Related to separation of Biomolecule, buffers are always used as the mobile phase to give a high resolution and efficient result (such as Reverse phase). However, the excess amount of salts in the HPLC column (Like 0.2 M to 0.4 M Sodium Sulfate) may be precipitated to block the Column or tubing seals. This would dramatically increase the system pressure from 15 to 80 bar, For that we are not able to run more than 10-12 cycle for one column (CHP07/10). Thus what methods or procedures can be applied after each HPLC sequence to rapidly remove salts from system?
What approaches can be used to remove the excess amount of salts from HPLC Column?
Thanks in Advance
Surya Bhushan Kumar
Regenerate columns in reverse flow at 1/2 maximum flowrate with 40-60C diH2O. This will absolutely get rid of precipitated salts from your column. If you don't have a column oven then microwave some di-H2O and degass before passing over your column. Many people recommend bypassing the flowcell if you expect a lot of junk to come off, but I keep it in line to watch what is coming off the column and ensure that I have a stable baseline with no salt or 280 nm leaching. Be aware that you might cause condensation in the flow cell with the heated water wash.
What kind of columns are you using? We routinely use PhenylHIC HPLC columns (Waters Biosuite) with 2M Ammonium Sulfate at room temp with little to no increase in system pressure (or DeltaP) over the elution. Often we have some precipitated protein and other junk (from marine extracts) non-specifically interacting with the resin, which increases back pressure and decreases resolution. I usually regenerate HIC columns after one or two injections in reverse flow first with water, then 0.5 M-1M NaOH and then back to water.
Instead of washing the column in the end, you might need to develop a better method. Usually, a pulse of back pressure occurs when a sample is injected. However, the back pressure continuously increased from run to run in your case, which is not good. The retention times of analytes might varied from run to run also. I don't understand why you need so high concentration of Na2SO4, which is not a buffer also. What will happen if you reduce the concentration to 20-50 mM.
If reduced the concentration of sodium sulfate the binding will be very strong and required more organic solvent to elute the protein. Here we are not using sodium sulfate as a buffer.
In less concentration of salt binding will strong and product also not that much pure.
We worked with NaOH/Warm WFI/Reverse Flow (as recommended by vendor) it will not happen.
- Badges
- Science method