Identifying Substances with Chromatography

Introduction

A chromatogram can do more than show that a mixture contains several substances: it can reveal which substances they are. By running an unknown sample beside a set of known substances, or by comparing Rf values with trusted data, chemists identify the ingredients of inks, food colourings, medicines and much more. This page shows how that matching is done and how scientists make sure a match is genuine.

Core explanation

Method 1: run references alongside. The most reliable approach is to put the unknown and several reference samples — known pure substances that might be present — on the same baseline and run them together. Afterwards:

- a spot in the unknown at the same height and colour as a reference spot is probably that reference substance; - a reference with no matching spot in the unknown is probably absent ; - a spot in the unknown that matches no reference is an unidentified substance.

Because everything ran under identical conditions, no calculation is needed; heights can be compared directly.

Method 2: compare Rf values. If references cannot be run on the same paper, the Rf value of each unknown spot is calculated and compared with Rf values recorded for known substances using the same solvent and paper . Matching Rf values suggest the same substance.

Making identification reliable. A single match is good evidence but not proof, because two different substances can have the same Rf value in one solvent. To increase confidence, chemists:

1. repeat the chromatogram with a different solvent — if the unknown still matches the reference, the identification is much stronger, since it is unlikely two different substances would co-run in both solvents; 2. check that colours match, not just heights; 3. use other tests too, such as melting point.

Colourless substances. Many substances, such as amino acids and sugars, are colourless. After the run the paper is dried and treated with a locating agent — a spray that reacts to form coloured spots — or viewed under ultraviolet light. Their positions can then be measured and matched in the usual way.

Step-by-step reasoning

To identify the substances in an unknown:

1. Spot the unknown and possible reference substances on one baseline. 2. Run and dry the chromatogram. 3. Calculate or compare the positions of all spots. 4. Match unknown spots to references with the same Rf and colour. 5. Confirm with a second solvent if the result matters.

Visual explanation

Picture a chromatogram with the unknown in the centre and four reference dyes arranged on either side. Horizontal dashed lines drawn from each reference spot run across the paper. Two of the lines pass straight through spots in the unknown; the other two cross empty paper.

Real-world analogy

Identifying substances this way is like an identity parade in a police station. The unknown spot is compared against a line-up of known "suspects". A match is strong evidence, and checking it a second way — a different solvent, like a second witness — makes the identification much more convincing.

Real-world example

Sports anti-doping laboratories use advanced chromatography to separate the substances in an athlete's urine sample. Each substance is identified by comparing how it behaves with certified reference samples of banned drugs run under the same conditions, and positive results are always confirmed by a second analysis.

Why?

Why is a match in two different solvents far more convincing than a match in one? Two different substances might, by chance, share the same Rf value in one solvent. It is very unlikely that they would also share the same Rf value in a second, different solvent, so a double match strongly suggests they are the same substance.

Common misconception

"If the unknown has a spot at the same height as a reference, it definitely is that reference." Matching height in one solvent shows the substances are probably the same, not certainly. Confirmation with another solvent or another test is needed before drawing a firm conclusion.

Worked example

Question: Using the same solvent, an unknown dye gives spots with Rf values 0.20 and 0.65. Recorded Rf values are: tartrazine (yellow) 0.65, sunset yellow 0.48, a blue dye 0.20. Which dyes are likely present?

Reasoning: Compare Rf values. 0.65 matches tartrazine; 0.20 matches the blue dye. No spot at 0.48, so sunset yellow is not detected.

Answer: The unknown probably contains tartrazine and the blue dye — giving a green colour — but not sunset yellow.

Quick check

1. What is a reference sample in chromatography? Answer: A known pure substance run under the same conditions so unknown spots can be compared with it.

Exam focus

Expect a diagram with an unknown and several known substances. Identify matches by same height (or Rf) and colour, state which references are absent, and note any unmatched spots. Explain that conditions must be identical for Rf comparisons, and that using a second solvent improves confidence.

Advanced insight

In professional laboratories chromatography is often linked directly to a mass spectrometer. The chromatography separates the mixture, and the mass spectrometer then measures the mass of each separated substance's molecules and fragments, giving an almost unique fingerprint. This combination is used routinely to identify drugs, pollutants and flavour compounds.

Summary

Unknown substances are identified by comparing their spots with known references run on the same chromatogram, or by comparing Rf values measured under identical conditions. A matching Rf and colour suggests the same substance; a match in a second solvent makes this much more certain. Colourless substances are revealed with a locating agent or ultraviolet light before matching.

Practice questions

1. How can you tell from a chromatogram that a reference substance is absent from an unknown? Answer: The unknown has no spot at the same height (Rf) and colour as the reference. 2. Why must Rf values be compared only when the same solvent was used? Answer: Rf depends on the solvent; a different solvent gives a different Rf value for the same substance. 3. An unknown spot matches reference X in water. Suggest how to make the identification more certain. Answer: Run both again using a different solvent; if they still match, the identification is much more reliable. 4. How can colourless amino acids be identified by paper chromatography? Answer: After running, the paper is treated with a locating agent (or viewed under UV) to reveal the spots, then their Rf values are compared with known amino acids.