SEC and IEX for Biomolecules: Column Selection and Troubleshooting

Size Exclusion and Ion Exchange are typically run in non-denaturing conditions. That’s one of the reasons they are the two most common types of HPLC used for biomolecules.

SEC separates based on size in solution. We will discuss calibration curves and how choosing the correct pore size allows you to work in the linear portion of your curve which will help you be successful using SEC. Buffer concentration, salt concentration, pH, and sample are also critical. These and other factors will be discussed as we explore things that can go wrong in the troubleshooting section.

Ion exchange separates based on differences in degree of charge. Column selection and conditions will depend on the pI of your protein. We’ll discuss those choices as well as what to do if you don’t know the pI of your protein. Another important thing to consider when selecting your column is whether resolution or robustness is your priority. Despite making the right column decision, things don’t always go the way we plan so we’ll look at how to identify the cause of a problem and ways to troubleshoot.

Presenter: Rita Steed (Application Engineer, Agilent Technologies, Inc.)

Rita Steed began supporting the LC column line for Agilent in 1999. As a Chromatography Specialist (first with Chromatography, Inc., then directly for Agilent), Rita worked on-site with researchers in Pharmaceutical and other industries presenting technical seminars and assisting researchers with troubleshooting and method development. Rita has over 20 years of Chromatography experience in the Biotechnology, Chemical, and Pharmaceutical industries. She has held positions in Research, Sales, and Technical Service. Rita has earned degrees in Microbiology and Life Sciences/Biochemistry. In her current position, she is an inside Application Engineer supporting LC columns.