Chromatography Forum

I have a 70 min gradient and nearly 20 peaks to come. My mobile phase is Buffer/ACN and the column temp is 60C.In this analysis we always have problems(baseline drifts, changing retention times, ghost peaks).My mobile phase is in room temp when i run(25C).I cannot thermostate the bottles. Do you believe that i should fix PH(must be 11) when the aqouas portion of M.P. is in 60C? Do you believe that the problems i am coping with, are due to this "temperature gradient" for my M.P.? Is there something i could do?

I really doubt that the method specifies a pH of 11 at 60 degrees. Yes, the pH will shift with temperature (and it will also change during the gradient), but it should do so the same way every time.

You describe three symptoms (baseline drift, ghost peaks, and changing retention time) which may well stem from different causes. I put together a quick presentation on drift and artifact peaks which may help (viewtopic.php?f=31&t=19085).

Changing retention times could be a temperature issue, or a mobile phase proportioning issue, or a buffer pH issue. If the changes are random, then I would be most likely to suspect the proportioning system. You can check that easily via the "stairstep test" (http://www.lcresources.com/resources/TSWiz/hs450.htm).

Temperature is a bit trickier, but you can use a thermocouple inserted in a PEEK tee plumbed into the transfer line just upstream of the column to check the *actual* temperature and see if it's changing. That will also tell you how effective your preheater is.

If the retention times are drifting continuously, then I would be inclined to suspect the buffer pH; at pH 11 you will absorb CO2 from the atmosphere. It's a good idea to use a CO2 trap on top of the jug to minimize the problem.

What kind of column are you using at pH 11 at 60°C?
Maybe you are degradating your column?

Ace

i 've made a mistake. PH of m.phase is 9. PH of the solvent is 10(before addition of the organic). i use x-terra ms C18 250x4.6. i think that these conditions destroy the column. After 3 batches the column is useless(for this analysis). But we have notice that if we forget this column for several months, maybe a year, it performs much better than the last time. Is it strange?

pH 9 for an XTerra MS should be fine, but it is still aggressive.
Also the 60°C make things worse.

Maybe you can try pH 9 at 45°C to rule the temperature out?
Or do some other tests like Tom describes.


Ace

Although Waters says that pH9 is no problem for XTerra, in reality it might be depending on the conditions. If column degradation really is the underlying problem, you might try some things:
- As Aceto already noted, higher temperatures speed up column degradation. Try to use a lower temp.
- What kind of buffer are you using? Inorganic buffers, especially phosphate, are far more aggressive than organic buffers. You might want to try something like glycine or piperazine. Borate is supposed to be less aggressive, too. Keep the buffer concentration as low as possible.
- According to my experience (and Waters advertisement ), XBridge columns are more alkali-resistant than XTerra. So a column switch might be an option. I've also made some good experience with the hybrid column from YMC (Triart).