Chromatography Forum

Hi there -

We're running an assay with no bells and whistles on an Acquity UPLC. We noticed an injection error where it was clear the instrument injected ~1.5x the amount specified (see picture below). After an examination, we replaced the seal in the injector and it seemed like the problem was fixed. After about 500 more injections, the same injection error occurred (~1.5x higher than it should have been). We spoke to a Waters rep. who suggested changing the needle height from the default value to 3 or 4mm. After that fix, the instrument behaved for ~1000 injections until the same injection error occurred again.

70% MPA: 4.3mM Octanesulfonic Acid + 0.17% TFA in water
30% MPB: 100% Methanol
Column: Waters BEH C18 1.7um, 2.1x50mm

We have an odd needle wash due to the solubility of our sample: 0.2%TFA in Water.

Has anyone seen this? Or, can anyone shed light on what we might do next to solve this problem?

Just wanted to update this in case anyone else sees a similar problem.

After months of root cause analysis (yay GMP), I finally narrowed this issue down to the following vial preparation parameters:
Note: we're using Kinesis amber screw cap vials and our diluent is basically water (with a little acid in it)
1) overtightened vial cap
2) slit-caps
3) vial has been inverted
4) vial is injected >1x; 2nd injection must occur at least 6 hours after 1st injection

ALL of the above parameters must be true in order for the overinjection to occur (e.g., if you replace the slit-cap with a normal cap and all of the other 3 parameters occur, there will be NO overinjection!)
N=35 Experiment with all 4 parameters above produced a 69% occurrence rate (24/35 vials).

I did a quick experiment varying organic (Methanol) in the diluent with all 4 vial parameters above. Results are below (>/= 20% Methanol does not produce overinjection)
1% MeOH - 80% Occurrence Rate
5% MeOH - 40% Occurrence Rate
10% MeOH - 20% Occurrence Rate
20% MeOH - 0% Occurrence Rate
50% MeOH - 0% Occurrence Rate

Contact me with any questions / comments - I've done a lot of work with this!

Its your sample solvent evaporating, or at least partially.
Inject the same vial with a long run time (~30 minutes) like 10 times. Watch the peak areas slowly climb. If you're not getting "over-injection" with methanol at less than 20% its because the methanol isn't evaporating out as readily because of the increase in water.

Here are some tips they don't teach you about anywhere:

• When you tighten your LC caps down, if the septum puckers, AT ALL, its too tight. You can push septa down into the vial, or pressurize the headspace causing some overdrawing of sample. It'll also help sample consistency. Tighten until it puckers a bit, then back the threading off until its *just* flat again.
• You should fill the vial to the base of the neck to minimize evaporation, but still allow for enough space that you're not creating a vacuum. Ideally, so the meniscus bows just under the neck, but the top is still stuck to the base of the neck. Not enough headspace can cause the needle to reflux back into the vial when it pulls through whatever headspace you do have. (I've seen this happen on Acquity H-Class/I Class and Agilent 1200/1260/1290; I'm sure its pretty much independent of make and model, and its just a consequence of physics)
• NEVER use a pre-split cap if you don't have to, ESPECIALLY if you're planning on using multiple injections from a single vial. Even water evaporates out of them over a few hours. I'm sure there has to be a useful application for a pre-split cap, but I can't think of anything that you'd want it for off the top of my head. I think they're mostly a waste of money, especially if you have light organic solvent for your samples.
• Adjusting the needle offset even +1 or +2mm can be helpful so you don't inadvertently damage the needle tip. (I see you've done this already, so good!)

[*]NEVER use a pre-split cap if you don't have to, ESPECIALLY if you're planning on using multiple injections from a single vial. Even water evaporates out of them over a few hours. I'm sure there has to be a useful application for a pre-split cap, but I can't think of anything that you'd want it for off the top of my head. I think they're mostly a waste of money, especially if you have light organic solvent for your samples.

I just want to point out that Waters supply a PEEK injection needle which does require pre-slit septas.

And I would agree with HippyLabRat that what you're observing is probably due to evaporative loss. Perhaps you can reduce the temperature in your Sample Manager.