PAG 06 - Chromatography or Electrophoesis

What is chromatography used for?

It is used to separate mixtures and identify substances (such as pigments, drugs, or contaminants).

What is the stationary phase in paper or thin-layer chromatography (TLC)?

• The chromatography paper or the TLC plate onto which the mixture is placed.

What is the mobile phase in chromatography, and why is it chosen?

The solvent that flows through the paper or plate. It is chosen because it can dissolve the molecules being tested.

What three factors determine how far different molecules travel up the paper or plate?

• The balance between their attraction to the stationary phase (paper/plate), their solubility in the solvent, and their size.

How can you make colourless spots visible on a TLC plate using light?

Use TLC plates that fluoresce under UV light, which makes the separated spots appear dark.

How can iodine be used to reveal colourless substances on a chromatography plate?

Leave the plate with iodine crystals in a sealed container.

What chemical is sprayed onto a chromatogram to make amino acids visible?

Ninhydrin.

What is the formula for calculating an \(R_{f}\) value?

$Rf$ = distance from starting line to pigment ÷ distance from starting line to solvent front

How do Rf values help identify unknown substances, and what are two real-world uses of chromatography?

Specific molecules have specific Rf values under the same conditions, allowing comparison against known standards.

• Real-world uses include testing urine for drugs and testing food or medicines for contaminants.

How do you prepare the TLC plate or chromatography paper before running it ( 2 steps)?

1 - Handle the plate by the edges only (wear gloves) to avoid contamination from skin oils/amino acids.

2 - Draw a pencil line (the origin) about 1–2 cm from the bottom edge

How should you apply the sample onto the origin line, and why?

Use a capillary tube to place a small, concentrated dot (under 2 mm wide) on the pencil line.

Allow the spot to dry completely between repeated applications (around 5–10 times) so the pigment stays concentrated and does not spread out or overlap.

How do you set up the chromatography tank/tube to run the chromatogram?

Place the plate/paper upright into a sealed container with a small amount of running solvent, ensuring the solvent level is below the pencil origin line.

Place a lid/bung on top to prevent the volatile solvent from evaporating.

What do you do when the solvent nears the top of the plate/paper?

Remove the chromatogram before the solvent reaches the very top edge and immediately mark the solvent front with a pencil before the solvent evaporates. Leave it to dry

Why must you draw the origin line and solvent front in pencil rather than pen?

Pencil graphite is insoluble in the solvent, whereas pen ink would dissolve and run up the chromatogram, interfering with the results.

Why must the solvent level in the tank stay below the sample spots on the origin line?

If the spots are submerged, the samples will dissolve directly into the solvent reservoir at the bottom instead of travelling up the stationary phase to separate.

How do you measure distances accurately to calculate an Rf value?

Measure in millimetres from the origin line to the centre of each spot, and divide by the distance from the origin line to the solvent front.

Name two key safety precautions required when carrying out PAG 06 chromatography.

1. Solvents (like propanone/petroleum ether) are highly flammable—keep away from naked flames.

2. Solvents are harmful/irritants—wear eye protection and work in a fume cupboard or well-ventilated room.