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How to Condition a CEC Column

Reference Number: AA-02511 Created: 12/19/2014 01:01 PM Last Updated: 12/19/2014 01:09 PM

It is very important that you condition the capillary properly before using it.
DO NOT CONDITION YOUR COLUMN LIKE A CE COLUMN:
IT WILL BE DESTROYED.

 

Instructions for Conditioning and Installation of Milli-phase CEC Columns:

Mobile Phase:

1.Begin with a similar mobile phase that you would use in HPLC for a particular separation you are approaching:
     •Typical CEC mobile phases in CEC are 80% Acetonitrile w/ 1-10mM Borate, MES, Tris etc. A mixture of organic solvent with a buffer is most widely used.
     •To generate sufficient EOF during the method, the pH of this solution should be between pH 3 and 9.
     •Filter this mobile phase solution and thoroughly sonicate with vacuum for at least 10 minutes. It is important to degas this mixture.


Installation:

2.Prepare the MicroSolvCEC Milli-phase column for Installation: 
•Remove the end caps (Teflon tubing) and the window protection sleeve from the column.
Installation into your Instrument:
3.Carefully install the Milli-phase column in the CE cartridge or directly into the instrument as you would an HPCE Capillary. 
•Be careful not to bend the capillary until the detection window has been properly secured and it will not bend. The window is very fragile.
Conditioning:
4.Fill the MicroSolvCEC Pump slowly with the mobile phase you selected in step no 1. 
•Hold the syringe upside down and up to a light source. If you find any bubbles, tap on the syringe with your finger until the bubbles have moved to the tip, push the plunger so to remove the bubbles.
•Be sure that any and all air bubbles have been removed from the syringe and the Teflon tubing.
Click here to see a picture of the MicroSolvCEC pump.
5.Carefully insert the inlet of the Milli-phase Column into the Teflon tubing about 3mm into the tubing.
6.Tighten the handle on the Pump to generate a moderate pressure (~ 500psi). 
•Lay the pump down.
•The pump will keep the pressure on the column for up to ½ an hour.
•Hand tighten the pump again if necessary.


Inspection:

7.Place the open section of the column into a microscope directly on light source under the field of view. 
•Use the open section on the outlet side of the column for inspection.
8.As the pressure from the syringe moves the mobile phase through the column.
•You will be able to see any air bubbles marching past your field of view through the inspection scope. Do not expect to see any droplets on the outlet of the column without a magnifier.
•The flow rate will be less than the rate of evaporation.
9.Allow the syringe to condition the column for two hours.
•Return to apply more pressure every ½ hour or so.
•Inspect the flow rate for five minutes after each time you apply more pressure.
10.After ten minutes when the air bubbles have completely disappeared, release the pressure on the syringe by loosening the handle on the pump. 
•Allow the pressure to dissipate for 5 minutes.
11.Cut excess capillary from the outlet end of the column if desired.
•NEVER CUT THE INLET SIDE OF THE COLUMN.
12.When you are satisfied that all air bubbles and dry spots have been removed, your column is ready to be used.


Equilibrate your Column:

13.Run your column on your instrument at a low voltage (less than 5kV) for 15 minutes with the mobile phase from step one.
14.Increase your voltage to the desired value of your method.
•A typical current for a 75mm ID x 25cm column packed with 3mm ODS running 80% Acetonitrile with a 4mM Sodium Borate Buffer is about 2mA at 20kV.

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