Chromatography Forum

We are currently having some issues with reproducibility for 100uL injections on our Agilent 1100 HPLCs. Specifically we are getting variable area counts for standards out of the same vials. Has anyone else experienced this and if so did you determine the cause? We had considered cross-port contamination, but given we are seeing it on a variety of instruments I do not believe this is an appropriate conclusion. Any assistance would be great.

How much variability (%RSD for how many injections) are you seeing?
What's the loop volume?
What's the syringe volume?

Injecting more than about half the nominal loop volume is known to hurt reproducibility because of laminar flow effects. Injecting less than about 10-20% of the syringe maximum volume can hurt by spreading the syringe drive's uncertainty over a small volume.

The sample loop we are using is 100uL, but I am not sure about the syringe.

In terms of the reproducibility, from what I understand the first injections out of a vial are at one level of area counts and then the second and all subsequent injections are about 3% lower and stay at about 3% lower areas with decent precision.

Can you recommend a reference for looking up your comment about laminar flow? Specifically, do you think that the composition of the sample and mobile phase would impact the effect? The reason I am asking is we have many methods that use the 100uL injection, but there only seems to be consistent problems with a few of them. The one that is particularly interesting is an assay and disso method where you use different dilutions of the same standard and inject at 40uL and 100uL respectively. The assay is generally fine, but standards often don;t compare for disso with the 100uL injection.

Any references or additional suggestions would be greatly appreciated.

Cheers

Check your autosampler vials. If the seal is very tight, subsequent injections are pulling against a vacuum and will have lower volumes injected. Test by doing replicate injections out of an open vial and comparing with single injections from replicate sealed vials and replicate injections from sealed vials.

Agilent 1100 AS's don't have loops--they have a syringe and a capillary seat which holds about 3 uL. Unless you add an extended capillary seat, (which is the cheaper, easier option) or change the syringe, 100 uL is the max you can inject. However, we have used 100 uL injections frequently, and without problems, providing the right vial cap is used (I like Snap caps, and will even use unlined ones if the sample is at low risk of evaporation).

Better check the linearity performance of your system.