Step 3: Set Up Routine Methods

Multi wavelength detection

Time-programmed wavelength switching traditionally is used to achieve low limits of detection and high selectivity in routine quantitative analysis. Such switching is difficult if compounds elute closely and require a change in excitation or emission wavelength. Peaks can be distorted and quantitation made impossible if wavelength switching occurs during the elution of a compound. Very often this happens with complex matrices, influencing the retention of compounds.

In spectral mode, the FLD can acquire up to four different signals simultaneously. All of them can be used for quantitative analysis. Apart from complex matrices, this is advantageous when watching for impurities at additional wavelengths. It is also advantageous for reaching low limits of detection or increasing selectivity through optimum wavelength settings at any time. The number of data points acquired per signal is reduced and thus limits of detection may be higher, depending on the detector settings compared to the signal mode.

PNA analysis, for example, can be performed with simultaneous multi wavelength detection instead of wavelength-switching. With four different wavelengths for emission, all 15  PNAs can be monitored (Simultaneous multi wavelength detection for PNA-analysis).

Previously, only diode array detectors and mass spectrometric detectors could deliver spectral information on-line to confirm peak identity as assigned by retention time.

Now, fluorescence detectors provide an additional tool for automated peak confirmation and purity control. No additional run is necessary after the quantitative analysis.

During method development, fluorescence excitation and emission spectra are collected from reference standards and entered into a library-at the choice of the method developer. All spectral data from unknown samples can then be compared automatically with library data. Peak confirmation using a fluorescence spectral library illustrates this principle using a PNA analysis. The match factor given in the report for each peak indicates the degree of similarity between the reference spectrum and the spectra from a peak. A match factor of 1,000 means identical spectra.

In addition, the purity of a peak can be investigated by comparing spectra obtained within a single peak. When a peak is calculated to be within the user-defined purity limits, the purity factor is the mean purity value of all spectra that are within the purity limits.

The reliability of the purity and the match factor depends on the quality of spectra recorded. Because of the lower number of data points available with the fluorescence detector in general, the match factors and purity data obtained show stronger deviations compared to data from the diode array detector, even if the compounds are identical.

Peak confirmation using a fluorescence spectral library shows an automated library search based on the emission spectra from a PNA reference sample.