measuring amount injected

Hello Melissa:

Let's see what other replies state.

However the question is are you interested in knowing the volume or the actual load (accuracy/recovery)?

For a ACQUITY UPLC sample manager you can be confident that you have an accurate map of the fluidics when you can repeat characterization of the loop and needle and get three identical values. This value corresponds to the size of the loop measured by following an air bubble in the fluidic path and uses the weak needlewash.

I will attach some thoughts on calibration of the system also.

However, the system will deliver different on column amounts depending on the injection mode. They modes work differently fluidically and so area counts will differ for the same nominal injection volume using different partial loop modes.

If you are using PLNO mode you will aspirate up 200 nL of sample, plus 14 uL of sample extra before the aliquot sample's leading edge of the aliquot and 1 uL after the tailing edge and so the amount delivered to the column will tend to be less dispersed and less diluted giving a bigger area for PLNO. For a given sample and method this will be reproducible across loops and systems. This is why PLNO mode was invented so that you can get similar area counts for a given method independent of the system or loop.

For Partial Loop with Pressure Assist Mode the sample is positioned with its leading edge at the end of the sample loop closest to the column (to reduce dispersion and keep maximum amount of sample in loop) Recovery depends upon Sample viscosity and the syringe draw rate and finally the sample volume vs. loop volume. Therefore, with PLPA injection mode recovery/accuracy/on column amount depends on an accurate “map” of volumes of the sampling needle, sample loop and connecting tubing. For small volumes such as 200 nL you are best to use a small syringe (50 uL) , loop (1 uL) and needle (15 uL). Thus areas/recovery will vary across the systems and will differs slightly across loops and systems, but again within one system and loop and method the areas/amounts will be reproducible. However, you cannot say one system is more accurate than another, it will be different.

Let's see what comments we get from the community, best regards,

Liz