Chromatography Forum

I am running Molecular weight standards on two new gel filtration columns with RI detector and PBS buffer. I have equilibrated the columns extensively and purged the RI detector over a number of hours but the tailing factor is greater than 1.5 and plate count is only at 800. I have changed the columns with the same results. This is a fully validated method so I cannot change the mobile phase or conditions but I am at a loss as to why the chromatography is so poor

Please do the initial QC test for your gel filtration columns as stated on the certificate of analysis. Use the same mobile phase and standards and test column by column. You should get nearly the same number of theoretical plates and assymetry factor as messured in QC of the manufacturer, otherwise the packing is destroyed. If that is the case please contact tech support of the manufacturer for quick action. In addition please check flow rate of your system, check injection system and check reference cell of your RI for any leackage. Column failure as you described can happen. Main reason could be that the box of the column dropped to the floor, mainly during transport. Check if the column box was ok. If you see that the column box was damaged please take a digital picture and send it to the tech support of the manufacturer. Good luck.

Also check for extra-column volume problems: excessively long runs of tubing, wrong diameter of tubing, improperly assembled fittings, etc.

Is the mobile phase Phosphate Buffered Saline or Phosphate Buffer?

Thank you for your help with this issue. I did everything from changing columns, length of tubing, changed fittings and systems. The issue turned out to be the coloumns we received from a certain supplier. As a final resort I ordered the exact same column from a different supplier and the method worked so all the columns we received originally were returned as they were all faulty. Thanks once again for all the suggestions

Please let us know that the column was packed by the manufacturer, or packed by the different suppliers. If it was packed by the manufacturer it might be a problem of storage, or transport or your first supplier had a faulty batch. In that case the problem can come up again with the other supplier as well. If the column was packed by the manufacturer you should contact them and provide them with the serial numbers. They should find out what was the problem with your columns. That should avoid such a problem in the future.