tailing problem part 2

Hello again.At last i am able to write.Thank you very much for interesting my problem.My compund is i think basic because it has amide group.HPLC method parameters are:Hypersil ODS (250x4,6 mm, 5 mcn)370 nm UV1 ml/min flow40 C column temperature10 mcl inj.volumeMP: 780 water/220 ACNSolvent : 250 water/750 meOHFirst i tried as a MP water/MeOH with different ratios at HPLC.With 350 MeOH/650 water i had a peak at 8.min.Tailing was ok.Then i began to try with UPLC with BEH C18 100 x 2,1 mm, 1,7 mcn.After lots of trying i decided to MP( and also solvent) as 800 Meoh/200 water, 0,25 flow and 30 C column temperature.RT is 0,8. Inj. volume was 2 mcl.Also i tried 1 mcl but tailing diidn`t changed.(Partial loop with needle over fill i chose) As i said before i tried some additives but it didn`t work.? have not the oppurtunity to try buffers yet. And by the way i didnt use ACQUITY UPLC Columns Calculator i have no idea if we have it so i must ask my friends from R&D. ? attached chromatograms both from HPLC and UPLC.i think that`s all i can tell.Thank you very much for your helps im waiting your comments.This friday i am going to a holiday for 2 weeks :) i need it so much. When i returned i will try your suggestions.Thank you very very much.Best RegardsHandan

Answers

  • The ACQUITY UPLC Columns Calculator is on the PC that is connected to your UPLC system. It was installed as part of the ACQUITY UPLC system installation. Look for a small calculator icon on your desktop or Start > Programs > Waters > ACQUITY UPLC Columns Calculator.

    Or, just download it from here: http://www3.waters.com/pdfs/acquity_columns_calculator_standalone_V1.1.1.exe

    Efficiency-wise, the HPLC column that you are using does not match the UPLC column you have selected. A better match would be a 2.1 x 100 mm UPLC column. Also, you may be overloading your UPLC column slightly, giving rise to tailing. A 10 uL injection on a 4.6 x 250 mm HPLC column corresponds to a 0.4 uL injection on a 2.1 x 50 mm UPLC column or a 0.8 uL injection on a 2.1 x 100 mm UPLC column.

    My suggestion would be to find/install the ACQUITY UPLC Columns Calculator and try what it suggests. It will suggest a longer UPLC column than what you have based upon the length to particle size (l/dp) ratios of the different columns. As I described above, the best match is a 100 mm length UPLC column and a 0.8 uL injection. Keep everything else the same: same mobile phase; same sample & concentration; same injection solvent; same temperature; same everything. Just change what the calculator tells you to change.

    You should not see chemical tailing of your peak on any of the BEH UPLC chemistries.

    I will let other comment on injection mode(s) and strong/weak needle wash solvents as this may also influence peak shape.

    Good luck and keep us posted.

    --Doug

  • I think you are using wrong compositions of mobile phase & diluent, as your mobile is 78% aqueous where diluent is 25%? & you can inject 2uL in PLNO its ok bcuz normally ACQUITY comes with 10uL loop with 30u injection, still you didn't tell me your compound name, it is weakly basic or strong? anyway if it is basic it is recommended to run mobile at lower pH for better protonation, at your mobile phase (water:acn) pH may be around 6.5 & without buffer it cant be stable.... just follow following UPLC conditions:

    Mobile phase: 75% aqueous (add 50% conce. H3PO4 drop wise in 500mL water to adjust pH 3.5)

    25% ACN

    Diluents: 50% MeOH

    Inj. vol: 2uL (sample conce. may be from 0.1 - 0.05mg/ml)

    Weak Wash: 20%MeOH, Strong Wash: 60%MeOH

    this is just one analyte sample so you can use 2.1x50 column if you have... at 40C temp.

    can I ask wavelength 370nm?, bcuz at this WL response is very low... anyhow you try above first

    ~~ Salman

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