Thin layer chromatography by Mr. Vinayak Bodhankar

Introduction
Theory
Principle
Applications
RF values

1. Y
Mr. Vinayak R. Bodhankar
M. Pharm. (QA)

2. Content
> Introduction
> Principle i

> Methodology, Rf values |

+

> Advantages is ntages and a)
an 1 gar

3. [11] mamopuoton

+ Chromatogrphic separation by Du — | of adsorbent coated on plate.

# The history of thin layer chronétomepteté. back to 1938 when Izmailov
and Shraiber separated plant: extracts wall 2mm thick and firm layer of
alumina set on glass guia == .

< In 1944, Gamal on ae Martin used filter pap

==

% In im | entifie PRÉ on filter paper Es later glass

‘coated with aluming

layer chromatography.

4. LE emo

Y Thin Layer Chromatography can _be-defined as a method of separation or
ne.

i 4
identification of a mixture of componentsinto-individual components by using

A

finely divided adsorbent eN spread over À chromatographic plate.

Y The mobile phase solvent flows through because of capillar

stam honak Ir TT

v The Sl i b ne
Y ar, ore E towards the stationary phase

“Th Cp" wi ae <a towards the stationa

À are separated on a thin

based on the affinity of the components towards the stationary phase.

5. LU) meruonoLocr |

1. Selection of stationary phase A

is u
2. Preparation of plate Jj es 2)

«|

i

3. Activation of plate

1

4. Purification of adsorbent _

à = Cr
5. Application of sample

rı

6. 1. Selection of stationary phase
Depends on type of chromatography i.e. NP or RP

Adsorbent: Water ratio

Silicagel H Silicagel without binder 1:1:5
Silicagel GF Silicagel + Binder + F 1:2
Silicagel G Silicagel + Gypsum 1:2
ALO, G Al,O, + Binder 1:2
Cellulose powder Cellulose powder without 1:5
binder
Cellulose powder G Cellulose powder with binder 1:6
Kieselguhr G Kieselguhr G + Binder 1:2

"id

7. 2. Preparation of plate
V The sizes of the glass plates for use with commercially available spreaders are

usually 20 X 20, 20 X 10 or 20 X5 cae

ES]
Y” In general, the glass plates BR ern. AM and should be withstand
temperatures i |
{
Methods:
au Pouring

Q Sing JA 4

3 ‘mr
si adin,
= Base

8. 3. Activation of plate
Air drying followed by drying in hot air oven so as to remove moisture.

“Plate is kept for drying in oven at
100°C.

"This step is called as activation of
plate.

“By doing this surface area of the

adsorbent increases,

elo

9. 5. Sample application
Y In order to get good spots, the concentration of the sample or standard

solution can be 2-5ul of a %1 solution ither standard or test sample is
spotting x 4 2
Y” using a capillary tube or ra Î

dom or equidistant from each other with
1

Y” The spots can be placed at r

markings. _

Y The spot should be kept aa “ees 2em above the bas

be immersed in th

| TLC Chamber

—TLC Plate

10. 6. Tank development

> Mobile phase should be below than 2cm line.
> Firstly tank is filled with mobile phasevonly and cover it with lid for 1 hr.

Is 4
> Volatile nature of solvent —————»«Vapors get settle and MP becomes
5
saturated. i

> Then place the TLC plate in Aaa a {

}-—— TLC Chamber

TLC Plate

11. 7. Selection of solvent
Selection of the mobile phase depends upon the below factors
= Nature of the substances to be separated"

= Nature of the stationary phase D nains LS
= Mode of chromatography a Phase or reverse phase)

List of solvents (of increasing polarity)
ES
Petroleum ether, Carbon tetrachloride, Cyclohexane,

Acetone, Benzene
+

ethanol, Wat

Da

12. 7. Detection of components

After the development of TLC plates, the spots should be visualized. Detecting
colored spots can be done visually. le el colorless spots, any one of
the following techniques can be usd ne”

a. Specific methods \ Î

|
In this method particular detecting agents are used to

M . — |
compounds or for identification purposes.

Examples are ”

= Dragendroff's reage r alkaloids |
E “4 > b
‚4- Dinitro azine -for aldehydes and ketones \

13. b. Nonspecific methods:
Where the number of spots can be detected, but not the exact nature or type of

compound. FL au

is x

Examples

2 ss

A

1. lodine chamber method: Where YE or amber spots are observed when the

TLC plates are kept in a tank with few iodine crystals at the bottom.

2. Sulphuric acid spray reagent: 70-80% v/v of sulphuric ith, fe
either potassium dichromate or potassium permangana
A

gis” sp

seen due to charring 0

heated in \ | pounds
3. Using flu ni ationary,phase: When the compound s are not
ae ha

fi “a fluo: escent stationary® hase is used. When the plates are viewed unde
chamber, dark-spoti re seen on a fluorescent background. 0
+ i

acid as oxidizing agent is u:

Examples of ‘such stationary phase is Silica gel GF

14. 1. It is a simple process with a short development time.

> : Een \ :
2. It helps with the visualization of separated compound spots easily.
3. It helps in isolating of most of A! compounds. |)

4. The separation process is faster i nd the selectivity for compounds is higher
|

(even small :
differences in chemistry is enough for clear separation)

rt É a
5. It is a cheaper chromato; e technique.

‘
. a icient separ:
and thi '

‘ is à of paper Za graphic separations have How been replac

on thai

15. Y” Automation is not possible

AA
Y” Only soluble components of mixture can be separated

v Separation is done in open “3 which can ape: by temperature or humidity

{ =

lycosides, e

‚Identification o;