What Is Chromatography?
Chromatography is a separation technique used to separate and identify different substances dissolved in a mixture. It works because the different substances in the mixture move at different speeds through a material, separating from each other.
Although there are many types of chromatography, the most common in school is paper chromatography, used to separate coloured dyes and inks.
How Paper Chromatography Works
The technique separates substances based on two competing forces:
- Stationary phase: The filter paper (or silica). Substances that are attracted more strongly to the paper travel a shorter distance.
- Mobile phase: The solvent (e.g. water, ethanol). Substances attracted more strongly to the solvent travel further.
Steps:
- Draw a pencil line near the bottom of the filter paper (baseline).
- Place small spots of the mixture on the baseline.
- Lower the paper into a solvent so the solvent level is below the spots.
- The solvent slowly rises up the paper by capillary action, carrying the dissolved substances with it.
- Substances that interact more with the solvent travel further up; those more attracted to the paper stay lower.
- Remove the paper before the solvent reaches the top. Mark the solvent front.
- Allow to dry and measure the distances.
You must use a pencil (not pen or marker) for the baseline, because ink from a pen would dissolve in the solvent and move up the paper, adding extra spots that confuse the results. Pencil marks are made of graphite, which is insoluble in most solvents.
The Rf Value
Each substance has a characteristic Rf value (retention factor) for a given solvent:
Rf values always fall between 0 and 1. By comparing Rf values with known substances, you can identify unknown ones.
Identifying a dye
The solvent front travels 8.0 cm. A dye spot travels 5.0 cm.
Rf = 5.0 ÷ 8.0 = 0.625
If the Rf of known amaranth dye in this solvent is also 0.625, the unknown dye is likely amaranth.
Using Chromatography to Test Purity
A pure substance gives a single spot on chromatography. A mixture gives two or more spots as the different components separate. This makes chromatography very useful for:
- Testing food colourings for undeclared dyes.
- Identifying drugs and poisons in forensic science.
- Quality control in pharmaceuticals.
Many coloured sweets and inks are actually mixtures of several dyes. A single black marker pen often contains 3–5 different coloured dyes. You can see this by placing a small spot of black ink on coffee filter paper, then adding a drop of water to the centre. The different dyes travel different distances, producing coloured rings.
Real world: Drug testing in sport uses chromatography
When athletes are tested for performance-enhancing drugs, their urine or blood samples undergo sophisticated chromatography (gas chromatography or liquid chromatography). Each banned substance has a characteristic Rf value (or retention time), which acts like a fingerprint. The sample is compared against known reference standards. This is why doping bans can be applied years later — frozen samples are retested when new detection methods are developed. Chromatography makes sports drug testing possible with extraordinary precision.
Real world: Food safety authorities test for undeclared dyes
Food standards agencies routinely analyse food colourings using chromatography. A single food dye that appears one colour might actually be a mixture of several dyes — paper or thin-layer chromatography separates them into individual spots, each with its own Rf value. If a food contains an undeclared or banned dye (like Sudan Red, used to adulterate chilli powder), chromatography reveals it. This is how food fraud is detected — the separated components betray the true mixture.
Real world: Forensic analysis of ink — which pen wrote the document?
Forensic document examiners use chromatography to determine if a signature or written text was genuine or altered. Different brands of black pen contain different mixtures of dyes. Paper chromatography of ink samples from a questioned document and from a suspect pen shows whether the Rf values of the separated dye spots match. Even if two inks look identical to the eye, their chromatographic profiles reveal them as different mixtures. This has helped solve forgery cases in wills, contracts, and financial documents.
Where do you see this in real life? Drug testing in sports (chromatography detects banned substances), food colouring verification (undeclared dyes in food products), forensic ink analysis (which pen wrote a document), pharmaceutical quality control (verifying drug purity) — chromatography's ability to separate a mixture into its components makes it one of the most powerful analytical tools in science.
Practice Problems
In a chromatography experiment, the solvent travels 10 cm and a compound travels 7 cm. Calculate the Rf value.
Show answer
A student runs chromatography on a sample of food colouring and gets three separate spots. What does this tell her about the food colouring?