M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.

Izmailov & Shraiber separated plant extracts using 2mm thick and firm layer of alumina set on glass plate.

In 1944, Consden, Gorden & Martin used filter papers for separating amino acids.

In 1950, Kirchner identified terpenes on filter paper

In 1958, Stahl developed standard equipment for analysing by TLC.

Adsorption Separation based on the affinity of the

compounds towards the stationary phase. Stationary phase: thin layer of adsorbent

coated on chromatographic plate. Mobile phase solvent flows through b’cos

of capillary action (against gravity) The components with more affinity

towards the stationary phase travels slower.

Lesser affinity towards the stationary phase travels faster.

1. Simple method & less expensive 2. Rapid technique 3. Separation upto µgms 4. Any type of compound can be analysed 5. Efficiency of separation: depends upon the

particle size ( small) flow rate ( less viscous) 6. Detection is easy and not tedious 7. Capacity of the thin layer can be altered. 8. Needs less solvent, stationary phase & time 9. Corrosive spray reagents can be used without

damaging the plates.

Stationary phasesGlass platesPreparation and activation of TLC

platesApplication of sampleDevelopment tankMobile phaseDevelopment techniqueDetecting or visualising agents

NAME COMPOSITION ADSORBENT: WATER RATIO

Silicagel H Silicagel without binder

1:1.5

Silicagel G Silicagel +CaSO4 1:2

Silicagel GF Silicagel+Binder+fluorescent indicator

1:2

Alumina Neutral, Basic & Acidic

Al2O3 without binder

1:1.1

Al2O3 G Al2O3 + binder 1:2

Cellulose powder

Cellulose without binder

1:2

Cellulose powder

Cellulose with binder

1:6

Kieselguhr G

Diatomaceous earth + binder

1:2

Polyamide powder

Polyamide 1:9 (CHCl3:CH3OH=2:3)

Glass plates 20cmX20cm(full plate), half plate (20x10), quarter plate (20x5)

These dimensions are used since the width of the TLC spreader is 20cm

Microscopic slides can be used for some applications like monitoring the progress of a chemical reaction

The development time is 5 min Good quality & should withstand

temperatures used for drying the plates

1. Pouring: slurry is prepared & poured on glass plate & spread uniformly

Demerit: uniformity in thickness cannot be ensured

2. Dipping: two plates are dipped into the slurry and are separated after removing from slurry and later dried.

Demerit: larger quantity of slurry is needed 3. Spraying: using a sprayer Demerit: layer thickness cannot be maintained

uniformly 4. Spreading : the best technique Normal thickness 0.25mm for analytical purposes 2mm thickness for preparative purposes Oven temperature for drying should be 100-120

for 1 hr.

To get good spots, the conc of the sample has to be minimum

2-5µl of a 1% solution is spotted using a capillary tube or micropipette.

Spots should be kept atleast 2cm above the base of the plate

Spotting area should not be immersed in the mobile phase

Glass beakers, specimen jars The tank should be lined inside with

filter paper moistened with mobile phase so as to saturate the atmosphere

If saturation is not done, edge effect occurs where the solvent front in the middle of TLC plate moves faster than that of the edge.

The solvent or mobile phase used depends upon

1. nature of the substances to be separated

2. nature of the stationary phase used 3. mode of chromatography ( normal /

reverse phase) 4. separation to be achieved ( analytical /

preparative) Eg: petroleum ether, CCl4, cyclohexane,

CS2, Ether, acetone, Benzene, Toluene, Ethylacetate, CHCl3, alcohols, water, pyridine, organic acids,

1. One dimensional development (vertical) 2. Two dimensional development 3. Horizontal development 4. Multiple development One dimensional development: The plates are

kept vertical & the solvent flows against gravity b’cos of capillary action.

Two dimensional development: This tech is used for complex mixtures First, the plates are developed in one axis & the

plates after drying are developed in the other axis.

After the development of TLC plates, the spots should be visualised.

Detecting coloured spots can be done visually.

For colourless spots 1. Non-specific methods: no. of spots can

be detected, but not the exact nature or type of compound

2. Specific methods: specific spray reagents or detecting agents or visualising agents are used to find out the nature of compounds or for identification purposes.

Where the no. of spots can be detected but not the exact nature / type of compound

1. Iodine chamber method: where brown or amber spots are observed

2. H2SO4 spray reagent: 70-80% of H2SO4 with few mg of either K2Cr2O7 or KMnO4 or few ml of HNO3 as oxidising agent is used.

After spraying on TLC plates is heated in an oven

Black spots are seen due to charring of compounds

3. UV chamber for fluorescent compounds: when compounds are viewed under UV chamber, at 254nm (short wavelength) or at 365nm (longer) fluorescent compounds can be detected. Bright spots are seen under a dark background.

4. Using fluorescent stationary phase: when the compounds are non fluorescent, a fluorescent stationary phase is used. When the plates are viewed under UV chamber, dark spots are seen on a fluorescent background. Eg: such stationary phase is Silicagel GF

Specific spray reagents or detecting agents or visualising agents are used to find out the nature of compounds or for identification purposes.

Ex: 1. Ninhydrin for amino acids 2. FeCl3 phenolic compounds & tannins 3. Dragendroff’s reagent for alkaloids 4. 3,5- Dinitrobenzoic acid cardiac

glycosides 5. 2,4-Dinitorphenylhydrazine aldehydes

& ketones

2 types 1. Destructive technique: samples are

destroyed Specific spray reagents, con H2SO4 2. Non-Destructive technique: Sample

is not destroyed even after detection. This is used in preparative TLC

Eg: UV-chamber method, Iodine chamber method, densitometric method

Measures the density of the spots When the optical density of the spots

for the standard & the test solution are measured , the quantity of the substance can be calculated.

The plates are neither destroyed nor eluted with solvents to get the compounds.

This method is also called in-situ method

1. Separation of mixtures of drugs of chemical or biological origin, plant extracts

2. Separation of carbohydrates, vitamins, antibiotics, proteins, alkaloids, glycosides

3. identification of drugs 4. identification of related compounds in

drugs 5. to detect the presence of foreign

substances in drugs 6. to detect decomposition products in

drugs

HPTLC is a sophisticated & automated form of TLC. Features: 1. the use of precoated plates with stationary phase

particle size of <10µ diameter. 2. wide choice of stationary phases like Silicagel, for

normal phase Reverse phase: C18, C8 3. Auto sampler instead of manual spotting and streaking

for preparative purposes. 4. New type of development chambers which requires less

amt of solvents for developing. 5. More efficiency b’cos smaller & uniform size of

adsorbents 6. The use of UV/Vis/Fluorescence scanner which scans the

entire chromatogram qualitatively & quantitatively. The scanner is an advanced type of densitometer. 7. Increased data processing capabilities by the use of

computers.

Rf value is the ratio of distance travelled by the solute to the distance travelled by the solvent front.

The Rf value ranges from 0 to 1.0 (0.3-0.8) Rx value: is nothing but the ratio of

distance travelled by the sample and the distance travelled by the standard.

Rx value is always closer to 1.0 Rm value is used in qualitative analysis to

find out whether the compounds belong to a homologous series.