Hello all!
I'm currently trying to resolve a problem of tailing peak when loading the calibration markers on a sephacryl S-300 HR gel filtration column. All the peaks corresponding to the elution of the markers (whichever the marker used) allways show a long tail. I'm using a PBS NaCl (0.2M, pH7.35)elution buffer, with a flow rate of 0.35 ml/min, and performed the experiments at room temperature. I loaded the manufacter recommended concentrations and volumes of the markers.If somebody has any solution for this problem, I would be glad to be introduced to him!!Thank you very much!
Juliette

Hi it's me again!
I just want to add that the column I'm using is a prepacked column so the tailing peaks are not a problem of an underpacked column (I hope so!!!)
Thanks
Juliette

Assuming that you are doing proteins (since you are using PBS, but why 0.2M rather than the usual 0.15M phosphate + 0.15M NaCl?): They have a tendency to do absorption chrom besides the size exclusion. You will have to play with the mobile phase (pH, ionic strength, org. modifyer), maybe even with the concentration (what does a lower conc. do?) and flow rate.
But, whatīs a long tail?? Have you seen sample chromatograms, are they better? Many, maybe most, SEC columns are quite disappointing.

Hi Mr Mueller!
Thank you for your answer! Well, about the NaCl 0.2M concentration, it was determined to be the most suitable for this column. I asked the customer Amersham service if my buffer was ok for the column and he said YES. Perhaps you're ok, I will try to play with the mobile phase.
My elution peaks are divided into a great vertical first part of the peak, a top (corresponding to one or two fractions of 0.7 ml), and the second part of the peak is less vertical than the first part and spreads out more fractions. The tailing peak seems to be a neverending one with a long almost (but not really) horizontal tail...
My explanations (without any diagram) are ot very clear, are they?!?
The ancient column (also a S-300 HR) showed with the same experiment conditions beautiful peaks! I really don't understand... Next week, I will try to load another kind of sample on this column.
THANK's a lot!

You may have seen my post on the excellent TSKgel Super SW 3000 column, namely, that tailing incresed permanently (partially) after contacting perfluoro acids. I still donīt know what causes that. Just play with mobile phases to see if you can get an improvement. ...
Would like to hear about results.