# Protein purification pressure limits confusion, about the X-MPa limit for FPLC?

I have always been a bit confused about column pressure limits. I have to elaborate a bit on the problem to get to my question:

In protein purification we use the äkta purification machines (e.g. äkta explorer), which always come with a high pressure and low pressure alarm setting for the column in their unicorn software. The most common column is the IMAC 5-mL HisTrap column to capture proteins that contain e.g. a 6xhistidine tag. When I check the column specifications I can see that the column has a flow limit of 0.3MPa which should be rounded up to 0.5MPa for the alarm (as the flow restrictor gives an additional 0.2 MPa pressure). The second type of column is the SEC column, one example is the analytical "superdex 200, 10/300 GL" for separation of protein between 10 and 300 kDa. The specification of this column is that the $$\Delta$$p is 1.5 MPa, however, its typical that most people are sceptical to go above 0.5 MPa for any type of column.

So my questions regarding pressure are as following:

• The 0,5MPa limit for a HisTrap column is what is called the $$\Delta$$p pressure limit?
• It always the specified $$\Delta$$p for each individual column that I have to care about? (not the pre or post column pressure limits)?
• Can I then assume that it is always safe to run the column all the way up to the specified $$\Delta$$p limit to e.g. equilibrate or wash the column? So for the mentioned SEC column, I could run in at a $$\Delta$$p value of 1,5MPa and assume that the column and protein are just fine?
• Proteins are fine at high pressure. Beads don’t like high pressure difference, and columns crack if total pressure is too high. So look up column limits ( different for plastic, glass and steel) and chromatography medium (material, bead size matters). – Karsten Theis Jun 27 at 13:43
• yes i know, thats why i specified the delta-p for the column, but your answer is then - yes i can be 100% sure that its okay to run the column up to the specified delta-p limit ? – CuriousTree Jun 27 at 14:03
• edited and formulated my questions a bit better – CuriousTree Jun 30 at 10:58