Chromatography Forum

Hi!

I´m developing a method for LC sugar separation and mass spectrometer detection of different sugars.
I want to know if this column is suitable to use with mass spectrometry detection because I can not detect single or mixed sugars after injection working with an isocratic protocol with 75% ACN 25% H2O (0,8ml/min flux) and adding or not 0,1% formic acid as buffer.
Some body can help me with suggestions or information?.
I´ll much appreciate the support.
Regards.

What is your ms method? I assume you started by infusing and could observe some signal in negative mode
but then could not see it while doing chromatography? Those conditions seem reasonable for hilic of a sugar.

What is your ms method? I assume you started by infusing and could observe some signal in negative mode
but then could not see it while doing chromatography? Those conditions seem reasonable for hilic of a sugar.

Thanks for your soon replay!

You right, I tested sugars with infusion an using other columns and are going well in detection (I use positive mode), sadly these columns are not good for sugar separation .
I have some suspicious that the column is not a good chance for mass detection (I can not find Agilent information), in fact in the ion source appear some withe precipitate over the cap that could be an ionization interference coming from column (silica maybe?) producing an ionization quenching and avoiding the signal (mass calibration ions are affected too) .
I´m little afraid about my mass detector integrity, so I stopped the analysis.

I have the chance to use a HILIC column, but my first approaches were that I can detect but not separate sugars, do you have some suggestion?

Thanks a lot.

You are running a hilic column already with your agilent column. It is silica based so you are also getting particles into your source.
Try an Shodex Asahipak or Supelco Aphera polymeric based amino if you want to quiet the baseline. I would try injecting lots of sugar
and watch with ELSD or RID until you figure out the problem. The method you have should work.

I would doubt your columns are bleeding off silica. The mobile phase you are using, 75/25 ACN/Aq 0.1% formic acid will prevent the silica from dissolving. Unless your water is basic (>pH I would not be afraid of running without any formic acid as most columns work well in the range of pH 2 to 8.
Dissolution of silica becomes a problem above pH 8.

The agilent website gives some info on this column
http://www.chem.agilent.com/en-US/Produ ... fault.aspx

None of the listed methods use formic acid in the mobile phase, it is all ACN/Water.
http://www.chem.agilent.com/en-US/Produ ... 58220.aspx

This page also states the pH range the column can work with is 2 to 8 , so a little formic acid should not hurt at all.
Unless the column you are using has been subject to non-recommended mobile phases, I would doubt it will cause you a problem with white ppt.

I have seen mobile phases containing ACN give a white or biege PPT in the source. You can test if this is happening in your case by just running moblephase for a day or two, with no column attached.

A method for sugars by lcms is found at
http://mass-spec.stanford.edu/assets/20 ... omeaux.pdf

It also uses an amino column and ACN/Water.

Alp

In theory I agree with Alp about particles from silica. In practice silica amino columns have about 10 times higher background by ELSD
than polymeric based. People attribute this to silica coming out of the columns. I haven't seen a good theoretical paper studying this
phenomenon but most people that use ELSD believe it is silica. I'm open to any theories. Thoughts?