Performing a Delay Calibration of Fraction Collector with a UV Detector via Lab Advisor

Answer

This Information Applies To: Agilent Analytical Fraction Collector G1364C/F and G5664A/B

Issue

The delay calibration determines the delay time between the detector and fraction collector in the system. The delay is used to compensate the time a compound needs to travel between the point of detection in the detector and the point of collection in the fraction collector (Figure 1). This time difference allows the calculation of the delay volume.

Figure 1. Scheme explaining Delay Volume Calibration
1. Injection of calibration sample (without column), 2. UV detector, 3. Fraction collector, 4. Delay sensor (FDS), 5. Waste

Background

Linking the delay volume to a pump allows the system to automatically change the delay time whenever the flow rate is changed. This is only valid for time and UV-based systems.

The delay volume is calculated as this formula V_delay = t_delay * F, where:

• V_delay is delay volume
• t_delay is delay time
• F is preparative flow

For an analytical fraction collector, a zero dead volume union is used.

Items Required

• Delay Sensor Calibrant (p/n G1946-85020)
• 1/16 in union, zero dead volume, stainless steel (p/n 0100-0900)
• Solvent A: water with 0.1% formic acid

Steps to Follow

1. Remove the column. Install a zero dead volume union.
2. Start OpenLab Acquisition or OpenLab ChemStation Online session and flush the system with 100% solvent A (water with 0.1% formic acid) for 5–10 minutes.
3. Exit the OpenLab software.
4. Make sure that both standard outlet and Fraction Collector’s flow delay sensor waste tubing go to the waste container.
5. Add at least 500 µL of the delay calibration solution into a 2 mL vial and place the vial in the position 1 of the autosampler.
6. Launch Agilent Lab Advisor. See article on How to Install Lab Advisor. 
7. Connect the HPLC to Lab Advisor. 
8. In Lab Advisor go to the Service & Diagnostics. From the list of modules, select your Fraction Collector. In the list on the right, click Delay Volume Calibration. Then click the Run button (Figure 2).

   
   
   Figure 2. Run Delay Volume Calibration in Lab Advisor
   1. Service & Diagnostics, 2. Fraction collector, 3. Delay volume calibration, 4. Run button
   
   

9. In the window that opens, select the following parameters.
   
   1. In Required Modules select the Pump, Detector, and Injector for the Delay Volume Calibration (Figure 3). The pump used for the delay volume calibration must be the same pump used for the fraction collector.
      

      
      
      Figure 3. Parameter Setting - Required Modules

   2. In the Pump section, set the Flow rate, Stoptime, and Solvent Channel (Figure 4).
      1. Flow rate: set the target flow rate for the pump. It can be from 0.1 to 5 mL/min. This flow rate will be used in the acquisition method.
      2. Stoptime: 1 minute by default
      3. Solvent Channel: select the corresponding pump
         

         
         
         Figure 4. Parameter Setting - Pump
         
         

   3. In the Detector section, leave the Use current wavelength, bandwidth, and data rate settings from the detector check box unchecked. This ensures that these settings are overwritten by the values from the delay calibration method, which are optimized for the calibrant. Mark the check box only if, for some exceptional reason, you want to retain the current detector settings for the delay calibration run (Figure 5).
      

      
      
      Figure 5. Parameter Setting - Detector 
      
      

   4. In Injector specify the injection Volume and the Vial position of the calibration sample (Figure 6).
      1. Volume: 25 µL
      2. Vial position: 1
         

         
         
         Figure 6. Parameter Setting - Injector
         
         

   5. Option for G1364A-D/G5664A fraction collector cluster:
      Clustering: Click the Selection Valve down-arrow in the Clustering section and select the valve that is used to switch between the clustered fraction collectors. The delay volume calibration is executed on the fraction collector that is connected to Port 1 of the selected valve. This action is not available for the Fraction Collector II Cluster.
      
10. Click the OK button to start the calibration.
11.  After the sample is injected, it is possible to click the Signals tab to see the UV detector and delay sensor signals (Figure 7).
    

    
    
    Figure 7. Checking the Signals tab
    
    

12. At the end of the procedure, the delay volume is calculated automatically for the specified instrument setup and is displayed in the Result section along with the delay volume currently stored in the fraction collector (Figure 8). Users can choose to replace the current value with the new delay volume by clicking the OK button.
    

    
    
    Figure 8. Checking the Result tab
    
    

13. After the calibration, the procedure offers to stop the main flow (recommended), to switch off UV lamp, etc. (Figure 9).
    

    
    
    Figure 9. Pop-up message after the delay volume calibration 
    
    

14. The delay volume must be positive otherwise the calibration is invalid. The delay volume is written to the Fraction Collector firmware and the value is coupled with the used UV detector and will be used for fraction collection. To check or change the value, go to Service & Diagnostics and select your Fraction Collector. In the list on the right side, click Delay Volume Tool.
15. Disconnect the HPLC from Lab Advisor.
16. Launch OpenLab Acquisition or OpenLab ChemStation Online session.
17. Go to the Instrument status, right click Fraction Collector, select Modify > Detector Delay Volumes. In the pop-up window, write down the obtained volume. Click the Ok button to exit the window (Figure 10).
    

    
    
    Figure 10. Modify the detector delay volume
    1. Right-click fraction collector, 2. Modify, 3. Detector Delay Volumes, 4. Modify the volume.
    
    

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