Aim:

To separate the mixture of amino acids (serine, phenylalanine, and lysine) by thin layer chromatography

Introduction:

Thin layer chromatography, abbreviated as TLC, is an analytical tool that is frequently used in chemistry laboratories to study the purity of organic compounds or to separate and analyze the components of complex mixtures. TLC is a solid-liquid form of chromatography i.e. the stationary phase is a solid while the mobile phase is a liquid. The stationary phase is prepared by coating a very thin layer of a polar adsorbent on a rectangular solid support. The solid support can be a glass, plastic, or a metal plate such as Aluminum. Silica (SiO₂· x H₂O) or alumina (Al₂O₃· x H₂O) are the most commonly used adsorbents (Figure 21.1) and are coated as uniformly thin layers on the solid supports. Analytical TLC plates usually have ~ 250 μm thick adsorbent layers. The plates used for preparatory separation can be up to 5 mm thick. A binding agent such as calcium sulfate or gypsum is usually incorporated to ensure the binding of the adsorbent to the solid substrate.

Figure 21.1: Structure of silica

A typical TLC experiment can be divided into following stages:

Preparation of plates

TLC plates are commercially available and are not very expensive. One can, however, prepare the TLC plates by oneself. The commercially available TLC plates usually have plastic or glass as the stationary phase support. The plates are sometimes purchased as large square sheets and one has to cut the desired size (usually a rectangular plate of 1–2 cm × 5 cm) from these large sheets. The plate of desired size can easily be cut using a scissors if the plates have plastic support. For glass backed TLC plates, one has to carefully cut the small plates using a diamond glass cutter. Cutting the glass plates is a straightforward procedure but requires some practice otherwise you can end up breaking the glass:

a. Take the large TLC plate and place it on a clean, smooth, and dry surface with the adsorbent layer facing down.
b. Take a ruler and draw parallel lines with a pencil to mark the places where you want the cut.

c. Place the ruler on the first line and firmly score the plate with one sweep of the diamond glass cutter (Note 1).

d. Place the plate with the score along the edge of your work-bench for the support and try to break the plate in a clean straight line.

e. Generate smaller plates repeating the steps a – d.

Once the plate of required dimensions is available, a line, parallel to the smaller side and at ~1 cm distance from one of the two small edges, is drawn using a pencil. The compound or the mixture of compounds to be analyzed is dissolved in an appropriate volatile solvent and spotted on the line. The spot on the plot is allowed to air-dry.