Clarify pre-column pressure vs. system pressure vs. backpressure for prepacked columns used with the äkta purification system

Question

Background: I am using the Äkta pure system for protein purification, with prepacked columns from GE-healthcare. Just to take one example, I use the HisTrap 5 mL column (IMAC: immobilized metal affinity chromatography). I understands the effects of over-exceeding the pressure limits of a column, and therefore vigorously set the pre-column pressure to 0.5MPa everytime I use the äkta. However, I am starting to question or how I should think about the different pressure, and if 0.5MPa is correct for the different columns I am using.

I was taught to always set the pre-columns pressure, which makes sense since as far as I can tell – this is the actual pressure inside the column (pressure felt by the resin). However, If I open the documentation on the HisTrap 5 mL column from GE-healthcare, I can’t seem to find much about the pre-column pressure limit. I also find the terms max-backpressure limit in the documentation for the column, and system pressure.

Questions: How is the system pressure different from the pre-columns pressure? Should I calculate the pre-column pressure myself as an effect of the size and length of the tubing leading to the column? Is there a good article or webpage that goes through this theory at a basic level?

It seems obvious, but how can backpressure be created in a system that is not closed and goes one way (or perhaps the Äkta is creating the pressure)?

Answer 1

Is there a good article or webpage that goes through this theory at a basic level?

Yes, Chapter 7 of the manual linked to by Andreas Tosstorff is a good source, and is also the source of the images below. The manuals for your ÄKTA system and the HisTrap columns discuss pressure in some detail as well.

How can backpressure be created in a system that is not closed and goes one way?

This is a physics question and, not being a physicist, I can't go into detail. However, I also use the ÄKTA system for FPLC and may be able to provide some form of an answer. Any fluid moving through tubing experiences some resistance from, for example, friction between the fluid and the tubing walls, bends in the tubing and obstructions like filters, mixers, valves, etc. Even though the system is open at one end, there is resistance to the flow of fluid through it which creates backpressure. The largest sources of backpressure in the FPLC system at low flow rates are filters, flow restrictors (if installed) and the column itself.

How is the system pressure different from the pre-columns pressure?

The system pressure for the ÄKTA is measured at the pump. This measurement in itself is not very informative. The pressures that are important as far as keeping your column safe are the pre- and post-column pressures (the pressure measured immediately before and after the column, respectively), and the difference between these two (the pressure across the column). Your column has two pressure limits:

1. Hardware pressure limit: this is the maximum pressure the column itself will withstand before there is a risk of failure. I know this from experience after a malfunctioning valve gave 4 times the maximum hardware pressure to my column. In my case, failure meant the top of the column blowing. The pre-column pressure should not exceed the hardware pressure limit.
2. Resin pressure limit: this is the maximum pressure the resin within the column can withstand before collapsing. The pressure across the column should not exceed the resin pressure limit.

Should I calculate the pre-column pressure myself as an effect of the size and length of the tubing leading to the column?

Some ÄKTA systems are capable of measuring pre- and post-column pressures directly. Mine is not, and I don't know about yours. In the case where pressure is measured at the pump, the pre-column pressure is equal to backpressure between the pump and the column (caused by the tubing/filter/etc.) subtracted from the measured system pressure. To determine this, you essentially disconnect the top of the column, run the system under your intended conditions and record the measured pressure (this is the backpressure generated before the column). The manual for my columns (HiTrap Q) explains this step by step, and yours may as well.

The manual also explains how to determine pressure across the column in a similar manner. However, in my experience, the flow path after the column is relatively unrestrictive (unless a flow restrictor is installed), and its contribution to backpressure is relatively be negligible. Thus the pressure across the column is reasonably equal to the pre-column pressure, which is also reasonably equal to the measured system pressure. Furthermore, listed in every manual I read is some statement to the effect of:

The maximum pressure over the packed bed (Δp) is an approximate limit. It is dependent on the characteristics of the chromatography medium and on sample/liquid viscosity. The measured value also includes the pressure generated by the column tubing. For many columns, the recommended flow rate is a better guideline for protecting the packed bed.

In fact, the manual for my columns doesn't even list the resin pressure limit and it took substantial digging to find out what it actually is. I always run at the recommended flow-rate and set the system pressure alarm at the resin pressure limit (0.3 mPa in my case versus 0.5 mPa hardware limit) without any of the corrections mentioned above. However, depending specifically on your system and usage, it may be necessary to make them.