Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
  • More
Consumables
Wiki Choosing the right pore size for size exclusion chromatography
  • Announcements
  • Forum
  • Files
  • Wiki
  • More
  • Cancel
  • New
Consumables requires membership for participation - click to join
  • Wiki
  • A Beginner’s Guide to Hydrophobic Interaction Chromatography
  • A simple tip to protect your columns
  • A Tip for Preparing Robust and Consistent Mobile Phases
  • Achieve accurate and repeatable gas flow meter measurements
  • AdvanceBio Columns Blog Series
  • Agilent 123 Meter Sodium probe maintenance
  • Agilent Collection of Columns, Supplies, and Standards Resources
  • Analysis of microplastics in the environment
  • Analyzing ADCs by HIC
  • Avoiding downtime in the lab: top tips for GC/MS success
  • Best Practices for Aqueous Mobile Phases
  • Best Practices for Making Good Connections
  • Bursting Tubing and Columns (GC and HPLC)
  • Calculating Column Volume
  • Cannabis Potency Testing: a Reliable, Cost-Effective Method
  • Checking Your Intuition - Sub 2 µm vs Superficially Porous
  • Choosing the right GC Injection Technique
  • Choosing the right pore size for size exclusion chromatography
  • Columns, Supplies, and Standards Knowledgebase
  • Consumables Applications and Workflows
  • Consumables Recommended Supplies Lists for Agilent.com
  • Custom Product Request
  • Extreme Makeover – Derivatizations in Chromatography – Part 1 GC
  • Extreme Makeover – Derivatizations in Chromatography – Part 2 LC
  • Fake It Until You Make It: When BioInert Isn’t an Option
  • Flipping Amino Acid Analysis on Its Head
  • Glycans at a glance:  Analyzing therapeutic glycoproteins
  • Handle and Care of Syringes
  • Help! My Peaks Look Strange - Fronting and Tailing in GC
  • Help! My Peaks Look Strange - Saddle Points - LC/GC Troubleshooting
  • Hydrophobic Interaction Chromatography of Proteins and mAbs
  • Importance of Silica Particle Strength for Sub-2 µm SEC Columns
  • KB: Ferrules recommended for GC self-tightening column nut
  • LC Method Translation - the Dwell Volume
  • Minimize spectroscopy workflow disruptions
  • Minimizing Metals for Best HILIC Results
  • More than just a drink: Analyzing the elemental composition of beer
  • Multi-Attribute Methods – Peptide Mapping Part IV
  • Must See Webinars
  • Nomenclature of CFC's/Freons/Halons/Coolants
  • Oligonucleotide Analysis - Unexpected Details Matter
  • Optimizing Bonding Chemistry for Sub-2 µm SEC Particles
  • Pass the Salt, Please – Mobile Phase Preparation for HIC
  • Pesticides and their stability during GC analyses
  • Pre-Columns - the forgotten art of using retention gaps
  • Problematic polar analytes? Hello HILIC…
  • Protecting your laboratory productivity
  • Recommended Reading
  • Sample Prep Pointers - Peptide Mapping Part I
  • Save your results with sample filtration
  • Simplified cone inspection with the new Agilent LED measuring magnifier
  • Software tool for the ADM Flow Meter (G6691A)
  • Software-Supported Method Development - the Scanview Program
  • Stay Safe: A Win-Win for Solvent Storage
  • Streamline your sample processing
  • The importance of chemical composition for vial performance
  • Tips & Tricks for Amino Acid Analysis – Part I
  • Tips & Tricks for Amino Acid Analysis – Part II
  • Tips & Tricks for Amino Acid Analysis – Part III
  • Tips & Tricks for Amino Acid Analysis – Part IV
  • Tips for Smooth Sailing with HIC
  • Troubleshooting HPLC autosamplers
  • Troubleshooting HPLC degassers
  • Troubleshooting Sequence Coverage – Peptide Mapping Part III
  • UV, MS, TFA, and Formic Acid – What to use? Peptide Mapping Part II

Choosing the right pore size for size exclusion chromatography

Hi everybody – for the next few weeks I have guest posts from my colleague Wu in R&D. This week he gives some good insight into how to choose the best SEC pore size for your sample. In the next couple of posts he’ll pull back the curtain on a couple of the things R&D considers when designing a column, and how they impact your separation.

 

Choosing the right pore size for size exclusion chromatography

For successful size exclusion chromatography, it is necessary to choose a column with the right pore size for the separation you are trying to achieve. Molecules that are too large to fit any of the pores will be excluded and elute first. Smaller molecules that can diffuse into the pore structure will take longer to elute from the column, and elute later:

 

A good rule of thumb is to choose a pore size that is 3x larger than the molecule you are trying to analyze. For a monoclonal antibody (mAb), the size of the molecule is around 5 nm. If you are trying to separate a mAb monomer from its aggregates (dimer, trimer, and higher-order species), the largest molecule could be approaching 10 nm in size. The most appropriate pore size column for this separation is therefore 30 nm, or 300 Å. Alternatively, if you are unsure of the size of your molecule, you may be able to use a column resolving range guide, such as the one below:

 

 

In this case you can see that a mAb of around 150 kDa in size fits in the middle of the resolving range of a 300 Å column. If you are studying a smaller protein such as insulin, you will need to choose a smaller pore size column such as 100 Å or 130 Å. It is important to remember that charts such as this are only a guide. Size exclusion chromatography relies in differences in size in solution, not molecular weight, so molecules that are cylindrical or elongated will elute earlier than expected, as that will have a larger than expected size compared to a compact, globular molecule.  Ideally you should choose a column that provides the maximum amount of pore volume and therefore maximum separation ability or resolving power. You can measure the pore volume by looking at the volume of mobile phase eluted between the exclusion point and the total permeation point.

 

 

 

Regularly running a mixture of standard proteins that covers the entire resolving range of the column you are using, like in the chromatogram above, ensures you can keep track of performance and has the added benefit of helping to detect problems early, allowing you to reduce instrument downtime.

 

Keywords: mAb, monoclonal antibody, ADC, antibody drug conjugates, SEC, size exclusion chromatography, biologics, biosimilars, insulin, molecular weight, aggregation, fragment, quality control, biopharmaceutical, CQA, critical quality attributes

  • mab
  • aggregation and fragment analysis
  • molecular weight
  • adc
  • quality control
  • biologics & biosimilars
  • insulin
  • release test
  • size exclusion chromatography
  • critical quality attributes
  • monoclonal antibody
  • pore size
  • Biopharma LC Columns & Consumables
  • sec
  • biopharma
  • cqa
  • antibody-drug conjugates
  • Share
  • History
  • More
  • Cancel
Anonymous
Related

Agilent Community Feedback

Agilent Community Feedback

×
We are glad this was helpful! We are sorry this was not helpful. If you still need assistance please create a community post or contact support. To help us improve, please provide any additional feedback. For full details of how we will treat your information, please view our privacy policy.
Submit Cancel
Submit Cancel
Recommended
Privacy Statement
Terms of Use
Contact Us
Site Help