THIN LAYER THIN LAYER CHROMATOGRAPHCHROMATOGRAPH

YYPn. Suryati Bt. SyafriPn. Suryati Bt. Syafri

20092009

Learning ObjectiveLearning Objective

1.1. State the definition of TLCState the definition of TLC2.2. Explain the phases used in TLCExplain the phases used in TLC3.3. List the materials & methods used List the materials & methods used

in TLCin TLC4.4. List the application of TLCList the application of TLC5.5. List the advanteges & List the advanteges &

disadavantages of TLCdisadavantages of TLC

Thin Layer ChromatographyThin Layer Chromatography

What is KLN?...What is KLN?...One of analysis method that is used to One of analysis method that is used to identify the unkonwn compounds and identify the unkonwn compounds and to determine the purity of mixture.to determine the purity of mixture.

This method is simple, rapid and cheapThis method is simple, rapid and cheap Widely used in pharmaceutical & food Widely used in pharmaceutical & food

stuff industry.stuff industry.

--A plate of TLC can be made from A plate of TLC can be made from aluminium or glass which is coated by a aluminium or glass which is coated by a solid matter as a stationary phase.solid matter as 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 florescence during the UV light exposure.exposure.

STATIONARY PHASESTATIONARY PHASE

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

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

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

Stationery phase Description Application

Silica gel G Silica gel with average particle size 15µm containing ca 13% calcium sulfate binding agent

Used in wide range pharmacopoeial test

Silica gel G254 Silica gel G with fluorescence added

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

Cellulose Cellulose powder of less than 30µm particle size.

Identification of tetracyclines

MOBILE PHASEMOBILE PHASE

The ability of mobile phase to move up is The ability of mobile phase to move up is depent on the polarity itselfdepent on the polarity itself

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

MOBILE PHASEMOBILE PHASE

SOLVENT POLARITY INDEX

Heksana 0

Butanol 3.9

Chloroform 4.1

Methanol 5.1

Ethanol 5.1

Acetonitrile 5.8

Air 9.0

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 0.5-1cm in dept from the bottomthe bottom

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

Prepare your size when Prepare your size when neccesaryneccesary

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

stick it using capillary pipe & stick it using capillary pipe & spott onto the line you have spott onto the line you have mademade

4.‘Develop the plate’4.‘Develop the plate’ after spotting, put the plate after spotting, put the plate

inside the chamber in the inside the chamber in the ascendant position ascendant position

Make sure that the dept of Make sure that the dept of solvent doesn’tb touch the spotssolvent doesn’tb 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 cahmber and let the solvent the cahmber and let the solvent be vaporizedbe vaporized

5. Detection of spots5. Detection of spots-- The color samples are The color samples are

easy to be seen and easy to be seen and no need to use UV no need to use UV lamp to detect themlamp to detect them

6. DETECTION OF SPOT6. DETECTION OF SPOT

1)1) Iodination-put the plate in which the spots face Iodination-put the plate in which the spots face to the iodine crystall and see what is the spot to the iodine crystall and see what is the spot color changingcolor changing

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

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

3)3) KMnOKMnO44 used to identify a reducing agent such as used to identify a reducing agent such as

glucose, fructose, vitamin C and others.glucose, fructose, vitamin C and others.4)4) Alkaline tetrazolium blueAlkaline tetrazolium blue specificaly used for corticosteroid identificationspecificaly used for corticosteroid identification

The use of Rf as The use of Rf as separation parameterseparation parameter

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

-- The distance taken trrough by the sample to move up will The distance taken trrough by the sample to move up will be assign as sample frontbe assign as sample front

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

RRff = = sample frontsample front solvent frontsolvent front

--

Thin-Layer Thin-Layer Chromatography: Chromatography:

Determination of Determination of RRff Values Valuessolvent front

component B

component A

origin

dSdB

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!

7. Quantitative determination of 7. Quantitative determination of known sampleknown sample

- Done by scratching the spot using Done by scratching the spot using spatula, and extract the compound spatula, and extract the compound using the suitable solventusing the suitable solvent

- The liquid extract can be determined The liquid extract can be determined its content using other method such its content using other method such as spectroscopy. as spectroscopy.

Prob;ems commonly occur in Prob;ems commonly occur in TLC and how to solveTLC and how to solve

a. The spot shape is too broada. The spot shape is too broad- Diameter is supposed to be < 1-2mm- Diameter is supposed to be < 1-2mm

b. The movement of solventb. The movement of solvent- should be straight up- should be straight up- unproportionality in stationary phase - unproportionality in stationary phase surface will inhibit the movement of surface will inhibit the movement of solventsolvent

c. streaking formationc. streaking formation- caused by too concentrated sample- caused by too concentrated sample

TLC Compared to Paper TLC Compared to Paper ChromatographyChromatography

1. Precise and effective1. Precise and effective2. More stable toward various organic 2. More stable toward various organic

solventssolvents

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

Identification unknown drugs using Identification unknown drugs using standard Referencestandard Reference

RujukanRujukan1.1. R.A.Day, Jr. A.L.Undewood(1987). R.A.Day, Jr. A.L.Undewood(1987).

Analisis Kualititatif. Edisi ke Empat, ms: Analisis Kualititatif. Edisi ke Empat, ms: 474-529.474-529.

2.2. David G. Watson(2005). Pharmaceutical David G. Watson(2005). Pharmaceutical analysis. Edisi ke-2, ms 315-331analysis. Edisi ke-2, ms 315-331

3.3. http//orgchem.colorado.edu/http//orgchem.colorado.edu/hndbooksupport/TLC/hndbooksupport/TLC/TLCprocedure.htmlTLCprocedure.html

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