Tailing after transfer to UPLC

<p>Hello Forum,</p><p>we have transfered an isocratic method for the measurement of S-adenosylmethionin from a Waters 2795/Quattro Micro to our UPLC/Premier. The column we use on both systems is a 100x2.1mm (3.5um) Symmetry Shield with the same type guard column. The HPLC method was transfered 1:1 since we arent using a "UPLC" column. MS parameters were slightly adapted to the Premier. However, on UPLC/Premier we get a clear peak tailing where there was none on the 2795/Quattro Micro System.</p><p>Any ideas were or how the tailing could be caused?</p>


  • Hello:

    Can you send a few more details, the weak and strong wash solvents and the injection method in particular and the injection volume. With this information I think our chemists can make some relevant suggestions.

    Many thanks


    Edited by: Elizabeth Hodgdon on Nov 5, 2008

  • Hello Sean,

    Possible source# 1 bad connection

    What fittings are you using on the UPLC? Are you using the "finger tight" (kit 700003139 high pressure reusable fitting set with tool) or the original "fixed" type of fittings supplied with the instrument? UPLC fitting are not compatible with the HPLC end fittings on your Symmetry Column. I recomend that you use either the 700003139 - reseated with a fresh ferrel or a standard finger tight fitting normally used for HPLC. It is important that you use the proper UPLC fittings at any point where they intersect with the instrument itself (injection valve, PDA bulkhead for instance) but you must use HPLC fittings appropriate for your Symmetry column at the inlet and the outlet of the column.

    Possible source #2 wash solvent incompatibility if running Pressure Assist Mode.

    1. To eliminate this try the mobile phase as weak wash solvent or solvent A if running a gradient

    2. or use PLUNO mode - no wash solvent is injected along with the sample

    Possible source #3 sample diluents not fully compatible with the sample or mobile phase.

    1. This issue sometimes arises when the sample components are "on the edge" of solubility in the sample diluents or there are other solvent incompatibilities present - such as injecting a bolus of acetonitrile onto a column running at low ACN concentrations. The HPLC system has a great deal more system volume between the injector and the head of the column to allow for dilution of the sample into the mobile phase before it hits the head of the column; the UPLC system has very little volume between the injector and the head of the column. You are putting essentially undiluted sample onto the head of the column and in some instances this is where a solvent incompatibly which has always existed, but never caused any issue, comes to light.

    2. Evaluate compatability of your sample diluents with the beginning of your gradient (in your case the mobile phase composition used in isocratic mode) and change the sample diluents if possible

    3. If it is not possible to change the sample diluents then use the weak wash solvent as a intermediary - If you select Pressure Assist mode tsome of the weak wash solvent will be injected along with the sample, it is possible to select the composition of the weak wash solvent so that it improves compatibility of the sample diluents with the beginning of the gradient/mobile phase.

    Message was edited by: Patty-SystemsMarketingLaboratory
    There are speaker notes on these slides - select "show comments" mode in attached pdf

  • Hello,

    I strongly agree with Patty's recommendations and troubleshooting plan. I would like to add a different perspective that is more associated to data collection rather than chemical incompatibilities or poor fitting connections. Typically I would follow all the recommendations Patty suggested first, but if you have issues locating the problem here are some things to look at. I had some experience in the past when transferring HPLC methods to UPLC instrumentation at the same time I was transferring methods from Quattro Micro to Quattro Premier. Currently you have two types of instrument changes taking place that you must be aware of.

    1) The HPLC to UPLC instrument transfer while keeping a HPLC column: Again, my suggestion is to be aware of the comments from Patty and Liz. Injection modes, fitting connections and solvent incompatibilities are probably the most common sources of a tailing problem. If you were running a gradient, then I would add dwell volume, residence time, injection volume and peak elution effects as other possible areas to explore. However you are running isocratically, so it makes it a little easier.

    2) The transfer from Quattro Micro to Quattro Premier: When I performed this transfer, I was on Masslynx version 4.0 for Quattro Micro/HPLC/UV and Masslynx 4.1 for Quattro Premier/UPLC/UV. I had to be very careful of the data collection rates when using both software because they had different defaults and subtle parameter differences. The electronics of both instruments are very different also. The Premier is a much faster instrument! When I created the same method for my application on Masslynx 4.1for Quattro Premier/UPLC/UV guided by the parameters written in Masslynx 4.0 for Quattro Micro/HPLC/UV, the UV data rates were too slow (2pts/sec) and the data filtering was far too high (1). This caused my UV peaks to be distorted and tailing at times. When I noticed and adjusted the problem to fix the UV peak, I quickly realized that I needed to take note of the MS method and scan rate being used. Most of the time it can work the same way on MS as in UV with my transferred gradient methods, but when transferring isocratic methods you may observe something happening that may have been masked by a slower instrument and slower scan rates. One thing you may find in your case was that the scan rate was slower in Masslynx 4.0 and hence the peak shape was not as accurately defined if you only had 6-7 or less scans. When using the Premier, the defaults and capabilities (and Waters training) may have caused you to increase the scan rate of the MS method in Masslynx 4.1 and hence you are getting a better defined peak because you getting the proper number of scans across the peak. I do use the term "better defined" in a sense that on some occasions the result may not be in your favor, hence tailing that was never seen before.

    Hopefully the guidance from the others alleviated your problem. If not, I hope this will be of some help to you.

    Best of Luck,

    Michael Jones

    Senior Applications Chemist

    Pharmaceutical Business Operations

    Waters Corporation