FDSC 450 - Food Analysis Lab Manual

Instructor: Dr. Camila Rosa

TLC - Thin Layer Chromatography for different food pigments

 It is routinely used by researchers in the field of phytochemicals,

biochemistry, and so forth, to identify the components in a compound
mixture, like alkaloids, phospholipids, and amino acids.

Principle

Similar to other chromatographic methods, thin-layer chromatography is also

based on the principle of separation.

1. The separation depends on the relative affinity of compounds towards

stationary and mobile phase.
2. The compounds under the influence of the mobile phase (driven by
capillary action) travel over the surface of the stationary phase. During this
movement, the compounds with higher affinity to the stationary phase
travel slowly while the others travel faster. Thus, the separation of
components in the mixture is achieved.
3. Once separation occurs, the individual components are visualized as
spots at a different level of travel on the plate. Their nature or character is
identified using suitable detection techniques.

TLC system components consist of

1. TLC plates, preferably ready-made with a stationary phase: These are

stable and chemically inert plates, where a thin layer of stationary phase is
applied on its whole surface layer. The stationary phase on the plates is of
uniform thickness and is in fine particle size.
2. TLC chamber. This is used for the development of the TLC plate. The
chamber maintains a stable environment inside for proper development of
 spots. It also prevents the evaporation of solvents and keeps the process
dust-free.
3. Mobile phase. This comprises of a solvent or solvent mixture. The mobile
phase used should be particulate-free and of the highest purity for proper
development of TLC spots. The solvents recommended are chemically
inert with the sample, a stationary phase.
4. A filter paper. This is moistened in the mobile phase, to be placed inside
the chamber. This helps develop a uniform rise in a mobile phase over the
length of the stationary phase.

Materials
● Capillary tubes (or syringes) (to apply samples to plates)
● Developing tank, with lid
● Food pigments
● Pencil
● Thin-layer chromatography plates: aluminium sheets silica gel 60 F 254, 0.2 mm
thick coating, 20 × 20 cm.
● Solvent: Chlorofom/Methanol (3:1).

Procedure

2. With a pencil, make a thin mark 2 cm from the bottom of the plate to apply the
sample spots.
3. Make marks with a pencil to divide the plate into 4 “lanes” of equal width.
4. Use capillary tubes. The application should be done as a streak across the
center of the lane origin.
5. Below the origin line, write the identity of the sample/standard in each lane.
6. Allow spots to dry.
7. Apply more sample to the same spot.
8. Write your name and the solvent used in the top right corner of the plate.
9. Write the name or numbers of the sample in the bottom of the lines.
10. Add a thin layer of solvent (Chloroform/Methanol – 3:1) in the chamber.
11. Place the plates with the sample line facing the mobile phase, such that the
sample spots are above the level of mobile phase (but not immersed in the
solvent) for development (figure 1).
12. Close the lid and observe the mobile phase travels over the stationary phase.
13. Calculate the retention factor (Rf) of all dots on the plate. The Rf is defined as
the ratio of the distance travelled by the solute to the distance travelled by the
solvent front (figure 2).

Figure 1: TLC system components.

Figure 2: Demonstration on how to calculate the retention factor (Rf)