Detector Control
Recording Fluorescence Spectra (RF2000)

Fluorescence detectors not only allow you to scan the entire range of the spectrum. Thus, spectra acquisition is possible for determining the optimum emission and excitation wavelengths.

It is not possible to record spectra simultaneously with the emission signal. Single spectra scans are performed, instead. To guarantee a stable status in the flow cell stop the pump flow while the scans are performed. The result of a scan is either an emission spectrum or an excitation spectrum.

How to record an emission/excitation spectrum

 Tip:

The duration of the scanning procedure depends on the wavelength range to be scanned and on the scan speed. You can add an LED or a color box to the Control Panel to visually indicate whether scanning is underway.

  1. Stop the pump flow via the Flow=0 command.

  2. Click F8 to open the Commands dialog box. Execute the ScanEmission command. Depending on when the pump flow was stopped and when the scan procedure was started, either a background spectrum or a peak spectrum is saved.

  3. Perform the analog procedure using the ScanExcitation command to determine an optimum excitation value.

 Tip:

While the RF2000 Fluorescence Detector is scanning, it cannot receive any other commands. The scanning process cannot be interrupted.

When the detector finishes scanning, the resulting spectrum is saved in the detector and overwritten with each new recording. To retain the data, Chromeleon saves the spectra automatically to a Spectra Library named  RF2000.LIB. The spectra library is saved in the timebase directory of the local datasource. If a scan is performed during sample processing, a library with the same name is saved in the associated sequence.

 

How to record a difference spectrum

To record a difference spectrum:

  1. Record a background spectrum as described above.

  2. Select Save Background Spectrum to save this spectrum separately in the detector.

  3. Reset the pump flow to the original value via the Flow= ... command. Continue until the maximum of the peak to detect is reached.

  4. Stop the pump flow and record a new spectrum.

    The two spectra now exist in the RF2000 spectra library and as individual spectra in two different storage locations within the detector.

  5. Execute the GetSpectraDifference command to receive a difference spectrum.

The detector behaves as if a Scan procedure was started. The result of the difference formation is also saved to the RF2000 spectra library. This spectrum indicates the optimum emission or excitation value.

 Tip:  

The basic requirement for forming difference spectra is the correspondence between the wavelength range defined by the start wavelength and the end wavelength, as well as the spectra type (Excitation or Emission).

For an example program, refer to  Determining the Optimum Emission Wavelength (RF2000).