Chromatography (GC, HPLC, IC) and Hyphenated Techniques
Chromatography (GC, HPLC, IC) and Hyphenated Techniques
This domain tests separation thermodynamics and kinetics, detector selection, and coupling of chromatography to mass spectrometry: GC, HPLC, ion chromatography, GC-MS and LC-MS.
Core rules
- Retention depends on stationary and mobile phase interactions. Selectivity α = k2/k1; resolution R = 2(t2 − t1)/(w1 + w2). Baseline resolution is generally R ≥ 1.5.
- Van Deemter: H = A + B/u + Cu. Smaller particles reduce A and C but raise pressure. GC uses capillary columns and temperature programming; HPLC commonly uses reversed-phase C18 and gradient elution.
- Ion chromatography uses ion-exchange columns and suppressed conductivity. Carbonate/bicarbonate eluents and suppression improve signal-to-noise.
- Hyphenation requires compatible flow, volatility for GC-MS, and ionization for LC-MS (ESI or APCI). Matrix effects and ion suppression are common in LC-MS.
- Use internal standards for volume and ionization variability; check carryover and peak purity.
Worked example
Two peaks elute at 12.40 and 13.10 min with baseline widths 0.35 and 0.42 min. R = 2(13.10 − 12.40)/(0.35 + 0.42) = 2 × 0.70/0.77 = 1.82, so they are resolved. If the second width increases to 0.55 min, R = 1.40 and integration becomes unreliable; reduce gradient slope or improve efficiency.
Traps
- Using retention time alone for identification.
- Reporting resolution without stating the width convention.
- Ignoring dead time and unretained peaks.
- Overloading columns, causing fronting and shifted retention.
- Neglecting ion suppression in LC-MS or thermal degradation in GC.