[Durgical](https://durgical.com/en/) › [DurgiPedia](https://durgical.com/en/durgipedia/) › [Biochemistry](https://durgical.com/en/biochemistry/) › Chromatography [Biochemistry](https://durgical.com/en/biochemistry/) · Topic # 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. ## In detail - 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 ## Clinical notes - 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. ## Related conditions - [Phenylketonuria (PKU)](https://durgical.com/en/conditions/phenylketonuria/) - [Maple syrup urine disease](https://durgical.com/en/conditions/maple-syrup-urine-disease/) - [Sickle cell disease](https://durgical.com/en/conditions/sickle-cell-disease/) - [Thalassemias (α and β)](https://durgical.com/en/conditions/thalassemias/) ## Related - [Plasma proteins and electrophoresis](https://durgical.com/en/biochemistry/topics/plasma-proteins-and-electrophoresis/) - [Hemoglobin variants (HbA, HbA2, HbF, HbS, HbC)](https://durgical.com/en/biochemistry/topics/hemoglobin-variants/) - [Amino acids: structure, charge and pKa](https://durgical.com/en/biochemistry/topics/amino-acids-structure-charge-and-pka/) - [Spectrophotometry and colorimetry (Beer–Lambert law)](https://durgical.com/en/biochemistry/topics/spectrophotometry-and-colorimetry/) - [Centrifugation and ultracentrifugation](https://durgical.com/en/biochemistry/topics/centrifugation-and-ultracentrifugation/) - [Newborn screening](https://durgical.com/en/biochemistry/topics/newborn-screening/) - [Glycated haemoglobin (HbA1c)](https://durgical.com/en/biochemistry/topics/glycated-haemoglobin/) ## Test yourself 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). [Add to my cards (5)](https://durgical.app/estudiar/?add=/en/biochemistry/topics/chromatography/) Educational information, not medical advice. For any symptom, see a health professional. Sources: [The Nobel Prize in Chemistry 1952 - NobelPrize.org](https://www.nobelprize.org/prizes/chemistry/1952/summary/) (NobelPrize.org (Nobel Prize Outreach), 2026); [Toxicology screen: MedlinePlus Medical Encyclopedia](https://medlineplus.gov/ency/article/003578.htm) (MedlinePlus, US National Library of Medicine, 2026); [Plasma amino acids: MedlinePlus Medical Encyclopedia](https://medlineplus.gov/ency/article/003361.htm) (MedlinePlus, US National Library of Medicine, 2026); [Newborn screening tests: MedlinePlus Medical Encyclopedia](https://medlineplus.gov/ency/article/007257.htm) (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). [← All of biochemistry](https://durgical.com/en/biochemistry/) ## **Spotted a mistake?** We are always improving for the community. Tell us what is wrong and our team will review it. Report a mistake Representative model, for educational purposes only. --- Source: https://durgical.com/en/biochemistry/topics/chromatography/ License: CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). Cite as: DurgiPedia (durgical.com), https://durgical.com/en/biochemistry/topics/chromatography/