Chromatography & Mass Spectrometry
These analytical techniques separate, identify, and quantify biological molecules. They are essential in clinical laboratories, drug development, proteomics, and metabolomics.
Principles of Chromatography
Separation based on differential distribution of molecules between a mobile phase (liquid or gas) and a stationary phase. Higher affinity for stationary phase → slower movement (higher retention time).
Types of Chromatography
- Ion-Exchange Chromatography: Stationary phase: charged resin. Separates by charge (ionic interactions). Cation exchange: positively charged molecules retained. Anion exchange: negatively charged molecules retained. Elution with salt gradient. Used for: Protein purification, amino acid analysis, HbA1c measurement (cation exchange — separates HbA from HbA1c).
- Size-Exclusion (Gel Filtration): Porous beads; large molecules cannot enter pores → elute first. Small molecules enter pores → elute later. Used for: Determining molecular weight, removing salts from proteins (desalting), separating aggregated proteins.
- Affinity Chromatography: Most specific. Ligand covalently bound to stationary phase; target molecule binds specifically; eluted by competitive ligand or pH/salt change. Examples: Protein A (antibody purification), Ni-NTA (His-tagged proteins), Streptavidin (biotinylated molecules), Lectin (glycoproteins), GST pull-down assay.
- HPLC (High-Performance Liquid Chromatography): High pressure forces mobile phase through tightly packed column → high resolution and speed. Reverse-phase HPLC (C18 column, hydrophobic interaction) most common. Quantitative; used for drug levels, vitamins, steroids, amino acids in plasma.
- Gas Chromatography (GC): Volatile compounds separated based on vapor pressure and affinity for stationary phase. Coupled to MS (GC-MS) for metabolomics, toxicology screen (drugs and alcohol), steroid analysis, amino acid analysis (after derivatization).
- TLC (Thin-Layer Chromatography): Quick, simple; separates lipids, vitamins, drugs. Rf value (ratio of distance moved by compound vs solvent front) is diagnostic.
Mass Spectrometry (MS)
Identifies and quantifies molecules by measuring their mass-to-charge ratio (m/z). Components: Ionization source → Mass analyzer → Detector.
- Ionization: ESI (Electrospray — for large molecules/proteins), MALDI (Matrix-Assisted Laser Desorption — for peptides/proteins), EI (Electron Impact — for volatile molecules in GC-MS)
- Mass Analyzers: Quadrupole (filtering), Time-of-Flight (TOF — measures flight time → m/z), Orbitrap (high resolution)
- Tandem MS (MS/MS): First MS selects ion → fragmentation → second MS analyzes fragments → protein sequencing, metabolite identification
Clinical Applications:
- Newborn screening (Tandem MS): Simultaneously detects >40 metabolic disorders from dried blood spot (amino acids, acylcarnitines, organic acids)
- Plasma amino acid analysis
- Drug monitoring (TDM — cyclosporine, tacrolimus)
- Toxicology screens
- Proteomics: MALDI-TOF for bacterial ID (VITEK MS, Bruker Biotyper) — rapid pathogen identification in clinical labs
- Metabolomics: Comprehensive analysis of all small molecules in a biological sample
Electrophoretic Methods (Clinical)
- Serum Protein Electrophoresis (SPEP): Separates albumin and globulins (α1, α2, β, γ). Monoclonal band (M-spike) in γ region → Multiple myeloma (MGUS)
- Hb Electrophoresis: Diagnostic for sickle cell, thalassemia, HbC, HbE
- Urine Protein Electrophoresis (UPEP): Bence-Jones proteins (light chains) in myeloma