Thin Layer Chromatography (TLC) By Aloo D. and Oloo B. - Principle of TLC -Components of TLC -Procedure of TLC -Interpretation of TLC Results -Advances in TLC -TLC Techniques - Coupling TLC -Thin-layer radiochromatography (TLRC) -Application of TLC

1. Thin Layer Chromatography (TLC)
By Aloo D. and Oloo B.

2. Definition
What is TLC?
A chromatography technique in which a liquid sample migrates by
capillarity through a solid adsorbent medium (such as alumina or silica
gel) which is arranged as a thin layer on a rigid support (such as a glass
plate). It is used to separate non-volatile mixtures.

3. Principle
Different compounds in the sample mixture travel at different rates due to the
differences in their attraction to the stationary phase and because of differences in
solubility in the solvent.
The separation depends on the relative affinity towards the stationery and the
mobile phase.
The compounds under the influence of mobile phase( driven by capillary action)
travel over the surface of the stationery phase. During this movement the
compounds with higher affinity towards stationary phase travels slower.
Components with less affinity towards stationary phase travels faster.Thus
separation of components in the mixture is achieved.
Once separation occurs, the individual components are visualized as spots at a
respective level of travel on the plate.

4. Components of TLC (1 of 2)
• TLC plates, preferably ready made with a stationary phase:These are stable and
chemically inert plates, where a thin layer of stationary phase is applied on its
whole surface layer.The stationary phase on the plates is of uniform thickness
and is in a fine particle size.
• TLC chamber- This is used for the development ofTLC plate.The chamber
maintains a uniform environment inside for proper development of spots. It also
prevents the evaporation of solvents, and keeps the process dust free.
• Mobile phase- This comprises of a solvent or solvent mixtureThe mobile phase
used should be particulate-free and of the highest purity for proper development
ofTLC spots.The solvents recommended are chemically inert with the sample, a
stationary phase.

5. Components of TLC (2 of 2)
• A filter paper- This is moistened in the mobile phase, to be placed inside the
chamber.This helps develop a uniform rise in a mobile phase over the length
of the stationary phase.

6. Procedure ofTLC
1.With a pencil, a thin mark is made at the bottom of the plate to
apply the sample spots.
2.Then, samples solutions are applied on the spots marked on the
line in equal distances.
3.The mobile phase is poured into the TLC chamber to a leveled
few centimeters above the chamber bottom.
4.A moistened filter paper in mobile phase is placed on the inner
wall of the chamber to maintain equal humidity (and also thereby
avoids edge effect).
5.Now, the plate prepared with sample spotting is placed in TLC
chamber so that the side of the plate with the sample line is
facing the mobile phase. Then the chamber is closed with a lid.

7. Procedure ofTLC ( Continued…)
6.The plate is then immersed, such that the sample spots are
well above the level of mobile phase (but not immersed in
the solvent) for development.
7.Sufficient time is given for the development of spots.
8.The plates are then removed and allowed to dry.
9.The sample spots are then seen in a suitable UV light
chamber, or any other methods as recommended for the
given sample.

8. Interpretation of the Results
• Once separation occurs, the individual components are visualized as
spots at a respective level of travel on the plate. Their nature or
character is identified by means of suitable detection techniques.
• Some common techniques for visualizing the results of aTLC plate
include: -UV light
-Iodine Staining
- KMnO4 stain (organic molecules)
-Ninhydrin Reagent
The behaviour of a compound on a TLC is usually described in terms of
its relative mobility or Retention/Retardation factor Rf value , given by
the formula:

10. Advances in TLC ( 1 of 5)
• TLCTechniques - CouplingTLC
Thin-layer chromatography can also be coupled on-line with
spectroscopic methods as well as with other analytical and
preparative separation techniques.This significantly increases
separation performance for complex mixtures and the reliability of
identification of individual substances.

11. Advances in HPLC ( 2 of 5)
• TLC Enhancements to HPTLC and PLC
TLC was recently instrumentalized to develop high-performanceTLC
(HPTLC) using a small particle size (5 μm) stationary phase distributed
in narrow and thin layers in the plate, increasing the speed of separation
and improving the limits of detection of this technique (Sherma, 2000).
Alternatively,TLC can be easily up-scaled to preparative-layer
chromatography (PLC). Preparative layer chromatography (PLC) on
0.5–2 mm layers of sorbent is used to isolate and purify material in
larger amounts than are normally chromatographed on 0.1–0.25 mm
analytical thin layers.

12. Advances in TLC ( 3 of 5)
New Sorbents ForThin-layer Chromatography
Surface-modified precoated plates
The reversed-phase (RP) supports in which non-polar alkyl groups
are chemically bonded to a silica gel skeleton.These hydrophobic
sorbents eliminate the problems of poor reproducibility and partial
elution of the liquid stationary phase, problems which were
previously difficult to overcome but inherent in the use of
impregnatedTLC supports.
The hydrophilic modified pre-coated plates also afford the same
advantages of improved reproducibility and outstanding stability
against various solvent systems.

13. Advances in HPLC ( 4 of 4)
Thin-layer radiochromatography (TLRC)
Enables direct detection and quantification of radiolabeled compounds
on thin layers by using autoradiography , liquid scintillation counting of
scraped zones, or a linear analyzer or phosphor-imaging plate analyzer
system for in situ measurement.

14. Advances in HPLC ( 5 of 5)
TLC as a Pilot Method for Column LC
• The introduction of chemically modified pre-coated RP plates enables the user for
the first time to optimize phase systems for liquid column chromatography (in
which approximately 80% of all separations are performed on RP phases), by
applyingTLC with chromatographically comparable support materials.
• This has led to considerable economies in terms both of time and cost.

15. Advantages ofThin Layer Chromatography (TLC)
• It is a simple process with a short development time.
• It helps with the visualization of separated compound spots easily.
• It helps in isolating of most of the compounds.
• The separation process is faster and the selectivity for compounds is higher
(even small differences in chemistry is enough for clear separation).
• The purity standards of the given sample can be assessed easily.
• It is a cheaper chromatographic technique.

16. Application of TLC (1 of 4)
• Purity of any sample:
used to check the purity of a sample.
Analysis of a compound is performed alongside an authentic reference.
Direct comparison is done between the sample and the standard or authentic
sample.
If any impurity is detected, then it shows extra spots and this can be detected
easily.

17. Application of TLC (2 of 4)
• Phytochemistry : Biological activities of plant compounds
to identify the nature of different plant compounds: anti-oxidative, antibacterial, or
antifungal.
To test the presence of antioxidants, theTLC plate can be sprayed with 2,2-diphenyl-
picrylhydrazyl (DPPH) and methanol.This compound, containing free radicals, is deep
purple in colour. However, after reaction with anti-oxidants, it turns to yellow colour,
identifying the presence of anti-oxidants.
To test the presence of anti-bacterial or antifungal compounds, theTLC
plate can be incubated with microorganisms.The plate can be
subsequently observed for inhibited growth of microorganisms.

18. Application of HPLC (3 of 4)
Examining Reaction Mixtures
used to study if a reaction is complete
At the beginning of a reaction, the entire spot is occupied by the starting
material.
During the progress of the reaction, the spot of the starting material keeps
reducing, while the spot of the products keeps increasing.
At the end of the reaction, the spot of the starting material is completely
gone, and only one spot of the product can be seen on the plate.
Thus, complete absence of the starting spot can be used to mark the end of a
reaction.

19. Application of TLC (4 of 4)
Other applications include:
• Analyzing ceramides and fatty acids
• Detection of pesticides or insecticides in food and water
• Analyzing the dye composition of fibers in forensics
• Assaying the radiochemical purity of radiopharmaceuticals

20. References
• https://microbenotes.com/thin-layer-chromatography/#:~
• Joseph Sherma ,(2003) in Recent Advances in theThin-Layer
Chromatography of Pesticides: A Review , Journal of AOAC International
Vol. 86, No. 3, 2003
• HarryW. Lewis & Christopher J. Moody (13 Jun 1989). Experimental Organic
Chemistry: Principles and Practice (Illustrated ed.). WileyBlackwell. pp. 159–
173.
• Jork, H., Funk,W., Fischer, W., Wimmer, H. (1990):Thin-Layer
Chromatography: Reagents and Detection Methods,Volume 1a,VCH,
Weinheim.
• https://www.sciencedirect.com/topics/biochemistry-genetics-and-
molecular-biology/retardation-factor
• Hoyoux-Roche, D. (2000) in Pesticide/Soil Interactions, J. Cornejo & P. Jamet
(Eds), Institut National de la Recherche Agronomique, Paris, France, pp 57–
63

21. References
• https://www.merriam-webster.com/dictionary/thin-layer%20chromatography
• https://link.springer.com/book/10.1007/978-1-4899-222-2
• www.sigmaaldrich.com/ thin layer chromatography techniques/
• https://www.news-medical.net/life-sciences/Applications-of-Thin-Layer-
Chromatography.aspx
• https://www.milesscientific.com/applications-of-thin-layer-chromatography/
• https://en.wikipedia.org/wiki/Thin-layer_chromatography