Chromatography Forum

Hi everyone..

I´m new to HPLC and trying to prepare a mobile phase for creatinine quantification in mice..

I need some help with my calculations of the relative amounts of the different components

MilliQ (96.6) : Acetonitrile (3) : Trifluoroacid (0.1)

in a total of 500 ml

Thanks

Well

Do you mean 96.9 instead of 96.6?

In that case 484.5 ml MilliQ, 15 ml Acetonitril, 0.5 ml acetonitril. What column do you use since creatinine has a low affionity for C18 HPLC columns.

PeterS

I've got my pedantic 5 minutes right now : 484.5 mL H20 + 15 mL ACN + 0.5 mL TFA will NOT give you a total volume of 500 mL.
BTW how do you measure 484.5 mL H2O volumetrically?

To the OP: Where did you get that mobile phase recipe from? Isn't there a decent decription on how to prepare it? Are you in a regulated environment?
Basically, this sounds like water/ACN 97:3 with 0.1% TFA. In absence of a decent recipe I'd mix 970 mL water with 30mL ACN and add 1mL TFA.
And yes, I wouldn't care if it's 970 or 969mL .
And yes, I'd be aware that the one who developed that method might have prepared his mobile phase in a different way. If the method's so sensitive (aka not robust) that you get severe chromatographic differences with mobile phases of 97:3 or 96:3 or 98:3 composition, it's not worth using it...