238 12 Chromatographic Separations

12.3.3 Procedure

1. Lay the chromatographic paper sheet flat on the rough filter paper using gloves.
2. Draw a base line 5 cm from one of the edges of the paper.
3. Draw another line perpendicular to the first line again 5 cm away from the
adjacent edge.
4. Apply 60 ml of the sample solution or given mixture containing unknown amino
acids at the point of intersection of these two lines. The sample should be
applied in small volumes at a time with the help of a micropipette with
intermittent drying to ensure that the zone of applied solution is as small as
possible.
5. Repeat the same procedure for a mixture of three standard amino acids using a
separate chromatographic sheet for each mixture. The composition of mixture
of standard amino acids should be such that each amino acid is present in at
least two different mixtures so that its identity can be established.
6. Hang the paper in the chromatographic tank whose interior has previously been
saturated with aqueous phase of solvent system No.1 (Butanol: Acetic acid:
Water mixture is 4:1:5).
7. After allowing an equilibration period of half an hour, pour the solvent No.1
into the trough of the chamber and let it run till t\it is about 10 cm from the
opposite edge of the paper.
8. Take the paper out, air dry it, turn it at 90
C angle and now develop the paper in
the second chromatographic chamber using the solvent system No.2 (Phenol:
Water).
9. Remove it when the solvent has travelled upto about 10 cm from the
opposite end.
10. Dry it at room temperature and spray it with ninhydrin reagent. After air drying
it, keep the chromatogram in an oven at 105
C for 10 min. Mark the blue and/or
purple coloured zones which appear on the paper.
11. Calculate the Rf value of the standard amino acids and those in given mixture as
given in previous experiment in both the solvents. From these values identify
the amino acids in the given mixture.

12.4 Identification of Lipids by TLC

12.4.1 Principle

Lipids generally exists as lipoprotein complexes and these need to be isolated.

Lipids, being soluble in non-polar organic solvents and proteins being soluble in
polar aqueous solvents, the efficient lipid extraction can be achieved only when an
aqueous solvent like ethanol or methanol is included in the non-polar organic
solvent like chloroform and diethyl ether. This would help in breaking the
12.4 Identification of Lipids by TLC 239

lipoprotein complexes. Extracted lipid components can be separated on TLC base

on their differential mobility along the porous stationary phase such as silica gel and
these can be located by spraying the plates with either 2’,7’-dichlorofluorescein or
50% sulfuric acid.

12.4.2 Reagents and Materials

1. TLC tank
2. Oven set at 110
C
3. Glass plates (20  20 cm) for TLC
4. Ultraviolet lamp
5. Solvent system: Petroleum ether (b.p. 60–70
C) or hexane: diethyl ether: glacial
acetic acid (80:20:1, u/v)
6. Lipid standards: Various lipids such as cholesterol acetate, vitamin A palmitate,
triacyl glycerol (e.g., trioleate, tripalmitate, tristearate)
7. Sulfuric acid (50%, u/v)
8. 2’,7’-dichlorofluorescein: Prepare 0.2% solution of 2’,7’-dichlorofluorescein in
95% (u/v) ethanol

12.4.3 Procedure

1. Extraction of lipids from sample: Grind 1 g of the tissue in the extraction solvent
[either ethyl ether: ethanol (3:1) or chloroform: methanol (2:1)] in pestle and
mortar. Transfer the homogenate to a separating funnel. Shake the contents
vigorously and allow it to stand till the two phases have completely separated.
Drain out the lower organic layer which contains the lipids. Evaporate the
solvent under organic layer which contains the lipids. Evaporate the solvent
under vacuum and keep the concentrated lipid extract protected from light under
N2 almosphere.
2. Prepare the TLC plates using Silica Gel G as the adsorbent as described in
previous experiment.
3. Activate the TLC plates at 110
C for 30 min, cool them in a desiccator and spot
the lipid samples, standards as well as unknown sample.
4. Develop the plates in the solvent system consisting of petroleum ether or hexane:
ethyl ether: glacial acetic acid (80:20:1) till the solvent has travelled upto 1 cm
from the opposite side of the plate.
5. Remove the plate and allow it to air dry.
6. Locate the lipid spots by either of the following methods:
(i) Spray the plate with 2’,7’-dichlorofluorescein and examine it under UV light.
Lipids show up as green fluorescent regions against the dark background.
240 12 Chromatographic Separations

(ii) Spray the plate carefully with 50% H2SO4 and heat it in an oven at 110
C
for 10 min. Areas containing lipids gel charred and appear as black spots.
7. Calculate the Rf value of the lipid components in the sample and identify them
by comparing their Rf values with lipid standards.

12.5 Separation of Pigments by Adsorption Column

Chromatography

12.5.1 Principle

Different pigments get adsorbed to alumina to different extents. They can be selec-
tively desorbed by using mobile phase of increasing polarity in a stepwise manner.

12.5.2 Materials and Reagents

1. Leaves/flowers
2. Pestle and mortar
3. Glass column
4. Whatman No.1 filter paper
5. Alumina
6. Benzene: methanol (2:1)
7. Sodium sulphate (anhydrous)
8. Acetone

12.5.3 Procedure

(a) Preparation of extract:

1. Homogenize 5 g leaves or flowers in a pestle and mortar, using sand as an
abrasive in 20 ml of benzene: methanol (2:1) adding a small amount of this
extractant at a time.
2. Filter the extract through Whatman No.1 filter paper and transfer the filtrate
to a separating funnel.
3. Add 10 ml of water to the filtrate and after shaking the contents and allowing
the phase to separate out, drain out the lower aqueous methanol layer. Repeat
this step. Avoid very vigorous shaking.
4. Collect the benzene layer in a beaker and add small amount of solid
anhydrous Na2SO4 to remove the traces of moisture.
5. Decant the clear benzene layer to another beaker and concentrate the extract
by evaporating the solvent on a boiling water bath.