2ul loop calibration

<p><span>I am looking for some information with regards to chracterization of a 2ul loop. Without exception everytime I place a 2ul loop on my Acquity it will take me the better part of half a day to calibrate the loop. If I prime my weak and strong wash with methanol I can calibrate t0n he first or second try but when I switch back to my normal weak wash (3% ACN in water) I cannot calibrate the loop. I ran every leak and pressure check I can and the system passes. I calibrated my bubble sensor as well and it passes yet I still cannot calibrate the 2ul loop. Anything larger then 2ul presents no problem. Has anybody else experienced this problem and if so have you found a way to correct it?</span></p>


  • lizh


    This does seem a long time, but it can take this long for the smaller loops.

    It would be good to know what is in your washes contain. Here is a post from Peyton in our Evaluation lab that may apply.

    Here is the answer that our evaluation lab has supplied...

    The characterized after installation of a new 1µL loop took in excess of 25 minutes. Usually when I've tested with 2 and 5µL loops it is much shorter than this. Is there any underlying reasons why this might have taken so long this time around?

    Firstly, times do vary depending on the loop and needle installed. The characterization process must be done correctly to ensure accurate injection volumes, thus Sample Manager runs a slow syringe speed (10 µL/min) and checks the volume of the loop by locating the position of aspirated air bubble twice, once with the loop offline and once with the loop online. The 1 µL loop is a very small loop and the ID is smaller than the other loops, (larger loops are manufactured using 0.009 stainless steel) this means to transport the solvent through the loop is more difficult and the process is slower, like sucking on a small straw. The volume that the air gap needs to travel for a 30 µL needle and to the volume detection device is about 40 µL, so that is 4 minutes, or 8 minutes for the repeats.

    The second reason is that if the Sample Manager gets an unsatisfactory answer with its first attempt it repeats, so I wonder if the process may have repeated. This uncertainty of detection of the aspirated air gap can arise if the mobile phase is UV absorbing, if contains TFA for example.

    I have data on times for the complete preparation of the Sample Manager at my fingertips, rather than characterization alone. At solvent changeover when one must perform:

    • Prime wash syringes (weak and strong)
    • Prime sample syringe
    • Characterize loop and needle volume

    The time for the default 30?L needle & 10?L loop is about 18 minutes and the time for the default 30?L needle & 2?L loop about 21 minutes.

  • JohnS

    Thanks for your reply. In answer to your question about my wash solvents the weak wash is 3% acetonitrile in water and the strong a 1:1:1 mix of IPA:Methanol:ACN. I see in your answer that the time required for calibration varies with needle and loop size as I expect it should. However, I never get to that point. I cannot calibrate a 2ul loop even if I tried all day so there must be something else going on. Additionally, this is not only one 1 Acquity but on 3 that I am running! The suggestion from Waters was to calibrate my bubble sensor 3 consecutive times then calibrate the loop...doesn't work. Is anybody else having problems like this with the 1 and 2ul loops?

  • lizh


    The increase in time is the one that crops up. I am still awaiting replies on what else to check.

    Can you use a 5 uL loop in the mean time. I wonder if you have a really small ID loop?

    Finally, what are the messages in the log saying (just send the sample manager ones), there amy be a clue there.

    I will be in touch as soon as I have better guidance for you.


  • We use 2 uL loops regularly, and don't seem to have any problems with them. They typically take around 8+ minutes to characterize. One thing you may want to check is the distance at which the ferrules are seated on the loop. When installing a new loop, if it isn't seated properly when the fittings are tightened you'll have problems with the characterization. I attached a picture to show what I mean (a pictures worth a 1000 words...) In the picture, the bottom loop is the good one, the top loop is bad; it would not characterize at all.

  • JohnS

    Elizabeth. Thanks. That is one thing I make certain of when I install a new loop of any size. My loop looks like the bottom set in your attached picture so I'm pretty certain I am okay. What size needle and syringe are you using? What is your draw rate and weak wash? I could see if I have this problem on only one instrument and/or 1 loop but I am seing it consistently on 3 different Acquity's and with both new and old loops (which worked just fine but were removed temporarily in favor of a larger volume loop). Once they're off they are very difficult to put back on and calibrate even when I physically mark the loop positions in the injector.

  • Our systems have the standard 30 uL peek needle and 100 uL syringe installed on them. We run a couple different methods with the 2 uL loop. One uses 10% MeCN, 90% H2O as the weak wash with the default draw rate, the other uses 20% MeOH, 80% H2O as the weak wash with a syringe draw rate of 75 uL/min.

  • RMJ


    I have a 2-uL loop that has been removed and reused at least 2 times, and it characterized within about 15 to 20 minutes when I did it on 09-July-2009. It takes longer than what I recall with the 10-uL loop, but it characterized OK. The needle is the 30-uL peek needle.

    Weak needle wash is 10%ACN/90%Water, and strong needle wash is 50/50 ACN/MeOH on my system.

  • lizh


    Here is the comprehensive answer from the firmware engineer for thr Sample Manager.

    If the issue is not as we suspect that the syringe draw rate is to low, we have a fault that is causing the issue ( possible the Volume Detection Device).

    The engineer suspects you must be downloading a method with a lower draw rate than 30 ul/min. Or that you have the wrong configurations (e.g. 1 ul loop or the 15 ul needle selected...please confirm).

    If the user has downloaded a method with a custom draw rate, the SM will use this user defined draw rate to characterize the needle and loop volumes. Otherwise, it will use internal defaults (defined by the needle and loop volumes).

    Approximate calculation on how much it should take (assuming a 30 ul needle and a 2 ul loop):

    1. Calibrate air sensor: (40 +8) ul / 30 ul/min = 1.6 min (8= air sensor volume + safety margin)

    2, loop w/ no pressure (w/ loop): (42+6)/30 = 1.6 min

    3, loop w/ no pressure (w/ no loop): (40 + 6) = 1.53 min

    4. loop w/ pressure (w/ loop): (15+4)/30 = 0.63

    5. loop w/ no pressure (w/ no loop): (15+4)/30 = 0.63

    6. Needle wash: about 2 mins

    Approximate total time: 6 minutes + wash needle time (the wash time is approximately (5*300/1000 = 1.5 min to fill wash syringe + .5 min to wash ==> about 2 minutes)

    It could have up to 2 tries: 8 *2 = about 16 +/- 3 minutes.

    So worst case under 20 mins.

    Let us know how this goes.