I'm been running Agilent OPA amino acids method but recently my column loss resolution for some of the peaks and will not resolved to baseline. After some discussion with others people in the field, I suspect this to be a column problem since OPA and borate buffer is known to degenerate column quickly.
I have always washed my column by the end of day with 50:50 ACN:H2O for 20 minutes.
Is that anyway I can regenerate or recondition my column again? What kinds of procedures and solvent would you recommend?
Additional Info
- Instrument modules: AGILENT 1200 WITH G1379B DEGASSER, G1312B BINARY PUMP, G1367C AUTOSAMPLER, G1316B TCC, G1315C DAD
- Column: AGILENT Poroshell 120 HPH C18, 4.6 x 100 mm, 2.7 μm
- Chromatograph shown represent a 100 pmol amino acids standard derivatized with OPA. You may notice intensity difference due to presence of air bubble in the microinsert which caused incorrect volume drawn, but I rectified the problem since then, air bubble is not the concern here anymore.
For injector program, DAD wavelength and gradient program used, please see application note: https://www.agilent.com/cs/library/applications/5991-5571EN.pdf
- Chee asked: "Is that anyway I can regenerate or recondition my column again?
No. This is an extremely common issue with AA analysis and the quick answer is to dispose of the column and replace it with a new one when the performance spec can no longer be met. None of the routine column cleaning suggestions (as others unfamiliar with AA analysis) will restore operation. AA analysis using derivatization chemicals such as OPA have very specific and well documented problems. The derivatization method used damages the column over time and no known method of "re-generation" exists that can replace the lost functional groups or restore the surface. Dispose of the column. It is not uncommon when using this particular method to only get 200 injection (samples) per column, before reaching the limit. This added cost must be factored into the method.
- "I have always washed my column by the end of day with 50:50 ACN:H2O for 20 minutes."
- Any wash solution of this type should include methanol, not ACN so switching over to a wash gradient method with water and methanol may help.
A few things that can help extend column lifetimes include: (1) make sure you regularly flush the mobile phase out of the column and HPLC system every day, after use (replaced by a solution that is safe for the column, HPLC system and has some organic solvent in it to prevent the growth of "bugs". Never leave the system sitting in the mobile phase. Use FRESH derivatization chemicals, use ultra high purity water, replace the solvent pickup frits in the mobile phase bottles every month (watch out for algae growth in the lines too!), replace the pump's PTFE frit every month too. Keep the system super clean. Run cal check stds often to monitor the column performance.
The application note I read recently suggest to wash and store the column after used with the mobile phase B (ACN:MET:H2O, 45:45:10) for 40 minutes.
Do you think this will work equally well with water and methanol only?
Not really (unless you have a LOT of time to waste!). Just get a 'new' column and protect it with a guard column.
It also depends on your sample matrix and the other 'crap' your injecting (even after SPE clean-up and ultrafiltration!).
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