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October 17, 2017

High pressure stressing you out?

By Severine Lebarq, Global Product Marketing Manager, Protein Purification at GE Healthcare

Are your chromatography system’s pressure alarms and slow flow rates stressing you out? Read on to learn how to quickly identify possible causes and how to fix them.


If your runs keep stopping because your chromatography system’s pressure alarm keeps going off, it’s time to look at a few possible causes. Three possible causes of back pressure increases are:

  • Precipitated protein in the column filter and/or at the top of the bed
  • Clogged in-line filters
  • Turbid sample

Likewise, if your flow rate keeps decreasing, you may be encountering problems such as bed compression or a clogged column filter. 

The most common reasons for reduced flow or back pressure increases, and how to fix them, are summarized by GE R&D protein purification experts in the following tables.

Possible cause

Feature

Bed compressed

  • Clean column according to cleaning procedures and/or change filter
  • Check sample preparation. If sample is too viscous, back pressure will be high. Dilutesample or reduce flow rate
  • Repack column, pack new column, or use prepacked column

Microbal growth has occurred in column

  • Clean column using recommended methods
  • Prepare and use predefined column and system maintenance methods. Make it a habit to include these methods in a method queue
  • Always filter samples and buffers. Choose low protein binding membranes such as Whatman™ regenerated cellulose
  • Store in presence of 20% ethanol when not in use

Clogged end-piece, adapter, or tubing

  • Remove and clean or replace if possible

Outlet closed or pumps not working

  • Ensure that column outlet is open
  • Check pumps for signs of leakage. If using a peristaltic pump, also check tubing

Too small system tubing i.d. for flow rate used

  • Change tubing to larger inner diameter (i.d.)

Sample too viscous

  • Dilute sample with buffer
  • Maintain protein concentration below 50 mg/mL
  • Reduce flow rate during sample loading using pressure-flow regulation functionality available on most modern chromatography systems

Sample not filtered properly

  • Clean column, filter sample with a low protein binding filter (e.g. Whatman SPARTAN™ filter), and repeat

Clogged column filter

  • If possible, replace filter or clean column with reversed flow according to cleaning procedures
  • Always filter samples and buffers before use. Choose low protein binding membranes such as Whatman regenerated cellulose
  • Reduce flow rate during sample loading using pressure-flow regulation functionality available on most modern chromatography systems
Possible cause

Remedy

Precipitated protein in column filter and/or at top of bed

  • Clean using recommended methods
  • Prepare and use predefined column and system maintenance methods
  • If possible, exchange or clean filter or use new column
  • If additives were used for initial sample solubilization, include them in running buffer

Clogged in-line filters

  • Change inlet filter or clean, if possible. Some systems have a filter on top in the mixer

Turbid sample

  • Extend the lysis time or change lysis method
  • Improve sample solubility by adding ethylene glycol, urea, detergents, or organic solvents. See resin or column instructions

Watch out for the next article about protein purification troubleshooting regarding purity and resolution.

Can’t wait for the next post? Download our Protein purification troubleshooting guide

This guide helps you quickly identify root causes for many of the common issues encountered during protein purification. It gives practical advice on small changes that you can make to fix these problems.