Semester 7
BP701T
Gas Chromatography (GC) & High-Performance Liquid Chromatography (HPLC)
This unit covers the two most powerful instrumental chromatographic techniques used in pharmaceutical analysis. Gas Chromatography (GC) — for analysis of volatile and thermally stable compounds, with detailed study of theory, instrumentation (columns, detectors, carrier gas), derivatization, and temperature programming. High-Performance Liquid Chromatography (HPLC) — the most versatile and widely used technique, covering theory, instrumentation (pump, injector, column, detectors), and pharmaceutical applications.
Syllabus & Topics
- 1Gas Chromatography – Introduction & Theory: GC: separation technique where mobile phase is a GAS (carrier gas) and stationary phase is either a solid (GSC — gas-solid chromatography) or a liquid coated on a solid support (GLC — gas-liquid chromatography, most common). Requirement: analytes must be VOLATILE (vaporizable) and THERMALLY STABLE at operating temperatures. Theory: separation governed by: distribution coefficient (K = Cs/Cm), column efficiency (N — number of theoretical plates), and selectivity (α). Van Deemter equation: H = A + B/u + Cu. H = plate height (HETP). A = Eddy diffusion (multiple paths through packing — reduced in capillary columns). B/u = longitudinal diffusion (∝ 1/flow rate — significant at low flow rates). Cu = mass transfer resistance (∝ flow rate — significant at high flow rates). Optimal flow rate (uopt): where H is minimum → best efficiency.
- 2GC – Instrumentation Overview: Block diagram: Carrier gas (supply + flow controller) → Injector (sample introduction) → Column (separation — in oven) → Detector → Data system/Recorder. Carrier gas: inert gas that carries sample through column. Types: Helium (most common — inert, suitable for all detectors), Nitrogen (cheap, slightly less efficient), Hydrogen (fastest, best efficiency but flammable — safety concerns). Requirements: high purity, dry, oxygen-free (use molecular sieve traps). Flow rate: typically 1-25 mL/min for packed columns, 1-2 mL/min for capillary columns. Injector: sample injected (μL) through rubber septum into heated injection port (vaporization). Split injection: for capillary columns (only fraction enters column — for concentrated samples). Splitless injection: entire sample enters (for dilute/trace analysis).
- 3GC – Columns: (1) Packed columns: glass or stainless steel tubes (1.5-3 m length, 2-4 mm ID) packed with solid support (Chromosorb W, diatomaceous earth) coated with liquid stationary phase. Lower efficiency (~3,000-5,000 plates per meter). Higher sample capacity. (2) Capillary (open tubular) columns: fused silica tubes (15-60 m length, 0.25-0.53 mm ID) — stationary phase coated on wall. Types: WCOT (Wall-Coated Open Tubular — liquid phase coated directly on wall, most common), SCOT (Support-Coated Open Tubular — support particles coated on wall then coated with liquid), PLOT (Porous-Layer Open Tubular — porous solid on wall). Capillary columns: much higher efficiency (~3,000-5,000 plates per meter × 30-60 m = 100,000-300,000 total plates). Common stationary phases: non-polar — polydimethylsiloxane (DB-1, OV-1); polar — polyethylene glycol (Carbowax, DB-WAX); intermediate polarity — phenyl-methylpolysiloxane (DB-5, OV-17).
- 4GC – Detectors: (1) Flame Ionization Detector (FID): sample burns in H₂/air flame → organic compounds produce ions → current measured. UNIVERSAL for organics. Sensitive (pg level), wide linear range. Does NOT detect: H₂O, CO₂, N₂, CS₂, inorganic gases. Most commonly used GC detector. (2) Thermal Conductivity Detector (TCD): measures thermal conductivity difference between carrier gas and carrier gas + analyte. NON-DESTRUCTIVE, truly universal (detects all compounds). Less sensitive than FID. Used for: permanent gases, water, inorganic gases. (3) Electron Capture Detector (ECD): β-emitter (⁶³Ni) ionizes carrier gas → standing current. Electron-capturing compounds (halogenated, nitro) reduce current. Extremely sensitive for halogenated pesticides, PCBs (ppt level). Very selective. (4) Mass Spectrometer (MS): GC-MS — most powerful for identification. Mass spectrum = molecular fingerprint.
- 5GC – Derivatization & Temperature Programming: Derivatization: chemical modification of analyte to make it suitable for GC (volatile, thermally stable, detectable). Types: (1) Silylation: replace active H (–OH, –NH, –SH) with trimethylsilyl (TMS) group using BSTFA/MSTFA. Most common. Makes compounds more volatile, less polar. (2) Acylation: convert –OH, –NH to esters/amides using acetic anhydride, TFAA. (3) Alkylation: convert –OH, –COOH to ethers/esters using BF₃/methanol (methylation). Why derivatize? Non-volatile compounds (sugars, amino acids, steroids) → volatile TMS derivatives. Thermally unstable → stable derivatives. Poor peak shape (tailing from –OH) → symmetric peaks. Temperature programming: column temperature increased during the run (e.g., 50°C → 300°C at 10°C/min). Why? Analytes with wide boiling point range: isothermal → early peaks overlap (fast), late peaks broad (slow). Temperature program → all peaks elute with good resolution. Like gradient elution in HPLC.
- 6GC – Applications: (1) Residual solvents in pharmaceuticals (ICH Q3C): GC with headspace sampling → identify and quantify Class 1, 2, 3 residual solvents (ethanol, methanol, acetone, dichloromethane, benzene). (2) Essential oil analysis: GC-FID and GC-MS for composition of volatile oils (menthol, eugenol, linalool). (3) Fatty acid profiling: FAME (fatty acid methyl esters) by GC-FID. (4) Pesticide residue analysis: GC-ECD or GC-MS for organochlorine/organophosphate pesticides. (5) Blood alcohol determination (forensic): headspace GC-FID. (6) Petroleum analysis. (7) Environmental monitoring (VOCs). Advantages: high resolution (capillary), fast, sensitive (FID, ECD), GC-MS for identification. Disadvantages: only volatile/thermally stable compounds, derivatization needed for many pharma compounds, not suitable for thermally labile biomolecules.
- 7HPLC – Introduction & Theory: HPLC: High-Performance (or High-Pressure) Liquid Chromatography. Liquid mobile phase pumped at HIGH PRESSURE (50-400 bar) through a column packed with fine stationary phase particles (3-10 μm). Result: superior resolution, speed, and sensitivity compared to classical column chromatography. Modes: (1) Normal phase: polar stationary phase (silica) + non-polar mobile phase. Polar compounds retained longer. (2) Reverse phase (RP-HPLC): non-polar stationary phase (C₁₈, C₈ bonded silica) + polar mobile phase (water/MeOH, water/ACN). Most commonly used mode (~80% of all HPLC). Non-polar compounds retained longer. (3) Ion exchange, size exclusion, affinity (covered in Unit 5). Theory: same as GC — described by Van Deemter equation, theoretical plates, resolution.
- 8HPLC – Instrumentation: (1) Solvent reservoir and degasser: mobile phase stored; degassed to remove dissolved air (bubbles cause noise). Online degassers or helium sparging. (2) Pump: delivers mobile phase at constant flow rate (0.5-2 mL/min) and high pressure. Reciprocating piston pump (most common): dual-piston for pulseless flow. Isocratic: constant mobile phase composition. Gradient: composition changes during run (e.g., 10% → 90% ACN). (3) Injector: Rheodyne manual injector (sample loop: 5-100 μL) or autosampler (automated, precise, for large sample batches). (4) Column: heart of HPLC. Stainless steel tube (10-25 cm × 4.6 mm ID) packed with stationary phase. C₁₈ (ODS — octadecylsilane) bonded silica = most common RP column. Guard column: short protective pre-column.
- 9HPLC – Detectors & Applications: Detectors: (1) UV detector: most common. Measures UV absorption at set wavelength. Fixed wavelength, variable wavelength, or PDA (photodiode array — full spectrum at every time point). (2) Fluorescence detector: for naturally fluorescent analytes or derivatized compounds. More selective and sensitive than UV. (3) Refractive Index (RI) detector: measures difference in RI between mobile phase and eluate. Universal but low sensitivity. Used for sugars, polymers. (4) Electrochemical detector: for electroactive compounds (catecholamines, vitamins). (5) MS detector (LC-MS): most powerful — mass spectrum for identification. HPLC Applications: (1) Drug assay in formulations (most common QC application). (2) Related substances/impurity profiling (ICH). (3) Dissolution testing. (4) Content uniformity. (5) Stability studies (stability-indicating methods). (6) Biological samples (pharmacokinetics, TDM). (7) Chiral HPLC for enantiomeric purity. Advantages: high resolution, wide applicability (non-volatile, thermally labile OK), quantitative, automated. Disadvantages: expensive (instrument + columns + solvents), requires trained operator.
Learning Objectives
Van Deemter Equation: Explain the Van Deemter equation terms and how each affects column efficiency.
GC Detectors: Compare FID, TCD, ECD, and MS detectors by principle, selectivity, sensitivity, and applications.
Derivatization: Explain why derivatization is needed in GC and describe silylation with an example.
HPLC Instrumentation: Draw the block diagram of an HPLC system and describe each component.
RP-HPLC: Explain why reverse-phase HPLC with C₁₈ column is the most widely used mode in pharmaceutical analysis.
Exam Prep Questions
Q1. Why is HPLC more widely used than GC in pharmaceutical analysis?
Most pharmaceutical compounds are NON-VOLATILE (cannot be vaporized without decomposition) → unsuitable for GC. HPLC works with compounds in SOLUTION → any compound that dissolves can be analyzed, regardless of volatility or thermal stability. Additionally, many drugs are thermally labile (decompose on heating), polar, or have high molecular weight — all handled easily by HPLC but not GC. GC is reserved for specific applications: residual solvents, essential oils, gases, and compounds that are naturally volatile.
Q2. What is the difference between Isocratic and Gradient elution?
Isocratic: mobile phase composition stays CONSTANT throughout the run (e.g., 60% MeOH:40% water). Simple, reproducible, but if sample has compounds with wide polarity range, early peaks cluster while late peaks are broad.
Gradient: mobile phase composition CHANGES during the run (e.g., start at 10% ACN:90% water → end at 90% ACN:10% water). Progressively stronger eluent sweeps off more retained compounds. All peaks are sharp and well-resolved. Similar to temperature programming in GC. Gradient is preferred for complex samples.
Q3. What makes Capillary columns superior to Packed columns in GC?
Capillary columns are open tubes (no packing) → dramatically lower resistance to flow → can be made MUCH longer (30–60 m vs 1–3 m for packed). Longer column = more theoretical plates = better resolution. The “A” term (Eddy diffusion) in Van Deemter equation is essentially ZERO (no multiple paths since there’s no packing). Typical efficiency: capillary ~100,000–300,000 plates vs packed ~3,000–15,000 plates. Trade-off: capillary columns have lower sample capacity and require split injection.