1. THIN LAYER CHROMATOGRAPHY(TLC)

2. INDEX INTRODUCTION PRINCIPLE ADVANTAGES TECHNIQUE OF TLC
PRAPARATION OF TLC PLATE
STATIONARY AND MOBILE PHASE
ACTIVATION OF TLC PLATES
APPLICATION OF SAMPLE
DEVELOPMENT OF CHAMBER
EVALUATION
PROBLEMS IN TLC
APPLICATIONS

3. INTRODUCTION
Thinlayer chromatography is a planar form of chromatography
Thin LayerChromatography (TLC) is a sensitive, fast, simple and inexpensive analytical technique.
It is a micro technique; as little as 10-9g of material can be detected
Qualitative and quantitative analysis

4. PRINCIPLE
Chromatography is a sophisticated method of separating mixtures of two or morecompounds.
The separation is accomplished by the distribution of the mixture between two phases:
one that is stationary and one that is moving.
Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned.

5. ADSORPTION CHROMATOGRAPHY
PARTITION CHROMATOGRAPHY
SEPERATION LARGELY OCCUR BY

6. ADVANTAGES Simple Equipment Short development Time
Wide choice of stationary phase
Quick recovery of separated constituents
Separation effects
Easy visualization of separated compounds
Trace analysis

7. TECHNIQUE OF THIN LAYER CHROMATOGRAPHY

8. PREPARATION OF TLC PLATES
POURING
DIPPING
SPRAYING
SPREADING
PRE-COATED PLATES

9. STATIONARY PHASE CHOICE OF ADSORBENT Solubility of the substance
Nature of the compound
Reactivity of the compound
Chemical reactivity of compound

10. ADSORBENTS Silica N No binder, no fluorescence Silica G
Gypsum , without indicator
Silica G-HR
Gypsum without indicator
Silica p
Preparative grade with indicator
Aluminium oxide G
Aluminium oxide N
No binder
Cellulose MN 301
Standard grade
Cellulose MN 301 HR
High purity grade

11. Inorganic adsorbent
Seperation
Aluminium silicate
Sterols
Bentonite
Vit D
Calicium carbonate
xanthophylls
Fullers earth
aminoacids
Silica gel
fattyacids
Calicium hydroxide
Carotenoids

12. ORGANIC ADSORBENTS
CELLULOSE
CHARCOAL
ION EXCHANGE RESINS

13. MOBILE PHASE CHOICE OF MOBILE PHASE Nature of the constituent
Nature of the process
Rate of migration

14. Least Eluting Power (alumina as adsorbent)
Petroleum ether (hexane; pentane)
Cyclohexane
Carbon tetrachloride
Benzene
DichIoromethane
Chloroform
Methanol
Water
Pyridine
Greatest Eluting Power (alumina as adsorbent)
Organic acids

15. ACTIVATION OF TLC PLATES
AIR DRYING
HOT- AIR OVEN

16. PURIFICATION OF ADSORBENTS
The iron present as an impurity in silicagel-G cause distortion of the plate.
Iron free adsorbent can be achieved by using pre-coated and air dried plates.
Use of methanol and conc. HCl.
Purified plates are dried and activated at 110 degree.

17. APPLICATION OF SAMPLE ,

18. Sample is applied as solution in a
non-polar solvent , if we use polar
solvent
spreading out of startingspot
Affect Rf value
volatile solvent
Area of application should be small
Size of the spot should be small

19. DEVELOPMENT OF TLC PLATES

20. DEVELOPMENT TECHNIQUES
ASCENDING-TECHNIQUE
MULTIPLE DEVELOPMENT
TWO- DIMENSIONAL
CENTRIFUGAL CHROMATOGRAPHY
HORIZONTAL TLC
PREPARATIVE TLC

21. ASCENDING TECHNIQUE TLC after development chamber drying e.g.beaker
with a lid or a closed jar

22. CENTRIFUGAL CHROMATOGRAPHY

23. DETECTION REAGENTS REAGENT DETECTION COMPOUND RHODAMINE 6 B LIPIDS
Silver nitrate and H2O2
Halogenated hydrocarbons
Thymol
sugars
Uv-light fluorescence
FLORESCENCE CONTANING COMPOUNDS
Bromocresol green
Organic acids
Chloranil reagent
phenols
Emerson reagent
Ninhydrin
amino acids
Bials reagent
glycosides

24. EVALUATION OF TLC PLATES
Qualitative evaluation
Rf value = distance traveled by substance
distance traveled by solvent front

25. The Rf (=retardation factor) depends on the following parameters:
solvent system
absorbent (grain size, water content, thickness)
amount of material spotted
temperature
Ideal 0.2 ≤Rf ≤0.8
Rf’s increase with increasing polarity
Methanol >ethyl acetate>Methylene chloride (aka dichloromethane)> hexane

26. QUANTITATIVE ANALYSIS
Direct method
measurement of spot area
Densitometry
spectrophotometry
Indirect methods
UV-spectrophotometry
Colorimetry

27. PROBLEMS IN TLC Over-large spots Uneven advance of solvent front
Streaking

28. APPLICATIONS Purity of sample Examination of reactions
Identification of compounds
Biochemical analysis
Quality testing
Pharmaceutical industry

29. .
SHABANA
08171S0410
M.PHARM ANALYSIS
BHARAT COLLEGE OF
PHARMACY