Chromatogram

The information obtained from a chromatographic experiment is contained in the chromatogram. When the elution mode is used this consists of a plot of the concentration or mass profile of the sample components as a function of the flow of the mobile phase or as a function of time. Typically the yy-axis will be detector response and the x-axis time or volume of mobile phase in column chromatography or migration distance in planar chromatography. The position of each peak in the chromatogram is characteristic of the identity of the compound and the area under the peak is a function of the concentration or amount of each compound. Peak widths in the chromatogram are controlled by solute-dependent kinetic factors, which in turn can be used to deduce values for

Figure 4 Calculation of the RF value in planar chromatography. ZX distance moved by the sample from the sample origin; Z0, distance between the solvent entry position and the sample origin; Zf, distance between the solvent entry position and the solvent front.

characteristic physical properties of either the solute or the mobile and stationary phases.

The position of a peak in the chromatogram is made up of two contributions: (1) the time (or volume of mobile phase) required by a compound that does not interact with the stationary phase to reach the detector from the sample inlet, called the column hold-up time or dead time; and (2) the time that individual compounds spend in the stationary phase (all compounds spend the same time in the mobile phase). The column hold-up time is a feature of the experimental system and is not fundamentally related to solute properties. Because of this, retention time is not a useful parameter for column comparisons. A more useful term is the retention factor (previously known as the capacity factor), k, defined as the ratio of the time the solute spends in the stationary phase to the time it spends in the mobile phase. The ratio of the retention factors for two solutes is called the separation factor, a, which by convention is always expressed with the larger retention factor in the numerator (a > 1). The separation factor expresses the ease with which the chromatographic system can separate two compounds, and is directly related to the difference in free energy for the interactions of the two compounds in the chromatographic system. It is a major optimization parameter, as we shall see later. In planar chromatography retention is usually expressed as the retardation factor, RF, equiva lent to the ratio of the distance migrated by the solute zone, ZX, to the distance moved by the solvent front, Zf — Z0, measured from the sample application position, (1>RF>0), as illustrated in Figure 4. The planar chromatographic retardation factor and the column retention factor are simply related by k =

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