PLNO vs. FL

<p><span>Hi everybody,</span></p><p></p><p><span>here is another interesting question on Acquity injection mode.</span></p><p><span>Have you ever tried to inject same solutions, same injection volume, same method in PLNO vs. FL mode?</span></p><p><span>I</span></p><p><span> did this and results told me that a difference around 30%in area appeared in PLNO (5µL injection volume on 10µL sample loop) respect to a 5µL injection on a 5µL sample loop.</span></p><p><span>PLNO clearly showed around 30% less sensitivity with respect to FL.</span></p><p><span>Is it normal? have you an idea about this?</span></p><p></p><p><span>Both sample sets contained a calibration curve and samples and data showed perfect correlation on standards samples (R2 >0.999) and very good accuracy (both with weighted and unweighted curves) in both injection modes.</span></p><p></p><p><span>Thank you in advance</span></p>

Answers

  • What is the volume of your 5 µL loop? This volume can be higher than 5 µL. This volume has to determined after changing a loop. You can find it in the logs.

    good luck!

  • Yes it is normal, this is not a matter of sensitivity, in PLNO sample injection is from 20 - 75% where in full-loop SM injects 100% of sample, in your case if SM injects 70% of your sample so your area is 30% less in PLNO,but in any case FL is more precise than PLNO as

    < 0.3%RSD with full-loop in standard 10u loop where

    < 1.0%RSD with PLNO within 20-75% of loop volume with 5u, 10u loops

    as you see in PLNO sample ranges from 10-75% (1u to 50u loop) so their should be big difference in area when you switch from 10u to 5u loops

    ~~ Salman

  • Hi Salman,

    thank you for your answer.

    I suspect this was the reason because area shows same less area at all levels of concentration: if it was an adsorption of poor soluble compound on needle this will affect more low level samples than higher level ones.

    Now I am checking the real volume of 5µL loop. The 10µl has a 11.2µL volume. I will let you know if all things fit.

    Bye

    Paolo

  • Hi Salman,

    I made some controls and these are the results.

    1) You are right about different precision of PLNO and FL modes

    2 ) What do you mean with "sample injection is from 20 - 75%": when I set 5µL both in PLNO and FL injector must inject 5µL (apart from intrinsic error) (Maybe I misunderstand what you are saying)

    3) I checked the "real" loop volumes: 11.2µ for 10µL loop and 6.5µL for 5µL

    I think that this is the real reason, confimed as I said by high reproducbility of "less sensitivity" (always around 30%): a simply difference in loop volumes.

    The reason why I have not noticed that is that commonly I perform start-up of the system in the morning comprising needle and loop check and all this made not possible to see real volumes in the log list. Do you know if it is possible to see this hidden log list?

    Thank you

    Paolo

  • Hi,

    As you could see in the Salman Q&A the reason is loop volume!

    Thank you for your troubleshoot.

    Bye

    Paolo

  • All:

    This will be a long explanation, so maybe read over the weekend! I ahve attached two items I hope will be useful.

    The Sample Manager has 3 modes and because they work to load the loop differently they deliver different volumes and must be treated as if you were using three types of injector. They should not be used in the same calibrations curve. Each mode is linear and reproducible, but indeed they will give different areas even for the same method and sample. This is because of the unfairness of the universe and physics of laminar flow. I always tell people to embrace this concept. It is very odd if you ahve used say a Alliance or HPLC system as usually they have only one injection method so the discussion does not arise.

    In general Full loop mode gives greatest area, followed by PLNO and last PLPA.

    There is no best mode, only the one that is best for the method and the application.

    What the last person meant was that you are always limited in partial loop mode to injecting only 10 to 50% of the loop being used for Partial Loop Uses Pressure assist mode or 10% to 75% in Partial Loop Uses Needle Overfill PLNO. So in a 10 uL loop this is 1 to 5 uL in PLPA and 1 to 7.5 uL in PLNO.

    But there are other practical chromatography issues to consider - as another person learned on a thread earlier - usually 10 uL is too large an injection for a 2.1 x 50 mm as it is too close to overlaoding the column, especially if your diluent is stronger than initial gradient conditions.

  • Many thanks for all your explanations.

    I greatly aprreciate everybody answers.

    Bye

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