Why would percent aggregates increase as I increase my buffer molarity when performing an SEC method? - WKB229751

ENVIRONMENT

• Size exclusion chromatography (SEC)
• High molecular weight species (HMWS)
• Aggregates
• Dimer
• Trimer

ANSWER

As salt molarity increases, hydrophobic interactions increase, causing proteins to aggregate at an increased rate.

ADDITIONAL INFORMATION

Use the Autoblend function on the Alliance system or other Waters LC System that has this feature to perform pH and molarity studies to see how changes affect protein behavior.

Use a constant 20mM phosphate buffer at pH 6.0
At pH 6.0:
Run a mobile phase of 100mM sodium chloride - monitor results
Run a mobile phase of 150mM sodium chloride - monitor results
Run a mobile phase of 200mM sodium chloride - monitor results
Run a mobile phase of 250mM sodium chloride - monitor results

Use a constant 20mM phosphate buffer at pH 6.5
At pH 6.5:
Run a mobile phase of 100mM sodium chloride - monitor results
Run a mobile phase of 150mM sodium chloride - monitor results
Run a mobile phase of 200mM sodium chloride - monitor results
Run a mobile phase of 250mM sodium chloride - monitor results

Use a constant 20mM phosphate buffer at pH 7.0
At pH 7.0:
Run a mobile phase of 100mM sodium chloride - monitor results
Run a mobile phase of 150mM sodium chloride - monitor results
Run a mobile phase of 200mM sodium chloride - monitor results​​​​​​​
Run a mobile phase of 250mM sodium chloride - monitor results

Use a constant 20mM phosphate buffer at pH 7.5
At pH 7.5:
Run a mobile phase of 100mM sodium chloride - monitor results
Run a mobile phase of 150mM sodium chloride - monitor results
Run a mobile phase of 200mM sodium chloride - monitor results​​​​​​​
Run a mobile phase of 250mM sodium chloride - monitor results

This is a good way to truly determine which pH and NaCl molarity will provide the best results.