You already learned the basics of column packing. When moving to more automated system using low pressure liquid chromatography systems, you can use pre-packed columns. But in order to compare several resins in specific conditions, and also to save money, you might need to pack your own low pressure columns.
The art of packing a column is to distribute all the particles in the column in such a way that you obtain an homogeneous packed bed in a reproducible manner. The following lines might seem a little bit dry, but see it as if you were backing a pie. You can start bringing your own ideas when you become an expert, but the first time you bake it, it is better to stick to the recipe. So let’s start with the ingredients.
- The lab-size columns: These are all similar—a tubing in polymer or glass, a bottom frit which can be fixed or adjustable, and a top frit that can be moved along the column through the adapter.
- The resin: It will most of the time be delivered as a suspension of chromatographic beads in 20% ethanol.
- The packing buffer: You should choose this empirically because the optimal buffer will vary with your specific application. In general, the mobile phase with the highest ionic strength to be used in the separation (including the cleaning and sanitization steps) is the best starting point.
- A pump system: This is probably already a part of your chromatography equipment.
2. Slurry preparation
After de-fining the resin, the slurry is prepared by re-suspending the resin in the packing buffer.
The slurry concentration should then be adjusted for packing the column. The slurry concentration is calculated as the volume of settled gel divided by the total volume of the slurry, and the slurry concentration is adjusted as follows:
- Re-suspend the resin slurry in the de-fining vessel and transfer the homogeneous slurry to a graduated cylinder.
- Allow the slurry to settle overnight (>12 hours) for best results.
- Determine the settled resin volume, and adjust the slurry concentration to 30–50 % by adding or removing packing buffer.
3. Packing the column
Before you start, make sure that the total volume of your column is sufficient to contain the whole slurry volume. If not, add a reservoir on top of it.
- Ensure that the column is leveled prior to packing. Wet the bottom frit in the column with buffer. Allow the buffer to drain a few seconds to remove any air bubbles. Plug the outlet of the column and leave 1 – 2 cm of buffer in the bottom of the column.
- Re-suspend the resin slurry to assure homogeneity. The best way to re-suspend the slurry and to eliminate air bubbles is to place it for a couple of minutes in an ultrasonic bath.
- Carefully pour the resin slurry down along the inside wall of the column. Pouring along the wall prevents air from being trapped in the resin slurry.
- After the resin slurry is transferred to the column, rinse the inside walls of the column using a squirt bottle containing packing buffer.
- Immediately place the flow adapter of the column onto the resin slurry. There should be no trapped air between the flow adapter and the buffer.
- Open the column outlet, and start the pump. Start slowly to flow packing buffer through the column.
- Slowly increase to the final flow rate. This prevents hydraulic shock to the forming bed and prevents uneven packing of the column bed. The flow rate can be ramped up in several incremental changes. These increments will be determined by the size of the column and target flow rate. Some examples are listed in the following table:
|Column Size (ID x L)||Target Flow Rate (mL/min)||Increment (mL/min)||Hold time (min)|
|2.2 cm x 60 cm||2||0.5||0.5|
|9 cm x 30 cm||300||50||2|
|25 cm x 30 cm||2,000||400||3|
- After the bed has fully formed, shut off the pump, and close the column outlet. If you do not use a packing reservoir on top of your column, go directly to (m).
- The entire bed should reside in the lower column section if using a packing reservoir. Using a pipette or pump, siphon the supernatant from the upper reservoir. Remove the upper reservoir and the coupling ring.
- Carefully place the flow adapter into the column, approximately 2-3 cm away from the consolidated bed. Avoid introduction of air into the column.
- Secure the flow adapter in place, begin the pump as described in step g, and open the column outlet.
- The bed will compress further. When compression is complete and pressure is stable, stop the pump and close the column outlet.
- Carefully loosen the flow adapter seal and lower the adapter near to the resin bed. Take care not to disturb the resin bed when moving the flow adapter.
- Repeat steps l) – m), until there is no further compression of the resin bed from the flow adapter. It will usually take 2-3 iterations until the bed is stable.
- In the final step lower the adapter 1 – 5 mm into the bed.
The column is now packed, but not yet ready to use! Before moving on with the real interesting part of your work—performing the chromatographic separation of your target biomolecule—remember to evaluate the quality of your packing! By evaluating your column, you will be able to ensure reproducible results and to avoid wasting your precious compounds by injecting them on a badly packed column.
Stay tuned if you want to learn more about how to evaluate how well you packed your first column! This and more will be covered in Part 2 of this tutorial.