Performing Capillary Alignment in the Controller Software for Agilent Parallel Capillary Electrophoresis Instruments

Answer

This Information Applies To: Agilent 5200/5300/5400 Fragment Analyzer, and Femto Pulse

Issue

Perform a capillary alignment when a new array is installed in your Agilent Parallel Capillary Electrophoresis instrument. An alignment may also be performed during troubleshooting.

Steps to Follow

Tip: An optical alignment can only be performed without dye for a 12- or 48-capillary array.

1. Select No when from the Capillary Alignment Prep window (Figure 1). The real-time view of the Capillary Alignment window will open (Figure 2). 

   
   
   Figure 1. Capillary Alignment Prep window. 

   
   
   Figure 2. Capillary alignment pop-up window. This screenshot is from a 5200 Fragment Analyzer.
   

2. Right-click the blue display area and select Reset All to reset the camera array window.
3. Adjust the contrast slide bar to the left to brighten the display (Figure 3, Figure 4)
4. Draw a box around the capillary array display area. Left-click and drag the appropriate area (Figure 3, Figure 4)

   Tips: Avoid the Top Red CCD Camera Reference Area and the Capillary Alignment References.

   
   
   Figure 3. Capillary alignment display. This screenshot is from a 5200 Fragment Analyzer.
   1. Contrast slider. 2. CCD Camera reference. 3. Box drawn by left-clicking and dragging. 4. Capillary alignment references.
   

   
   
   Figure 4. Capillary alignment display. This screenshot is from a Femto Pulse. 
   1. Contrast slides. 2. Box drawn by left-clicking and dragging. 3. Capillary alignment references.
    

5. After the box is drawn, right-click and select Set Camera Window. 
6. If you are using a Fragment Analyzer, adjust the height to 14. If you are using a Femto Pulse, adjust the height to 100.                                                                                                                                  
7. Adjust the red baseline seen in Figure 2 until a red dot is observed above each capillary peak. The baseline position determines which capillary peaks are selected. It is important to ensure that this red line is above the baseline.
8. Select Align from the menu above the capillary alignment display area (Figure 2 and Table 1). A blue vertical line will be placed through the center of each capillary. If the blue lines are not in the exact center of each peak, adjust the lines by left-clicking and dragging to the desired location.                                                                                                                                         

   Icon	Description
   Align	Aligns cursors to peaks.
   Save	Saves changes to the alignment and exits the window.
   Cancel	Cancels any actions and closes the file.
   Auto Space	Auto locates the capillary positions based off the first capillary position. Positions will need manual adjustment.
   Read raw	Opens the Align from File window allowing the user to complete the capillary alignment using a previously run file.

   Table 1. Capillary alignment display menu options. 
   
   

9. Select Align every time the red baseline is moved. This alignment ensures that the peaks are selected for integration and places a blue vertical alignment line in the middle of each peak (corresponding to the location of the red dots).
10. To save the capillary locations and close the Capillary Alignment window, select Save from the menu above the capillary alignment display area (Figure 2 and Table 1). 

Learn how to effectively maintain and operate your Agilent Parallel Capillary Electrophoresis systems: 5200, 5300, and 5400 Fragment Analyzer System Manual on Agilent.com Femto Pulse User Manual on Agilent.com

For Research Use Only. Not for use in diagnostic procedures.

PR7001-1728