Chromatography Forum

I found a literature method for analysis of a pharmaceutical that I want to try and validate. The B Mobile Phase is listed as 41:18:10 MeOH:ACN:THF.

This may be a "stupid" question, but the total of these does not add up to 100. For 2L of Mobile Phase I end up with 820 mL of MeOH, 360 mL ACN, and 200 mL of THF, which is actually only 1380 mL of solution. If I back calculate percentages from there my MP is actually 59.4:26.1:14.5 MeOH:ACN:THF.

Am I making this more complicated than it actually is? If I list the solution as 41:18:10 MeOH:ACN:THF with the volumes are my quality/regulatory teams going to flag this?

Thanks in advance!

Hi ,
I could understand that this ratios are not percentage so the sum of them don't must be 100 % , for each 100 mL or don't have to be 1000 mL for each 1L ,
Just please note that not all internet published analytical methods are valid and about 20 % of what you can find on internet may not be applicable just don't be disappointed if it happened

Remember, volumes may shrink (because of intercalation) and are not additive! 1L of Methanol and 1L of Water do not give you 2L of mobile phase. Thus, solvents are weighed or volumes listed BEFORE mixing.

Hi Rachel,

The numbers 41:18:10 is the relative volumes between the solvents. They are noted this way, and not 59.4:26.1:14.5, to keep the numbers without decimals (it is quite difficult to add 59.4 mL of a solvent, or doing fast math in the lab with such a number).

Hello

I understand that for solvents mix this method of presenting ratio can be right (volumes ratio) but I dont believe that mobile phase cannot be optimized to present it without decimal places.
Why can't it be optimised and presented as 60:26:14 % to avoid confusion? I can't believe that mobile phase compostion must be that tight (59.4 rather than 60%)

If it is method from USP or other magic book I wouldn't be surprised

Regards

Tomasz Kubowicz

I've already optimized the method (chromatography looks beautiful), I'm just curious as to the write up.

Is it more logical to leave it at the 41:18:10 as was listed in the original method, or switch to the 59:26:15 for clarity. I'm leaving it as v/v/v. Thanks!

Hello

I understand that for solvents mix this method of presenting ratio can be right (volumes ratio) but I dont believe that mobile phase cannot be optimized to present it without decimal places.
Why can't it be optimised and presented as 60:26:14 % to avoid confusion? I can't believe that mobile phase compostion must be that tight (59.4 rather than 60%)

If it is method from USP or other magic book I wouldn't be surprised

Regards

Tomasz Kubowicz

Yes very unusual ratio. Ive never seen ACN AND MEOh as %B, usually one or other. THF is nasty and should be avoided on HPLC if possible. Ive seen leak sensors turn black and ruined with THF. I dont know what your analyzing, but you could try 100% ACN as B and probably get similar chromatography.
%B in reverse phase is typically straight ACN or with 0.1% formic or TFA.
Agreed. USP is not the holy grail or practical method

you could try 100% ACN as B and probably get similar chromatography.

The catch is that "similar" does not mean "identical". THF provides selectivity that is different from that of ACN or MeOH and cannot be duplicated by simply blending ACN + MeOH.

USP is not the holy grail or practical method

No, but USP methods *are* considered to be validated for regulatory purposes.