Estimation of drugs by thin-layer-chromatography (1).ppt

Estimation of drugs by

1. THIN LAYER THIN LAYER
CHROMATOGRAPHYCHROMATOGRAPHY
Dr P V SureshDr P V Suresh

2. Learning ObjectiveLearning Objective
1.1.State the definition of TLCState the definition of TLC
2.2.Explain the phases used in TLCExplain the phases used in TLC
3.3.List the materials & methods used in List the materials & methods used in
TLCTLC
4.4.List the application of TLCList the application of TLC
5.5.List the advanteges & disadavantages List the advanteges & disadavantages
of TLCof TLC

3. Thin Layer ChromatographyThin Layer Chromatography

What is KLN?...What is KLN?...
One of analysis method that is used to One of analysis method that is used to
identify the unkonwn compounds and to identify the unkonwn compounds and to
determine the purity of mixture.determine the purity of mixture.

This method is simple, rapid and cheapThis method is simple, rapid and cheap

Widely used in pharmaceutical & food Widely used in pharmaceutical & food
stuff industry.stuff industry.

4. --A plate of TLC can be made from aluminium A plate of TLC can be made from aluminium
or glass which is coated by a solid matter as or glass which is coated by a solid matter as
a stationary phase.a stationary phase.
- The coated material has 0.1-0.3mm in - The coated material has 0.1-0.3mm in
thicknessthickness
-some of them has been added by -some of them has been added by
fluorescent indicator that will make it fluorescent indicator that will make it
florescence during the UV light exposure.florescence during the UV light exposure.

5. STATIONARY PHASESTATIONARY PHASE

Silica is commonly used as stationary Silica is commonly used as stationary
phasephase

The separation of sample mixture will be The separation of sample mixture will be
depent on the polarity of sample.depent on the polarity of sample.

Some modified silica is also used in Some modified silica is also used in
certain purposes.certain purposes.

6. Stationery phase Description Application
Silica gel G Silica gel with average
particle size 15µm
containing ca 13%
calcium sulfate binding
agent
Used in wide range
pharmacopoeial test
Silica gel G
254
Silica gel G with
fluorescence added
Same application with
Silica gel G where
visualization is to be
carried out under UV
light.
Cellulose Cellulose powder of
less than 30µm particle
size.
Identification of
tetracyclines

7. MOBILE PHASEMOBILE PHASE

The ability of mobile phase to move up is The ability of mobile phase to move up is
depent on the polarity itselfdepent on the polarity itself

Volatile organic solvents is preferably used as Volatile organic solvents is preferably used as
as mobile phase.as mobile phase.

8. MOBILE PHASEMOBILE PHASE
SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0

9. MATERIALSMATERIALS
•TLC plateTLC plate
•‘‘Developing container’Developing container’
- chamber/ jar/ glass beaker- chamber/ jar/ glass beaker
•PencilPencil
•RulerRuler
•Capillary pipeCapillary pipe
•Solvents / mobile phaseSolvents / mobile phase
- organic solvents- organic solvents
•UV lampUV lamp

10. METHODMETHOD

11. 1.Developing Container 1.Developing Container
PreparationPreparation
Solvent is transferred Solvent is transferred
into the container with into the container with
0.5-1cm in dept from the 0.5-1cm in dept from the
bottombottom

12. 
Commercialy obtained with Commercialy obtained with
5cm x 20cm in size5cm x 20cm in size

Prepare your size when Prepare your size when
neccesaryneccesary

Line 1 cm from the bottom Line 1 cm from the bottom
with a pencil as a part should with a pencil as a part should
be spotted.be spotted.

2. TLC Plate Preparation2. TLC Plate Preparation

13. 3.Spotting’ TLC plates3.Spotting’ TLC plates

Make sure that your sample is Make sure that your sample is
liquified already.liquified already.

stick it using capillary pipe & stick it using capillary pipe &
spott onto the line you have madespott onto the line you have made


14. 4.‘Develop the plate’4.‘Develop the plate’

after spotting, put the plate inside after spotting, put the plate inside
the chamber in the ascendant the chamber in the ascendant
position position

Make sure that the dept of solvent Make sure that the dept of solvent
doesn’tb touch the spotsdoesn’tb touch the spots

Let it develop up to the 1cm from Let it develop up to the 1cm from
the top of platethe top of plate

After that, pull out the plate from After that, pull out the plate from
the cahmber and let the solvent be the cahmber and let the solvent be
vaporizedvaporized

15. 5. Detection of spots5. Detection of spots
--The color samples are The color samples are
easy to be seen and no easy to be seen and no
need to use UV lamp to need to use UV lamp to
detect themdetect them

16. 6. DETECTION OF SPOT6. DETECTION OF SPOT
1)1)Iodination-put the plate in which the spots face to Iodination-put the plate in which the spots face to
the iodine crystall and see what is the spot color the iodine crystall and see what is the spot color
changingchanging
2)2)Ninhydrin: Ninhydrin:
-spesific identification of amino acid compounds.-spesific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when it - Ninhydrin solution will show a purple spot when it
is sprayed to the amino acid spot.is sprayed to the amino acid spot.
3)3)KMnOKMnO
44
used to identify a reducing agent such as glucose, used to identify a reducing agent such as glucose,
fructose, vitamin C and others.fructose, vitamin C and others.
4)4)Alkaline tetrazolium blueAlkaline tetrazolium blue
specificaly used for corticosteroid identificationspecificaly used for corticosteroid identification

17. The use of Rf as separation The use of Rf as separation
parameterparameter
- The distance taken through by the solvent to move up will be assigned as - The distance taken through by the solvent to move up will be assigned as
solfent frontsolfent front
--The distance taken trrough by the sample to move up will be assign as The distance taken trrough by the sample to move up will be assign as
sample frontsample front
--Rf value is obtained by dividing the sample front toward solvent frontRf value is obtained by dividing the sample front toward solvent front
RR
ff = = sample frontsample front
solvent frontsolvent front
--

18. Thin-Layer Chromatography: Thin-Layer Chromatography:
Determination of Determination of RR
ff Values Values
solvent front
component B
component A
origin
d
S
d
B
d
A
R
f
of component A =
d
A
d
S
R
f
of component B =
d
B
d
S
The R
f
value is a decimal
fraction, generally only
reported to two decimal
places
More polar!
Less polar!

19. 7. Quantitative determination of known sample7. Quantitative determination of known sample
-Done by scratching the spot using spatula, and Done by scratching the spot using spatula, and
extract the compound using the suitable solventextract the compound using the suitable solvent
-The liquid extract can be determined its content The liquid extract can be determined its content
using other method such as spectroscopy. using other method such as spectroscopy.

20. Prob;ems commonly occur in TLC and Prob;ems commonly occur in TLC and
how to solvehow to solve
a. The spot shape is too broada. The spot shape is too broad
- Diameter is supposed to be < 1-2mm- Diameter is supposed to be < 1-2mm
b. The movement of solventb. The movement of solvent
- should be straight up- should be straight up
- unproportionality in stationary phase surface will - unproportionality in stationary phase surface will
inhibit the movement of solventinhibit the movement of solvent
c. streaking formationc. streaking formation
- caused by too concentrated sample- caused by too concentrated sample

21. TLC Compared to Paper TLC Compared to Paper
ChromatographyChromatography
1. Precise and effective1. Precise and effective
2. More stable toward various organic solvents2. More stable toward various organic solvents

22. AdvantagesAdvantages

CheapCheap

SimpleSimple

The developing can be monitored visually The developing can be monitored visually

Able to use various chemical as a detectorAble to use various chemical as a detector

23. Identification unknown drugs using standard Identification unknown drugs using standard
ReferenceReference

24. Thank youThank you