Chromatography Forum

Dear experts,
I would really like to get your recommendations on the following issue:

Generally I do not combine the aqueous/organic components of my mobile phase manually. I put them into separate reservoirs on the HPLC and let the instrument do the combining. I do not have any gradients running at the moment.

When it comes to making up some mobile phase for dissolving standards or diluting samples I am concerned that I am not matching the mobile phase accurately. Now while this probably does not have the potential to cause a great deal of difference I am curious to know what the best technique is.

Conscious that this may be a simplistic query:oops: but

Do you:

Measure out the individual volumes in separate measuring cylinders and then combine ...

Measure out the aqueous component (say 60mL) and add the organic component to the 100mL mark...

Measure out the organic component (40mL) and add the aqueous component to the 100mL mark....

I have read many conflicting opinions on this and am keen to know what the expert practice is.

Many thanks
Martina

There is a two-page article on specifically this subject by Veronika Meyer in her "Fallstricke und Fehlerquellen der HPLC in Bildern", published by Huethig. There is an English version of the book as well, "Pittfalls and Errors of HPLC in Pictures", published by the same publisher, Huethig. I also included this subject in my HPLC Troubleshooting collection.

When an HPLC instrument makes up a mobile phase, it adds a predetermined volume of component A to a predetermined volume of component B. This is also how one shoud do this manually: you measure separately X mL A and combine it with Y mL B. The same applies if you make more complicated mixtures.

If you adhere to this principle, the mixing by the HPLC instrument and your manual mixing is done in exactly the same way, and you get the same selectivity of the separation, no matter if you do this manually or if the instrument does this for you.

When you mix methanol and acetonitrile with water, you get contraction and temperature changes. Due to this, you would get a different composition if you start with water and add the organic solvent to the 1L mark in your graduate, or if you start with the organic and add water to the 1L mark. Therefore this approach (i.e. filling the graduate to the 1L mark) is a no-no...

There remains a subtle difference between a low-pressure mixing system and a high-pressure mixing system of the classical design. If you combine the solvents before the pump, the flow rate will be whatever you have selected. If you do the same thing with a high-pressure mixing system, the composition will be the same, but the flow rate will be less due to the solvent contraction. This can create differences in retention, but not differences in selectivity compared to the low pressure mixing or proper manual mixing.

More (and pictures) in the references...

If the composition is critical, we do the measurements by weight. Modern digital balances are wonderful. For instance the mobile phase for our column QA test is "70:30 v/v" MeCN:H2O, but we make it 546:300 w/w assuming densities of 0.79 and 1.00 for acetonitrile and water. The other nice thing is you don't need any volumetric glassware; any clean bottle will do.