Required Practical 4: Chromatography

Question 1

Independent variable

Answer 1

Pigment solubility

Question 2

Dependent variable

Answer 2

Distance moved by pigment on chromatography paper

Question 3

Method

Answer 3

1) A mixture is dissolved in a fluid (called the mobile phase) and passed through a static material (called the stationary phase)

2) The different components of the mixture travel at different speeds, causing them to separate

3) A retention factor can then be calculated (Rf value = distance component travels ÷ distance solvent travels)

Question 4

What are the two common techniques for separating photosynthetic pigments?

Answer 4

-Paper chromatography – uses paper (cellulose) as the stationary bed

-Thin layer chromatography – uses a thin layer of adsorbent (e.g. silica gel) which runs faster and has better separation

Question 5

Conclusion/explanation

Answer 5

-If you did a number of chromatographic separations, each for a different length of time, the pigments would migrate a different distance on each run.

-However, the migration of each pigment relative to the migration of the solvent would not change.

-This migration of pigment relative to migration of solvent is expressed as a constant, Rf (Reference front). It can be calculated by using the formula:

Rf = distance pigment migrated/ distance solvent front migrated

Question 6

What is chromatography?

Answer 6

The process of separating chemicals in a mixture, where some pigments are more soluble than others and the distance they travel is contingent upon that.

Question 7

Diagram showing a simplification of two chromatography results

Answer 7

Question 8

What can you conclude from the results in Figure 2?

Answer 8

Species 2 does not contain phaeophytin, but species 1 does.

Question 9

Safety

Answer 9

-A lab coat and eye protection must be worn at all times.

-You will need to follow your teacher’s instructions for using the alcohol solvent: as well as wearing protective clothing, you should work in a well-ventilated area.

-Do not inhale the solvent vapours.

Question 10

Diagram of apparatus

Answer 10

Question 11

More in depth method (from Kognity)

Answer 11

-Grind up a leaf in a mortar and pestle, using a small amount of sand to help grind up the sample and break down the cell walls - this will release thepigments without too much damage to the pigment molecules

-Add a small amount of solvent and grind up the mixture further to dissolve the pigments in the solvent.

-Once the solvent is dark green, let the solids settle and pour the solvent (now containing the sample of pigments) into a small beaker and allow to dry.

-Using a toothpick or small paintbrush, apply the pigment sample to a spot that is at least 20 mm from the bottom of your paper or TLC strip.

-Ensure that the pigment sample is above the solvent at the beginning of the experiment

-Carefully remove the stationary phase when the solvent line is nearing the top of the strip. Do not let the solvent pass the top of the strip. If this occurs, it is likely that the pigments, which were separated as they travelled up the stationary phase, will move closer together again at the top as the solvent continues to move them up the stationary phase: thus it would be more difficult to measure their different distances.

-Remove the paper or TLC strip from the mobile phase.

Question 12

Diagram of mistakes to avoid in set-up

Answer 12

Question 13

What observations should you describe?

Answer 13

You should record observations of the colour of each of the spots, as this may help you identify the pigments.

Question 14

What do you do after you’ve found the Rf value?

Answer 14

-Use this value in your analysis.

-You might try to identify the pigments based on their Rf values and their colors.