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

Thin Layer Chromatography


Thin Layer Chromatography

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

 Discuss the principle involved various in Thin layer

 Explain the procedure involved various in Thin layer 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


• 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


• 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


• Only used for small scale preparative work

of TLC

• 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


• Common adsorbents are




Used to separate

Silica gel


Adsorption and Partition

Acidic and neutral substance



Adsorption and Partition

Basic and neutral substance




Strongly hydrophilic substance

Cellulose powder



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

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


 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

• 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

• 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

Detection of

• Methods used for paper chromatography applicable for TLC

• Colored substances directly

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


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

365nm  —- long

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


• 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




Layer of sorbent





High due to smaller particle size generated



Use of UV/visible, fluorescense. Scanner is an advanced type of

Not possible


Sample spotting

Auto sampler

Manual spotting

Analysis time

Shorter migration distance and the analysis time is greatly reduced



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



 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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