PLANAR

CHROMATOGRAPHY

Ferosekhan . S
FNB-41
 Chromatography
Chromatography is a technique for separating mixtures
into their components in order to analyze, identify, purify,
and/or quantify the mixture or components.

• Analyze
Separate • Identify
• Purify

Mixture Components
• Quantify
Mechanisms Of Separation

 Partitioning equilibrium ( SP: liquid, MP: liquid/gas )

 Adsorption equilibrium ( SP: solid, MP: liquid )

 Exclusion equilibrium ( SP&MP: liquid )

 Ion Exchange equilibrium ( SP: solid ion-exchanger,

MP: liquid electrolyte )
 Affinity equilibrium ( SP: immobilised ligand, MP:
liquid)
 Classification Of Chromatography
chromatography

Gas chromatography
Liquid chromatography

Flat chromatography Column chromatography

Open column e.g

Partition chromotography
Paper Thin layer Adsorption chromotography
High performance
chromatography chromatography Ion exchange liquid
TLC chromotography
Gel filtrtion chromotography chromotography
Affinity chromotography

Ascending descending Circular Two dimensionlal

 Classification

 Column Chromatography

the stationary phase is held in a narrow tube through

which the mobile phase is forced under pressure or by
gravity.

 Planar Chromatography

the stationary phase is supported on a flat plate or

the interstices of a paper and the mobile phase moves
through the stationary phase by capillary action or by
gravity.
 Planar Chromatography - Types
 Thin layer chromatography (TLC)
separates dried liquid samples with a liquid solvent
(mobile phase) and a glass plate covered with a thin
layer of alumina or silica gel (stationary phase)

 Paper Chromatography (PC)

separates dried liquid samples with a liquid solvent
(mobile phase) and a paper strip (stationary phase)
 THIN LAYER
CHROMATOGRAPHY
(TLC)
 Thin layer chromatography (TLC)

 In TLC, any substance that can be finely divided

and formed into a uniform layer can be used.

 Both organic and inorganic substances can be

used to form a uniform layer for TLC.

 Organic substances include: cellulose,

polyamide, polyethylene

 Inorganic: silica gel, aluminum oxide and

magnesium silicate
Cont…

 TLC to separate lipids

cellulose Al2O3
 Surface of the plate - very thin layer silica – SP

 Silica – polar (stationary phase)

 Spot the material at the bottom of the TLC plate

Cont…

 Place the plate into a glass jar - small amount of

a solvent

 This solvent - moving phase.

 Remove the plate from the bottle when the

solvent is close to the top of the plate.
Thin-Layer Chromatography: A Two-
Component Mixture
solvent front

component B Less polar!

solvent front

component B

component A More polar!

component A

origin mixture origin origin

solvent front

Increasing Development Time

 Thin Layer Chromatography

silica gel - silicon dioxide (SiO2)x TLC plate

(a common, inexpensive stationary phase)

O O O
| | |
O−
−Si−O−
Si−O−
Si−O−
H
| | | These exposed OH units
O O O
give silica gel a
| | |
O−
−Si−O−
Si−O−
Si− Hrelatively polar surface.
O−
| | |
O O O

bulk (SiO2)x surface

 Four Stages in TLC

1. Sample Application - Capillary used to spot solution of each sample.

2. Development - This is when the separation actually occurs.

3. Visualization - viewed under UV light.

4. Interpretation of Result - Comparison of retention factors.

 1. Sample Application (spotting)
TLC plate
A. Draw “guide lines”
lightly with pencil “finishing line”
} 1 cm.

B. Dissolve solid
sample in MeOH

C. Use TLC capillary

to transfer and spot
dissolved sample “starting line”
Sample A B
Ref. Ref.
C
Ref.
} 1 cm.
 2. Development of TLC
Plate
{keep capped}

A. Place spotted TLC plate TLC plate

in developing chamber

B. Developing solution
is drawn up the plate
by capillary action

C. Remove TLC plate

when solvent
reaches top line

NOTE: During this ~20 min.

developing stage, compounds
Developing
solution
}
in the original spots are being (mobile phase) TLC Developing Chamber
pulled through the silica gel.
(just a glass jar with solvent in it!)
 3. Visualization of TLC Results

A. Allow solvent to evaporate

from surface of TLC plate.
B. View results under UV light.
look for grayish spots on the UV
fluorescent green background

C. Mark spots with a pencil

while viewing under UV.
4. Interpretation of TLC Results

A. Determine retention factors

(Rf) for each spot detected.

distance spot has moved

_______________________ X
Rf = distance solvent has moved = Y
- - - - - - - - - - - -1- - - - - - - - - - - - - - - - - - -
B. Use Rf’s of reference spots to
identify the other components.
?
- - - - - - - - - - - - - 2- - - - - - - - - - - - - -
Y
How do you interpret X1 3 ?
any other spots? X2
- - - -4 - - - - - - - - - - - - - - - - - - -
X3
 Applications
1. Separation of carbohydrates:

Mobile phase:
acetonitrile : water (85:15)

Detection:
sulfuric acid : methanol (1:3)
heat for 10 min at 110 C to see
brown spots
 Separation of Total Lipid into
different Classes

Cholesteryl esters

TAG

Free fatty acids

Cholesterol

1,3-DAG
1,2-DAG

Monoacyl glycerols

Phospholipids

Mobile Phase: hexane: diethyl ether: formic acid (80:20:2)

 Separation of Triacylglycerols

Tristearin

2-oleodistearin

1-stereodiolein

Triolein

Trolinolein

With HUFA

Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1)

 Paper Chromatography
 Purpose

Use the technique of paper chromatography to separate a

homogeneous mixture into its individual components

 Uses

Separation
Identification

 Chromatography paper

Stationary phase

 Solvent

Mobile phase
Paper Chromatography
Cont…
 Paper chromatography is a variant of partition
chromatography procedure in which the
cellulose support is in the form of a sheet or
paper

 Cellulose contain a large amount of bound water

even when extensively dried

 Partitioning occurs between the bound water

and the developing solvent
Cont…

 In paper chromatography the mixture to be

separated is spotted onto the paper and dried

 Then the solvent flows along the sheet either by

gravity ( descending chromatography ) or
capillary attraction (ascending chromatography )
 Procedure
 Place 25 mL of solvent in a 600 mL beaker. Cover
the beaker and set it aside.
 Obtain a piece of chromatography
paper and draw a line 1 cm from the
bottom with a pencil.
 Place a small spot of each indicator
on the line. 25 mL

2 mm

1 cm
Cont…
 Spot and label each of the four indicators and one
of the unknowns.
 The spots should be about 2 cm apart.
 When the spots have dried, re-spot each one.

2 cm
Cont…
 When the spots have dried, form the
paper into a cylinder with the spots
facing out. Staple the edges together
being careful to keep them straight
and not allowing them to touch.

 Place the cylinder into the 600 mL

beaker and replace the cover. Be
sure the cylinder is not touching the
sides of the beaker.
Cont…
 Let the chromatogram develop until the
solvent is 2 cm from the top of the paper.
 Remove the chromatogram from the
beaker and immediately mark the
solvent front with a pencil.
 Allow the chromatogram to dry before
going to the next step.
Cont…
 Take the chromatogram to the
hood and lightly mist it with water.
Place it in the ammonia
chamber.

 Remove the cylinder

from the ammonia
chamber and unroll it.
Immediately circle
the colored regions
with a pencil.
Cont…

 Determine the RF values for

each colored spot in the
knowns and the unknown.
a
RF(a) = d
d c

 Use your computed RF b

a
values to identify the
components of your
unknown.
 Paper Chromatography-Applications

 Separation of amino acids

Mobile phase: butanol : acetic acid: water(4:1:1)

Detection: spray with ninhydrin reagent

 Separation of carbohydrates:

Mobile phase: ethylacetate : pyridine water(10:4:3)

Detection: 1. silver nitrate (1 ml in 200 ml of acetone)
2. 40% NaOH in methanol gives brown
spots