Sắc ký: kỹ thuật HPLC và GC

The Principle of Chromatography

Chromatography is a separation technique in which components of a mixture are distributed between a stationary phase (a solid or liquid coating) and a mobile phase (a liquid or gas) that moves through or past the stationary phase. Different components interact differently with the two phases — those with stronger affinity for the stationary phase travel more slowly, while those preferring the mobile phase travel faster. This differential migration separates the mixture into individual components.

All chromatographic techniques share the same fundamental theory. The key parameters are: - Retention time (t_R): Time from injection to peak maximum for a given compound - Dead time (t_M): Time for unretained compounds to travel through the system - Retention factor (k'): k' = (t_R − t_M) / t_M — measures how long a compound is retained relative to an unretained compound - Resolution (R_s): Measure of how well two adjacent peaks are separated; R_s ≥ 1.5 indicates baseline separation

Gas Chromatography (GC)

Overview

Gas chromatography (GC) separates volatile or semi-volatile organic compounds using an inert carrier gas (usually helium or nitrogen) as the mobile phase and a liquid or solid stationary phase coated inside a capillary column. The sample is vaporized at a heated inlet (injector) and carried through the column by the mobile gas.

The GC Column

Modern GC uses fused silica capillary columns, typically: - Length: 15–60 m - Inner diameter: 0.25–0.53 mm - Film thickness: 0.1–5 μm of stationary phase

Stationary phases range from nonpolar (polydimethylsiloxane — separates by boiling point) to polar (polyethylene glycol/Carbowax — separates polar compounds more strongly). The rule "like dissolves like" applies: polar stationary phases retain polar analytes more strongly.

Temperature Programming

Because a mixture may contain compounds with widely different boiling points, GC runs often use temperature programming: the oven temperature rises during the run. Low-boiling compounds separate at low temperatures; high-boiling compounds elute as temperature increases, reducing total run time and sharpening late-eluting peaks.

Detectors

• Flame Ionization Detector (FID): Burns eluting compounds in a hydrogen-air flame, producing ions detected as a current. Near-universal response to carbon-containing compounds; excellent sensitivity and linear range. The most common GC detector.
• Thermal Conductivity Detector (TCD): Measures change in thermal conductivity of the carrier gas stream when a compound elutes. Universal but less sensitive than FID.
• Electron Capture Detector (ECD): Highly selective for electronegative compounds (halogens, nitro groups). Essential for pesticide residue analysis and environmental PCB monitoring.
• Mass Spectrometer (MS): The gold standard — identifies compounds by fragmentation pattern and provides quantitation. GC-MS is the reference method for environmental, forensic, and flavor/fragrance analysis.

Applications of GC

• Fuel analysis: Characterizing gasoline, diesel, and jet fuel composition
• Environmental: VOCs in drinking water (EPA Method 524), pesticides in food (QuEChERS extraction + GC-MS)
• Forensic toxicology: Blood alcohol determination (headspace GC-FID)
• Flavor and fragrance: Identifying aroma compounds in wines, coffees, and perfumes
• Petroleum refining: Simulated distillation of crude oil fractions

High-Performance Liquid Chromatography (HPLC)

Overview

High-performance liquid chromatography (HPLC) uses a liquid mobile phase pumped at high pressure (up to 400 bar) through a column packed with small-diameter particles (2–10 μm). Because the mobile phase is liquid, HPLC can analyze non-volatile, thermally labile, and highly polar compounds that cannot be vaporized for GC — making it the preferred technique for pharmaceuticals, proteins, food additives, and environmental pollutants.

Reversed-Phase HPLC

Reversed-phase HPLC (RP-HPLC) is the dominant mode, accounting for ~70% of HPLC applications. The stationary phase is nonpolar (typically C18 — octadecylsilane chains bonded to silica), and the mobile phase is a polar aqueous–organic mixture (water + methanol or acetonitrile).

• Nonpolar compounds are retained strongly (long t_R)
• Polar compounds elute first
• Gradient elution: Starting with high-water mobile phase and gradually increasing organic solvent concentration progressively elutes compounds in order of increasing hydrophobicity

Normal-Phase HPLC

A polar stationary phase (silica, amino, cyano) with a nonpolar mobile phase (hexane/isopropanol). Used for isomer separations and non-polar compounds not retained in RP mode.

HPLC Detectors

• UV-Vis detector: Most common; measures absorbance at fixed wavelength or scans a spectrum (diode array detector, DAD/PDA). Sensitive for UV-absorbing compounds.
• Fluorescence detector (FLD): 10–1000× more sensitive than UV for fluorescent compounds (PAHs, aflatoxins, derivatized amino acids).
• Refractive index detector (RID): Universal but insensitive; used for sugars, polymers.
• Evaporative light scattering detector (ELSD): For non-UV-absorbing analytes (lipids, carbohydrates).
• Mass spectrometer (LC-MS/MS): Provides structural identification and ultra-sensitive quantitation; increasingly the standard in pharmaceutical and clinical labs.

Ultra-High Performance Liquid Chromatography (UHPLC)

UHPLC uses sub-2-μm particles and pressures up to 1000+ bar. Smaller particles improve resolution and speed dramatically — a 30-minute HPLC method can become a 3-minute UHPLC method with equivalent or better separation.

Applications of HPLC

• Pharmaceutical QC: Assaying active pharmaceutical ingredients, detecting impurities and degradation products (ICH guidelines mandate HPLC purity testing)
• Biopharmaceuticals: Characterizing monoclonal antibodies, peptides (reversed-phase), proteins (size-exclusion, ion exchange)
• Food safety: Detecting mycotoxins, veterinary drug residues, food colorants, vitamins
• Clinical: Measuring immunosuppressants (tacrolimus, cyclosporine) in transplant patients' blood
• Environmental: PAHs in drinking water, herbicides in groundwater

Comparing GC and HPLC

Chromatography — whether gas or liquid — is inseparable from modern analytical chemistry. Nearly every quality control laboratory, environmental agency, hospital, and research institution relies on chromatographic methods daily to ensure product quality, patient safety, and regulatory compliance.