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

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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	Nature	Principle	Used to separate
Silica gel	Acidic	Adsorption and Partition	Acidic and neutral substance
Alumina	Basic	Adsorption and Partition	Basic and neutral substance
Keisulghur	Neutral	Partition	Strongly hydrophilic substance
Cellulose powder	Neutral	Partition	Water soluble compounds

• 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

	HPTLC	TLC
Layer of sorbent	100ům	250üm
Efficiency	High due to smaller particle size generated	Less
Scanning	Use of UV/visible, fluorescense. Scanner is an advanced type of densitometer	Not possible
Sample spotting	Auto sampler	Manual spotting
Analysis time	Shorter migration distance and the analysis time is greatly reduced	slower
Solid support	Wide choice of st.phases like silica gel for normal ph and c8,C18 for reverse ph modes	Silica gel, keiselguhr, alumina
Development chamber	Less amount of mobile phase	More amount