6.1 Identification of the amino acids in a protein using paper chromatography

Aims

• To separate amino acids in a mixture

• To identify amino acids using their Rf values

• To understand how substances move differently based on solubility and interactions

Independent variable (what you change)

Type of amino acid present in the mixture

Dependent variable (what you measure)

• Distance travelled by each amino acid spot

• Rf value calculated from distances

Control variables (what you keep the same)

• Amount of sample applied

• Time left in solvent

• Temperature

Limitations

• Uneven spotting → inconsistent results

• Paper contamination (touching paper affects results)

Improvements

• Use a sealed container to reduce solvent evaporation

• Repeat experiment and calculate mean Rf values

• Use capillary tubes carefully for consistent spot size

• Keep environment controlled (temperature, humidity)

Chromatography paper

• Acts as the stationary phase

• Allows separation based on different movement rates

Chromatography solvent

• Acts as the mobile phase

• Carries amino acids up the paper

Gas jar (chromatography chamber)

• Contains solvent and creates a saturated atmosphere

• Prevents solvent evaporation and ensures even movement

Pencil

• Used to draw lines because it is insoluble in solvent

• Prevents interference with results

Capillary tubing

Applies small, precise spots of sample

Ninhydrin spray

• Reacts with amino acids to produce purple spots

• Makes otherwise invisible amino acids visible

Hair dryer / oven (110°C)

• Speeds up drying and colour development

• Intensifies visibility of spots

Ruler

Measures distances for Rf calculations

Gloves

Prevent contamination and protect from hazardous chemical