How To Select a Pore Size for C18 Reversed Phase Chromatography

Caleb Ferguson

How To Select a Pore Size for C18 Reversed Phase Chromatography

Mar 23, 2022 12:00:00 PM / by Caleb Ferguson

Reversed-phase chromatography is an essential step in any drug discovery workflow, particularly as a desalting step prior to MS/MALDI analysis. One of the first choices you must make when you design your purification protocol is resin selection, and there are several options to choose from depending on your sample type and downstream application. One important variable to consider among different resin types is pore size.

Pore size refers to small holes in the silica particles that make up the resin. These pores greatly increase the surface area of the resin, to the extent that 99% of the surface area lies within the pores. As a result, access to the pores is essential for the analyte to bind efficiently. Diffusion of the sample into the pores is the rate limiting step of the binding reaction and is determined by the relationship between the size of the sample and the size of the pore.

  Pore Size Drawing

 Image Credit: Chromatography Online

The larger your sample is and the closer it is to the size of the pore, the more slowly it diffuses into the particle. This results in lessened affinity to the resin. At the extreme end, targets larger than the pore size will be completely excluded from the binding reaction. 

 

Resin 90 Å 300 Å
Starting (μg) 233 182
Yield (μg) 58.5 38.6
% Recovery 25.1% 21.2%

C18 PhyTip Columns Processing Results in Low loss of Peptide Sample

Small pores (~100A) exclude proteins and large peptides but bind well with small to medium peptides. This makes small pore resins ideal for purifying digested peptides prior to peptide mapping. Undigested protein will not bind which increases purity of the final sample. Purification of proteins or large peptides requires a larger pore size (~300A).

Pore size exclusion new copy

For more details regarding the benefits of using C18 PhyTip Columns for MS analysis, check out our application note below!Biotage offers both 90A and 300A pore size C18 resins. These two pore sizes allow the flexibility to purify a variety of proteins and peptides. If a different pore size is required, be sure to take a look at our information on custom packing.

Download Application Note

 

 

 

Topics: Reversed-phase, mass spec, protein purification, pore size, C18, C18 (300 Å), chromatography

Caleb Ferguson

Written by Caleb Ferguson

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