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3=Thin Layer Chromatography (TLC)

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II. Thin Layer Chromatography
(TLC)
• Describtion
- Identifying substances and testing purity
of compounds.
- Quick and needs small amount of
sample.
- Stationary phase is a thin layer of Silica
gel or Alumina.
- Mobile phase is usually an organic
solvent.
1
Separation of a mixture using TLC
1. Preparing the chamber:
i. Add the mobile phase (1 cm in height)
to the developing chamber (Jar).
ii. Place a filter paper in the jar
(contacted to the walls and immersed in
the solvent)
iii. Close the jar for 30 minutes. WHY?
2
Question
This jar has been standing for 30 minutes. Why
is it not ready to be used in a TLC experiment?
3
Answer
Although the jar has been standing for 30
minutes, the atmosphere inside it is not
saturated with solvent vapor because it was
not covered. Any solvent vapor would
escape the jar into the room.
4
2. Preparing the plates for development:
i. Mark the TLC plate with a pencil at 2
cm from its bottom.
ii. Spot the samples on the plate with a
capillary glass tube.
iii. If the sample amount is not enough,
add more sample.
5
Question
What is wrong with the plate shown below?
6
Answer
The samples were spotted on the plate
too closely together. During the
development of the plate, the spots will
run together, causing interference.
7
3. Developing the plates:
i. Hold the plates only from its edges.
ii. Insert the plate in the chamber
quickly. WHY?
iii. Sample spots are developed
(separated).
iv. Remove the plate and quickly mark
the solvent front with a pencil.
8
Question
Predict what will happen when this plate is
developed in the chamber.
9
Answer
The samples were spotted too low on the
plate, and the chamber is too full with the
developing solvent. Therefore, when the plate
is placed into the chamber, the spots will
dissolve in the solvent. The plate cannot be
developed.
10
4. Identifying the spots:
i. If spots can be seen, mark them with
a pencil.
ii. If no spots appear, put the plate under
an Ultraviolet lamp. When the spots
appear, mark them with a pencil.
11
Commercial TLC plate after
development
in normal light
Same TLC plate held under a UV
lamp. Note the appearance of
additional spots.
12
5. Interpreting the data:
i. Rf values are calculated as in paper
chromatography, where:
13
Question
Calculate the Rf values for the spots on the
TLC slide below.
14
Answer
Use the equation:
Measure the distances from the starting point
to the solvent front and from the starting point
to the center of each spot, and calculate the Rf
values:
15
Continue …
16
Question
Which of the samples spotted on the TLC plate
below are composed of more than one
substance?
17
Answer
Samples 1 and 3 are composed of more than
one substance, because during the TLC
experiment two or more spots separated from
the original sample. Sample 2 appear to be
composed of one substance.
18
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