Chromatography !

Chromatography separates the parts of a mixture as it flows over a material that holds each part with a different strength.

Updated 2026-11-01

Key facts

Principle: a mobile phase (liquid or gas) carries the sample over a stationary phase; molecules that bind it more weakly move faster and separate.
Partition (paper, thin-layer, gas–liquid, reverse-phase HPLC): separates by solubility between two phases; Martin and Synge won the 1952 Nobel Prize for it.
Ion exchange: separates by charge (amino acid analysers, HbA1c by HPLC). Gel filtration (size exclusion): large molecules come out first.
Affinity: a specific ligand (antibody, substrate, metal) holds only the target protein, which is then eluted; the most selective method for purifying proteins.
Clinical uses: HPLC for HbA1c and haemoglobin variants, amino acid profiles, drug and toxin screens (GC or LC with mass spectrometry), tandem MS newborn screening.

1. Paper and thin-layer chromatography: the solvent rises by capillarity; each spot's Rf = distance moved by the substance ÷ distance moved by the solvent; amino acids are seen with ninhydrin (purple).
2. Column chromatography: the sample is loaded at the top of a packed column and fractions are collected at the bottom.
3. Ion exchange: cation exchangers (negative resin) hold positive molecules; elution with rising salt or changing pH releases them in order of charge.
4. Gel filtration: porous beads trap small molecules, which take a longer path; large molecules leave first. It also estimates molecular weight.
5. HPLC pushes the mobile phase at high pressure through fine particles: fast, sharp and quantitative separation.
6. Gas chromatography separates volatile compounds (alcohols, drugs, fatty acids) and is often coupled to mass spectrometry (GC-MS) for identification.

Also called: Paper and thin-layer chromatography, Ion-exchange, gel filtration and affinity chromatography, HPLC and gas chromatography

Confirming a positive drug screen uses GC-MS or LC-MS/MS, which identify the exact compound.
Tandem mass spectrometry in newborn screening detects amino acid and acylcarnitine patterns of PKU, MSUD and fatty acid oxidation defects.
HPLC separates HbA, HbA2, HbF and HbS and measures HbA1c.

Think of the answer, then tap the question to see it.

In gel filtration chromatography, which molecules come out first and why?

The largest, because they cannot enter the pores of the beads and take the shortest path through the column.

What property separates molecules in ion-exchange chromatography?

Their net charge, which depends on pH; they are eluted by changing salt concentration or pH.

What is the Rf value in paper or thin-layer chromatography?

Distance travelled by the substance divided by distance travelled by the solvent front.

Which chromatography gives the highest selectivity for purifying a single protein?

Affinity chromatography, which uses a ligand such as an antibody or substrate that binds only that protein.

Which technique confirms the identity of a drug found on a screening test?

Gas or liquid chromatography coupled to mass spectrometry (GC-MS or LC-MS/MS).

Educational information, not medical advice. For any symptom, see a health professional.

Sources: The Nobel Prize in Chemistry 1952 - NobelPrize.org (NobelPrize.org (Nobel Prize Outreach), 2026); Toxicology screen: MedlinePlus Medical Encyclopedia (MedlinePlus, US National Library of Medicine, 2026); Plasma amino acids: MedlinePlus Medical Encyclopedia (MedlinePlus, US National Library of Medicine, 2026); Newborn screening tests: MedlinePlus Medical Encyclopedia (MedlinePlus, US National Library of Medicine, 2026); Textbook of Biochemistry for Medical Students, 6th ed. (Jaypee Brothers Medical Publishers (Vasudevan DM, Sreekumari S, Vaidyanathan K), 2011).

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Representative model, for educational purposes only.