Unit 2: Chromatography
Analytical Biochemistry notes · PTU syllabus (BMLS302-18)
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Unit summary
Chromatography separates mixtures by how components distribute between a stationary and a mobile phase. This unit covers paper, thin-layer (including HPTLC), column, gas, ion-exchange and gel chromatography — their principles, instrumentation and applications.
After this unit you can
- Explain the general principle and types of chromatography
- Describe paper and thin-layer chromatography and Rf values
- Describe column, ion-exchange and gel chromatography
- Describe gas chromatography
PTU syllabus topics
- Introduction
- definition and types of chromatography — paper chromatography
- thin-layer chromatography (including HPTLC)
- column chromatography
- gas chromatography
- ion-exchange chromatography
- gel chromatography — principle
- instrumentation and applications of each
Paper / TLC
Partition and adsorption
Amino acids, sugars
Column
Adsorption or size
Purifying proteins
Gas
Volatility
Alcohol, drugs
Ion exchange
Charge
Haemoglobin variants, proteins
Gel filtration
Molecular size
Protein separation
Topic 1
Principle and types of chromatography
- Chromatography (Tswett, 1906): components of a mixture are separated as a mobile phase (liquid or gas) carries them over a stationary phase (solid or liquid); components that bind more strongly move more slowly.
Adsorption
Silica or alumina — TLC, column
Partition
Liquid on a support — paper, gas–liquid
Ion exchange
Charged resin
Gel filtration (size exclusion)
Porous beads
Affinity
Specific binding (antibody–antigen)
Topic 2
Paper chromatography
- 1Spot the sample on Whatman filter paper above the solvent line
- 2Place in a closed tank with solvent (e.g., butanol–acetic acid–water)
- 3Solvent rises by capillary action (ascending) or descends (descending)
- 4Dry and spray with a locating reagent (ninhydrin for amino acids)
- 5Measure distances and calculate Rf
Rf
Distance moved by the substance ÷ distance moved by the solvent front (always less than 1)
- Applications: amino acids in urine (aminoacidurias), sugars. Two-dimensional chromatography uses two solvents at right angles for better separation.
Topic 3
Thin-layer chromatography and HPTLC
- A thin layer of silica gel or alumina on glass, plastic or aluminium is the stationary phase; separation is faster and sharper than paper.
- HPTLC (high-performance TLC): finer, uniform particles, automated sample application and densitometric scanning for quantitation.
- Applications: lipids, drugs and toxins (drug screening), sugars, amino acids, purity checks.
Topic 4
Column chromatography
- A glass column packed with adsorbent (silica, alumina); the sample is added on top and eluted with solvents of increasing polarity; fractions are collected. HPLC pumps solvent at high pressure through fine particles for fast, high-resolution separation — HbA1c, vitamins, drugs, catecholamines.
Topic 5
Ion-exchange chromatography
- Principle: charged molecules bind to oppositely charged groups on a resin — cation exchangers (e.g., carboxymethyl, negatively charged) bind positive molecules; anion exchangers (DEAE, positively charged) bind negative molecules. Bound molecules are eluted by changing pH or salt concentration.
- Applications: separating proteins, amino acids, haemoglobin variants (HbA1c and HbA2 by HPLC), water deionisation.
Topic 6
Gel (size-exclusion) chromatography
- Principle: porous beads (Sephadex, agarose) let small molecules enter pores and take a longer path, so large molecules elute first. Used to separate proteins by size, estimate molecular weight and desalt samples.
Topic 7
Gas chromatography
- 1
Carrier gas (nitrogen, helium)
- 2
Injector vaporises the sample
- 3
Column (packed or capillary) in an oven
- 4
Components separate by volatility and interaction
- 5
Detector (flame ionisation, thermal conductivity, mass spectrometer)
- 6
Chromatogram — retention time identifies, peak area quantifies
- Applications: blood alcohol, volatile poisons, drugs of abuse (GC–MS), fatty acids, steroids.
Paper
Partition
Amino acids, sugars
TLC and HPTLC
Adsorption
Drugs, lipids
Column and HPLC
Adsorption or partition
HbA1c, vitamins
Ion exchange
Charge
Haemoglobin variants, proteins
Gel filtration
Size
Protein separation, desalting
Gas
Volatility
Alcohol, toxicology
Key terms
- Stationary phase
- Fixed phase in chromatography
- Mobile phase
- Moving phase carrying the sample
- Rf value
- Ratio of solute distance to solvent distance
- Ion exchanger
- Resin with charged groups
- Retention time
- Time for a component to pass through a GC or HPLC column
Quick revision
- Principle; adsorption, partition, ion exchange, gel, affinity.
- Paper chromatography; Rf; ninhydrin; two-dimensional.
- TLC, HPTLC; column and HPLC.
- Ion exchange (cation, anion); gel filtration (large first).
- GC components and detectors; clinical applications.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Who introduced chromatography?
- Q2.Define Rf value.
- Q3.Which reagent locates amino acids?
- Q4.In gel filtration, which molecules elute first?
- Q5.What is DEAE?
- Q6.Give two uses of gas chromatography.
Long-answer questions
- Q1.Explain the principle and types of chromatography.
- Q2.Describe paper and thin-layer chromatography.
- Q3.Describe ion-exchange and gel chromatography.
- Q4.Describe gas chromatography with its instrumentation.
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