Chromatography Forum

First off I am far from an expert in the field so please excuse any lack of detail here. I can try and provide any extra detail you need to help me though.
I am running SEC to check for antibody purity on an Agilent 1260 and am using a a Biosep SEC s3000 column for the run. The absorbance for the run is a280 at 1ml/min. Bandwith is set at 8.
The mobile phase for the run is 50mM NaPO4 NaN3.

I work in a manufacturing laboratory (not research). To meet our in house specifications the Monoclonal Antibodies must have retention times that fall between set positions according to the Bio-Rad Standards. In this case Thyroglobulin (MW670,000) and gamma-globulin (MW158,000). Generally the first 2 of the 5 peaks you expect to get when using this standard.

The issue I am having is that the standards are appearing unresolved and not separated on my traces (See Attached Link). In simple terms a big blob with some peaks at the top. You will also see an image of a 2mg/ml BSA standard I ran for reference.

If anyone has any troubleshooting tips for this issue it would be a huge help to me. I have plenty experience running the instrument, but little in terms of method creation or research.

https://imgur.com/a/zsU4eTZ

Hi Azide95, please see this link for the manufacturer purchasing guide blob:https://www.phenomenex.com/cb473e51-b55c-4aff-9fb9-d2966c687a25

on pg11 you can see they have run a similar protein mix under similar but slightly different conditions (MP=100mM sodium phosphate pH 7.0, 300mM sodium chloride) if you are able I would advise trying your separation under these conditions.

as a general troubleshoot however SEC separations are most definitely pH dependant and you havn't mentioned a pH in your post so I would advise checking/controlling the pH of your mobile phase. Looking through the above guide most separations on this column seem to be pH 6.8 or 7.0 however the column can handle a wider range.
You also have not mentioned an injection volume or standard concentration, column overloading could also lead to poor resolution between standards so while I assume you are injecting at an appropriate on column concentration I would look at these as well.

Good luck, and let us know how you get on,
Chromavore

Hey thank you so much for the reply. I will let you know how I get on.


The pH of the solution should be 7 +\- 0.1. I will re check this though.
The standards as far as I am aware https://www.bio-rad.com/webroot/web/pdf ... T-102E.pdf
Are used at a set concentration provided by the manufacturer. We add 0.5ml to our 18mg vials.
Finally we are loading currently 10ul on to our column.

Again thanks for the help