L2 - Chromatography

Chromatography - General Theory

Chromatography is used to separate and analyse small amounts of mixtures

Methods involve a stationary phase and a mobile phase.

There are several forms of chromatography:

Paper Chromatography

Comparison can be a problem if…

a) components have similar R_f values

b) the unknown substance is new and there is no previous chemical to compare it with

Thin-Layer Chromatography

Column Chromatography

Summary Videos and Practice Practical

Advanced Gas Chromatography

Gas chromatography is a separation technique in which the components of a sample partition between two phases:

1. The stationary phase.
2. The mobile gas phase.

According to the state of the stationary phase, gas chromatography can be classified in gas-solid chromatography (GSC), where the stationary phase is a solid, and gas-liquid chromatography (GLC) that uses a liquid as stationary phase. GLC is to a great extent more widely used than GSC.

During a GC separation, the sample is vaporized and carried by the mobile gas phase (i.e., the carrier gas) through the column. Separation of the different components is achieved based on their relative vapor pressure and affinities for the stationary phase. The affinity of a substance towards the stationary phase can be described in chemical terms as an equilibrium constant called the distribution constant Kc, also known as the partition coefficient, where [A]s is the concentration of compound A in the stationary phase and [A]m is the concentration of compound A in the stationary phase.

The distribution constant (Kc) controls the movement of the different compounds through the column, therefore differences in the distribution constant allow for the chromatographic separation. Figure 1 below shows a schematic representation of the chromatographic process. Kc is temperature dependent, and also depends on the chemical nature of the stationary phase. Thus, temperature can be used as a way to improve the separation of different compounds through the column, or a different stationary phase.

Typical Gas Chromatograms

Figure 2 shows a chromatogram of the analysis of residual methanol in biodiesel, which is one of the required properties that must be measured to ensure the quality of the product at the time and place of delivery.

Chromatogram (Figure 2 A) shows a standard solution of methanol with 2-propanol as the internal standard. From the figure it can be seen that methanol has a higher affinity for the mobile phase (lower Kc) than 2-propanol (iso-propanol), and therefore elutes first. Chromatograms (Figure 2 B & C) show two samples of biodiesel, one with methanol (Figure 2 B) and another with no methanol detection. The internal standard was added to both samples for quantitation purposes.

Interpreting Gas Chromatographs Quantitatively

• Most modern GC instruments allow for integration of the peaks in a GC spectrum with the click of a button.
• Peak integrations are useful because it is possible to correlate the area under a peak to the quantity of material present in a sample.
• Note it is the area of a peak that is relevant, not the height.
• A tall, narrow peak may have a smaller area than a short, wide one.
• It would be very useful if peak areas would directly reflect mixture composition, but that is unfortunately not always the case.
• The reason for this has to do with how compounds are detected upon exit from a column.