TLC, thin layer chromatography

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THIN LAYER

CHROMATOGRAPHY

(TLC)

by by Mr. Shaise Jacob Mr. Shaise Jacob

Faculty, Nirmala College of Pharmacy

Muvattupuzha

Kerala, India

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Chromatography

There are two basic types of

chromatography

² ² GasGas ² ² LiquidLiquid

Liquid includes TLC and high

performance liquid chromatography

(HPLC)

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Introduction

TL

TLC i

C is a fo

s a form o

rm of li

f liqui

quid c

d chr

hrom

omat

atog

ogra

raph

phy

y

consisting of:

²

² A mobilA mobile phae phase (dese (developiveloping ng solvesolvent) ant) andnd ²

² A statiA stationary onary phase (phase (a plate oa plate or strip cr strip coated wioated with ath a form of silica gel)

form of silica gel) ²

² AnalAnalysis ysis is perfois performed rmed on a flaon a flat surft surface uace undernder atmospheric pressure and room temperature atmospheric pressure and room temperature

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Michael Tswett is credited as being the

father of liquid c

hromatography. Tswett

developed his ideas in the early 1900·s.

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TLC

Th

The

e two

two mo

most

st co

commo

mmon c

n clas

lasses

ses of

of TLC

TLC are

are::

²

² No

Norm

rmal p

al pha

hase

se

²

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Normal Phase

No

Norma

rmal p

l phas

hase is

e is the

the ter

termin

minolo

ology us

gy used

ed wh

when t

en the

he

stationary phase is polar

; for example silica gel,

and the

and the mobile phase is an organic solvent

mobile phase is an organic solvent

or a

mixture of organic solvents which is less polar

than the stationary

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Reversed Phase

Rev

Revers

ersed

ed ph

phase i

ase is th

s the te

e termi

rmino

nolo

logy us

gy used

ed whe

when

n

the

the stationary phase is a silica bonded with an

stationary phase is a silica bonded with an

organic substrate

such as a long chain aliphatic

such as a long chain

aliphatic

acid like C-18 and the

acid like C-18 and the mobile phase is a mixture

mobile phase is a mixture

of water and organic solvent which is more

polar than the stationary

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THIN LAYER

CHROMATOGRAPHY

Chromatography is used to separate mixtures

of substances into their components

.

Similar to P.C, except that a thin layer of

some inert material, i.e. Aluminium oxide,

mag.oxid. , sili.oxid

mag.oxid. , sili.oxide is

e is

used instead of

paper.

A layer of any one of these oxide is made from

a slurry of power in a suitable

inert solvent.

Slurry is spread over a flat

Slurry is spread over a flat surface ( glass, metal

surface ( glass, metal

or rigid plastic ) & dried

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P

RINCI

PP

LE



 ADSOR ADSOR PP TION TION

 The component with more affinity towards the S. The component with more affinity towards the S.PP travelstravels

slower

 The component with lesser affinity towards the S. The component with lesser affinity towards the S.PP _travels_travels

faster

ADVANTAGES OF TLC

simple mtd. & cost of the equipment is low

rapid technique & not time consuming like C.C

separation of µg of the substances can be achieved

any type of compound can be analyzed

corrosive spray reagents can be used without

damaging the plate & needs less solvent

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Steps in TLC Analysis

Th

The f

e foll

ollow

owing ar

ing are

e the

the imp

import

ortant

ant com

compo

pone

nents of

nts of

a typical TLC system:

²

² ApparApparatus atus (devel(developing oping chambchamber)er) ²

² StatiStationary onary phase phase layelayer and mobr and mobile pile phasehase ²

² AppApplilicatcation ion of sof sampamplele ²

² DevDeveloelopmepment nt of thof the ple plateate ²

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General Procedure (1)

²

² Decide Decide if you if you are goare going ing to do to do NormaNormal orl or Reversed phase chromatography

Reversed phase chromatography ²

² PPrepare a plate or select a plate with the properrepare a plate or select a plate with the proper

sorbent material sorbent material ²

² PP_{repare the mobile phase}_{repare the mobile phase}

²

² MaMark rk ththe pe plalatete ²

² ApApplply ty the she samamplplee ²

² DeDevevelolop tp the phe plalatete ²

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PRACTICAL

REQUIREME

NTS

1

1.. SSTTAATTIIOONNAARRY Y PHPH ASE ASE

Adsorbents mixed with water or other

Adsorbents mixed with water or other solvents slurrsolvents slurry y

Silica gel

Silica gel HH _{( Silica gel with out binder )}_{( Silica gel with out binder )}

Silica gel G ( Silica gel +

Silica gel G ( Silica gel + CaSO4 )CaSO4 )

Silica GF (Silica gel + binder +

Silica GF (Silica gel + binder + fluorescent indicator)fluorescent indicator)

Alumina, Cellul

Alumina, Cellulose powder, ose powder, Kieselguhr G( DiatomaceousKieselguhr G( Diatomaceous

earth + binder)

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Coater

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2

. GLASS

PP

LATE

Specific

dimensions-20

20cm cm  2020cmcm, 2, 20c0cm m  1010cmcm, 2, 20c0cm m  5c5cmm

Microscopic slides can also be used

Plates should be of good quality & withstand high

temperatures

3.

PP

RE

PP

ARATION & ACTIVATION OF TLC

P

LATES



 PPouring ouring ( simplest methods )( simplest methods )



 DiDippppiing ng (used for small plates )(used for small plates )

Spraying

Spraying ( difficult to get uniform layers )( difficult to get uniform layers )



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Activation of

PP

lates



 After spAfter spreadireading  ng  Air dry (5 tAir dry (5 to 10 minuo 10 minutes)tes)



 ActivActivated by hated by heatineating at abg at about 100Üout 100ÜC for 30 miC for 30 min.n.

Then plates may be kept in desiccators

4. A

4. APPPP_{LICATION OF SAM}_{LICATION OF SAM}PP_LE_LE

» Using capillary tube or micropipette

» Spotting area should not be immersed in

» Spotting area should not be immersed in the mobilethe mobile

phase

5. DEVELO

5. DEVELOPPMENT TANK MENT TANK



 Better to deveBetter to develop ilop in glass bean glass beakers, jkers, jars to avoid mars to avoid moreore

wastage of solvents



 When stWhen standard andard method imethod is used, use twis used, use twin trougn trough tanksh tanks



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6

. MOBILE

PHPH

ASE

M.

PP

used depends upon various factors



Nature of the substance



Nature of the S.

PP



Mode of

Chromatograph

y



Separation to be achieved,

Analytical/

PP

reparative

e.g. 

pyridine, pet. ether,

carbon tetrachloride,

acetone, water, glycerol, ethanol,

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7

. DEVELO

PP

MENT TEC

HH

NIQUE

1.

1. O

O

n

e

di

me

n

si

o

n

al

de

ve

lo

p

me

n

t

2

.

T

w

o

d

i

m

e

n

s

i

o

n

a

l

d

e

v

e

l

o

p

m

e

n

t

3.

HH

orizontal

developmen

t

4

4 .

.

M

u

l

t

i

p

l

e

d

e

v

e

l

o

p

m

e

n

t

8.

8. DETECTING

DETECTING

OR

VISUALISING

AGENTS

a

a)) NNoon sn sppeecciiffiic c mmeetthhooddss

Iodine chamber method

Sulphuric acid spray reagent

UV chamber for fluorescent compounds

Using fluorescent stationary phase

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Specific methods

Spray reagents or Detecting agents or

Visualizing

agents

 

Same as

PP

.C

QUALITATIVE ANALYSIS

1

1 .

.

R

x

v

a

l

u

e

²

The ratio of distance traveled by the

sample & the distance traveled by the standard.

2.

2. R

R

f f

value -

value

-QUANTITATIVE ANALYSIS

QUANTITATIVE ANALYSIS

>

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APPLICATION

S OF

TLC

»

PP

urity of sample

»Examination of reaction

»Identification of

compounds

»Biochemical analysis

»In pharmaceutical industry

»Separation of multicomponent

pharmaceutical

formulations

»In food and cosmetic industry

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