1. Thin Layer Chromatography
Rabia Perveen FA22-MPH-47
Rameen Waseem FA22-MPH-49
Subject: Chromatographic Techniques
Dr. Shafia Iftikhar
2. Thin Layer Chromatography
Thin layer chromatography is a chromatographic technique in which a liquid
sample migrates by capillary action 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.
Like other chromatographic techniques, thin-layer chromatography (TLC)
depends on the separation principle. The separation relies on the relative
affinity of compounds towards both the phases. The compounds in the mobile
phase move over the surface of the stationary phase. The movement occurs in
such a way that the compounds which have a higher affinity to the stationary
phase move slowly while the other compounds travel fast. Therefore,
the separation of the mixture is attained. On completion of the separation
process, the individual components from the mixture appear as spots at
respective levels on the plates. Their character and nature are identified by
suitable detection techniques.
TLC system components consists of:
1. TLC plates: preferably ready made
with a stationary phase: These are
stable and chemically inert plates,
where a thin layer of adsorbent (i.e.
silica gel or alumina) is applied on an
unreactive sheet of glass, aluminum
foil or plastic. The stationary phase on
the plates is of uniform thickness and
is in a fine particle size.
2. TLC chamber- This is used for the
development of TLC 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.
1. Mobile phase- Mobile phase is the one that moves, and it comprises of a
solvent or solvent mixture. The mobile phase used should be particulate-free
and of the highest purity for proper development of TLC spots. The solvents
recommended are chemically inert with the sample, a stationary phase.
Commonly used mobile phase are chloroform, diethyl ether, ethanol etc.
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.
• The process starts by making a thin mark on the TLC plate’s bottom with a
pencil. It helps in the application of sample spots. These spots are kept at
• The sample is then applied to these spots made on the line.
• Then the TLC chamber is filled with the mobile phase up to a few centimetres
of its bottom.
• After pouring the mobile phase, the moistened filter paper is placed along
with the inside of the chamber wall. This helps to avoid the edge effect by
maintaining equal humidity.
• Finally, the prepared stationary phase plate is put inside the chamber. At this
point, the sample spots are kept on the mobile phase’s side.
• The chamber is then closed after placing the plate into it.
• Once enough time has elapsed for the process, the plate is taken out and
allowed to dry.
• The sample spots are then seen in a suitable UV light chamber, or any other
methods as recommended for the given sample.
• Some common techniques for visualizing the results of a TLC plate include
1. UV light
2. Iodine Staining: is very useful in detecting carbohydrates since it turns black
on contact with Iodine
6. Rf value
When you decide on a developing solvent and do thin-layer
chromatography, you get the Rf value. Rf value means the distance
that the compound has moved. The Rf value can be easily obtained
by calculating the distance between the developing solvent and the
distance the compound moved.
Rf value =
𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒖𝒕𝒆 (𝒔𝒐𝒍𝒖𝒕𝒆 𝒇𝒓𝒐𝒏𝒕)
𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 (𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒇𝒓𝒐𝒏𝒕)
For example, in the following conditions, the Rf values are 0.6 and
The more polar a compound is,
the more it interacts with the
silica gel. Since it is strongly
adsorbed by silica gel, it is
difficult to move forward, and
the Rf value decreases. On the
other hand, highly hydrophobic
compounds are less likely to be
adsorbed by silica gel, so their
Rf value is higher.
8. Applications of TLC
The applications of TLC are described below:
• Being a separation process, TLC proves to be highly effective for
separating pharmaceutical formulations that consist of multiple
• The process can be used to examine a given product’s purity.
• Medicines like local anesthetics, analgesics, sedatives, hypnotics,
anticonvulsant tranquilizers, and steroids go through the TLC procedure for
their qualitative testing.
• The cosmetic industry also uses TLC for checking the presence of
preservatives in the products.
• A given compound can be purified using TLC and then compared with a
• TLC also finds its use in Biochemical analysis. Here, it can be used for
biochemical metabolites’ separation from urine, blood plasma, serum, and
• Just like the cosmetic industry, the food industry also utilizes TLC for the
detection of preservatives, artificial colours, and sweetening agents.
• 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.
• The TLC procedure can not be used for lower detection limit experiments
because it has a high detection limit.
• It does not give reproducible results.
• TLC is limited to qualitative analysis, and it can not be used for
• TLC plates do not have long stationary phases. Therefore, the length of
separation is limited compared to other chromatographic techniques.