Recording Fluorescence Spectra (FLD-3x00RS): Program Examples

Detector Control
Recording Fluorescence Spectra (FLD-3x00RS): Program Examples

The Dionex Ultimate3000 FLD-3x00(RS) fluorescence detectors allow you to record the fluorescence spectra of substances used in an analysis. Spectrum scans can be performed manually by executing a command (for more information, refer to Recording Fluorescence Spectra) or in a Program. Below are several examples for spectrum scanning in a program.

Tip:

Record the baseline spectrum before the scan, at a time when no peak occurs. It is stored in the detector and automatically subtracted from the recorded spectra. It is not mandatory to perform a baseline scan prior to every excitation/emission/synchro scan. However, you must record a new baseline spectrum before the scan whenever you change the scan settings. The ClearBaseline command deletes the current baseline spectrum.

Emission scan with preceding baseline scan

Run an emission scan to record the emission spectrum of an analyte. The excitation wavelength remains constant while the emission wavelength is scanned over a defined wavelength range. The spectrum maximum indicates the optimum emission wavelength.

The program example below shows a simple emission scan program, with a preceding baseline scan. Adjust the parameters as needed for your application.

0.100	ScanStartExWavelength =	250.0 [nm] ;defines the constant excitation wavelength
	ScanStartEmWavelength =	340.0 [nm] ;defines the lower emission scan limit
	ScanEndEmWavelength =	480.0 [nm] ;defines the upper emission scan limit
	ScanPMT =	Pmt1 ;defines which PMT is used during the scan (only for detectors with second PMT)
	ScanSpeed =	Fast ;defines the speed of any following scan
	ScanSensitivity =	5 ;defines the sensitivity of the PMT during the scan
	ScanFilterWheel =	280nm ;selects the position of the filter wheel (only for FLD-3400RS)
	ScanSignalType =	Standard ;determines if the recorded signal will be corrected regarding the xenon lamp spectrum. Standard will provide the best signal-to-noise ratio in method development.
	BaselineBehavior =	Append ;defines that the baseline is appended to the previous signal after a wavelength, sensitivity, PMT, or filter switch
	BaselineScanEmission	;executes a baseline scan
0.500	ScanEmission	;executes an emission scan

Excitation scan with preceding baseline scan

The program example below shows an example of an excitation scan with preceding baseline scan. Note that some commands are similar to an emission scan. Adjust the parameters as needed for your application.

0.100	ScanStartEmWavelength =	480.0 [nm] ;defines the constant emission wavelength
	ScanStartExWavelength =	250.0 [nm] ;defines the lower excitation scan limit
	ScanEndExWavelength =	450.0 [nm] ;defines the upper excitation scan limit
	ScanPMT =	Pmt1 ;defines which PMT is used during the scan (only for detectors with second PMT)
	ScanSpeed =	Fast ;defines the speed of any following scan
	ScanSensitivity =	5 ;defines the sensitivity of the PMT during the scan
	ScanFilterWheel =	280nm ;selects the position of the filter wheel (only for FLD-3400RS)
	ScanSignalType =	Standard ;determines if the recorded signal will be corrected regarding the xenon lamp spectrum. Standard will provide the best signal-to-noise ratio in method development.
	BaselineBehavior =	Append ;defines that the baseline is appended to the previous signal after a wavelength, sensitivity, PMT, or filter switch
	BaselineScanExcitation	;executes a baseline scan
0.500	ScanExcitation	;executes an excitation scan

Synchro scan with preceding baseline scan

The program example below is an example of a synchro scan with preceding baseline scan. A synchro scan alternates both the excitation and the emission wavelengths across a defined wavelength range. During the scan, the offset between excitation and emission is kept constant. This scan type is useful to determine a functioning wavelength pair. Thereby, the maximum of the synchro spectrum indicates the best wavelength combination at the given offset. Conversely, the synchro spectrum does typically not indicate the optimum wavelength combination. Adjust the parameters as needed for your application.

0.100	ScanStartExWavelength =	250.0 [nm] ;defines the lower excitation scan limit
	ScanEndExWavelength =	450.0 [nm] ;defines the upper excitation scan limit
	ScanSyncOffset =	50.0 [nm] ;defines the offset between Ex and Em wavelength. The Em range starts at StartExWavelength + 50 nm (= 300 nm) and ends at EndExWavelegnth + 50 nm (= 500 nm).
	ScanPMT =	Pmt1 ;defines which PMT is used during the scan (only for detectors with second PMT)
	ScanSpeed =	Fast ;defines the speed of any following scan
	ScanSensitivity =	5 ;defines the sensitivity of the PMT during the scan
	ScanFilterWheel =	280nm ;selects the position of the filter wheel (only for FLD-3400RS)
	ScanSignalType =	Standard ;determines if the recorded signal will be corrected regarding the xenon lamp spectrum. Standard will provide the best signal-to-noise ratio in method development.
	BaselineBehavior =	Append ;defines that the baseline is appended to the previous signal after a wavelength, sensitivity, PMT, or filter switch
	BaselineScanSynchronous	;executes a baseline scan
0.500	ScanSynchronous	;executes an synchro scan

Emission scan using a trigger

The trigger example below shows a Trigger that starts a scan at a signal change of 250.000 counts/second or when reaching 2.500.000 counts. The trigger automatically reduces the flow to zero, defines the scan conditions, performs the scan and then increases the flow rate to the initial value. The trigger is activated each time the conditions become true. The trigger can be used to record spectra for any peaks independent of the retention time.

You can add this trigger example to your program by adjusting the values as needed.

;Trigger Control
1.000	Trigger PEAK (Emission_1.Delta > 250000) OR (Emission_1 > 2500000) ;activates the trigger when the signal increases for more than 250,000 counts/second or total signal level exceeds 2,500,000 counts
	MaximumFlowRampDown =	Infinite ;defines the speed with which the pump reduces the flow rate
	Flow =	0.000 [ml/min] ;sets the pump flow to zero to park the peak in flow cell during scan
	FLD.BaselineBehavior =	Append ;defines that after a wavelength, sensitivity, PMT, or filter switch, the baseline is appended to the previous signal
	FLD.ScanSpeed =	Fast ;defines the emission scan speed
	FLD.ScanStartExWavelength =	255.0 [nm] ;defines the constant excitation wavelength
	FLD.ScanStartEmWavelength =	275.0 [nm] ;defines the lower emission scan limit
	FLD.ScanEndEmWavelength =	600.0 [nm] ;defines the upper emission scan limit
	FLD.ScanSignalType =	Standard ;determines if the recorded signal will be corrected regarding the xenon lamp spectrum. Standard will provide the best signal-to-noise ratio in method development.
	FLD.ScanEmission	;starts the emission spectrum scan
	Wait FLD.Ready	;highly important - restarts pump flow only AFTER scan has finished
	MaximumFlowRampUp =	10 [ml/min²] ;defines the speed with which the pump increases the flow rate ;outside trigger control you can set MaximumFlowRampUp to a different value (e.g., to slowly increase the flow at system startup)
	Flow =	1.000 [ml/min];sets the pump flow to the initial value.
	EndTrigger
;End of Trigger

Excitation scan using a trigger

The trigger example below shows an excitation scan with a trigger as described above. You can add this trigger example to your program by adjusting the values as needed.

;Trigger Control
1.000	Trigger PEAK (Emission_1.Delta > 250000) OR (Emission_1 > 2500000) ;activates the trigger when the signal increases for more than 250,000 counts/second or total signal level exceeds 2,500,000 counts
	MaximumFlowRampDown =	Infinite ;defines the speed with which the pump reduces the flow rate
	Flow =	0.000 [ml/min] ;sets the pump flow to zero to park the peak in flow cell during scan
	FLD.BaselineBehavior =	Append ;defines that after a wavelength, sensitivity, PMT, or filter switch, the baseline is appended to the previous signal
	FLD.ScanSpeed =	Fast ;defines the emission scan speed
	FLD.ScanStartEmWavelength =	500.0 [nm] ;defines the constant emission wavelength
	FLD.ScanStartExWavelength =	250.0 [nm] ;defines the lower excitation scan limit
	FLD.ScanEndExWavelength =	450.0 [nm] ;defines the upper excitation scan limit
	FLD.ScanSignalType =	Standard ;determines if the recorded signal will be corrected regarding the xenon lamp spectrum. Standard will provide the best signal-to-noise ratio in method development.
	FLD.ScanExcitation	;starts the excitation spectrum scan
	Wait FLD.Ready	;highly important - restarts pump flow only AFTER scan has finished
	MaximumFlowRampUp =	10 [ml/min²] ;defines the speed with which the pump increases the flow rate ;outside trigger control you can set MaximumFlowRampUp to a different value (e.g., to slowly increase the flow at system startup)
	Flow =	1.000 [ml/min];sets the pump flow to the initial value.
	EndTrigger
;End of Trigger

Synchro scan using a trigger

The trigger example below shows a synchro scan with a trigger as described above. You can add this trigger example to your program by adjusting the values as needed.

;Trigger Control
1.000	Trigger PEAK (Emission_1.Delta > 250000) OR (Emission_1 > 2500000) ;activates the trigger when the signal increases for more than 250,000 counts/second or total signal level exceeds 2,500,000 counts
	MaximumFlowRampDown =	Infinite ;defines the speed with which the pump reduces the flow rate
	Flow =	0.000 [ml/min] ;sets the pump flow to zero to park the peak in flow cell during scan
	FLD.BaselineBehavior =	Append ;defines that after a wavelength, sensitivity, PMT, or filter switch, the baseline is appended to the previous signal
	FLD.ScanSpeed =	Fast ;defines the emission scan speed
	FLD.ScanStartExWavelength =	250.0 [nm] ;defines the lower excitation scan limit
	FLD.ScanEndExWavelength =	450.0 [nm] ;defines the upper excitation scan limit
	FLD.ScanSyncOffset =	50.0 [nm] ;defines the offset between Ex and Em wavelength. The Em range starts at StartExWavelength + 50 nm (= 300 nm) and ends at EndExWavelegnth + 50 nm (= 500 nm).
	FLD.ScanSignalType =	Standard ;determines if the recorded signal will be corrected regarding the xenon lamp spectrum. Standard will provide the best signal-to-noise ratio in method development.
	FLD.ScanSynchronous	;starts the synchronous spectrum scan
	Wait FLD.Ready	;highly important - restarts pump flow only AFTER scan has finished
	MaximumFlowRampUp =	10 [ml/min²] ;defines the speed with which the pump increases the flow rate ;outside trigger control you can set MaximumFlowRampUp to a different value (e.g., to slowly increase the flow at system startup)
	Flow =	1.000 [ml/min];sets the pump flow to the initial value.
	EndTrigger
;End of Trigger

For a list of all fluorescence detector commands, refer to Dionex UltiMate 3000 Series Fluorescence Detectors.