By definition, pressure is inherent in HPLC systems because solvent is being pumped through a packed bed of small particles. It is important for chromatographers to understand what “normal” pressure is for their HPLC column and system (and system pressure without HPLC column installed), so when there is excess pressure, they can start troubleshooting. Excess pressure in HPLC systems typically comes from particulates building up in the HPLC flow path and on the inlet of the HPLC column. There are three main sources of particulates in an HPLC system: sample, mobile phase, and components in the fluid path that wear over time.
For the purpose of this blog, we are assuming that you accurately isolated the source of the high pressure coming to your HPLC column. Please note that clogged tubing is another common source of elevated HPLC pressure and can easily be identified by systematically removing components one at a time and resolving the issue.
Reducing Column Pressure
HPLC columns have an inlet and outlet frit to hold the packing inside the column. Many times, column pressure is caused from the inlet frit clogging. It is possible to attempt to backflush the HPLC column to get back to your “normal” operating pressure.
How to backflush an HPLC column
- Reverse HPLC column direction
First, remove the column from the HPLC system and attach the “outlet” side of the column to the tubing that normally is connected to the inlet. The side of the column that is normally the inlet is now the outlet, and this new outlet flow should be directly connected to the waste container. Take care, as you must NOT backflush into the detector (or the contaminants from the frit/column will end up in your detector, and you will have more problems to troubleshoot).
- Solvent wash the column
Wash solvents are selected based on the suspected contamination and the column media. If you suspect particulate, wash the column with a solvent at a high flow rate based on your column manufacturer’s recommended protocol of solvents. If you suspect something is adsorbed to your column packing, wash the column with varying solvents at a low flow rate, based on your column manufacturer’s recommended protocol of solvents.
- Reconnect the column in the original direction
Initiate your desired method. If your column pressure and chromatography are back to your method’s “normal,” you have likely successfully cleaned your column. If things are not back to normal, you can consider a longer/more aggressive backflush, but many times it is most economical to replace the HPLC column at this point (and try to prevent excess column pressure in the future).
How to prevent excess column pressure
“An ounce of prevention is worth a pound of cure.”—Benjamin Franklin.
Whenever possible, it is recommended to consider filtering your sample prior to analysis. Syringe filters, filter vials, filter plates, and centrifuge filters are popular options. Effective sample preparation can save you money in the long run, as it can help extend column lifetime as well as system uptime.
Even when samples are filtered, the mobile phase and wear and tear on instrument parts can wreak havoc on your HPLC column. Therefore, using inline filters (and changing the frit regularly as part of routine maintenance) and/or guard columns (and changing the guard regularly as part of routine maintenance) can minimize the particulate that would reach your HPLC column. Adhering to a preventative maintenance schedule allows you to replace items that eventually wear down and can also help prevent excess system pressure. Preventative maintenance typically includes replacing piston seals, rotors, and needles seats, to name a few.
In summary, it is best to minimize the chance of particulate entering your HPLC system and reaching your HPLC column. Filtering samples, using high-quality solvents (or filter solvent), and routine system maintenance are all recommended to prevent excess pressure in your HPLC column and system. If you have diagnosed that the excess system pressure is coming from your HPLC column, it is recommended to attempt backflushing the column, and if that is not successful, replacing the column.