How to do a NaOH Flush on an Agilent Fragment Analyzer

Answer

This Information Applies To: 5200/5300/5400 Fragment Analyzer, Fragment Analyzer controller software

Issue:

Over time the dynamic coating polymer on the capillaries needs to be refreshed. Failure to do so can lead to a loss of performance. Indicators that the dynamic coating needs refreshing include:

• Delayed migration
• Peak tailing

Also, if capillaries are stored dry or the Storage Solution is not replaced they can clog. Indicators of clogged capillaries include no flow through the capillaries leading to no signal. Flushing the capillaries with sodium hydroxide (NaOH) followed by Conditioning Solution rejuvenates the dynamic coating and unclogs capillaries restoring instrument performance.

Recommended maintenance schedule:  Agilent recommends performing the NaOH flush as part of troubleshooting procedures and your routine maintenance to restore separation performance. You can add it as part of a regular cleaning schedule weekly or biweekly to ensure optimal performance.

Steps to follow:

Tips: Further information on the NaOH flush procedure is available in the Fragment Analyzer System Manual Appendix. To remove dried gel or Storage Solution, consider performing Method B before the NaOH flush: submerse capillary ends and electrodes in hot water (65–93 °C). Sometimes, the reservoir vent valve needs to be cleaned as well (for more details, see here).

Warning: Sodium hydroxide is corrosive and can cause severe burns when it contacts skin. Always wear appropriate personal protective equipment when handling sodium hydroxide.

1. Prepare 0.5 N NaOH solution. Minimum solution volume required is 15 mL for priming all three lines twice plus the volume required for ten-minute flush as follows:

   ≥12 mL	for a 12-capillary array
   ≥25 mL	for a 48-capillary array
   ≥50 mL	for a 96-capillary array

2. Place a NaOH solution sequentially in the Gel1, Conditioning Solution, and Gel2 positions and prime the tubing twice to clean out potential debris (click Utilities > Prime). Volume needed per prime step: 2.5 mL. You may leave the tube with NaOH in the Gel2 position. 
3. Place a bottle or tube containing fresh 1x Conditioning Solution in the Conditioning position and prime the tubing.
4. Place a bottle or tube containing freshly prepared gel-dye mix in the Gel1 position and prime the tubing.
5. Perform a 0.5N NaOH flush:

   1. In the main screen Operation tab > Capillary Array - Conditioning click Add to queue 

   2. From the Method drop down menu, select Method C Flush - 0.5 N NaOH - 10min 200psi.mthdc (you can check the parameters by clicking Edit). 

   3. Select OK and run the method from the queue (see Figure 1)

      
      Figure 1. How to select Method C Flush
      

       

       
6. If the Gel2 position is not needed otherwise, you may leave the NaOH there. It will not damage the tubing. 

   Caution: If NaOH is not washed out of the capillaries immediately after the NaOH flush, it can damage the capillary array.

7. Perform at least a Full Conditioning (see Figure 2) or a separation method with freshly prepared reagents and 24 µL of blank solution (for quantitative kits), marker only, or samples. This step is crucial to flush out residual NaOH and restore the capillaries. 

 
Figure 2. Where to find the Full Conditioning method

Learn how to effectively maintain your Agilent Fragment Analyzer: Fragment Analyzer system manual

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

 PR7000-8016