Hello !
I'm separating a mixture of very hydrophilic and small biomolecules by normal phase chromatography
in CN-silica. The retention is good but I would
like to improve the separation. Can you give an
advice or a reference to optimize separation in
normal phase ?
Thank you.

I assume that you work with a solvent system of primarily a hydrophobic solvent such as methylene chloride and a polar solvent like isopropanol to adjust the retention. You can adjust the selectivity of the separation by choosing another polar solvent or using other polar additives.
What is your cuurrent mobile phase?

Thank you Uwe. My solvents are acetonitrile (A)and
water(B). I use a gradiente from A to B.

OK, this is not normal-phase chromatography. This is HILIC, I guess. You can get different selectivities by either using methanol or playing with the pH, just as in RPLC.

Thank you Uwe. I will try it.
What's the difference between normal phase and
HILIC ? More polar solvents, more polar gels, both ?

Actually, both. Normal phase is considered everything polar stationary phases and non-polar mobile phases. HILIC is the extension of this to water/organic mixtures in the mobile phase. Of course, the transition is grey, just as between reversed-phase and HIC (hydrophobic interaction chromatography).

I agree with Uwe, chromatography is littered with semantic traps, probably brought about by confusion of biochemists 'dabbling in the black art'. On my soap box I would suggest biochemist use (and sometimes to very good effect) techniques without understanding how they work (and why should they, if it works?) but then try to adopt these techniques, and describe them in terms which, although appropriate to biochemistry, only leads to confusion for other people trying to figure out 'how to'.
Examples are many, the use of the word 'buffer' by biochemists to indicate anything liquid, numerous words to describe elution and eluate. HIC is another example, derived, no doubt, from the hydrophobic moieties of a protein, interacting with a lipophilic stationary phases.
I accept that language is dynamic, but if one uses words or phrases which are difficult to interpret and ambiguous, surely, by definition, we are not using language to communicate effectively, and therefore it becomes redundant.
Sorry, I'll get off my soap box now...
hasta la victoria siempre!
Cheers
Che

Thank you very much Uwe.

Muchas gracias, Che. Un saludo desde Cuba.

Hello!
Now I am having serious problems with reproducibility during separation in HILIC.
The pumps and the controler work well. Solvent quality is good (Acetonitrile and water). Temperature is constant (37 C).
What can be the source of irreproducibility ?
The sample, the column... ?
Thank you in advance.

Hi,
If you can I would try to move away from the CN column. Is your sample ionic? is it a base. You may want to try exploring different ph's and a more stable column, maybe the Waters Xterra RP8. If your analyte is basic try a high pH like 11. The more detailed information you give about the sample the better sugestions people will give. Hope this helps
Daren

Thank you Daren. My column is not CN but DIOL, I
made that mistake at the begining of the conversation. I don't know exactly the nature of
the mixture, probably this is a mixture of nucleosides, nucleotides and small peptides and maybe some sugars. They are not retained even in C18 columns (Vydac or J.T Baker), in the DIOL column they are retained, but the profiles change.
Once more, thank you for your help.

Hi,
Do the chromatogram profiles change injection to injection or sample to sample? If it's sample to sample you may just have too much going on in your samples and may require some clean-up steps. If it's injection variability I would try a C8 with an embedded polar group and start with 100% water and see what type of retention you can achieve. I have been moving most of my method development in this direction (finding very stable/ versatile columns and exhausting the development process with them until moving on to others). I have developed a few methods with diol and phenyl columns and have continuosly come across retention variations with lot to lot changes of the packing material. The selectivity that some of these columns offer is unique and sometimes necessary, but I've found the majority of separations can be achieved with a "good" C8 or C18.
Daren

Thank you Daren. Profiles change from injection to injection. I should have soon a C18 column of the type that you are refering, from Vydac. I won't hesitate to use it. At this moment I don't
have any similar, C8 or C18. The column that I use is LiChrosorb DIOL, quite old, maybe I am asking it for much.
I am trying to develop the HILIC experiment with that LiChrosorb DIOL column. It has been said to me that HILIC technology is from the 90's. I don't know the technological improvement made from normal phase to HILIC. Can water be a problem for reproducibility if I don't use those new stationary phases ?
Thank you in advance.

Reproducibility of HILIC. In some messages there are concerns on the reproducibility in HILIC mode.
One should consider the selection of stationary phase carefully since native silica or diol columns will give strong hydrogen bonding and adsorbtion effects. Ion Exchangers sometimes require high salt concentration to minimise electrostatic binding. There are only few columns designed for HILIC separations from PolyLC and the new one from Sequant.