CHAPTER 7B

BASIC CHROMATOGRAPHY
Planar Chromatography (PC & TLC)

JAMIL MOHAMED SAPARI

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CONTINUE…

1 2 3
Planar Planar
Chromatography Advantages of
Chromatography
TLC over PC
- Paper - TLC

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 1. PLANAR CHROMATOGRAPHY

Paper chromatography (PC)

A sheet or a narrow A thin film of a
strip of paper serves as stationary phase of

Thin-layer chromatography (TLC)

the stationary phase. solid particles bound
together for
mechanical strength
with a binder, such as
calcium sulfate, coated
on a glass plate or
plastic or metal sheet.

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a) PAPER
CHROMATOGRAPHY

• Use the technique of • Separation • Cheap, fast , sensitive, • Reproducibility of the

paper chromatography • Identification wide uses. results is low.
to separate a • Cannot withstand
homogeneous mixture corrosive chemicals.
into its individual
components

Purpose Uses Advantages Disadvantages

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 Stationary water molecules trapped in the paper or paper
phase strip
• Made up of cellulose fibers (made-up of hydroxyl groups).
• The trapped water acts as the stationary phase and the filter paper
itself is the support.

Mobile an organic solvent, such as a mixture of ethanol

phase and water.
• The mobile phase liquid will be less polar than stationary phase.
• Principle of separation: differences in the partition coefficients of
components of a mixture between two liquids

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 Paper is of type partition chromatography
chromatography

• The component to be separated are partitioned between the stationary

phase (water adsorbed on paper) and the mobile phase (the solvent).

Paper is a liquid-liquid chromatography

chromatography
• If a component is more soluble in the mobile phase, the component will
be carried further up on the paper and will be separated from the other
components.

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Principles and techniques of PC

2 Development of
HOW chromatogram

1 3 Measuring Retention
Sample Preparation
& Application factor (Rf) value
 • A sample is dissolved in a volatile solvent.
The sample is spotted using the capillary
tube on a line previously drawn on one
end of the PC.

Spotted
sample

• The spot should be dried first before the

second time as to make sure that the
diameter of the spot is less than 5 mm.
Origin line

• Chromatograph is "developed by
dipping one end in the mobile
phase.
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2) Development of chromatogram

The development
All spots are This is to make sure The solvent will carry the
tank should be
allowed to dry first that the spots various spots upward at
close so that the different rate until the
before it is dipped move upward
development solvent front is about ¾
into the smoothly by of the PC. The paper is
chamber is
development capillary action removed from the tank
saturated with the
solvent (mobile without and the solvent front is
development marked.
phase). interruption.
solvent.

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Place small a spot of the mixture
to be analysed. Dip the paper in
the solvent.

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3) MEASURING
Rf = distance solute moves (b)
RETENTION
FACTOR (RF) distance solvent front moves (a)
VALUE
Solvent front

(b) (a)

Origin line

✓ The distance the solute moves, b is measured at the

center of the solute spot or at its maximum density.
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 ✓ By comparing the Rf of samples to that ✓ Sample with same Rf value with the
of standards, the identity of the samples standard confirms its presence.
can be determined.

Measuring
Retention
factor (Rf) value

spots move
Solvent:
Filter upward by
mobile
paper: capillary
phase
stationary action
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phase
• Example of calculation of Rf value:

❖ Values below 0.1 is considered

poor (the spots are too close to
origin)
❖ Values of 0.1-0.8 (good and
any other spots (impurities)
or other active are resolved
form each other)
❖ Above 0.8 (poor, spots may be
too broad or distorted)

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TRY THIS !!!
• Calculate the Rf values for blue, purple and yellow spots.

Solvent front

blue

purple
10 cm
8 cm
6 cm
yellow

2 cm

Origin line
 a) THIN LAYER
CHROMATOGRAPHY
• TLC: A type of adsorption chromatography where the mobile
phase is a liquid (volatile solvent) and the stationary phase is a
glass plate, a thick aluminium foil or a plastic sheet coated which
with solid adsorbent such as silica gel (SiO2), alumina (Al2O3) or
cellulose.

• Thin layer chromatography is of the type planar

chromatography.

• In this technique, a small quantity of solution

of mixture to be analyzed is deposited as a
small spot on a TLC plate which consists of a
thin layer of silica gel 16
- Can be obtained with small additive of CaSO4
(increases the binding to the plates) and
flourescent indicator. Silica gel
- For separation of acidic and basic substances.

- Less polar than silica gel

Kieselguhr - Less strongly adsorbent

Solid
Adsorbents
of TLC
- Degree of adsorption depends
on its pH and the amount of
Alumina
activation.
- The spots are sharper on TLC than paper and - For separation of basic
give better separation. substances
- Available with or without binder. Cellulose
- For separation of polar substances

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 Principles of separation using a TLC
The separations of compounds
depend on the attraction The more strongly a compound
between the compound and is adsorbed, the less distance it
those of the solvent and the can travel up the plate.
stationary phase.

If the mobile phase is a polar solvent and

stationary phase is a non-polar.

- The more polar compound is more soluble in the mobile phase. So, it will be
travelled faster up the plate.
- The less polar compound will strongly adsorbed to the stationary phase and will
travel slower up the plate. 18
• If the mobile phase is a non-polar solvent
and stationary phase is a polar.

✓ The less polar compound is more soluble in the

mobile phase. So, it will be travelled faster up the
plate.

✓ The more polar compound will strongly adsorbed to

the stationary phase and will travel slower up the
plate.

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 Proses in Thin Layer Chromatography (TLC)
(a) Preparing and activating TLC plates Pre coated TLC plates are
available commercially.
(b) Spotting the TLC plates with sample

(c) Developing the TLC plates

(d) Drying the plates

(e) Visualizing the substance spots

(f) Measuring the Rf value

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Preparing TLC plates The steps involved are as follows:

1 2 3
The glass plates An aqueous The slurry is
are cleaned slurry of spread on the
with soaps and powder (SiO2 or plate to form a
any trace of Al2O3) (in water) thin film (0.1–
grease/oil on is prepared by 0.3 mm thick)
the plates is mixing with a analytical
removed by binder such as purposes and
using acetone. plaster of paris, around 0.5 –
gypsum, or poly 2.0 mm for
(viny alcohol) to preparative TLC
help it adhere by using a
to the backing spreader to
material. assure uniform
thickness.

spreader
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The plates are
air dried for ½
hour

then are heated

in the oven at
100°C for
another ½ hour

They are cooled

in a desiccators
before use.

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(b) Spotting the TLC plates with sample
1. Spotting a TLC plate first
involves preparing a glass
spotting capillary (available
commercially).

2. These capillaries are made using

7. Allow the solvent to melting point capillary which has been
evaporate from the spot. elongated and thinned down to a small
diameter capillary tube by heating.

3. To spot a TLC plate, obtain a silica gel

6. The sample mixture will be
TLC plate (4 cm x 10 cm), draw a straight
deposited on the plate as a
line parallel to the short dimension of the
small spot.
plate with a pencil.

5. Hold vertically the capillary

tube containing the sample mixture 4. Load the glass capillary
close to the line drawn on the tube with the sample
TLC plate and slowly let the tube mixture solution.
touches the plate.

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(c) Developing the TLC plates

Prepare the solvent system/ the mobile phase.

Example: For small scale prepare about 10 mL of solvent Developing the TLC
✓n-hexane: dichloromethane (9: 1) plates
✓Pet. Ether: methanol (8.5: 1.5)
✓H2O: NH3 (8: 2)

Mix the solvent well and pour the solvent system into a
closed jar.
By using forceps, place the TLC plate that has been spotted TLC Developing Chambers
inside the jar. Make sure the solvent level is below the
starting line (below the spot).
 (e) Visualizing the substance spots
If the components are colourless, the spots on TLC plates can be
UV lamp visualise by using this method:

▪ Exposing the plate to iodine vapour.

▪ Spraying the plate with suitable chemicals such as ninhydrin.
▪ Spraying the plate with conc. H2SO4 and heat.
▪ Using fluorescence indicator TLC plates which can be visualize
under UV light.

TLC plate under TLC plate under

UV light (365 nm) UV light (254 nm)
TLC plate inside the Spraying TLC plate
iodine chamber with ninhydrin
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Advantages of TLC over PC

The separation occurs much
faster & better. 1 2
Have choice between
different adsorbents.
Spots are more discrete.
Can withstand corrosive
detection reagents, without
harming the adsorbent.
4 3
 TRY THIS !!!

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TRY THIS !!!
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