Thin Layer Chromatography

1. Chromatography: Chromatography is physical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary (stationary phase), the other one is mobile
(mobile phase).
Types of chromatographic technique:
Technique Stationary Mobile
Column/Adsorption chromatography Solid Liquid
Partition chromatography Liquid Liquid
Paper Chromatography Liquid Liquid
TLC Liquid/ Solid Liquid
Gas Liquid Chromatography Liquid Gas
Gas Solid Chromatography Solid Gas
Ion Exchange Chromatography Solid Liqud

2. Thin-layer Chromatography

3. Introduction
TLC is one of the most simplest, fastest, easiest and least expensive of several chromatographic techniques
Used in qualitative and quantitative analysis to separate organic compounds and to test the purity of
Compound
TLC is the form of liquid chromatography consisting of:
• A mobile phase
• A stationary phase
• Analysis is performed on a flat surface under constant atmospheric pressure

4. Definition
Thin Layer Chromatography can be defined as a method of separation or identification of a mixture
of components into individual components by using finely divided adsorbent solid/liquid spread over
a glass plate (or other material) and liquid as mobile phase

5. Principle of TLC
It is based on the principle of adsorption chromatography or partition chromatography or combination of both,
Depending on adsorbent, its treatment and nature of solvents employed
The components with more affinity towards stationary phase travels slower, The components with less affinity towards
stationary phase travels faster

6. • The two most common classes of TLC are:
– Normal phase
– Reversed phase
• Normal phase is the terminology used when the stationary phase is polar; for example silica gel, and the mobile
phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase.
• Reversed phase is the terminology used when the stationary phase is a silica bonded with an organic substrate
such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent
which is more polar than the stationary phase.

7. • The following are the important components of a typical TLC system:
– Apparatus (developing chamber)
– Stationary phase layer and mobile phase
– Application of sample
– Development of the plate
– Detection of analyte
Important Steps in TLC process

8. 1. Stationary phase
Adsorbents mixed with water or other solvents→ slurry
Silica gel H ( Silica gel with out binder )
Silica gel G ( Silica gel + CaSO4 )
Silica GF (Silica gel + binder + fluorescent indicator)
Alumina, Cellulose powder, Kieselguhr G( Diatomaceous earth + binder)
GLASS PLATE
Specific dimensions-
20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm
Microscopic slides can also be used
Plates should be of good quality & withstand high temperatures

9. Pouring ( simplest methods )
Dipping (used for small plates )
Spraying ( difficult to get uniform layers )
Spreading (Best technique ) TLC Spreader
Activation:
○ After spreading → Air dry (5 to 10 minutes)
○ Activated by heating at about 100˚C for 30 min.
Then plates may be kept in desiccators

10. Application of sample:
» Using capillary tube or micropipette
» Spotting area should not be immersed in the mobile phase
Development tank
▫ Better to develop in glass beakers, jars to avoid more wastage of solvents
▫ When standard method is used, use twin trough tanks
▫ Do chamber saturation to avoid “edge effect”

11. Mobile phase used depends upon various factors
► Nature of the substance
► Nature of the S.P
►Mode of Chromatography
►Separation to be achieved, Analytical/Preparative
e.g. → pyridine, pet. ether, carbon tetrachloride, acetone, water, glycerol, ethanol,
benzene….

12. Iodine chamber
method Sulphuric
acid spray reagent
UV chamber for fluorescent
compounds Using fluorescent
stationary phase
Detection or visualizing the agent
Non specific method :
Specific method :
1) Ferric chloride - for phenolic compounds and tannins
2) Ninhydrin in acetone – for amino acids
3) Dragondroff’s reagent – for alkaloids
4) 3,5-dinitro benzoic acid – For cardiac glycoside
5) 2,4-dinitrophenyl hydrazine – For aldehyde and ketones

13. Detecting techniques can also categorised as:
1) Destructive technique: eg. Specific spray reagents, sulphuric acid spray reagent, etc. where
the samples are destroyed for detection.
2) Non-destructive technique: like uv chamber method, iodine chamber method,
densitometric method etc where the sample is not destroyed even after detection.
these detection techniques are used in TLC method development and preparative TLC

14. • Rf value – Rf value is the ratio of distance travelled by the solute to the distance travelled by the solvent front
the range of Rf value from 0 to 1. But ideal values are from 0.3 to 0.8. Rf value is specific and constant for every
compound in particular combination of stationary and mobile phase
• Rx value – The ratio of distance travelled by the sample & the distance travelled by the standard. Always closer to 0 to 1
Qualitative analysis
Rf =
D
distance travelled by the solute
distance travelled by the solvent front

15. Application:
 Purity of sample
 Examination of reaction
 Identification of compounds
 Biochemical analysis
 In pharmaceutical industry
 Separation of multicomponent pharmaceutical
 Formulations
 In food and cosmetic industry

16. High-performance thin-layer chromatography (HPTLC)

17. Principle:
• Separation my result due to adsorption or partition or by both phenomenon depending upon the nature of adsorbents
used on plates and solvents system used for development.
• The mobile phase flows through the plate because of capillary action. the components move according to their
affinites toward the adsorbent .
• The component with more affinity towards stationary phase travels slower. the component lesser affinity towards
stationary phase travel faster

18. Sample preparation
Application of sample
Chromatography development
Detection of spots
Scanning and documentation
Selection of chromatography layer
Pre washing
Preconditioning
Steps involving in HPTLC

19. Selection Of Chromatography Plates
•Hand Made Plates
•Pre Coated Plates
•Hand Made Plates
 Cellulose
 Cellulose with binder starch
 Silica gel with starch
 Acetylated cellulose + CaSO4 ½ H2O

20. Precoated Layer Of HPTLC
•Different support materials are used
•Glass
•Polyester sheets
•Aluminium
•Silica gel 60F
•Aluminium oxide: Basic substances ,alkaloid and steroids.
•RP,RP8,RP18: Nonpolar substances ,fatty acids,carotenoids,cholesterol.
•Preservatives,barbiturates,analgesic and phenothiazines-Hybrid plates RP18WF25S

21. Sample And Standard Preparation
•Sample and standard should dissolved in the same solvent to ensure comparable distribution at stationary zones.
•For normal phase chromatography solvent for dissolving the sample should be non polar.
•For reverse phase chromatography polar solvents are used.

22. Pre Washing Of Pre Coated Plates
•To avoid any possible interference due to impurities it is recommended to wash the plates is
called pre washing.
•Methods used for pre washing are :
Dipping
Ascending
Solvents used for washing are:
 Chloroform in methanol(1:1)
 Methylene chloride – methanol(1:1)
 1%ammonia or 1% acetic acid

23. Activation Of Pre-coated Plates
•Freshly open box of plates do not require activation.
•Plates exposed to high humidity or kept on hand for long time to be activated
•By placing in oven at 110-120ºc for 30 minutes prior to spotting.
•Aluminium sheets should be kept in between two glass plates and placing in oven at 110-115ºc for 15 minutes.

24. Application Of Standard And Sample
•Selection of sample application and devices used depends on
•Sample volume
•Number of samples to be applied
•Samples is applied by use of automatic devices and graduated capillaries.
•Volume recommended for HPTLC 0.5-5μl
•Sample should not excess or not low
•Over loading can be over come by applying sample as band.

25. Selection Of Mobile Phase
•Normal phase –
•Stationary phase is polar
•Mobile phase is non polar
•Non polar compounds eluted first because of lower affinity with stationary phase .
•polar compound retained because of higher affinity with the stationary phase
•Reversed phase
•Stationary phase is non polar
•Mobile phase is polar
•polar compound eluted first because of lower affinity with stationary phase non polar compounds
retained because of higher affinity with the stationary phase.

26. Detection and visualization
•Detection under UV light is first choice –non destructive spots of fluorescent compounds can be seen at 254nm(near UV range)

27. Quantification
•Sample and standard should be chromatographed on sample plate after development chromatogram is scanned
•Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode.
•Scanning speed is selectable up to 100 mm/s –spectra recording is fast -36 tracks with up to 100 peak windows can be evaluated.
•Calibration of single and multiple levels with linear or nonlinear regressions are possible. When target values are to be verified
such as stability testing and dissolution profile single level calibration is suitable.

28. Application of HPTLC
•Pharmaceutical Industry : quality control, content uniformity, identity/purity check.
•Food Analysis: quality control, additives, pesticides, stability testing.
•Clinical Application :metabolism studies, drug screening, stability testing etc.
•Industrial Application: process development and optimization, In-process check, validation etc.
•Forensic : poisoning investigation.
•Finger print analysis.

29. Difference between HPTLC & TLC