glutamic acid separation

[guilingverity]

I am attempting to analyze oxidation products of amino acids reacting with something else, but my first task is to identify the starting compound, glutamic acid. As of yet I have not been able to see a peak. Glutamic acid is in phosphate buffer, with 200uM glutamic acid and 10mM phosphate. I am using a C18, reversed phase column and have tried a number of combinations of water, acid, methanol, and acetonitrile for my mobile phase. Does anyone have any advice on what I can do? Again, I haven't even seen a glutamic acid peak! Programs that I've found from literature on amino acid separation just hasn't worked for me. I do not know if glutamic acid is so polar that it's coming off the column at the same time as my buffer, or if it's taking so long to come off that I haven't been able to see it (running samples for ~an hour). Any advice at all would be very helpful!

[Markus Laeubli, Metrohm]

Glutamic acid is a pretty strong acid pK1 = 2.2, pK2 = 4.3.
I assume that it is almost not retained on RP columns.

[Tomtotom]

Which wavelength did you use? I would assume that glutamic acid absorbes at around 210 nm. If you use longer wavelength you would probably miss it.

[guilingverity]

Thanks for answering!!

Yes I have been trying to keep both of those things in mind. When I run the UV-Vis of glu, it peaks somewhere out of range (at a lower wavelength than 190nm), but starts going up around 200ish. The HPLC automatically monitors at 254nm, but I can go back through the saved data and look across the range of 209 to 394nm, and haven't really found anything.

As for coming off quickly, it may be coming out with the buffer, which is the only peak I can find (which is always less than 5 minutes, regardless of sample combo). I know it's buffer because I also always run a buffer-only sample, and it's exactly the same each time for buffer and for glu.

Is there anything else I may be missing?

[guilingverity]

Also should I not be using reversed phase?

[chromatographer1]

yes, you should not be using reversed phase chromatography.

Literature is available for methods of these compounds. Call a column vendor for help if you don't have a library available.

best wishes,

Rod