The powerpoint presentation is about the Thin layer chromatography it's uses and techniques with special reference to amino acids, carbohydrates and organic compounds.

1. ST. ANDREW’S DEGREE COLLEGE
NAME: NITIK KALRA
CLASS: M.Sc. IV SEMESTER (CHEMISTRY)
ROLL NO: 2313097530006

3. Seminar
M.Sc. Chemistry
(IV SEMESTER)
UNDER THE GUIDANCE OF PROF. M.H. KHAN
AND PROF. AMIT MASIH

4. Seminar
M.Sc. Chemistry
(IV SEMESTER)
UNDER THE GUIDANCE OF PROF. AMIT MASIH
YOU READY?

5. Table of contents
01
03
02
04
Introduction to
Chromatography
Thin-Layer
Chromatograhy
Chemistry
behind TLC
Application and
Uses
SO LET’S
BEGIN

6. Table of contents
01 02
03
Introduction to
Chromatography
Thin-Layer
Chromatograhy
Application and
Uses Of TLC

7. Introduction to
Chromatography
01

8. What is Chromatography?
• The term chromatography is derived from
Greek, chroma meaning, “color,” and
graphene meaning, “to write.”
• Chromatography is a process for separating
components of a mixture.
• To get the process started, the mixture is
dissolved in a substance called the mobile
phase, which carries it through a second
substance called the stationary phase.

9. What is Chromatography?
• The term chromatography is derived from
Greek, chroma meaning, “color,” and
graphene meaning, “to write.”
• Chromatography is a process for separating
components of a mixture.
• To get the process started, the mixture is
dissolved in a substance called the mobile
phase, which carries it through a second
substance called the stationary phase.
Mikhail S. Tsvet

10. Terms related to Chromatography

11. Terms related to Chromatography
Stationary Phase: substance that stays fixed inside the column
Mobile phase: Solvent moving through column
Eluent: fluid entering the column.
Eluate: fluid exiting the column
Analyte: mixture whose individual components have to be
separated and analyzed.

12. Applications of chromatography
• The chromatographic techniques is used for separation of
amino acids, proteins and carbohydrates.
• Used for analysis of drugs, hormones, vitamins.
• Helpful for qualitative and quantitative analysis of
complex mixtures.
• Useful for determination of molecular weight of proteins.

13. Different types of chromatography
 Paper Chromatography
 Thin Layer Chromatography
 Gel Chromatography
 Column Chromatography
 Ion exchange Chromatography
 Gel filtration Chromatography
 Gas Liquid Chromatography
 Affinity Chromatography

14. Different types of chromatography
 Paper Chromatography
 Thin Layer Chromatography
 Gel Chromatography
 Column Chromatography
 Ion exchange Chromatography
 Gel filtration Chromatography
 Gas Liquid Chromatography
 Affinity Chromatography

15. Thin Layer
Chromatography

16. INTRODUCTION
TLC is one of the simplest, fastest, easiest and least expensive of several
chromatographic techniques.
Separation or identification of mixtures of components by using finely
divided adsorbent liquid/solid spread over a glass plate and liquid as
mobile phase.
TLC is a form of liquid chromatography consisting of :
 A mobile phase (developing solvent).
 A stationary phase (a glass slide coated with a layer of silica)
 Separation of adsorbed substances by mobile phase

17. Principle
• The separation principle of the TLC procedure is based
on the given compound’s relative affinity towards the
mobile and stationary phase.
• The mobile phase moves over the stationary phase
surface.
• During this the compounds that are more attracted
towards stationary phase secure their positions at lower
levels while others move towards higher level resulting
in separation.

18. MATERIALS REQUIRED TO PERFORM TLC:-
 Glass Slide/Plate
 Watch glass / Jar cover
 Stationary Phase (Silica Gel SiO2)
 Mobile Phase
 Oven
 Developing Chamber
 Storage facility for prepared plates.

19. EXPERIMENTAL PROCEDURE
 TLC plate preparation
 Activation of TLC plate
 Spotting the TLC plate
 Development of plate
 Visualize the spots.

20. TLC PLATE PREPARATION
• TLC plates are prepared by mixing the adsorbent
(stationary phase) such as silica gel with water to from a
slurry.
• This slurry is spread uniformly on the glass slide creating
a layer of uniform thickness.
• The thickness of the adsorbent layer is typically around
0.5 – 2.0 mm.

21. Activation of TLC Plate
Plate is kept for drying in oven at 100 deg Celsius
for 30 mins.
This step is called activation of plate.
By doing this surface area of adsorbent increases.
TLC PLATE

22. SPOTTING THE TLC PLATE
• Take the prepared TLC slide and draw a line with pencil
near the bottom of plate. (2cm above)
• Spot the plate on the marked line using capillary tube
• In a jar containing the solvent insert the plate vertically,
the spot should not be immersed in the solvent.
• Cover the jar and the solvent will begin to rise up.
• Remove the plate before the solvent hits the top of plate.
APPLY
SAMPLE HERE

24. Solvent front
Separation of
components
VISUALIZATION
(On application of visualizing reagent)

25. Rf value (Retention factor)
It is defined as the ratio of distance travelled by the
compound mixtures to the distance travelled by the
solvent front.
Rf= distance travelled by compound
distance travelled by solvent front

26. Rf value (Retention factor)
It is defined as the ratio of distance travelled by the
compound mixtures to the distance travelled by the
solvent front.
The Rf value is always less than one.
Rf= distance travelled by compound
distance travelled by solvent front

27. Advantages of TLC
• Separation is sharper in TLC.
• TLC is much more rapid.
• Non volatile compounds can be separated.
• It is more accurate.
• The components of complex mixtures easily separate.

28. Fields of application of TLC
Biomedical
use
Pharmacy Food industry
30% 18%
25%
12%
Cosmetics

29. • Mark two straight lines on the activated TLC plate.
• Spot the amino acids samples.
• Place the TLC plate in the jar with appropriate solvent.
• Allow the solvent to reach the top line.
• Dry the TLC plate and apply the visualizing reagent.
• Look for different colored spots and calculate their Rf value.
SEPARATION OF AMINO ACIDS BY TLC

30. • The ninhydrin reaction is used to detect the presence of amino acids.
• Amino acids contain a free amino and carboxyl group which reacts together with ninhydrin to
produce a characteristic blue color (or occasionally pale yellow).
• In this reaction first an amino group is attached to the first or alpha carbon of the amino acid’s
carbon chain and then the nitrogen atom of the amino group reacts with ninhydrin to give a blue-
purple product known as Ruhemann’s purple.
• Some amino acids (e.g. proline, secondary amine) give yellow-orange colour.
SEPARATION OF AMINO ACIDS BY TLC

31. Amino
acids
Rf value
Developing
Solvent
Locating
reagent
Spot colour
Alanine 0.34
Butanol:Acetic
acid:water
(4:1:1)
Ninhydrin Purple
Glycine 0.45
Butanol: Acetic acid:
water
(4:1:1)
Ninhydrin
Purple
Serine 0.56
Butanol: Acetic acid:
water
(4:1:1)
Ninhydrin
Purple
Valine 0.71
Butanol: Acetic acid:
water
(4:1:1)
Ninhydrin
Purple
Threonine 0.82
Butanol: Acetic acid:
water
(4:1:1)
Ninhydrin
Purple

32. Developing
Solvent
Locating
reagent
Spot colour
Chloroform:methanol:water P-Anisidine hydrochloride Green brown
Chloroform:methanol:water P-Anisidine hydrochloride Green brown
Chloroform:methanol:water P-Anisidine hydrochloride Green brown
SEPARATION OF CARBOHYDRATES

33. Organic
compounds
Rf value
Developing
Solvent
Locating
reagent
Spot colour
Acetic acid 0.34 Butanol:water Iodine vapours Tan brown
Citric acid 0.45
Ethyl acetate: acetic acid:
water
Iodine vapours Tan brown
Oxalic acid 0.56
Ethyl acetate: acetic acid:
water
Iodine vapours Tan brown
SEPARATION OF ORGANIC COMPOUNDS

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