Understanding Volume Characterization and Air Gaps

<p>Greetings, </p><p></p><p>An issue arrived in my e-mail the other day and reminded me that I wanted to cover the subject in a discussion. One of our Field Service Engineers, in Australia, was having difficulties getting an ACQUITY system to, reliably, characterize the needle and sample loop volumes. I believe the key to this problem was the use of 100% MeOH as the Weak Wash Solvent in the Sample Manager. </p><p></p><p>The ability of ACQUITY to measure and map the internal volumes of the Sample Manager is a significant benefit and should never be overlooked when trying to improve injection performance. An accurate characterization is critical for all three Injections Modes. The ability of the system to "know" the volumes of the loop and the needle is, essentially, mandatory for the utilization of the Partial Loop with Needle Overfill (PLNO) injection mode. This injection mode absolutely depends on the accurate location (in volume) of the interface between the injection valve and the upstream side (port 1) of the sample loop. The sample is drawn through the injection valve and then "backed" into the loop from the upstream (syringe) side of the valve. If the exact location of the injection valve and Sample Loop interface is not accurate, the system will not meter the correct amount of sample into the Sample Loop. When changing mobile phase systems, I often characterize more than once to ensure that I do not see differences greater than 0.1 uL between characterizations. This is especially true when I am evaluating, or depending upon, the very highest level of performance from the Sample Manager. Accurate characterization requires that the Volume Detection Device (the VDD, which is the little black box attached to the injection valve) is able to detect and recognize small air gaps that are introduced during characterization procedure. </p><p></p><p>The situation in Australia presented me an opportunity to make a suggestion. Methanol (and most other organic solvents) has a much higher gas load capacity than water (or predominantly aqueous based solvents). This means methanol is much more able to dissolve gases (air). This is why we do not recommend using 100% organic to characterize the system. However, the issue can almost always be resolved, even with 100% organic, by employing larger air gaps. A method should be created that sets customized air gaps to between 6 uL and 8 uL (the automatic value is 4 uL). This method should then be downloaded to the system. Once this is done, the characterization will use the conditions, of the last valid (downloaded) method, to perform the characterization. The larger air gaps prevent the organic solvent from, completely, dissolving the air gaps. I always utilize this technique when I run normal phase chromatography on ACQUITY. If the Sample Manager is already in an error state, due to a characterization failure, it will not accept the new parameters (this has just been corrected in ACQUITY v1.40). It will be necessary to first prime with an aqueous Weak Wash Solvent, characterize with this solvent, then return to the organic wash solvent and download the larger air gaps. The size of the custom air gaps depends on the size (volumes) of the needle and Sample Loop. The larger the needle and loops, the larger the internal diameters. Larger diameters make the air gaps "shorter" and easier for organic solvents to dissolve. The 6 uL to 8 uL range is what I recommend for a "standard" configuration of a 30 uL needle and a 10 uL Sample Loop. </p><p></p><p>It might occur to some of you to characterize with an aqueous solvent and utilize that characterization no matter what Weak Wash Solvent is in use. This strategy is acceptable (mostly) if you are employing the PLNO injection mode, but not for the Full Loop or Pressure Assist modes. The PLNO mode positions the sample into the Sample Loop under atmospheric pressure, but the other modes place the sample into the loop under ele...


  • Jim


    I'm very interested in this subject. Can you comment more on the statement you made in your last paragraph?

    We have been using the 15 uL stainless steel needle with a 1 uL sample loop and operate in the full loop mode. I adjust the syringe draw rate to 10 uL/min and use the over fill setting. I have water in my weak wash and IPA/ACN in the strong wash. Using these conditions I have been able to successfully characterize my needle. My analyses require the BEST injection precision possible, and typically in the full loop mode the peak area RSD is ~0.1% (UV, isocratic, caffeine). Can you mention conditions that might be better to use when characterizing under these conditions?



  • Greetings Jim,

    From what you describe, I believe you have already arrived at some pretty favorable conditions. I know that I, previously, commented on the issues that organics pose during characterization, but you might try adding 10% ACN to your weak wash. I say this because these are the exact conditions of our qualification tests and the conditions most frequently evaluated (hence characterized). Though we try not to become too focused on any one set of conditions, if the characterization routine is "tuned" to any single weak wash solvent, it would be 90% water and 10% ACN. Pairing up the 15 uL needle and the 1 uL sample loop is highly desirable as the internal diameters (ID) of the two are much more closely matched than for the 30 uL needle and the 1 uL sample loop. This reduces mixing and dispersion of the sample (and air gaps). Your aspiration speed is conservatively, but appropriately, low for good metering through the small ID of the 15 uL needle. Air gaps are not as critical in your configuration, because the small needle and sample loop have the effect of "elongating" the air gaps and making them less likely to be dissolved. You should be fine with the automatic (4 uL) air gaps.

    The upcoming release of ACQUITY drivers, v1.40 is going to allow larger full loop Overfill Factors. This corrects a small defect that existed which impacted the very small (1 uL and 2 uL) sample loops. Prior to this correction, the maximum custom Overfill Factor was 4X. However, the automatic Overfill Factor was, actually, 5.8X for the 1 uL sample loop. So, the best possible optimization of Overfill Factor for the 1 uL sample loop was "automatic". The new drivers will allow Overfill Factors up to 25X and will protect the system against "over drawing" when larger loops are installed.

    I must tell you that 0.1% RSD area precision for full loop injections with a 1 uL sample loop is about as good as I have seen or can, personally, achieve. However, there is a little "trick" that might squeeze out slightly better performance. The software (ACQUITY drivers and Empower) will allow you to perform a "full loop" injection utilizing the Partial Loop with Needle Overfill (PLNO) injection mode. To do this, simply create a PLNO version of your current method. Then, in the Sample Set or Sample List, request an injection volume that is X times larger than your, measured, sample loop volume. The system will, very happily, push that volume through the sample loop and make the injection. For example, if your 1 uL sample loop actually measures 1.4 uL, then request an injection volume of 14 uL in the Sample Set. This will, effectively, create a 10X overfill. The excess sample will be pushed into the needle and be flushed during the next needle wash.

    Sorry I didn't have much to add,


  • Hello,

    One thing that works well with the smaller injection volumes is to change the sampling syringe to the 50uL syringe from the standard 100uL sampling syringe. I have noticed even better area RSD's.

    Best Regards,

    M. Jones

  • Excellent information. Thanks.

    I suspect that the system which prompted this discussion is located in our QC Lab!.

    I dont think we have heard back from our field engineer on the problem, so i'll be able to pretend i just had an idea of what the problem is :-)

  • Greetings Lusby,

    Glad I could offer some little bit of useful advice.