hello together,

its my first time to involve a new methode
what my question is, can anyone help me
to find a way how i could separate plant pigments
in a LiChroCart RP-18 (5 ĩm) 4mm x 10 cm Column, or is that column too small?
Another question, i`ve tried to buffer Acetonitril:Methanol 87:10 with 1M Tris ph 8 or Kaliumphosphate buffer ph 7, in both liquids something precipitatet but why ?
I need a good methode, i don`t know what to do...

please help me

greetings,
sabrina

Sabrina,

If I understand you correctly, you are trying to utilize a system with 97% organic content with a 3% added buffer (with ionic strength as high as one molar). Your problem is connected to the limited solubility of electrolytes in eluents containing high percentages of organic solvent. Do you really need to use such a high percentage of acetonitrile and methanol for your chromatography or is this just so that you can add the same buffer to both a high organic content eluent and an aqueous eluent which will be mixed together by the pump? If you really need this level of organic for your chromatography, you will need to greatly reduce the ionic strength of your buffer in order to avoid the precipitation problem. On the other hand, if you are simply mixing two different eluents together in the instrument, a better solution would be to add the buffer only to the aqueous eluent. You can construct a gradient using three eluent containers: one for aqueous buffer, a totally aqueous eluent and a third with the organic solvents. If you keep the buffer percentage at a constant level and simply vary the ratio of aqueous to organic solvent, this will avoid the necessity of adding buffer to your organic solvent. However, you still need to manually prepare the highest organic content eluent to make sure that the buffer does not precipitate under these conditions. Otherwise you'll have precipitation problems in the column and/or pump! Perhaps you are adding buffer to the organic solvent because you believe that you must adjust the pH separately in the organic solvent and in the aqueous buffer solution. I would suggest that this is a mistake. The pH electrode doesn't really work correctly in high solvent content liquids so it's probably not a very good idea to try to measure the pH and adjust it in the presence of 97% solvent. It's true that solvent has an effect upon pH but I'm not sure it's all that crucial that you know precisely what the pH is at any solvent content as long as your results are reproducible. Anyway, there isn't a direct linear relationship between the solvent effect on pH and the solvent content so even if you know the pH at high solvent content and at low solvent content, you still don't know the pH for intermediate solvent compositions.

Have you tried searhing the literature for a good method. Its usually the best place to start.

hello Chris,

thank you very much for this information!
i think, this method i found was for three pumps.
they still working with it - with only two.
ok, i donīt work with any buffer right now.
so i neednīt ask me furthermore.

bye,
Sabrina

hi

pl any one explain what eaxacly
A) POST INJECTION VOLUME
A) ISOCLUTROPIC STRENGTH

thanks