Method/Column for Neutral and large Polar compounds

<p>I HAVE POSTED FOLLOWING MESSAGE FOR METHOD TRANSFER/DEVELOPMENT BUT STILL I DID'NT GET ANY HELP.... I REQUEST WATERS CO.WORKERS & UPLC USERS TO ASSIST ME</p><p>THANKS,,,..........>>>></p><p style="height: 8pt"/><p>Jul 25, 2009 6:27 AM in response to: Doug McCabe </p><p>Re: Polar Compounds using UPLC BEH Shield RP18 Column in Highly Aqueous Mobile Phases</p><p></p><hr/><p style="height: 8pt"/><p>Dear Azimi </p><p></p><p>You both posted an interesting topic, i am in method transfer/development team and we are working to transfer/develop our two methods in highly aqueous mobile phase, but still i am little confused that i have to use either UPLC BEH Shield or UPLC HSS T3? Because these two HPLC methods are on Supelco ABZ+ & 65-75% aqueous in mobile. My samples are combination of 3-analytes & 2-analytes & details are</p><p></p><p style="height: 8pt"/><p>Sample #1 two neutral compounds & one polar</p><p>Sample #2 two polar but large in molecules (400-600 mw) </p><p style="height: 8pt"/><p style="height: 8pt"/><p>I also request Waters co-workers to sugest me which column is good for these two samples, i read HSS T3 is best for polar but not recommended for large molecules like 400-600 mw.</p><p></p><p style="height: 8pt"/><p>Mr. Azimi, i was reviewing your paper posted in this thread, polar embedded alkyl group columns are Supelco ABZ+ as well BEH Shield, according to this paper i can use Shield column for my samples?</p><p></p><p>I hope i will get satisfactory answer from Waters.</p><p style="height: 8pt"/><p>Thanks.</p><p style="height: 8pt"/><p></p><p>Sophia</p>


  • dougm

    If you are looking for a Waters <2 um UPLC column that will give you the best chance of having the most similar selectivity to your old ABZ+ HPLC column, I suggest the BEH Shield chemistry.

    If you are looking for a Waters <2 um UPLC column that will provide you with the maximum retentivity for all analytes (polar & non-polar, including the relatively low MWs that you mention (400-600)) I suggest the the HSS T3 chemistry.

    Could you please let me know where you heard that the HSS T3 chemistry is not recommended for the small molecule MWs that you mention? I've not heard that before.

    Both chemistries I suggest are compatible with mobile phases you describe. In fact, mobile phases with 65-75% aqueous content are compatible with all C18 chemistries.

    If you've additional questions, please let us know.


  • Its very nice to see Doug again, Dear Sophia, I am agree with Doug you can try BEH Shield for your methods, best chance to get best results.

    If you study more about chemistry of analytes you will be closure to get best method, if your samples are aromatic & phenolic then BEH Sheild could be the best.

    We have done too much work on BEH Shield, Xbridge & Xterra columns & no doubt, they are very good for polar as well as neutral compounds.

    MWs 400-600 does not mean too big, it can possible with HSS T3.

    I want to share one of my application for your knowledge, it was on BEH Shield for simultaneous assay of neutral & acidic analytes (check example #2), may be it will help you.

    ~~ Salman

  • Doug and Salman,

    Thanks for comments, we tried our samples for many days on BEH C18, but unhappy with results, after your suggestions, today we unpacked our BEH Shield RP18 (2.1x100mm) & started work on these samples.

    We dont have HSS T3 right now, but we are ordering ,we had received 3 columns; BEH C18, BEH C8 & BEH Shield with our UPLC package.

    Tomorow i will confirm my results, if any questions i will post you.

    Salman, we also reviewing your application certainly it will guide us.


  • Thanks Doug & Salman,

    its work, initially we are getting good separation for both samples with BEH Shield, now we are doing repeatability study & adjusting best parameters for our samples....,

    Doug i mean large MWs ( <300 MWs).

    but thanks again for your suggestions.

  • dougm


    With the BEH and HSS UPLC columns you can easily separate molecules with MWs up to 60000. In SEC, for example, with 120A pore size packing materials, molecules with MWs of up to 100K can be separated without issue.

    Good luck with the BEH Shield chemistry.


  • Dear Doug,

    we are getting very low response in Roxithromycin samples by using BEH columns even in high concentrations, before we worked on following HPLC column

    Purosphere Star RP-18e 5u (4.0x250)

    i have checked UPLC column selectivity chart, according to this HSS C18 can be used, but we don't have this right now. But why BEH C18 is not giving good response or absorbance for Roxithromycin


  • dougm


    I cannot explain why an analyte's UV response would be influenced by stationary phase, all things being equal. It simply does not make sense and consequently I suspect something else.

    Could you please do a couple things to help me figure this out?

    1) Would it be possible to post your HPLC method and your UPLC method? How did you scale the separations from HPLC to UPLC? Are the peak areas different as well?

    2) Also, is it possible to simply take your HPLC column and run the HPLC separation on your UPLC system? Do not change the mobile phase(s), standard(s) or wash solution(s) that you are presently using with the BEH C18 UPLC column. Assuming that you are using the same mobile phase(s) in both the HPLC and UPLC methods, simply run your HPLC method with your HPLC column on the UPLC system and report your findings.

    My goal here is to differentiate the system from the column in terms of its influence on peak response.


  • Hi Doug,

    Thanks for your answer, here are the HPLC conditions:

    Mobile phase: Buffer pH 6.8 (K2HPO4)MeCNH2O {21012718} mixture, then adjust pH 4.1

    Sample diluent: Buffer pH 6.8:ACN:H2O

    Flow= 1mL, WL= 220nm

    Column= RP-18e Purosphere Star, 5u (4x250)

    roxithromycin conce. 1mg/ml, (20u injection)

    UPLC conditions that we applying...

    Flow= 0.85mL, Column= BEH 2.1x50, Column temp=45`C

    10 p/s, SNW=70%ACN, WNW=20%ACN

    roxithromycin conce. 1.2mg/mL

    Inj. vol= 0.7u (PLNO)

    I am attaching one UPLC chromatogram of roxithromycin, I cant load HPLC picture bcuz it was done on old HPLC system.

    in this picture you will find at 1.2mg/ml conce. UV response is low, where we did same before & response was not that low i.e higher than this & peak shape also better.....

    we tried FL mode & even 7u (PLNO) but peak broadening (sample overload)....

    I hope you suggest me better solution otherwise we will order HSS C18 (acc. to UPLC calculator).


  • dougm

    I have a couple questions and a comment.

    First, phosphate is not a buffer at pH 4.1. I know you are likely using it because you are operating at 220 nm, but phosphate's pKa's are nowhere near 4.1 so you have little to no pH control.

    Second, have you been able to run your HPLC column and HPLC method on the UPLC system as I suggested previously? I suggest you do that and report back with your findings. Even if the HPLC column does not really fit inside the UPLC system column oven, this will tell us if your method and system are working.

    Third, how did you come up with the UPLC method? Did you use the ACQUITY UPLC Columns Calculator (not the Selectivity Chart) to help you transfer HPLC to a UPLC method? As best I can tell from your method, the ACQUITY UPLC method suggested is attached.

    If you cannot find your HPLC to UPLC Calculator just go to the Overview Tab here in the eCommunity, scroll down the page a bit and find the 'UPLC Basics' section. The HPLC to UPLC calculator is there for you to download and install. It is quite straightforward.

  • I am suspecting two things, 1- pH of your diluent & 2- pH of mobile phase

    I never suggest you to ammend something in your method I just suggest you two things

    1- As you are saying your sample diluent pH is 6.8 did you check pH of diluent before sample prep. if not pls assure pH must be 6.8 as in method.

    2- I think you used pre-mixed mobile phase of Buffer+MeCN, try Buffer pH 4.1(A1) & MeCN (B1) in composition of 65:35. By this you can adjust mobile phase composition slightly up & down for better separation & response, may be you can good peak at 62:38 ...!

    In my view you have bad peak because of pH, roxithromycin is not highly polar, its nearly neutral compound & this type of compounds are mostly pH depended slight change may affect seperation.

    I hope you got my point.

    ~ Salman

  • Dear ALL,

    I am really very thankful, for your comments & suggestions, it works, yes pH stability was the main reason behind bad separation.... what we did continuously in 12hrs work, we didn't change anything in our old HPLC method, but we monitored pH of mobile & diluent, as described in HPLC method, only run two mobile solvents separately A1=Buffer pH 64% & MeCN 36%... and we succeed pls check files attached...

    Dear Doug, we have UPLC calculator, but solvent composition is limited only for MeOH, MeCN & Water... that's the reason pressure & flow was not same as observe during run, so we satisfied at 1.0mL/m flow (10100psi) & have better results at 1.5u injection.

    At the end I am happy with my work, check my results, now will do equivalency test...



  • Great work,

    I’m still tending to think in your HPLC method, you worked on with phosphate2 (di-K) buffer, pKa=7.2, and adjusted pH of mobile phase to 4.1, where diluent pH was 6.8....? i am not sure exactly the pKa of roxithromycin, by structure it looks neutral or weakly base... should pKa from 6-9

    My view here that selecting right buffered mobile phase is very important in LC for optimal peak shape, good response & consistent retention.