Dear all,
I am studying the transformation of nitroaromatic compounds (essentially 4-amino-2,6-dinitrotoluene and 4-amino-2-nitrotoluene).
From these compounds, I am getting polar intermediates. I am expecting metabolites such as amino- and hydroxy-aromatic compounds. The problem is that I get a lot of polar compounds and that their respective pics are not well separated from each other.
I am using a Nova-Pack C18 3,9*300 mm column. The mobile phase is ACN/H3PO4 (10 mM, pH 3,2). I have tried an isocratic mode as well as a gradient mode (linear, convex or concave), but I did not observe any relevant changes.
Any tips woulod be very welcome!
Thanks in advance for your help

What was the % acetonitrile in the isocratic composition, and over which range did you vary the acetonitrile composition in the gradient?
Did you try to substitute methanol for acetonitrile?
Did you try to change the pH to pH 7?

M.G. Musolino et al. in J.Chromatogr A 818 (1998) 123-126 presents good resolution in "Liquid chromatographic separation of intermediates of the catalytic hydrogenation of 2,4-dinitrotoluene".

I recently had just the same problem, but ith other components and I solved it by changeing the column to a X-Terra RP18 and a pH of 8.

Thanks for your tips!
In reply to the first post, I used for the isocratic mode 50/50% ACN/H3PO4. THe pH always remained at 3.2. As suggested, I will change the pH to 7.0.
I will also try to change ACN for methanol.
However, I have the impression that I am just changing a parameter for an another, without any systematic/rationnal approach. Do you have any good references that could help me to solve my problem step by step? It would be worth to have something like 'First, change the pH to pH 7.0. If you do observe that, make that and so on...'.
Thank you very much

No, you are on the right track. You make a giant step to observe a difference. If you get an effect, you can work with an intermediate composition to finetune the separation.

Actually, reversed-phase HPLC method development is straightforward (albeit sometimes tedious!) to systematize.

There are really only a half-dozen or so factors you can use to change selectivity:
- mobile phase strength (isocratic %B or gradient steepness are equivalent in this regard)
- temperature
- organic modifier type (usually ACN/MeOH/THF)
- pH
- column type
- additives (some people subdivide this further into type and concentration).

The idea is to rank these variables in order of their expected effect on your separation, then pick a "best-guess" set of iniitial conditions, then make a *big* change in the first variable. If you see an effect on selectivity, then "finetune" as suggested by the post above.

It just so happens that the company I work for (LC Resources) makes software to streamline this process. Click on the LC Resources link in the upper left corner and follow the links to "DryLab".

-- Tom Jupille

Would anyone have any idea whether methotrexate or folic acid will dissolve to an appreciable level in any organic solvent

Thanks

Keifer

I would like to know the detail of X Terra RP18's structure, its price and where I could buy?

try waters web site: http://www.waters.com

Before buying Xterra check the complains on this board abour quality of Xterra, lot to lot reproducibility, etc. People are compalining and I had problems too, peak shape is not thta great. I would consider other alternatives (Luna, Zorbax)

PJ