIONARY PHASESTATIONARY PHASE

Silica is commonly used as stationary Silica is commonly used as stationary phasephase

The separation of sample mixture will be The separation of sample mixture will be depend on the polarity of sample.depend on the polarity of sample.

Some modified silica is also used in Some modified silica is also used in certain purposes.certain purposes.

MOBILE PHASEMOBILE PHASE

The ability of mobile phase to move up is The ability of mobile phase to move up is depend on the polarity itselfdepend on the polarity itself

Volatile organic solvents is preferably used as Volatile organic solvents is preferably used as mobile phase.mobile phase.

MATERIALSMATERIALS

• TLC plateTLC plate• ‘‘Developing container’Developing container’ - chamber/ jar/ glass beaker- chamber/ jar/ glass beaker• PencilPencil• RulerRuler• Capillary pipeCapillary pipe• Solvents / mobile phaseSolvents / mobile phase

- organic solvents- organic solvents• UV lampUV lamp

METHODMETHOD

1.Developing Container 1.Developing Container PreparationPreparation

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

Commercially obtained with Commercially obtained with 5cm x 20cm in size5cm x 20cm in size

Prepare your size when Prepare your size when necessarynecessary

Line 1 cm from the bottom Line 1 cm from the bottom with a pencil as a part should with a pencil as a part should be spotted.be spotted.

2. TLC Plate Preparation2. TLC Plate Preparation

3.Spotting’ TLC plates3.Spotting’ TLC plates

Make sure that your sample is Make sure that your sample is liquefied already.liquefied already.

stick it using capillary pipe & stick it using capillary pipe & spot onto the line you have madespot onto the line you have made

4.‘Develop the plate’4.‘Develop the plate’

after spotting, put the plate inside after spotting, put the plate inside the chamber in the ascendant the chamber in the ascendant position position

Make sure that the dept of solvent Make sure that the dept of solvent doesn’t touch the spotsdoesn’t touch the spots

Let it develop up to the 1cm from Let it develop up to the 1cm from the top of platethe top of plate

After that, pull out the plate from After that, pull out the plate from the chamber and let the solvent be the chamber and let the solvent be vaporizedvaporized

5. Detection of spots5. Detection of spots

The color samples are The color samples are easy to be seen and no easy to be seen and no need to use UV lamp to need to use UV lamp to detect themdetect them

6. DETECTION OF SPOT6. DETECTION OF SPOT

1)1) Iodination-put the plate in which the spots face to Iodination-put the plate in which the spots face to the iodine crystal and see what is the spot color the iodine crystal and see what is the spot color changingchanging

2)2) Ninhydrin: Ninhydrin: -specific identification of amino acid compounds.-specific identification of amino acid compounds.

- Ninhydrin solution will show a purple spot when it - Ninhydrin solution will show a purple spot when it is sprayed to the amino acid spot.is sprayed to the amino acid spot.

The use of Rf as separation The use of Rf as separation parameterparameter

- The distance taken through by the solvent to move up will be assigned as - The distance taken through by the solvent to move up will be assigned as solvent frontsolvent front

-- The distance taken trough by the sample to move up will be assign as The distance taken trough by the sample to move up will be assign as sample frontsample front

-- Rf value is obtained by dividing the sample front toward solvent frontRf value is obtained by dividing the sample front toward solvent front

RRff = = sample frontsample front

solvent frontsolvent front

Thin-Layer Chromatography: Thin-Layer Chromatography: Determination of Determination of RRff Values Values

solvent front

component B

component A

origin

dS

dB

dA

Rf of component A =

dA

dS

Rf of component B =

dB

dS

The Rf value is a decimal fraction, generally only reported to two decimal places

More polar!

Less polar!

ApplicationApplication

Quantitative determination of known sampleQuantitative determination of known sample- Done by scratching the spot using spatula, and Done by scratching the spot using spatula, and

extract the compound using the suitable solventextract the compound using the suitable solvent- The liquid extract can be determined its content The liquid extract can be determined its content

using other method such as spectroscopy. using other method such as spectroscopy.

AdvantagesAdvantages

CheapCheap SimpleSimple The developing can be monitored visually The developing can be monitored visually Able to use various chemical as a detectorAble to use various chemical as a detector

ThanksThanks