Required Practical 6 – Chromatography

Aim

Investigate the coloured components of a mixture using chromatography by comparison to known colours.

General Scientific Background

Chromatography is used to separate mixtures and help identify substances. Different chemicals have:

• Different solubilities in the solvent

• Different attractions to the chromatography paper

So they separate and travel different distances → creating separate spots.

A chemical that is more soluble in the solvent will travel further up the paper.
A chemical that is more strongly attracted to the paper will travel a shorter distance.

To compare substances we calculate the Rf value:

Rf values are always between 0 – 1, and substances with identical Rf values in the same solvent are likely to be the same compound.

Chromatography is a powerful way to test the purity of a substance.

✔ Pure substances

A pure substance contains only one chemical, so on a chromatogram it produces:

• Only ONE spot in a vertical column above the sample.

• No matter how many times the experiment is repeated.

• And the spot appears in the same position each time (same Rf value).

✔ Impure substances (mixtures)

A mixture contains more than one chemical, so it will produce:

• Two or more spots in a vertical column above the sample.

• Each with its own Rf value.

• Showing that the substance is made of different components.

Variables

Independent variable

Type of dye (A, B, C, D, X - unknown).

Dependent variable

Distance moved by each dye (mm) → used to calculate Rf.

Controlled variables

• Solvent volume

• Solvent type

• Size of the chromatography paper

• Starting spot size

• Same pencil baseline height

• Temperature of solvent

Apparatus

• 250 cm³ beaker

• Glass rod/wooden splint

• Chromatography paper

• Capillary tubes

• Solvent (e.g. water/ethanol)

• Known food colours (Sample 1–6)

• Unknown sample (X)

• Pencil

• Ruler

• Lid (to stop solvent from evaporating)

Method

1. Draw a horizontal pencil line 2 cm from the bottom of the chromatography paper.
Mark five pencil spots evenly spaced along the line.

2. Use capillary tubes to place small spots of food colouring 1–6 and X onto the pencil marks (each spot < 5 mm diameter). Allow to dry.

3. Pour water (solvent) into a beaker to a depth of no more than 1 cm.

4. Attach the top of the chromatography paper to a glass rod/wooden split using tape/paper clip and hang it in the beaker so:

• the baseline is above the solvent,

• the paper does not touch the sides.

5. Place the lid over the top of the beaker and leave the chromatogram until the solvent front travels about ¾ up the paper. Do not disturb the beaker.

6. Remove the paper and immediately mark the wet solvent front with pencil.

7. Allow the paper to dry and measure the following:

• Distance moved by solvent (baseline → solvent front).

• Distance moved by each spot (baseline → centre of each spot).

8. Calculate Rf values using:

X contains two substances:

• Orange (Rf 0.88)

• Red-medium (Rf 0.38)

✔ Substances that match X:

X Does one sample contain BOTH components of X?

No.

• Samples 2 and 4 have the orange spot, but not the red (middle height). And 4 has an extra blue spot which is not in X.

• Samples 3 and 5 have the medium red, but not the orange and sample 5 has an extra red spot (low height).

➡ Therefore: No sample fully matches X but it is a mixture of 2 and 3.

Recall

Apply

Challenge