Retention Time Instability Batch-To-Batch & Bottle to Bottle

[Xcalibur]

Hi everybody,

I have a problem regarding retention time instabilities in an multicomponent LC_MS/MS assay.

Column: Accucore RP-MS 15 cm, 3mm ID, 2.7 µm particles
Solvent A: Water/Methanol 95/5 v/v + 0.2 % FA + 10 µM LiOAc
Solvent B: Methanol + 10 µM LiOAc
Flow 0.4 mL/min
Column Temp: 35 °C
Dionex Ultimate 3000 quarternary low pressure gradient pump

Gradient:
0 min 100 % A
0.3 min 65 % A
4.5 min 5 % A
5 min 2 % A
10 min 2 % A
10.1 min 100 % A (flow to 0.8 ml/min)
15 min 100 %A

The fast initial gradient slope is due to a mismatch of injection solvent ( 95 % EtOH, not changeable), else the peakshape of the first eluter ( ~ 5 min ) is bad. Starting a linear ramp at higher % B does not work properly.

The phenomenon: LC runs of identical samples give a large variation in RT (more than a minute) on consecutive days.

An experiment for repeatability was set-up: 2 bottles of Solvent A were prepared by weighing in all components by the same person within one hour.

Analysis using either solvent A1 or A2 (on different channels on the HPLC) gave a retention time difference of 2 minutes!

I have no clue which screw to turn at the moment, does anybody have an idea?

Thanks
Georg

[Steve Reimer]

Is the pressure the same for A1 and A2? i.e. is your flow what it is supposed to be for both solvent bottles?
If you switch the pick up tube does the rt shift follow the solvent or the tube?

[Xcalibur]

Hi Steve,

yes, pressure is the same. I will redo the experiment, maybe something with mobile phase pre-mixing went wrong. Anyway, the same happens for the same mobile phase in the same channel on two consecutive days...

[Steve Reimer]

So it makes no difference if you switched the mobile phases in the bottles. If you switched tubes in the bottles, does it follow the tubes or the bottles? Do you have frits on the tubes?
Is your gradient what you think it is or is your low pressure mixing correct?
It's a place to start.

[Xcalibur]

Hi Steve,

I did another experiement with a linear gradient and various gradient speeds

A) 20-98B, 10 % B/min
B) 20-98B, 5% B/min
C) 20-98B, 2.5 %B/min.

With each run (5 consecutive runs). rts decreased, (drastically for early eluters, not so drastically for late eluters). Might maybe an equilibration problem (equil = 5 CV, will try to increase)

I have frits on the tubes. Will try to exchange tubes and bottles today!

BR Georg