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Detection

The different detectors


Many detectors can be used in HPLC. However, three detectors are mainly used today for detection in liquid chromatography:

The chromatogram


The chromatogram is the 2D representation of the signal analyzed by chromatography. The two dimensions commonly found on a chromatogram are time and signal intensity (e.g. absorbance, luminescence, conductivity, m/z). In this way, the eluted compounds are represented by peaks on the chromatogram (visible in the figure below):

View of a chromatogram. On the abscissa the retention time, on the ordinate the signal intensity. We can see multiple peaks. The chromatogram is marked UV signal acquired at 227nm.

Some detectors are capable of collecting several signals simultaneously (several wavelengths or m/z) enabling three-dimensional chromatograms to be plotted. However, 3D chromatograms are not very often used in chromatography because the signal processing is complex and requires advanced mathematical tools.

View of a chromatogram in 3D. Two axes retention time and m/z ratio form the base of the chromatogram, on which the peaks are located in the 3rd dimension, distributed at different positions on the first 2 axes.

Quantification


The quantification of the compounds analyzed in HPLC is based on the peaks visible on the chromatogram.

The useful parameter for quantification is either the area of the peak or its height. In general, quantification is preferred using peak area rather than peak height. The peak area is obtained using the integration software sold with chromatographic instruments.

On the chromatogram below you can see a peak that has been automatically integrated by the software. Despite the good quality of recent software, caution is advised for each peak integration as an incorrect integration can generate important errors in the quantification of the compounds.

Graph with time in min on the abscissa, and fluorescence in LU on the ordinate. The curve forms a peak which is distributed in 3 phases in time: the base line, the shoulder at the beginning of the peak, the peak itself and finally the shoulder at the end of the peak.
Chromatographic peak integration

For a quantification to be correct, some important points must be respected:

  • The integration must start before the pre-peak shoulder
  • The integration must finish after the end-peak shoulder
  • The integration must not be too far from these two shoulders
  • The integration line must be in line with the baseline of the chromatogram

If all these criteria are respected then the integration is correct and quantification can be done using the calibration method

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