Gas and high-performance liquid chromatography

A car is pulled over, the breath reading is suspicious, and a blood sample goes to the lab. There, 0.5 mL of blood is sealed in a vial with an internal-standard solution of 1-propanol, warmed, and the gas above it is injected into a gas chromatograph. Three minutes later the chromatogram shows two peaks, ethanol and 1-propanol, and the ratio of their areas decides whether the report reads “drink driving” or “drunk driving”. This lesson explains how that chromatogram is made and how it becomes a trustworthy number.

A gas chromatograph has five parts: the carrier gas from a cylinder (He, H₂ or N₂); an injector that flashes the sample into vapour (split/splitless, or headspace as above); a capillary column in a thermostatted oven (a fused-silica tube of 0.25 mm i.d. and 15–60 m, coated inside with a 0.1–5 µm polymer film); a detector; and the data system.

Each detector has its niche: the flame ionisation detector (FID) responds to C–H compounds almost in proportion to their carbon atoms, with a linear range of 10⁷, and does most quantitation; the thermal conductivity detector (TCD) sees everything but insensitively; the electron-capture detector (ECD) is exquisitely sensitive to halogenated pesticides; a mass spectrometer (MS) adds structural information.

Retention in GC depends first on vapour pressure: the less volatile a solute, the longer it stays in the film. On a nonpolar column lg k of the n-alkanes is a straight line in carbon number, and compounds elute roughly in boiling-point order. Other compounds are placed by their retention index I, slotted between the neighbouring n-alkanes — n-heptane is 700, n-octane 800. The difference between a compound’s indices on two phases (the McReynolds constants) measures the polarity of a phase.

Oven temperature is GC’s strongest knob: every 20–30 °C roughly halves k. When a mixture spans low and high boilers, no single temperature suits everyone — cool, and the early peaks separate but the late ones come out broad and slow; hot, and the late ones come out quickly but the early ones pile up. This is the general elution problem. Temperature programming ramps the oven linearly so each compound is driven off near “its own” temperature; the peaks come out evenly spaced and similarly narrow.

Compounds that cannot be vaporised or would decompose — drugs, proteins, sugars, dyes — go to high-performance liquid chromatography (HPLC). A high-pressure pump drives 0.2–2 mL/min of mobile phase through a column packed with 1.7–5 µm particles (tens to over a thousand bar), an injection valve delivers a few microlitres of sample, and a UV/diode-array, fluorescence, refractive-index or mass-spectrometric detector reads the column effluent.

Most work uses reversed phase: the stationary phase is C18 chains bonded to silica (nonpolar), the mobile phase a mixture of water with methanol or acetonitrile (polar). Hydrophobic molecules prefer the C18 and are retained longer; the larger the organic fraction φ, the stronger the eluent. Over a wide range lg k is a straight line in φ (the linear solvent strength model), with S ≈ 3–5 for small molecules: 10 % more methanol cuts k by about a half to two-thirds. The counterpart of temperature programming is gradient elution, raising φ during the run.

Peak area is proportional to the amount reaching the detector, but the constant differs between compounds (an FID responds less to ethanol than to 1-propanol) and every injection varies by a few per cent in volume. The internal-standard method fixes both: the same amount of an internal standard — a compound absent from the sample, chemically similar, and well separated — is added to every sample and standard, and quantitation uses the area ratio of analyte to standard. Inject a little more or less and both peaks change together; the ratio does not.

A standard of known concentration gives the response factor F, and the unknown’s area ratio then gives its concentration. Better still, make several standards and plot area ratio against concentration.