Chromatography and Paper Chromatography
Chromatography is a technique for separating the components of a mixture by letting them move at different speeds through a stationary phase carried by a moving solvent called the mobile phase. In paper chromatography the stationary phase is paper and the mobile phase is a solvent that climbs up the paper. In brief: a black ink spot placed on filter paper splits into several colored spots as water rises, which shows that the ink is a mixture of dyes.
Chromatography is a powerful way to separate and identify substances, even when only a tiny sample is available. It is used in forensic laboratories, food testing, medicine and environmental monitoring. This article explains how paper chromatography works, how to carry it out, how to calculate Rf values, a worked example with data, and the history and mistakes to avoid.
How does chromatography work?
Every chromatography setup has two phases. The stationary phase stays in place, and the mobile phase moves through it, carrying the mixture. Each component of the mixture is attracted to both phases to different degrees. A component that is strongly attracted to the solvent and weakly held by the paper moves far and fast. A component that is held strongly by the paper moves only a short distance.
Because each substance has its own balance of attractions, the components of a mixture spread out at different heights and form separate spots or bands. The movement of the solvent through the paper is caused by capillary action, the same effect that draws water up a paper towel.
How do you carry out paper chromatography?
- Draw a pencil line about 1.5 cm from the bottom of a strip of chromatography paper. Pencil is used because ink would run with the solvent.
- Place a small spot of the mixture on the line, let it dry, and add more layers if needed to make the spot concentrated.
- Pour solvent into a beaker to a depth of about 1 cm, so that the level is below the pencil line.
- Lower the paper into the beaker so that the bottom edge touches the solvent, and cover the beaker to keep the air saturated with vapor.
- Remove the paper when the solvent front is near the top, and immediately mark the position of the solvent front with a pencil.
- Let the paper dry and mark the center of each spot.
What is the Rf value?
The retention factor, Rf, describes how far a substance moves relative to the solvent:
Rf = distance moved by the substance ÷ distance moved by the solvent front
Both distances are measured from the starting line. The Rf value is always between 0 and 1 and has no units. For a given substance, solvent and type of paper under the same conditions, the Rf value is nearly constant, so it can be used to identify the substance by comparison with known values or reference samples.
Worked example: identifying a dye
A black marker ink is run beside two reference dyes. The solvent front travels 10.0 cm from the starting line.
| Spot | Distance moved (cm) | Rf value |
|---|---|---|
| Ink, spot 1 (yellow) | 8.0 | 0.80 |
| Ink, spot 2 (red) | 5.0 | 0.50 |
| Ink, spot 3 (blue) | 2.5 | 0.25 |
| Reference dye A | 5.0 | 0.50 |
| Reference dye B | 3.5 | 0.35 |
- Calculate each Rf. Spot 1: 8.0 ÷ 10.0 = 0.80. Spot 2: 5.0 ÷ 10.0 = 0.50. Spot 3: 2.5 ÷ 10.0 = 0.25.
- Compare with the references. The red spot in the ink has the same Rf (0.50) as reference dye A, so the ink probably contains dye A.
- Conclusion. The ink is a mixture of at least three dyes, and one of them is probably dye A. No spot matches reference dye B.
The matching of an Rf is evidence but not absolute proof, because different substances can have similar Rf values. Running the sample with the reference on the same paper makes the comparison more reliable.
Other types of chromatography
| Type | Stationary phase | Mobile phase | Typical use |
|---|---|---|---|
| Paper | Paper | Liquid solvent | Inks, plant pigments |
| Thin-layer (TLC) | Thin layer of silica on a plate | Liquid solvent | Checking reaction progress, purity |
| Column | Packed column of solid | Liquid solvent | Purifying compounds |
| Gas (GC) | Liquid or solid in a tube | Gas | Fuel, drugs and flavor analysis |
| High-performance liquid (HPLC) | Fine particles in a column | Liquid under pressure | Medicines, food, water testing |
Where is chromatography used?
- Forensic science: comparing inks, dyes and drugs found at a scene.
- Food safety: detecting banned dyes and pesticide residues.
- Medicine: measuring drug levels in blood and checking purity of medicines.
- Environment: finding pollutants in water and soil.
- Sports: testing for banned substances.
How was chromatography invented?
The Russian-Italian botanist Mikhail Tsvet developed the method in the early 1900s. He separated plant pigments by passing a plant extract through a column of calcium carbonate and observed colored bands, which inspired the name from the Greek words for color and writing. In 1941 Archer Martin and Richard Synge introduced partition chromatography, and they received the Nobel Prize in Chemistry in 1952 for it.
Which mistakes are common?
Mistake 1: Drawing the starting line in ink
The ink dissolves in the solvent and moves with it, which spoils the result. Always use a pencil.
Mistake 2: Letting the starting line sit below the solvent level
The sample would dissolve into the solvent in the beaker instead of climbing the paper.
Mistake 3: Measuring the Rf from the bottom of the paper
Distances must be measured from the starting line, not from the lower edge of the paper.
Mistake 4: Forgetting to mark the solvent front
Once the paper dries, the solvent front is no longer visible. Mark it right after removing the paper from the beaker.