Injection linearity Waters Acquity

[Mattias]

I have just installed a new loop (50 µl) and syringe (250 µl) on my Acquity, and as qualification I injected a peptide sample in a standard method using 5, 10, 25 and 50 µl injection. Loop and needle volume was measured and entered into the system. Same vial, column, mobile phases etc (it was just a one sample set). Injection mode: partial loop, needle overfill.

When the results came out, the area/ injected µl varied a lot (mean of five injectons, very low RSD)

50 µl: 27400
25 µl: 31300
10 µl: 20100
5 µl: 22800

I did not have any overloading problem, max peak height was 100 mAu. The injection linearity is not a demand in our qualifications, and when using a fixed volume for samples and standards, this is not so important.

I am just worried that the linearity is so bad, and why is there not any trend? Can there be something wrong with my system?

[danko]

I would qualify the shown results as a problem despite the fact that your intention is to inject a fixed volume only.

Best Regards

[Mattias]

One more thing,

I have just blown the whistle about adsorption of a peptide to the product vial. This came after I diluted the product (directly in the product vial) with 200 µl acetonitrile (product contains 1 ml). Then I injected the solution before and after addition of acetonitrile. The peak areas of the two samples were almost the same, despite the 20% dilution.

Everyone was very surprised, since this peptide is in high conc. Can this difference be an effect of the "partial loop, needle overfill" injection mode, injecting different volumes depending on the sample composition? (slightly off-topic but anyway)

[Mattias]

Danko> I agree. This does not look good.

Strange enough, the RSD is very low (0.1-0.2%), so there must be something wrong with the syringe levels.

[Chromatographer2010]

Would not try to inject the loop volume in a partial loop injection mode. Typically up to half the loop volume is a good rule of thumb for partial loop injections.

Also with the larger syringe did you lower the syringe draw rate? You can get some weird apparent linearity effects if your draw rate is high and your diluent viscosity changes, such as adding ACN to water.

Also you could have an issue with your syinge or fittings such as a leak or an air bubble in the syringe.

[aceto_81]

Did you recalibrate your needle and looop volumes?

Normally partial loop with needle overfill should show a good linearity.

Ace

[Mattias]

Chromatographer2010> I have not adjusted the syringe draw rate, but I assume that the system did this automatically when I entered the new volumes into Empower. Looking at the syringe, it seems to draw the sample very slowly. The sample is just a peptide in 0.9% NaCl (ie not viscous at all). I agree that injecting the full loop in partial loop mode was not very smart.

aceto_81> yes, the needle och loop was characterised (found loop volume was 46.6 µl)

I need to contact Waters about this. It is really strange that I get very low RSD for injections between 10 - 50 µl, but with such bad linearity.

[juddc]

What I might do here is this:

1. Check the thing for leaks on ANY fittings you touched. I'm sure you've done that; do it again.

2. Assuming you have no leaks, purge the beans out of the thing with neat MeOH, not MP. Purge it at least 2-3 times. Then perhaps purge it again.

3. Check the injector accuracy: Weigh a capped vial that contains ~1.2 ml water, do 6 x 50ul injections of DI water, then reweigh the vial. Did the machine pull ~300mg in total? You can also do this for other injection volumes, obviously - 12 inj x 25ul, etc...If you get the right injection volume and decent linearity here, the injector is most likely fine. Still, I'd move to #4.

4. Set up a set of standard test conditions with a simple separation (don't use peptides). Waters' PQ test mix, a mixture of parabens, any simple, rapid isocratic separation with the std dissolved in MP will do. Inject betewen 5-50-ul (5, 10, 25, 50) and do a system suitability (6X whatever inj vol you use most). What do you see?

Best of luck!

CJ