Hi All,
I need some ideas from you all to solve my chromatographyu problem. I was using an already validated HPCEC (cation exchange method) to separate some of the impurities from the main peptide drug peak. The original column was Tosohas (Sp-5PW, 7.5cmX7.5mm). Since they are not selling this column anymore we were using the same column under the brand name 'Protein Pack' (same column dim) from Waters and it was ok. However, the problem is suddenly a new column from Waters can not even give my main drug peak not to mention the impurity peaks. My drug smaple is ok and no questions about that. I tried injecting their recommeneded std (acetone); it is eluting at VV and it is not an ideal std to check ; they told me to use Cytidine but I dont have a RI detector. I tried to regenerae the column with their recommended 0.1NNaOH and 40%HAC and still no luck. (this column just bought in Aug. Any suggestions and comments are appreciated.

Before throwing the column away, make sure the composition of the mobile phase is correct and if you are using a gradient method, the gradient valve is working properly, It sounds to me that you are somehow getting more organic into your mobile phase and that is why you don't see your peak of interest.

Thanks for your comments. I have only 10% ACN in the mobile phase and we think the reason is this particular lot of columns comes under any brand names either TosoHass, Waters or Polymer Labs is bad. I found if I increase the salt gradient I am getting something that i am supposed to get.
Thanks for your comments. Hope your move of the instruments was not hefty:)~