is this method or system problem?

[flora0975]

I am transfering in a method for a racemate. I have two system recently done PM. It is first run on both systems.

The method has Mobile phase A: 10mM K2HO4, natual pH, Mobile phase B: ACN. XBridge phenyl column. UV 220nm.

The system suit injections are consistant on retention time, but RSD>10% on peak area. The same case on both systems. The standard curves ( prepare twice) did have large deviation too.

Is this method problem or system problem? Anyone can give me a clue? Thanks!!!

[DR]

pKa of racemates?
pH of mobile phase?

Rules of thumb re: pH, pKa:
If they're close, that may well be your problem.
RP columns in general + high pH are seldom a good combination.

[flora0975]

Thank you! No pH adjustment for Mobile phase A. The retention time is not drifting, is that normal for pH problem?

I don't have pKa inforamtion, the compound contain -OOH, -B(OH)2, and -SO2.

The method has good peak shape, it is better stick on K2HPO4 buffer. Are you suggesting high pH for MPA?

pKa of racemates?
pH of mobile phase?

Rules of thumb re: pH, pKa:
If they're close, that may well be your problem.
RP columns in general + high pH are seldom a good combination.

[HPLCaddict]

In order to verify if the system or the method is the problem, there's one easy way: Take out the method .
E.g. take out the column, insert a column blank (a capillary that gives you some backpressure), use pure methanol as eluent and do a series (e.g. 10) of injections. A decent autosampler should give you a peak area %RSD of <1% easily.
If it works, your method is the problem. If it doesn't, check your system.

[juddc]

pH issues will mess with your chromatography, but assuming that the chromatograms look consistent from a resolution and retention standpoint, you shouldn't see a ~10% RSD in peak area.

Was there any performance check done on either system post PM? Typically a standard PQ would be sufficient to see if there are any issues, especially if you have prior PQ data to compare against.

If no PQ was done and this is the first analytical run after the PM, I might suspect something as simple as air in your syringe(s).

Making the admitedly large assumption that the method is OK, I'd be somewhat suspiscious of your system(s) simply because they had been altered recently.

My advice:
1. Look your system(s) over carefully for leaks.
2. Purge your injector.
3. Purge your injector again.
4. Check your syringe for bubbles. If you find any, go back to step 1.
5. Check your injector accuracy - Fill an autosampler vial with water, weigh it, then inject 50-uL six times. Use a run time of 0.1 min and have the system pumping neat MeOH at 1.0 ml/min. Reweigh the vial. If the mass of the vial decreases by 294-mg to 306-mg, your injector is likely OK.
6. Run a PQ. If that passes, your system is fine and your method is no good. If it fails, your system is no good and your method may or may not be good.

Best of luck!

CJ

[flora0975]

Thank you so much for your reply.

After few runs investigation. I feel it is mobile phase A problem. The mobile phase A was prepared one day before the run, the pH was 7.9 ( checked on third day). While the fresh mobile phase A pH is 8.9 ( checked on first day). It is huge difference. The run with fresh MPA are good on SST.

I am using 10mM K2HPO4 buffer, is it very unstable?

[paulw]

There should not be a buffer stability issue. I find it curious that you have a pH difference of 1.0. The two reasons I can think of for this right offhand are variability in the actually amount of buffer used, or variability in the pH of the source water. I know that our DI system has some day to day variability in pH, so I tend to pH control my mobile phases when I develop a method. I also generally try and avoid using a mobile phase with a pH higher than 7.0 (I know that many columns claim they can handle it).

Paul