poor peak selectivity

[Pep]

Can someone please help me with the following. I have recently seen a change in the selectivity of my peaks in some methods even though my method has not changed. I am using a c18 reverse phased column from alltech and have recently made some modifications to my system due to the high back pressures I was experiencing. These changes included adding an in-line filter and changing to a hastelloy inlet filter. The method where the change is most noticible involves the threee aminos in question eluted at a quicker time with noticeablly less seperation between peaks. The mobile phase is a 0.01 M NaH2PO4 buffer in 95 % aqueous solution and 5 % MeOH ( pH 2.8 ). This change comes about even though there is no discernable difference in both my column's initial plate count and other methods where buffers are not as prevelant. My system is relatively new and i am struggling to know whether my issue is system related or maybe related to my mobile phase / column. I have recently changed suppliers of my NaH2PO4 and was wondering if this could cause a problem. I would trully appreciate any suggestions on this matter.

[HW Mueller]

Assuming that you only added a hastelloy filter and that your backpressure problem was due to blocked frits, my guess would be that your pH changed. Also, the buffering at 0.01M is not that hot.

[Mark Tracy]

Is your "buffer" a real buffer or simply a salt solution? That is, do you add some acid or base to your NaH2PO4? If it is simply a salt solution, the pH will wander significantly from one bottle of salt to the next. (Believe it not, I have seen validated, peer reviewed procedures with this error.)

[Chris Pohl]

Pep,

In addition to what was proposed above, another possibility is that your stationary phase has suffered (at least in part) from dewetting. Since your mobile phase contains only 5% methanol, this can be low enough organic solvent content to observe a drop in retention due to dewetting, depending upon your operating pressure and column dimensions. Also, the recently introduced filter may have exacerbated the problem unless it was thoroughly degassed prior to plumbing into your system. Try rinsing your column with 90% methanol and then return to your eluent system to see if your original chromatography returns.

[Uwe Neue]

Check the nature of your old and new sodium phosphate! The usual form is with 2 H2O, but there is also the anhydrous form.

[HW Mueller]

Mark,
the phosphate species present at a given pH are always the same, no matter how it was approached, except if you added gobs of other salts (non-phosphate, like NaCl....) such that the ionic strength is way off.
Anyway, something must to be wrong with his buffer.

[Uwe Neue]

Hans
I fully understand that the species is the same. I also understand that if the same molar amounts are used, it makes no difference whether the thing has water or not. But if I start with a recipe that requires X mg of stuff to make a y molar solution, it will make a difference whether the stuff has water in the crystal or not. These things tough to catch...

[HW Mueller]

Uwe, I was not referring to using the wrong molecular weight, or changed material. Rather, I was wondering about getting a desired pH by adding only a salt (here NaH2PO4) into H20 and getting the desired pH, without adding something else (here an acid). If you add only the "salt" NaH2PO4 you get a pH around 4, and only that. If you want a lower pH you have to add acid, etc. If you use H3PO4 at the same concentration you can not get a final wrong concentration, no matter how clumsy you are. If you use HCl you change the ionic strength (introducing Cl-), which might or might not effect things.....etc. etc.

[Mark Tracy]

HW,
You are quite right. I misread the original post and gave an inappropriate answer.

[Pep]

Just a quick note to thank you all for the time you have spent on giving suggestions to what could be the cause of my poor peak separation. The good news is that the problem has been solved. It appears the due to the high aqueous nature of my mobile phase my column was undergoing symptoms of de-wetting ( as suggested by Chris Pohl ). In order to make my system compatible with my phosphate buffer I was initially flushing with 100% water which was causing problems for my stationary phase. This has been rectified by increasing the organic solvent component to 20 % when flushing through my system.
Once again than you all for your time on this matter.

[HW Mueller]

You didn´t flush with pure H2O before adding the in-line filter?