Chromatography is the general name given to the methods by which two or more compounds in a mixture are physically separated by distributing between two phases: a stationary phase which can be a solid or liquid supported on a solid and a mobile phase, either a gas or a liquid which flows continuously around the stationary phase.
The separation of the individual components results from the relative difference in affinity for the stationary phase.
Gas chromatography (GC) and High Performance Liquid Chromatography (HPLC) are both separation techniques.
Mobile phase
HPLC uses a liquid mobile phase, these are generally mixtures of solvents of compatible polarities. Commonly for reversed phase chromatography this is a combination of water and either acetonitrile or methanol.
In gas chromatography the mobile phase is a single high purity gas either helium, hydrogen or nitrogen
Operating temperature
HPLC separations are mostly carried out at ambient temperatures whereas GC separations are carried out at elevated temperatures as the analyte needs to be in gaseous form.
Nature of compounds
GC separations are mainly carried out on compounds ranging in molecular weights up to a few hundred. Such compounds separate on differences in their volatilities as well as interactions with the column stationary phase and remain stable at high temperatures. On the other hand, compounds separated on HPLC have higher molecular weights ranging from a few hundred to several million for large polymers and biomolecules. Such compounds can be analysed at room temperature up to around 80C only because at elevated temperatures they tend to degrade.
Column Dimensions
Liquids used as carrier in HPLC generally have higher viscosity in comparison to gases used in GC, this results in increased column back pressures in HPLC. It is for this reason that HPLC columns are much shorter and have wider diameters in comparison to GC columns which are much longer and narrower – typically HPLC columns range in length from 50mm to 250mm and internal diameters of 2.0mm to 4.6mm versus 15m to 100m and 0.25mm to 0.53mm respectively for GC. Increased column length improves resolution between closely spaced peaks. As the trend is towards faster analysis, columns for HPLC are now as short as 10 mm in length.
Column Packings
Column packings offer greater resistance to flow of liquids in comparison to gases. Gases also have a higher permeability within solid supports compared to liquids. The retention mode of HPLC columns depends on polarity differences or molecular sizes whereas GC separations are based on differences in volatilities of compounds.
Detection principles
HPLC detection is commonly based on non-destructive detection such as Ultra Violet (UV) or Refractive Index (RI) (Evaporative Light Scattering Detector (ELSD) is an exception to this). On the other hand GC detection is based largely on destructive principles such as Flame ionisation detection (where the sample is burnt). Mass spectrometry detectors common to both LC and GC are destructive in nature.
Samples
- Compounds of interest must be extracted out of the samples and into solution.
- Extracted samples must be free from particulates and precipitates.
- Ideal solvents for GC methods are volatile solvents such as hexane, diethyl ether or dichloromethane.
- For HPLC methods more polar solvents such as water and water mixtures, methanol and acetonitrile may be used.
In general, for GC substances that vaporize below 300 °C (and therefore are stable up to that temperature) can be measured quantitatively. The samples are also required to be salt-free; they should not contain ions. Very minute amounts of a substance can be measured, but it is often required that the sample must be measured in comparison to a sample containing the pure, suspected substance known as a reference standard.