Zero Order Mode; Scans - Thermo Scientific Dionex UltiMate 3000 Series Operating Instructions Manual

UltiMate 3000 Series:
Fluorescence Detectors FLD-3100 and FLD-3400RS

5.6.3 Zero Order Mode

In Zero Order Mode, the grating of the emission monochromator is set to the position of
zero order. In this position, the monochromator lets the entire spectrum pass, rather than
only a certain wavelength. The excitation monochromator is set to one wavelength as
usual. The FLD-3400RS allows you to cut off undesired wavelengths using the filter wheel
setting (→ page 85). To activate the Zero Order Mode in Chromeleon, set the
EmWavelength property to ZeroOrder.
Use this mode during method development, if you do not know the retention times of the
various substances in your sample. As the entire range of emission wavelengths is acquired,
you can determine the retention times of all substances in a single run.
The Zero Order Mode is also suitable for samples that emit light at a broad spectral
bandwitdh. In this case, the measured intensity is higher in Zero Order Mode than under
normal operation. This allows you to measure samples with a lower concentration.
5.6.4 2D Scans
With the FLD-3x00(RS) detectors, you can also record several types of 2D spectra. The
excitation monochromator or emission monochromator (or both simultaneously) moves
over a settable wavelength range, while the intensity of the fluorescence signals is
measured and recorded continuously for each wavelength. This allows you, for example, to
record spectra for determining the optimum emission and excitation wavelengths.
During all scans, stop the pump flow or pump at a very low flow rate to achieve stable
conditions in the flow cell of the fluorescence detector.
A baseline spectrum with the same parameters can be recorded for all scan modes. The
baseline spectrum is stored and automatically subtracted from the recorded spectra. The result
is a difference spectrum. If the parameters for a scan change, the background spectrum is
automatically deleted and a new spectrum must be recorded. You can delete a baseline
spectrum any time by using the ClearBaseLine command.
Chromeleon automatically saves the spectra, including the baseline spectrum, to a spectra
library named FLD3000.LIB. The spectra library is saved in the timebase directory on the
local data source. If a scan is performed during sample processing, the library is saved in the
associated sequence.
There are three different scan types:
Excitation Scan
The wavelength on the emission monochromator is kept constant, while the excitation
monochromator scans a wavelength range. The result is an excitation spectrum of the
sample.
Page 74 Operating Instructions