Chromatography Forum

hi..
i see a lot of retention time variation when i use different preparations of mobile phase on RP-HPLC. the mobile phase composition is same for all of my runs..
but is there a specific way to measure or mix mobile phase with acetonitrile and water as a mix, and then addition of TFA as ionpairing agent.

how i do it is take measured water say 800 ml - add Acetonitrile say 200 ml and then add TFA in required amount. Should i be adding acetonitrile first and then water? should these impact my volume measurements?

i wish to discuss the compressibility and volume measurement accuracy for acetonitrile and water. also what is an appropriate way to mix these - are they easily miscible?
when they are mixed the solution becomes cold. are these miscible at this low tempeartaure or should i leave the mix at room temperature and then use it.

thanx!!

The real issue is to make the mobile phase up CONSISTENTLY. There are too many ways to make phase up, the instructions must be definitive, and exact. For example, ACN and water are completely miscible. But adding 800ml water and 200ml ACN will be somewhat different volume than if you added 800ml water to a 1000ml volumetric flask, then mixed and diluted to volume with water. And adding 200ml ACN then mixing and diluting to volume with water would be different still. With modern quaternary-pump HPLCs, these mix your solvents on a volume to volume basis, so that's how we detail ours. We detail how many ml water get mixed with how many ml ACN; if we were pre-mixing (rare for us) we would not worry what the total volume ends up.

For your TFA, you could add the same concentration of TFA to both the ACN and the water, then even if you tweak the ACN:water, your TFA would remain same concentration.

If you have a buffer that calls for a certain pH, better if you adjust the pH of the aquous phase, then mix in the organic, as pH of aqueous-organic can be tricky, and the key is to do this reproducibly. Actually, I like best figuring out how much acid or whatever is required to adjust to my desired pH, then adding that amount by volume or weight, and making the instructions read that way.

And, if you pre-mix your mobile phase, over time (due to selective evaporation) your water:organic ratio will change, causing retention time variability.

Also, you already know this, we always membrane-filter any mobile ohases that contain a dissolved solid, even though the solvents are pre-filtered HPLC grade.

Water and acetonitrile are 100% miscible, and TFA (at any reasonable level!) is 100% soluble in those mixtures. There *is* a volume change (decrease) on mixing. The order of addition is not important. The temperature decrease is normal (in contrast to methanol/water for which the temperature increases).

If you have a non-robust method (where separation varies strongly with mobile phase composition), you have to be *very* careful with your mobile phase prep. In general, you will get more reproducible results if you measure by weight rather than by volume.

It is the mixing that causes the drop in temperature, if it wouldn´t mix it wouldn´t get cold. This system needs energy to mix, that is why the temp. goes down. For the chromatography you should make sure that the mobile phase´s temp. doesn´t change.
One does not need to know the final volume in this setting, one should just measure the volumes separatly then mix them together. One can not add one solvent to a graduated cylinder and then top it off with the second solvent.
On the compressibility: I can imagine that it could be something to be considered if one mixes two solvent on the pressure side, but since one usually does these things by trial and error the compressibility may be ignored, even though it could be responsible for not knowing what the solvent ratio is.

The best decription of what is happening when mixing solvents can be found in Veronika Meyer's "Tricks and Pittfalls". I have copied this comparison in my "HPLC Troubleshooting", of which I can send a copy to anyone interested.

In principle, you should always measure the solvents separately, and then mix them. Due to solvent contraction, the volume is always smaller than the sum of the volumes that you put together. This can create some headaches for a classical high-pressure mixing system: while you get the exact same solvent composition from the high-pressure mixer and manually mixed solvent, the retention time with the premixed solvent is slightly lower, since the flow rate is lower for the on-line high-pressure mixed solvent. This is not the case for a binary UPLC system, which compensates for this with a sequential piston design.

thank you people i get my answers!

uwe, could you please send this to me? thanks!

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