Thin Layer Chromatography (TLC) – Principle, Instrumentation and Application – Pharmacognosy and Phytochemistry II B. Pharma 5th semester PDF Notes

Thin Layer Chromatography

Objective

Thin Layer Chromatography

• At the end of this lecture, student will be able to

 Discuss the principle involved various in Thin layer chromatography

 Explain the procedure involved various in Thin layer chromatography

Chromatography – Thin Layer Chromatography

 Similar to paper chromatography

 But a thin 0.25mm layer of some inert material Alumina, Magnesium oxide, silica (Al2O3, MgO, SiO2 )

 Slurry is made with the material using inert solvent

 Slurry is spread evenly on a surface and dried

 Spreading – manually or mechanically

Thin layer chromatography TLC – Principle

• Component – more affinity -stationary phase – travels slower – eluted later

• Component – less affinity -stationary phase – travels faster – eluted first

• No two components have same affinity for the given set of st.phase and mobile phase

Thin layer chromatography TLC – Advantages

• Technique – superior to CC and PC

 Simple equipment and simple procedure

 Its speed

 Short development time – CC – several hours or days

 Wide choice of stationary phase – inert St. phase – use heat or corrosive spraying reagents like sulphuric acid

• Sensitivity – Trace amount of substance can be detected – 100 folds compared to PC

• Resolution – separation is more efficient

• Needs less solvent

• Detection is easy not tedious

• Easy recovery of separated components

Limitations

• Only used for small-scale preparative work

Requirements of Thin layer chromatography

• An adsorbent/ coating material

• Glass plates

• Preparation of thin layers on plates

• Activation of plates

• Sample application

• Choice of solvents

• Chamber saturation

• Development techniques

• Detection

• Applications

Adsorbent

• Common adsorbents are

• Adsorbent are selected – based on the character of the compounds to be separated

• Check the solubility – decide whether it is basic or acidic or amphoteric

• Mostly – silica gel – calcium phosphate, magnesium sulphate, polyamide, ferric oxide hydrate etc

• Adsorbent – as such will not adhere to glass plate – binders – used

• Binders – Gypsum (plaster of Paris), starch, hydrated silicon oxide

• Commercially – Silica gel G

• Inert fluorescent indicator – Zinc silicate

• Exposure – UV light – glows green fluorescent – non fluorescent – but UV absorbing substances appear – dark spots

• Silica gel GF

Preparation of thin layers on plates

• Adsorbent – slurry – water

• Thin layers are prepared by

a. Pouring – slurry is poured on the plates

b. Dipping – developed – Peifer (1962) – sand witched plates are dipped in slurry

c. Spraying – Bekersky (1963) – sprayed – spray gun – uniformity is not there

• Spreading – spreader or applicator

• Slurry – placed applicator

• Applicator – is moved over stationary plate – or – held constant – plate is pushed or pulled through

• Stahl’s original applicator – 0.25mm thickness

Applicator

 Precoated plates

 Precoated on glass or plastic sheets

 Thickness varied from -0.1-0.2 mm

 High resolution

Activation of Plates

• Removal of water from thin layers

• Adsorbent – many adsorption sites and retain water – minimized by drying

• 110˚ C – 30 MIN – layers active

• Very active layers – prepared – by heating 150˚ C – 4 hrs

• Too high drying – avoided – chemically alter the layers

Purification of silica gel G layers

• Iron impurities – distortion of the chromatograph

• Purified – preliminary development – methanol: conc. HCl (9:1)

• Iron get migrated – solvent front

• Again plates – dried – activated – 110˚C

• Generally – binder – dissolves – methanol: HCl – silica gel – mixed with binder

Sample application

• Capillaries – micropipettes – microsyringe

• Sample – dissolved – suitable solvent

• Quantitative work – Microsyringe

• Qualitative work – capillaries

Choice of Solvent

Depends upon

• Nature of substance to be separated

• St.phase on which the separation to be carried out

• Determined by trial or error method / literature

• Chemical nature of sample – known – stahl’s triangle

Chamber Saturation

• Chamber – saturated – solvent vapours – edge effect will occur

• Proper saturation – will reduce development time

• Rf values are less

• Good resolution of spots

Development Technique

 Ascending technique

 Multiple development

 Two dimensional technique

Ascending Technique

Multiple Development

• Chromatogram is development – with mobile phase

• Development takes place repeatedly – same solvent and in same direction, each time after drying

Two Dimensional Development

Detection of components

• Methods used for paper chromatography applicable for TLC also

• Colored substances directly

• Colorless substances – under UV light or by treating with visualizing agent

Detection

Qualitative analysis

                       Distance travelled by the solute

Rf Value  = ——————————————–

                      Distance travelled by the solvent front

Rf  – 0.3 – 0.8

Non Specific Methods

Iodine chamber method

Iodine crystals  — amber or brown-colored spots

UV chamber for fluorescent compounds

254 nm  — short wave length

365nm  —- long wave length

Specific methods/ Destructive Technique

Specific spray methods

Ferric chloride – phenolic compounds

Ninhydrin in acetone – amino acids

Dragendroff’s reagent  – Alkaloids

3,5 – Dinitro benzoic acid – cardiac glycosides

2,4 – dinitrophenyl hydrazine – aldehydes and ketones

Radioactive compounds – Autoradiography

Antibiotics – chromatogram  – layered – nutrient agar – zone of inhibition  – measured

Quantitative Analysis

Direct technique

Densitometer – measures quantitatively – density of spots

In – situ method

Indirect method

Spots – cut – eluted – solvents

Solution – analyzed – conventional techniques – Spectrophotometry, electrochemical methods

Indirect method

Spot area – scooped – craig tube – dissolve in suitable solvent – adsorbent – removed – centrifuge

Microanalysis of resultant eluate can be done

• Gravimetry

• UV – spectroscopy

• Fluorimetry

• Calorimetry

• Polarography

• Flame photometry

Thin layer chromatography Applications

• Separation of mixture of drugs or biological samples or plant extracts

• Separation of carbohydrates (sugars), vitamins, antibiotics, proteins, alkaloids, glycosides, amino acids etc

• Identification of drugs

• Identification of impurities

• Analysis of metabolites of drugs in blood, urine etc

Analysis of components of Foodstuffs by TLC

• Fruits – aminoacids – Silica gel 0.25 – Chloroform, pentanol, acetic acid, 3:30:1, ninhydrin

• Milk – aminoacids – cellulose 0.25, methanol, chloroform, conc NH3, 2:2:1, ninhydrin

• Apple – sorbitol – kieselghur G – isopropanol, ethylacetoperidoate, acetic acid, water 54:7:1:2

Summary

 Thin layer chromatography – Thin layer of Inert material is coated in a support

 Coating materials may be alumina, silica, kieselghur, cellulose powder etc

 Plate preparation – Pouring, dipping, spraying, spreading

 The developed plate is activated and developed in suitable solvent system

 Detected by visualizing agents or UV- visible light and evaluated by qualitative and quantitative methods

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