API analysis on UPLC

<p><span>Hello UPLC Community,</span></p><p><span>I am thinking about a method transfer for an active pharmaceutical ingredient (API) from a 1100 HPLC to UPLC. The HPLC method runs (~30mins, isocratic) on a classical ODS Hypersil column (250x4). The UV chromatogram shows many related compounds (known and unknown) and I expect them to shift in retention time when changing to the new BEH C18 material.</span></p><p><span>Has anybody here done such a critical transfer? What are your experiences?</span></p>


  • lizh


    We will see what everyone else's comments are, but try the Waters Selectivity chart (you can download from the eCommunity) and if you are worried about Relative RT you could try and find a column with a closer match such as tone he HSS options rather than BEH.


  • AJA

    As Liz said, try and match, as best you can, your current column and an ACQUITY column from the selectivity chart. Following that use a systematic method transfer strategy (use the ACQUITY columns calculator to help) to move your method over. There are a number of documents on the web site that describe this process.

    HPLC to UPLC Migration: Taking the Guesswork out of Transfer and Development
    Transfer and Subsequent Redevelopment of a USP-Related Substance HPLC Method to the ACQUITY UPLC System

    Transfer of the USP Human Insulin Related Compounds HPLC Method to the ACQUITY UPLC System

    I also have seen that there are a number of web based learning available (check the web site)

    Method Transfer from HP to PC - Web-based

    As lab personnel make the transition from High Performance Liquid Chromatography (HP) to Ultra Performance Liquid Chromatography (PC) there are several things which must be considered. In this live on-line session, we will discuss transferring and optimizing methods as one makes this transition. Learn More (audiovisual presentation)

    I have done quite a number of these type of transfers, you will get RT differences and you will get relative RT shifts, but, you will gain separating power and end up with a shorter better method in the end.

  • rajnil


    I agree with responce given by LIZ and AJA. Follow waters column selectivity chart for selection of right Acquity column. Pls go through presentation given in Waters web site. It might help you.



  • lizh

    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.