Chromatography Forum

I´ve been trying to reproduce a previously reported separation method. The column is a Discovery HS F5 (supelco), and the samples are iron complexes of an aminated macrocycle, called desferrioxamine. They are polar, highly soluble in water.

Here is the fragment about the method:

"LC-MS analysis of tris-hydroxamates in culture supernatants... ...An Agilent 1100 HPLC instrument
equipped with a binary pump and a diode array detector was used
for HPLC analysis. Samples were analysed on a Supelco Discovery
HSF5 column (150x4.6 mm, 5 mm i.d., column temp. 20 uC) and
eluted with 10 mM ammonium carbonate, pH 7.0 (solvent A)/
MeOH (solvent B) (10 : 90) at 1 ml/min for 10 min, followed by a
gradient to 100 : 0 A/B over 8 min, 10 min isocratic conditions at
100 : 0 A/B, a gradient to 10 : 90 A/B over 8 min and isocratic conditions
at 10 : 90 A/B for 4 min. Ferric-tris-hydroxamate complexes
were detected by monitoring A(435nm)."

The problem is that I am getting no separation!!! I can´t reproduce the cromatograms reported in the article. Everything elutes in the front...

First, I thought it was the mobile phase, so I changed the very hygroscopic carbonate by a new one, carfully checking the pH. Then I evaluated the column under other conditions and analytes (from the catalog) and I was able to achieve a separation... so, the column works.
Then I tried a 1:1 dilution of the sample with the mobile phase... still nothing. Everything still elutes in the front.

I ran out of ideas... please, enlighten me!!!

Have you contacted Supelco Tech Service?

Try 800-359-3041

If that doesn't work, let me know.

hdz -
based on what you have written, the mobile phase appears to be backwards. It is rare that one would use these components in a HILIC (high organic modifier) system. I would, however, expect this system to work using the F5 in a reversed-phase mode. Can you provide the citation to the article you reference? You may also contact Supelco Tech Service via email at techservice@sial.com

thank you for your answers.
Well, in fact, I have already contacted Supelco. They suggested to equilibrate the column overnight, which I did, with a nice baseline noise reduction, but still no separation. They also are concerned about the sample solubility, but it works fine when I tried to run a 1:1 diluted sample in mobile phase.
Actually, I could not try a different gradient. I am not allowed, because the method was supposed to work that way as one of the authors (who is working with me, but is not the one who knows chromatography! ) claims.
The article reference is:
Microbiology (2006) 152 3355-3366
I am open to suggestions and I will try the reversed phase gradient... but I am still intrigued about this method and why it does not work with me