Measuring the loop volume

lizh

<p>How does the ACQUITY UPLC System measure the loop volume?</p>

lizh

The ACQUITY UPLC System uses a "Volume Detection Device" (VDD), see figure below, to detect the movement of an air bubble generated by the needle as it travels through the needle and sample injection valve. The air gap is transported until the bubble detector senses the bubble. The VDD has a light source and a window portion, when the air gap crosses the window the light is defracted and the sensor is tripped. This happens twice: once with the sample loop in the load position and once with the loop in the inject position. The measured volumes are then compared and the sample loop apparent volume and the needle apparent volumes are determined. These are called apparent volumes since there are many fluid dynamics in play within this measurement. However, the measurement does simulate the actual transport process and the measured volumes correspond to the amount that the sample needs to be transported in terms of motor steps etc. One factor that should be considered is that the VDD device measures volume based on the air gap. Since bubbles are somewhat sensitive to surface tension, there will be some additional variance associated with the method and the mobile phase used. If the calibration is done in a solvent with high organic modifier or with a compressible solvent like Hexane, the size of the air gap may need to be increased to get consistent results. Assuming that the measured volumes are consistent, the calibration volumes become a "map" for the Sample Manager as it positions the sample in the loop for partial loop injections (PLNO and Pressure Assisted). The volumes for the loop and the needle are inserted into the positioning algorithm that the Sample Manager uses to ensure that the sample is placed in the loop correctly. The loop volume should be characterized once prior to setting up a given method with fresh mobile phase. This can be done automatically by the system when it is primed and prepared for the analysis initially, using the "Sys Prep" function in the Console Software. The user selects this in the appropriate Characterization tab of the SysPrep setup menu.

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estherd

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lizh

I have received a few corrections on this document amongst others - especially spelling. Any additions or comments that I should add?

Vic37

Do you know about any issues if doing this test with 100% Organic Mobile Phase (Acetonitrile) as Strong Wash Soln. I'm having problems trying to Characterize the volumes after replacing an Injector Pod and Sample Loop. My Strong Wash is 0.1% Formic Acid in Acetonitrile.

ehenning

Hi Liz,

     I'm actually seeing an issue when using the "Startup System" applet.  I was wondering if you or anyone else has seen this?  ICOP is at 1.50.  If the characterize needle seal and characterize needle and loop volumes boxes are checked when performing the startup system functions, the needle and loop volumes portion fails every time.  If a user tries to run samples, "z axis not calibrated" error occurs.  If I look in the Waters Instrument folder, all calibration/characterization values are missing.  If I perform calibrate and characterizations from the "maintain" pull down, everything is fine, all values are populated in the waters instruments folder, the log shows same values for diagnostics, and the instrument performs flawlessly.  If at any point after, a user chooses startup system and checks the characterize boxes, the failure returns.  I've confirmed that the system is sufficiently primed and that there are no bubbles in sample or wash syringes during both situations.  I'm assuming a reload of ICOP and firmware is in order, but have not been able to perform yet due to GMP concerns.  This is only on our binary systems.  H-Class at 1.45 and 1.50 do not seem to be affected.  Thanks!

Eric

rune

Hi Victor

Volume characterization is done with the weak wash solution in the sample syringe and flow path , so the composition of the strong wash solution should not affect this measurement. Have you checked that all fittings on the injection valve are seated correctly, this is the most common cause of the characterization failing, especially since you say you have changed the POD and the loop.

best regards

Rune