Planar Chromatography

Presented by FEROSEKHAN. S , CIFE, MUMBAI

1. PLANAR
CHROMATOGRAPHY
Ferosekhan . S
FNB-41

2. Chromatography
Chromatography is a technique for separating mixtures
into their components in order to analyze, identify, purify,
and/or quantify the mixture or components.
Separate
• Analyze
• Identify
• Purify
• Quantify
ComponentsMixture

3. 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)

4. Classification Of Chromatography
chromatography
Liquid chromatography
Gas chromatography
Flat chromatography Column chromatography
Paper
chromatography
Thin layer
chromatography
TLC
Ascending Two dimensionlal Circulardescending
Open column e.g
Partition chromotography
Adsorption chromotography
Ion exchange
chromotography
Gel filtrtion chromotography
Affinity chromotography
High performance
liquid
chromotography

5. 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.

6. 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)

7. THIN LAYER
CHROMATOGRAPHY
(TLC)

8. 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

10. Cont…
TLC to separate lipids
 Surface of the plate - very thin layer silica – SP
 Silica – polar (stationary phase)
 Spot the material at the bottom of the TLC plate

cellulose
Al2O3

11. 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.

12. Thin-Layer Chromatography: A Two-
Component Mixture
More polar!
Less polar!
solvent front
originmixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time

13. Thin Layer Chromatography
TLC plate
O O O
| | |
-O-Si-O-Si-O-Si-O-H
| | |
O O O
| | |
-O-Si-O-Si-O-Si-O-H
| | |
O O O
silica gel - silicon dioxide (SiO
2
)
x
(a common, inexpensive stationary phase)
bulk (SiO
2
)
x

These exposed OH units
give silica gel a
relatively polar surface.
surface

14. 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.

15. TLC plate
“finishing line”
}
1 cm.
A. Draw “guide lines”
lightly with pencil
“starting line”
}
1 cm.
B. Dissolve solid
sample in MeOH
C. Use TLC capillary
to transfer and spot
dissolved sample
Sample A B C
Ref. Ref. Ref.
1. Sample Application (spotting)

16. 2. Development of TLC
Plate
TLC plate
TLC Developing Chamber
(just a glass jar with solvent in it!)
A. Place spotted 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
Developing
solution
(mobile phase)
}
{keep capped}
NOTE: During this ~20 min.
developing stage, compounds
in the original spots are being
pulled through the silica gel.

17. 3. Visualization of TLC Results
A. Allow solvent to evaporate
from surface of TLC plate.
C. Mark spots with a pencil
while viewing under UV.
UV
B. View results under UV light.
look for grayish spots on the
fluorescent green background

18. 4. Interpretation of TLC Results
A. Determine retention factors
(R
f
) for each spot detected.
B. Use R
f
’s of reference spots to
identify the other components.
distance spot has moved
distance solvent has moved
_______________________
R
f
= =
X
Y
Y
- - - - - - - - - - - - - - - - - - - - - - -
X
3
- - - - - - - - - - - - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
X
1
X
2
How do you interpret
any other spots?
4
1
3
2
?
?

19. 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

20. Separation of Total Lipid into
different Classes
Mobile Phase: hexane: diethyl ether: formic acid (80:20:2)
Cholesteryl esters
TAG
Free fatty acids
Cholesterol
1,3-DAG
1,2-DAG
Monoacyl glycerols
Phospholipids

21. Separation of Triacylglycerols
Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1)
Tristearin
2-oleodistearin
1-stereodiolein
Triolein
Trolinolein
With HUFA

23. 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

24. Paper Chromatography

25. 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

26. 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 )

27. Place 25 mL of solvent in a 600 mL beaker. Cover
the beaker and set it aside.
25 mL
1 cm
2 mm
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.
Procedure

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

29. 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…

30. 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…

31. 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…

32. Cont…
Determine the R
F
values for
each colored spot in the
knowns and the unknown.
a
b
c
dRR
F(a)F(a) = =
aa
dd
Use your computed R
F

values to identify the
components of your
unknown.

33. 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