Interpreting chromatograms

Make sure you know the direction of the horizontal axis of the chromatogram (usually, either volume or time) - it may run from right to left or vice versa - and make a note of the detector sensitivity on the vertical axis. Ideally, the base line should be 'flat' between peaks, but it may drift up or down owing to a number of factors including:

• changes in the composition of the mobile phase (e.g. in gradient elution);
• tailing of material from previous peaks;
• carry-over of material from previous samples; this can be avoided by efficient cleaning of columns between runs - allow sufficient time for the previous sample to pass through the column before you introduce the next sample;
• loss of the stationary phase from the column (column 'bleed'), caused by extreme elution conditions;
• air bubbles (in liquid chromatography); if the buffers used in the mobile phase are not effectively degassed, air bubbles may build up in the flow cell of the detector, leading to a gradual upward drift of the base line, followed by a sharp fall when the accumulated air is released. Small air bubbles that do not become trapped may give spurious small peaks as they pass through the detector.

Peak close to the origin may be due to non-retained sample molecules, flowing at the same rate as the mobile phase, or to artefacts, e.g. air (GC) or solvent (HPLC) in the sample. Whatever its origin, this peak can be used to measure the void volume and dead time of the column. No peaks from genuine sample components should appear before this type of peak.

Peaks can be denoted on the basis of their elution volume (used mainly in liquid chromatography) or their retention times (mainly in GC). If the peaks are not narrow and symmetrical, they may contain more than one component. Where peaks are more curved on the trailing side compared with the leading side, this may indicate too great an association between the component and the stationary phase, or overloading of the column.