A.

Preparing the TLC Chamber

Prepare the chromatographic chamber as
shown in Figure 7.1.

Prepare the solvent system, mix 25 mL of Ethyl Acetate

with 1 mL each of ethanol and acetic acid in a 50-mL
graduated cylinder

Fill a 400-mL beaker with 0.5-cm high of the

solvent system

Cut a rectangular filter paper that would cover three-

fourths of the circumference of the beaker and its
height should be below the mouth of the beaker

Thoroughly moist the filter paper with development solvent.

Adjust the depth of the solvent system to 0.5 cm.

Place the filter paper into the beaker and allow it to

stick to the walls of the beaker

Cover the beaker with a watch glass or an aluminum

foil. Set aside the prepared chamber
B. Preparing the TLC Plates

Obtain two 10 cm x 5 cm TLC plates. Only hold the plates

by their edges (Do not touch the side coated with silica gel)

Place the plate’s shiny side down on paper towel

Using a pencil, draw a light straight line 0.5-cm above

one of the widths of the plate. (Solvent front)

Draw another light straight line 1-cm above the other

width of the plate. (Sample spotting position)

Make 3 evenly spaced marks along the line – one for

each reference analgesic and one for the unknown
C. Sample preparation and TLC Spotting
Place a pinch of each reference sample and unknown into
separate, clean vials and dissolve each with 5 mL Ethanol

Using clean capillary micropipettes, spot the solutions

separately on the sample marks made in the TLC plate

Label the marks spotted with the sample above the solvent
front with a pencil. (Each spot should be at least 1-mm in
diameter after spotting it three times with the sample solution

D. Developing the TLC Plate and Unknown Identification

Using a pair of tweezers, place carefully the TLC plate inside the TLC
chamber. (Do not lay any part of the TLC plate on the filter paper)

Cover the chamber with a watch glass and allow the

solvent to reach the solvent front

Remove the TLC plate and allow it to dry in air.

View the spots using UV lamp and trace the spots

lightly with a pencil.

Calculate the retention factor (Rf) of each spot based

on the center of each spot