Thin layer chromatography.

1. Thin layer
chromatography
Dr. Amany M. Elshamy
Lecturer of Medical Laboratory Science
PH.D of Biochemistry and Molecular biology
MLS, AUC, Cairo

2. Outlines

4. Introduction
• Thin-layer
chromatography (TLC)
is different from
column
chromatography.
• TLC is most
commonly used as a
semiquantitative
screening test.

5. Thin-layer
chromatography
(TLC)
• Thin Layer Chromatography is a
technique used to isolate non-volatile
mixtures.

6. Stationary phase
• Many stationary phases are available, including
(1) silica
(2) cellulose
(3) alumina

7. The experiment is conducted on a sheet of aluminum
foil, plastic, or glass which is coated with a thin layer
of adsorbent material.

8. Thin-layer
chromatography
• Separation is normal-
phase partition
chromatography between
the nonpolar mobile
phase and the polar
stationary phase.

9. Thin-layer
chromatography
(TLC)
• Mobile phase is a solvent usually
placed in a closed container until
the atmosphere is saturated with
solvent vapor.

13. Thin-layer chromatography
In TLC the stationary phase,
a thin Layer of particles of a
material such as silica gel is
spread uniformity on a
glass plate or a plastic or
aluminum sheet.
When the thin layer consists
of particles of small
diameter (4.5 μm), the
technique is known as high
performance thin-layer
chromatography (HP LC).

14. Procedure
• In TLC, the sample is applied as a small spot or band near
the bottom edge of the plate (1.5-2 cm).
• Then, the plate is placed in a closed tank containing the
mobile phase, which migrates up the plate by capillary
action.
• After the mobile phase travels a desired distance (2/3 of
plate), the plate is removed from the tank and is dried.
Solutes are visualized directly by fluorescent or by
performing aurora radiography or radioactive compounds.

15. • Capillary dropper

16. The solvent moves up the thin layer o adsorbent by capillary action.

17. Thin-layer chromatography

18. Separation
• Separation can be achieved by any
of the four processes previously
described in lect 1 , depending on
the sorbent (thin layer) and
solvent chosen.
• After the solvent reaches a
predetermined height, the plate is
removed and dried (2/3 of the
plate).
• Sample components are identified
by comparison with standards on
the same plate.

19. Retention factor (Rf):
• The distance a component migrates, compared with the distance the
solvent front moves, is called the retention factor, Rf:
• Rf is a the distance traveled by compound(solute)/ distance traveled
by solvent front
• Rf values are affected by chamber saturation,
temperature, humidity, and composition of the solvent.

20. Each sample component Rf is compared with the Rf of standards.

21. Example for
Retention
factor (Rf):
• standard A has an Rf value of 0.4,
• standard B has an Rf value of 0.6,
• standard C has an Rf value of 0.8.
• The first unknown contains A and C, because
the Rf values are the same.
• Because Rf values may overlap for some
components, further identifying
information is obtained by spraying
different stains on the dried plate and
comparing colors of the standards.

23. Separation
• The solute components move at
different rates because of solubility in
the mobile phase and electrostatic
forces of the stationary phase that
retard solute movement.
• These two phases work together to
provide solute resolution and
separation.
• a. Solute will stay with the solvent
front if solvent is too polar for the
solute.
• b. Solute will remain at origin if
solvent is insufficiently polar.

24. Application

25. Application
• A popular type of planar
chromatography use in the
clinical laboratory is latera
flow affinity
chromatography, which uses
a membrane such as
nitrocellulose as the
stationary phase with one or
more zones of immobilize
antibody or antigen.

26. Application

27. Application
• Using a pregnancy test as an example, a
urine or serum specimen is applied to the
membrane and serves as the mobile phase.
• Human chorionic gonadotropin (hCG) in the
mobile phase is capture by specific
antibodies immobilize in small zones on the
membrane (S.P).
• Detection of bound hCG is achieve by
binding of colored particles coupled to
antibody versus hCG.
• If hCG is present, the antibody-conjugate
particles forms a visible zone.
• If no hCG is present, the particles do not
concentrate in the detection zone.

32. Sandwich-
type Assay
• Detection of specific antibodies is achieved
with analogous Sandwich-type assays using
capture and detection antigens.
• Small molecules, such as drugs of abuse, are
too small to accommodate binding of two
antibodies or a sandwich-type assay.
• CK isoenzymes are measured by
electrophoresis, ion-exchange
chromatography, and several types of
immunoassays.

34. Thin Layer Chromatography
Applications
The qualitative testing of
Various medicines such as
sedatives, local anaesthetics,
anticonvulsant tranquilisers,
analgesics, antihistamines,
steroids, hypnotics is done by
TLC.
TLC is a technique used
clinically for urine drug
screening.

35. Thin Layer Chromatography
Applications
TLC is extremely useful in
Biochemical analysis such
as separation or isolation of
biochemical metabolites
from its blood plasma, urine,
body fluids, serum, etc.
Thin layer chromatography
can be used to identify
natural products like
essential oils or volatile oil,
fixed oil, glycosides, waxes,
alkaloids, etc.

36. Thin Layer Chromatography
Applications
It is widely used in
separating
multicomponent
pharmaceutical
formulations.
It is used for the
purification of
samples.
It is used in the food
industry, to separate
and identify
colours, preservatives
and cosmetic industry.

37. Qs

38. • Which phase of chromatography carries the sample?
• a. Stationary phase
• b. Mobile phase
• c. Retention phase
• d. Column phase

39. • Which of the following statements concerning chromatography is correct?
• a. High-performance liquid chromatography (HPLC) involves the use o support
particles coated on a piece of paper or glass.
• b. In affinity chromatography, a hormone can bin to its receptor that is coated on glass
or resin beads.
• c. Thin- layer chromatography ( LC) is considered to be a quantitative type of
chromatography.
• d. Ion-exchange chromatography uses the principle that particles compete or binding
sites in the stationary phase

40. Spotting a specimen on a silica-coated glass plate an calculating the retention
actor (Rf) or migrate substances in a sample is referred to as LC. What type of
chromatography is this?
a. Column chromatography
b. Affinity chromatography
c. Planar chromatography
d. Liquid chromatography

41. • Some point-of-care tests use chromatography as a processing step in the
assay. In point-of-care pregnancy testing, or example, the stationary phase is
nitrocellulose paper and the hCG antigen is present in a urine samp e, which is
considered the mobile phase. The antigen in the mobile phase binds with an
antibody in the stationary phase. This is an example of planar chromatography
using which of the of owing as the separation mechanism?
• a. Partition
• b. Ion exchange
• c. Affinity
• d. Steric exclusion (size)

42. Which of the following statements is TRUE?
a. Partition chromatography is most appropriate for identifying analytes that may be distributed between two
liquid phases.
b. Steric-size exclusion chromatography best separates analytes based on their solubility in the mobile solvent.
c. In liquid–solid chromatography, the stationary phase separates analytes based on size, shape, and polarity.
d. Ion-exchange chromatography has a resin phase that is soluble to water, and separation of the mixture is
based on magnitude and charge of ionic species.
e. The partition coefficient is measured and compared with standards in thin-layer chromatography.

43. • CK isoenzymes is measured by
• A. electrophoresis
• B. ion-exchange chromatography
• C. immunoassays
• D. all of the above
• Most drugs can be quantified using immunoassay techniques or chromatography (e.g.,
GC and HPLC).
A. true B. False

44. • Given the following information on a particular compound that has been visualized by means of
thin-layer chromatography, calculate the Rf of the compound.
Distance from origin to spot center = 48 mm
Distance from spot center to solvent front = 93 mm
Distance from origin to solvent front = 141 mm
A. 0.29
B. 0.34
C. 0.52
D. 0.66

45. • Which of the following chromatography systems is characterized by a
stationary phase of silica gel on a piece of glass and a moving phase of liquid?
• A. Thin-layer
• B. Ion-exchange
• C. Gas-liquid
• D. Partition

46. • Ion-exchange chromatography separates solutes in a sample based on the
• A. Solubility of the solutes
• B. Sign and magnitude of the ionic charge
• C. Adsorption ability of the solutes
• D. Molecular size

47. • Which of the following statements concerning chromatography is correct?
a) High-performance liquid chromatography (HPLC) involves the use of support particles
coated on a piece of paper or glass
b) In affinity chromatography, a hormone can bind to its receptor that is coated on glass or
resin beads
c) Thin-layer chromatography (TLC) is considered to be a quantitative type of chromatography
d) Ion-exchange chromatography uses the principle that particles compete for binding sites in
the stationary phase

48. • Spotting a specimen on a silica-coated glass plate and calculating the retention factor
(Rf) for migrated substances in a sample is referred to as TLC. What type of
chromatography is this?
a) Column chromatography
b) Affinity chromatography
c) Planar chromatography
d) Liquid chromatography

49. Ion-exchange chromatography separates solutes in a sample based on
the:
• a. solubility of the solutes
• b. sign and magnitude of the charge on the molecule
• c. adsorption ability of the solutes
• d. molecular size and shape