junk at the end of amino acid run

[pink05]

I run a Shimadzu HPLC system and am having trouble separating my Arginine peak from junk that comes out at the very end of my 80 minute run. I've discovered, by doing a NUL injection, that this "junk" is part of my mobile phase since it is still there during this particular injection. I'm currently trying to play with my gradient times to see if I can get better separation. Does anyone have any suggestions? I would greatly appreciate any help.

[SIELC_Tech]

What is your mobile phase, column, detection technique and sample matrix? Are you trying to quantitate arginine in the mixture? What other compounds do you have in your sample?

Regards,

Vlad

[pink05]

My mobile phase is a mix of sodium eluants, one at pH 3.28 and one at pH 7.40, bought from Pickering. The column is a sodium cation exchange column from Pickering. UV-Vis is my detector. Samples are diluted with a sodium diluent also from Pickering at pH 2.20 and additionally filtered before analysis. I'm trying to quantitate arginine along with 17 other amino acids that are present in my standard. I am fairly new to HPLC as I just graduated from college in May. Let me know if you need any other information.

[tom jupille]

Try doing the following test:
1. Run a NUL gradient (to wash accumulated junk off the column)
2. Equilibrate the column for the normal time and run another NUL gradient
3. Equilibrate the column for three times longer than nomal and run another NUL gradient

If the junk is coming from your "A" mobile phase, the junk peaks will be about 3X larger in run 3 than they were in run 2. If that's the case, the best approach is to keep the junk from getting in there in the first place. Some things to look at:
- water system: how old are the cartridges?
- buffers, additives etc. purity? age?
- did you stick a pH electrode directly in your mobile phase (buffer leakage, carryover)
- was your glassware clean? any residual detergent? fingerprints on the inside of the beaker?

If the problem persists, consider filtering your A solvent through a C18 impregnated filter .

[Mark Tracy]

Is this junk a series of closely spaced peaks? If so, it is oxidation of the phenol use as a preservative in the pH 7.4 eluent. Try a newer lot. Some years ago when I worked for them, they had a bad batch of citrate that somehow promoted oxidation over time and caused the symptom you describe.