Post Air Gaps in PLNO Injections

<p>Without writing an essay on why.... i want to reduce the amount of contact time between my analyte and the mobile phase during the PLNO injection sequence. </p><p></p><p>Due to the small sample loop and slow draw rate, the sample is in contact with the mobile phase for some time (relatively) between being pushed into the sample loop and when valve spins to push the sample onto the column. I was thinking of using a post air gap to isolate the sample from the mobile phase. </p><p></p><p>My questions regarding this are: </p><p></p><ul><li>will this adversely affect precision </li><li>what volume air gap should i use </li><li>any other suggestions to minimise pre-injection mobile phase contact </li></ul><p></p><p>Full loop mode is not an option as the injection volume is less than 1 uL. </p><p></p><p>Thanks in advance for any help</p>


  • The default air gap in the PLNO mode is 1 uL and it is a pre sample air gap (in PLNO only the leading edge of the sample has any chance to contact solvent). Further, the sample would not be in contact with the mobile phase during the draw cycle (without air gaps it would contact the weak wash solvent in the needle), the only time it would contact mobile phase is as it enters the loop. If you wish, you may increase the pre air gap (through the advanced settings) in the method editor.

    To answer your questions:

    will this adversely affect precision - no, precision will remain unaffected in this mode by increasing the air gap what volume air gap should i use - The default value is one, try two any other suggestions to minimize pre-injection mobile phase contact - in this mode, the only sample contact with mobile phase is as the sample enters the loop, there is no way to prevent this contact. During the sample transfer from the vial, through the needle to just before the sample would enter the loop, the only contact would be with the weak wash and there is an air gap to minimize this contact.

  • You are correct AJA, the mobile phase only contacts the analyte as it is pushed into the sample loop (as i stated in original post). This is the contact i am trying to minimise.

    I was not refering to the pre sample air gap & disagree that there is no way to prevent contact with mobile phase during the injection....there is an option to put in a post sample air gap in PLNO mode (its just the default setting is zero) which presumably could prevent contact with mobile phase until the sample is pushed out of the sample loop.

    I am concerned if the software can cope with this configuration, i.e. when it loads the sample loop will it allow for the post sample air gap, or will i have to add the air gap to my injection volume?.

    Has anyone tried this, or is a POST air gap a big no-no in PLNO injection mode?.


  • Liz Wrote....

    "Thank you everyone, but also we would like to understand more from your statement...

    "I want to reduce the amount of contact time between my analyte and the mobile phase during the PLNO injection sequence." We have a few questions! If this does not apply let us know, but we wanted to know what was worrying you.

    (1) Does this mean that the sample precipitates in the mobile phase?

    (2)Does it mean that the sample reacts with the mobile phase? if it does, why won't it react on column?

    (3) Is the time that the sample is being loaded into the sample loop and the injection is being synchronized long compared to the separation time?

    There is only a small (1 uL) lead air gap injected into the sample loop in a PLUNO injection. Even if it were larger you still have the problem that air gaps dissolve (Henry's Law) at system pressure and whatever "bad" things happen to the sample when it comes in contact with the mobile phase begin as soon as the inject valve goes to INJECT.

    (4)If a partial loop injection is desired and air gaps are needed, try partial loop with pressure assist.

    a. it's faster

    b. it's got air gaps

    c. you can choose a weak wash solvent that does not "react" with your sample.

    Hope this applies.

    Best regards, Liz RDE and Evaluation."

    Thanks Liz,

    (1) Yes the sample can precipitate in initial conditions for the method. It doesnt seem to be a super quick precipitation, so contact in the mobile phase stream from the injector to the column where it gets focused anyway doesn't seem to be a problem. I just want to shield the analyte during the actual sample loop loading, which is slow at the draw rates we are using compared to the time it takes to get from injector to column head.

    (2) The analyte doesn't react with the mobile phase thankfully.

    (3) No, its a relatively long method for a UPLC

    You make a good point about the air gaps dissolving at system pressure, but i don't think this is an issue, as the sample is focused on the column head before it gets a chance to precipitate between the injector and the column.

    (4) I had considered using this mode, but am concerned about the precision. So i was going to introduce a post air gap in the PLNO mode, but don't know how the system/software would cope with this (see previous post).

    Thanks again for your reply Liz.

  • Lusby

    The post sample air gap does not put are air gap where you think it does. The sequence goes something like this:

    weak wash

    air gap

    pre sample volume (13 uL I think)

    sample volume

    post sample volume (2 or 3 uL I think)

    Post air gap (which by default is about the needle volume in size, so changing this value does nothing)

    So what gets pushed into the loop is only sample, and yes it does contact the mobile phase in the loop.

    Even if you could put a gap there, as Liz pointed out, as soon as the loop is a system pressure, the air gap is dissolved.

    Its not a no-no, it just doesnt do anything in this mode.


  • Yes, the post sample buffer of 1-2 uL seems to ruin the plan & I presume the post sample buffer volume is not configurable.

    I will give the pressure assisted mode a go, am i being optimistic wanting precision of <1% RSD in this mode.

  • There is no such constraint in the method and an post air gap is configurable in the "Advanced tab". It will add time that's all. In PLPA it will be added.

    You should absolutely get better than 1% with PLPA. If you do not, often its due to the syringe speed being too high which can happen if your sample diluent is viscocity is high (DMSO would be an example).


  • Thanks,

    the Full Loop mode works well, the %RSD is routinely <0.1%. Unfortunately both the Partial Loop modes give %RSD >2.0%. This leaves me with the problem that this is a validated method with an injection volume of 0.4 uL.

    Can custom sized sample loops be ordered? (i doubt it - no provision in ICOP software).

    Re-validating using full loop mode is an option ;-( but the problem there is the variation in the loop volumes, i checked 3 of our instruments and the 2 uL loops vary between 2.4 and 3.2 uL making validation 'interesting' . What are the specifications for sample loops, if i do have to validate using a 1 or 2 uL loop, what volume range can i expect?.

    Any suggestions gratefully received.

  • If Full loop works - likely there is no instrument issue. However, if the sample can precipitate in initial conditions for the method (it begs the question of why this was chosen?) you have to fix this one way or another and in PLPA mode you are far more sensitized to this as the sample diluent and washes are injected together, if you get bad %RSD's clearly the issue is impacting perfromance and it has to be addressed.

    You say Full loop is OK and so in this case I think you may have to go to PLNO mode. In PLNO mode the loop is rinsed with extra sample diluent. But we shall see. Please list starting gradient mobile, sample diluent and your two needlewash conditions. Ideally can we see the gradient table?

    I do realize that your method is validated so that fixing initial conditions is not an option, but we may have to change the modes and the washes and use them to assist solubilizing the sample. Then we can give some better guidance. But honestly the air gaps are not going to make much difference if there is a solubility issue.


  • Hi,

    can i just say that it is only a theory that the sample is precipitating in the sample loop. This type of method is well used by us, and has only become troublesome for precision when moved to UPLC.

    The precision is poor when using PLNO and PLPA modes (for whatever reason), but is excellent in FL mode. The fact that the precision was poor (approx 1.7 %RSD) when using PLPA is evidence against the precipitation theory.

    Our metrology team have been working on this for some time, trying various advanced settings in PLNO mode without success. The only consistent cure is full loop mode. I would love find the fix, and use PLNO mode so i am grateful for any suggestions.

    Sample Matrix: 1% formic acid in water

    Strong Needle Wash: 0.1% formic Acid (200 uL)

    Weak Needle Wash: 10% acetonitrile in water (600 uL)

    Buffer Composition: 10 mM Ammonium Formate, pH 9.6

    Organic Modifier: acetonitrile

    Gradient: 10-60% over 18.5 minutes

    Flowrate: 0.15 mL/min

    Column: BEHC18, 1.0 x 100 mm

    Injection Volume: 0.4 uL

    The reason for the small injection problem is that it helps an early eluting compounds peak shape.

    The obvious fix is to move to full loop and revalidate the method, but i need to know the variation that i can expect for 1 and 2 uL loops so that this option may be assessed. Any ideas?.

    Cheers for the help

  • Thank you again for the analysis details. We do not think your choice of strong wash is appropriate if the chromatography uses a basic buffer and 10%-65% organic modifier. Can you try 70/15/15 ACN/IPA/Water as an alternate.

    Also check that your loop is installed correctly as in PLNO that can cause issues.

    Finally, what loop have you used 1 or 2 uL? It may be that if you select a smaller syringe - 50 uL that will help with sub 1 uL injections.

    Best regards,


  • Have tried the suggested needle wash. Reproducability looks good so far, %RSD = 0.4%, when previously was >1%.

    We use a 2 uL loop and 100 uL sample syringe. I did not realise the 50 uL syringe was avalaible for non Nano-UPLC sample managers.

    Not sure why the acidic needle wash was chosen, personallly i use unbuffered initial conditions for my weak wash, and unbuffered final gradient conditions for my strong wash unless carryover is suggesting something more agressive is needed.

    Thanks for all the help, its good to (hopefully) have the method working well,

    kind regards,


  • Jason:

    Well that is great news, if you are on the 2 uL loop then do not bother with the syringe and if at 0.4% RSD's now. The 50 Ul is nice to have for the 1 uL loop as the volume is so small.

    You are absolutely right, avoid salts in the needlewash, unless you need the selectivity to amnage a carryover issue with something sticky.


  • I just noticed your bit map attachment and I was wondering - is that the number of injection since you received the system and have you ahd your injector valve changed?

    Many TX for this

    Apologies, I was not very observant!


  • Hi Liz,

    Yes that is the true number of injections. That was our first UPLC (installed 2005 - replacing 3 HPLCs), it is used for screening puposes with a combination of 1 and 2 minute methods, and has the optional sample changer. The injector pod was changed when a new version of the valve was released a few years back, and again when an operator cross-threaded the samle loop. Neither was due to a failure of the valve itself.

    I wish that i could tell you how many injections were done with each valve, but the instrument is located in one of our less regulated departments, and the record keeping for maintenance isn't fantastic. As a very rough guess, i'd say that one of the valves would have done ~ 80000 injections without failing.

    kind regards,


  • Many thanks for this - good to know how things are working.

    Unless you reset the counter the injections will continue to be counted.

    Many TX again,


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