Unit A23 5.4 Chromatography

Question 1

5.4.1/5.4.3 - Carrying out paper and thin-layer chromatography and measure the Rf values of the components and interpret the chromatograms

One-way chromatography

Answer 1

• Draw a base line using a pencil close to the bottom of the paper/thin-layer plate
• Spot the samples onto the paper/thin-layer plate using a capillary tube. Allow the spots to dry. Repeat to make the spots concentrated.
• Place the paper in the tank containing a shallow amount of solvent, cover with a lid, and allow the solvent to run up over the spots until the solvent almost reaches the top of the paper/thin layer plate. Mark the solvent front. Allow to dry.
• If the substances to be separated are colourless, the spots are made visible by spraying with a locating agent e.g. ninhydrin
• Measure the distance travelled by the solvent front, and the distance moved by each spot (to centre of spot) and calculate the retardation factor, Rf.

Question 2

5.4.1/5.4.3 - Carrying out paper and thin-layer chromatography and measure the Rf values of the components and interpret the chromatograms

Two-way chromatography

Answer 2

• Carry out the method for one-way chromatography
• Rotate the paper/thin-layer plate through 90°
• Run in a second solvent - place the paper in the tank containing a shallow amount of a different solvent, and allow the solvent to run up over the spots until the solvent reaches the top of the paper. Mark the solvent front
• If the substances to be separated are colourless, the spots are made visible by spraying with a locating agent e.g. ninhydrin
• Measure the distance travelled by the solvent front, and the distance moved by each spot (to centre of spot) and calculate the retardation factor, Rf for this solvent.