Equilibration issue

Record: 850

Category: Applications
Product: NH2P
Sample:

Question
I’ve been having issues separating my sugar standards using the column we purchased. I am detecting glucose at less than 2 min, although the flow rate and mobile phases are all in line with what you suggested in the certificate of analysis. The pressure was at ~3 Mpa, which is in line with what was proposed. Our LCMS machine is new and therefore there is no reason to suspect that the flow rate is not accurate. Am I missing something? Please tell us how we can improve our separation using this column. By the way, I followed all the guidelines in the manual included, including using ammonium acetate to equilibrate the column. But, even after that sucrose is being detected very early.

Answer
Could you give me a little more detail as to the conditions and eluent used? If you could send the flow rate, eluent, and chromatogram it would help our discussion.

I am assuming that the glucose is not retained on the column and this is your concern. I’m not sure what your eluent conditions are; however the addition of additional acetonitrile will retain the sugars for a greater amount of time. Please see the link for information on the retention time with addition of ACN:
http://www.shodex.net/index.php?applic=598

Also, keep in mind the elution volume of saccharides found on our website are analyzed for the analytical column (4.6 mm ID x 250 mm) not the micro sized column (2.0 x 150). The retention time on the micro column will elute earlier than the analytical column:
http://www.shodex.net/index.php?applic=593

Please disregard the column equilibration method. We had been shipping the column in 75% ACN in water and believed it was necessary. Currently, we ship the columns in 100% ACN and the equilibration is not needed. It will not harm the packing material, it is just unnecessary. Should you need to regenerate the column at a later date, please use the regeneration procedure for the line filter found on page 20 of the operation manual and run several column volumes rather than the amounts listed.

Please let me know some of the details, so I can better help you.

 
 
Additional Comments

Download or Print PDF