Connecting and Preparing Your Column For Use
Stephen Aspey
When connecting a column to a HPLC instrument it is important to minimize the volume in the tubing and end-fittings in order to reduce extra-column peak broadening. The narrowest tubing appropriate for a particular flow rate and shortest tubing lengths should be used. All tubing should also be carefully cut at 90 degrees to ensure that additional volume does not occur at unions.
Poly-ethylene ether ketone (PEEK) and stainless-steel fittings are available for connecting tubing to the column and the appropriate fitting should be selected according the mobile phase composition and temperature used in the separation. Most columns use a standard 1/16” diameter 10-32 thread connection. The Tubing and Connection Selection guide provides additional help in all aspects of tubing and column selection.
Guard Columns and Filters
Screw direct connect (Uniguard) guard into the analytical column inlet using finger-tight pressure only. See the section Extending Column Lifetime for more information.
Purging the System
Prepare fresh mobile phases where possible and degas using helium sparging, ultrasonication or an on-line vacuum degasser. The accumulation of air in the system can lead to problems pumping the mobile phase as well as erroneous chromatographic peaks and baseline irregularities. With the column disconnected, purge the system to flush out any old solvent. This should be carried out for each solvent line to be used. Ensure that the solvent in the tubing is miscible with any new solvent to be introduced . The solvent miscibility chart in this guide will help you with this.
It is important to ensure that a mobile phase with a high organic content is not used with a column that contains buffer, or vice-versa. To do so would risk precipitating the buffer and blocking the column. If precipitation is suspected then flush the column first with 100% water to redissolve the buffer, then 100% acetonitrile to re-saturate the stationary phase.
Attaching the Column
Select the appropriate tubing and fittings for the application in hand. See the section Tubing and Connection Selection for more information.
PEEK finger-tight fittings are available with integral, and also separate ferrules. Stainless-steel fittings use a separate ferrule. Where a separate ferrule is used, this should be seated in the screw fitting before tightening.
Feed the screw fitting and ferrule (PEEK or stainless-steel) onto the tubing and push the tubing as far into the column inlet as possible. Then, while holding the tubing in the column inlet, screw the fitting into place. If this is a PEEK fitting then use secure finger-tight pressure only. If this is stainless steel then apply additional wrench pressure – normally an additional 1/8 of a turn.
Equilibration
Ensure that the solvent in the column is miscible with the mobile phase to be used with it. The solvent miscibility chart in this guide will help you with this.
If the mobile phase to be used is immiscible then flush the column with a co-solvent for at least one hundred column volumes, typically 2-4 hours. Propan-2-ol and ethanol are useful co-solvents since they are miscible with non-polar normal phase solvents and also polar reverse-phase solvents. Stationary phases such amino, cyano or diol can be used in normal-phase and reverse-phase mode, and these are normally shipped for immediate use with normal phase solvents.
Having ensured that the solvent in the column is miscible with the mobile phase, the column should be equilibrated for at least ten column volumes of mobile phase. This is done using the appropriate flow rate compatible with the diameter of the column - see the Scaling a Separation section in this guide. Then equilibrate for a period of time dependent on the length of the column: 30mm for 3 minutes, 50 mm for 5 minute, 100 mm for 10 minutes, and so forth.
