Lab Manual

CBB 20303 Biochemistry

EXPERIMENT 8: Thin Layer Chromatography (TLC)

Objective:
To detect lipid and plant pigments using TLC
Pre-lab Reading:

Principles of TLC
Mobile phase and stationary phase
Principle steps in TLC separation
Application of TLC

Procedure:
(A) Preparation of TLC plate
1. Wear gloves when handling the TLC plates.
2. Cut the TLC plate into strips of 2 cm 10 cm in size.
3. With a pencil, draw a line approximately 1.5 cm from the short edge of the TLC plate.
Mark another line approximately 1.0 cm from the edge of the other side. Be careful to not
scratch the surface of the plate too deeply.

1.5cm
m
(B) Lipid detection

1 cm

1. Mix 26mL acetone, 4 mL toluene, 4 mL water, and 1 mL ammonium hydroxide.

2. Pour 10 mL of this solution into the TLC chamber.
3. Dip a micropipette tip into triglyceride solution and then gently touch the end of it onto
the 1.5 cm line on TLC plate. Make sure each spot is as small as possible, about 1 mm in
diameter.
4. Dry the spot with hair dryer. Repeat step 3 on the same spot until the spot is clearly
visible
5. Develop the plate in the closed TLC chamber until the solvent front reaches to within 1
cm from the top of the TLC plate.
6. Dry the plate in 60 C oven for 3 minutes.
7. Spray bromophenol blue solution on the developed plate.
8. Observe the spot and calculate its Rf value.

Lab Manual

CBB 20303 Biochemistry

(C) Plant pigment detection

The pigments in vegetables, flowers and leaves can be separated and identified by using thinlayer chromatography. Green pigments, known as chlorophylls, serve as the main photoreceptor
molecules of plants. Carotenoids, yellow pigments, aid the plant in the photosynthesis process.
In addition, xanthophyll is contained in the chloroplasts which can be isolated and identified
using chromatographic techniques.
1. Place the sample in a mortar.
2. In the fume hood, add approximately 5 ml of acetone and carefully grind the sample with
the pestle. Continue grinding until a concentrated extract is obtained. More acetone may
be added to compensate for evaporation.
3. Dip a micropipette tip into the solution and then gently touch the end of it onto the 1.5 cm
line on TLC plate. Make sure each spot is as small as possible, about 1 mm in diameter.
4. Dry the spot with hair dryer. Repeat step 3 on the same spot if necessary.
5. Develop the plate in the TLC chamber with mobile phase (70:30 hexane-acetone solvent)
until the solvent front reaches to within 1 cm from the top of the TLC plate.
6. Dry the plate in 60 C oven for 3 minutes. Lightly circle the individual spots with a
pencil because the pigment will eventually fade.
7. Observe the spot and calculate its Rf value. Identify each spot based on table below.
Pigment

Color

Rf value

carotene

yellow-orange

0.93

pheophytin a

grey

0.55

pheophytin b

light grey (may not be visible)

chlorophyll a

blue-green

0.46

chlorophyll b

green

0.42

xanthophylls

yellow

0.41

xanthophylls

yellow

0.31

xanthophylls

yellow

0.17

0.47-0.54