Quick answer: Amines are organic bases because the lone pair on nitrogen accepts a proton, and amino acids contain both an amine and an acid group so they form zwitterions. This Chemistry 11-12 lab on amines and polymers gives the aim, the equipment, the safety points, a step-by-step method, sample results and 7 analysis questions; the answers are on a separate page and the lab sheet is a free printable PDF.
Colourless amino acids are hard to tell apart by eye. In this lab you separate them by paper chromatography, develop the spots with ninhydrin and use Rf values to identify the amino acids in an unknown mixture.
Level: Chemistry grades 11-12 (A Level · IB · Class 11-12) · Curriculum: A Level 3.3.11-3.3.13
Aim
To separate and identify the amino acids in an unknown mixture by paper chromatography and Rf values.
Equipment
- Chromatography paper, 20 cm × 12 cm
- Glass tank or large beaker with a lid
- Solvent: butan-1-ol, ethanoic acid and water mixed 4 : 1 : 1 by volume
- 0.1 mol/dm³ solutions of glycine, alanine, valine and leucine
- Unknown solution X
- Capillary tubes, pencil, ruler
- Ninhydrin spray (0.2 % in propanone) and a hot air dryer
- Disposable gloves and a fume cupboard
Safety
Wear gloves and eye protection. Butan-1-ol, ethanoic acid and propanone are flammable and irritant, so keep away from flames. Spray ninhydrin only in a fume cupboard; it stains skin. Wash hands after the lab.
Method
- Draw a pencil baseline 2.0 cm from the bottom of the paper and mark five points along it.
- Use a clean capillary for each solution and place a small spot at each point, drying between additions to keep spots small.
- Pour solvent into the tank to a depth of 1.0 cm, below the baseline, and put the lid on for 5 minutes.
- Hang the paper in the tank and replace the lid. Let the solvent rise until it is 2.0 cm from the top.
- Remove the paper, mark the solvent front at once in pencil and dry it in a fume cupboard.
- Spray with ninhydrin, warm gently until purple spots appear and mark their centres.
- Measure the distance from the baseline to each spot centre and to the solvent front.
Results
The solvent front moved 12.0 cm from the baseline. Sample measurements:
| Sample | Distance moved by spot / cm | Rf = distance ÷ 12.0 |
|---|---|---|
| Glycine | 3.1 | 0.26 |
| Alanine | 4.6 | 0.38 |
| Valine | 6.6 | 0.55 |
| Leucine | 8.8 | 0.73 |
| Unknown X | 4.6 and 8.8 | to be calculated |
Analysis questions
Question 1 Calculate Rf for X
Calculate the two Rf values for unknown X and compare them with the table.Question 2 Identify X
Identify the amino acids in X and justify your decision.Question 3 Pencil and ninhydrin
Explain why the baseline is drawn in pencil and why the paper is sprayed with ninhydrin.Question 4 Solvent level
Explain why the solvent level must be below the baseline at the start.Question 5 Why Rf varies
State two factors that change the Rf of an amino acid and one way to reduce their effect.Question 6 A close pair
Serine runs 3.3 cm in the same system. Calculate its Rf and say whether it could be confused with glycine.Question 7 Improve the lab
Suggest two improvements so that the identification of X is more reliable.
