Chromatography Forum

Hi!
I'm trying to separate structural monosaccharides in neutralized plant hydrolisates by using HPLC-UV coupled with pre-column PMP derivatization. I'm having trouble with chromatographic reproducibility... Experience with this technique, anyone?
Thanks!

Derivatization is always not repeatability proces. To get good results you need to use a Internal Standard for sample preparation and use peak area as a relative peak area to peak area of internal standard.

Are you using some compound as a Internal Standard?

Not at the moment, still just trying to separate the standard mix. But I will definitely use the internal standard on "real samples", thanks for the tip! There is a lot of literature available describing separation parameters - from different mobile phases, different buffers (pH values, concentration,...) so I've tried some of these variations and for my compounds of interest (glucose, galactose, arabinose, xylose, rhamnose, mannose and galacturonic acid). I have found that potassium phosphate (K2HPO4, 10mM) at pH 8.5 in gradient with AcN (from 10% to 15% AcN at the end of the run) gives the best peak shapes, satisfactory retentions and a very nice separation, and these are not often used conditions in the literature, but they work for me the best. I'm using Zorbax Extent C18 column and Agilent 1200 system. During the sequence, from run to run, the retentions are changing (sometimes increasing, sometimes decreasing) and the backpressure increases significantly. For the later, I'm aware that it might be due to salt precipitation (but it's low concentration salt and the content of AcN is only 15%...when I mix it in a glass, I observe no precipitation (??)). So, this is the problem, the stability of the chrom. process upon repeated injections... I think my equilibration time after the gradient is good - 7' isocratic after reaching the initial ratio, and the pressure returns to the initial values as well.
Thank you in advance for your comments!
Best,
Aleks

try maybe increasing ACN lvl to 60-80% after each run. Maybe something is retain on column after each injection that needs to be flushed.