Chromatography Forum

If I read the following recipe of a Mobile Phase:

Acetonitrile (99.9%), acetic acid (100%) and distilled water (100:1:899)

how I have to prepare it?

A.- Mixing 100mL of acetonitril + 1mL of acetic acid and distilled water e.q.f 1000mL
B.- Mixing 1mL of acetic acid + 999mL of distilled water and programme 10% of acetonitril and 90% of the mixture on my HPLC

A or B will change a lot the results????

Thank you!!!

That recipe is ambiguous, for just the reason you cite. The "most correct" (which is an oxymoron!) would be:

C. Add 100 ml of ACN + 1 mL of acetic acid + 899 mL/water.

However, with mobile phase prep, correctness is less critical than consistency. Hopefully the original method references an explicit procedure that was used to prep the mobile phase.

Thanks Tom,

But unfortunately the original method references doesn't explicit procedure used to prep the mobile phase

Perera V et al. Caffeine and paraxanthine HPLC assay for CYP1A2 phenotype assessment using saliva and plasma. Biomed Chromatogr. 2010 Oct;24(10):1136-44.

I'm trying to quantificate caffeine and paraxanthine... but I couldn't yet!!!

That recipe is ambiguous, for just the reason you cite. The "most correct" (which is an oxymoron!) would be:

C. Add 100 ml of ACN + 1 mL of acetic acid + 899 mL/water.

However, with mobile phase prep, correctness is less critical than consistency. Hopefully the original method references an explicit procedure that was used to prep the mobile phase.

Agree with Tom, precise un-ambiguous instructions are the desired target, but each writer knows what "he" intends, but may be confusing to a new reader. In my first publication, a reviewer asked if my filtration was by vacuum or by gravity, and of course since I had done the procedure myself so many times, I "knew" what I intended, but was not clear to reviewer.

I like "C" as well, as any mix done by an HPLC pump is by volumes of each stream.

Even more confusing can be when mobile phases containing organics are taken to a pH. I prefer adding modifier (like acid) by volume or weight, more reproducible in my thinking.

If you do the technically correct procedure, "C" above, you will have to make up that mobile phase often, as the ACN will evaporate faster than your other components. On the other hand, the ACN will keep the microbe count in your mobile phase down.

On the other hand, if you do "B" (assuming your LC pump is well-calibrated with consistent mixing), the 100% ACN will be good for a very long time, the 0.1% acetic acid will be good reasonably long, and you will always be delivering 10.0% organic to the column.

I'm trying to quantificate caffeine and paraxanthine... but I couldn't yet!!!

So try it both ways and see which one gives results that meet system suitability. And then put *that* procedure in as an addendum to the method for future reference.

With it reading 100:1:899, I would measure 100mL of ACN, I would measure out 899 water, and then add 1 mL of acetic acid. I would never qs to a final volume with either organic or aqueous as that will give you a different percentage depending on which you start with.

Also I would change B to:
Mixing 1 mL of acetic acid + 899 mL of distilled water and program 10% ACN and 90% of the mixture on the HPLC. That would just be me being picky, the absolute concentration of the acetic acid probably doesn't make that much difference, it looks to just be there to set the acidity of the mobile phase.

Paul

Can anyone tell me a way of volumetrically measuring 899 ml of water using the equipment in a average HPLC lab ?

Peter

Peter, someone, somewhere, is religiously filling a 1L measuring cylinder to the 900mL mark, and removing one mL with a pipette. Utterly pointless, but I'd bet you a large bar of chocolate that someone is really doing it.

Peter, someone, somewhere, is religiously filling a 1L measuring cylinder to the 900mL mark, and removing one mL with a pipette. Utterly pointless, but I'd bet you a large bar of chocolate that someone is really doing it.

I'll take that bet -- if you will give me odds of about a million to one !

Acetonitrile (99.9%), acetic acid (100%) and distilled water (100:1:899)

I really can not understand the insistency of some chromatographers to complete the final volume to round to 1000 or 100, especially when it causes insignificant figures like 899.

Acetonitrile (99.9%), acetic acid (100%) and distilled water (100:1:899)

I really can not understand the insistency of some chromatographers to complete the final volume to round to 1000 or 100, especially when it causes insignificant figures like 899.

And I cannot understand how deeply rooted is the assumption that everything has to be in volumes. Nobody would think twice about a recipe containing 899 grams of one thing and 1 gram of another. For all we know, given the somewhat sketchy details provided, the mobile phase composition could have been given as masses.

Peter

In all honesty if I had to absolutely keep that ratio, I would just measure 1000 mL water then add 1110uL of acetic acid to it. Then I would do the 100:900 of ACN to aqueous. I was more pointing out that I would never dilute to volume with one half or the other.

I agree with you Peter that the desire for the 1000 is obscene. I do like using nice big fat numbers for the major components, but my mobile phases often have a total of 1000 for the organic+aqueous with some random bit left over. I have no issue making 1001 if I just want to add a mL of an acid to set pH. Also though I think lmh would still win the single chocolate bar, what would you do if you won and ended up with a million large chocolate bars?

The reason for volume is partially because if we do the ratios in volume it can allow the option to do pump mixing of the two components on the HPLC. The pumps operate by volume, not by weight. That said, I hate pump mixing as it can make each HPLC slightly different.

Some of our assays utilize mobile phase made up by using automatic pure water station to deliver 4.0 liters water into a bottle (or use a full 4-liter bottle of HPLC water) then add small amount of acetic acid to that. Yes, we know the final volume is more than 4.00000 liters. Our robustness testing used this component at +/- 25% of the target acidity, with no differences.

Are the odds still 899 to 1 on the chocolate bet?

There is an AOCS (Oil Chemists) test procedure for glycerin in soap where one is directed to add 91 ml CHCl3 from a buret, the thinking is that the 10 grams of soap will produce about 9 ml of fatty acids from the acidity in the aqueous phase when all is taken to 1000 ml. After the CHCl3 separates and settles to the bottom, and aliquot of the 900 ml (supposedly) aqueous portion is taken for analysis/reaction with periodic acid. I changed that for our company to 88 - 102 ml CHCl3 from a graduated cylinder back in the 1970s, after the match shows that 25 ml aliquot/898 ml and 25 ml aliquot/902 ml were statistically equivalent for the accuracy required.

In all honesty if I had to absolutely keep that ratio, I would just measure 1000 mL water then add 1110uL of acetic acid to it. Then I would do the 100:900 of ACN to aqueous. I was more pointing out that I would never dilute to volume with one half or the other.

I agree with you Peter that the desire for the 1000 is obscene That was prepcolumn, not me. I have no problem with 1000, or any other number that can be measured in sensible units. I do like using nice big fat numbers for the major components, but my mobile phases often have a total of 1000 for the organic+aqueous with some random bit left over. I have no issue making 1001 if I just want to add a mL of an acid to set pH. Also though I think lmh would still win the single chocolate bar, what would you do if you won and ended up with a million large chocolate bars I think that lmh would win as well. that's why I wanted the generous odds. Of course the burden of proof is on him - what would be a reasonable time limit for him to come up with the evidence ?? ?

The reason for volume is partially because if we do the ratios in volume it can allow the option to do pump mixing of the two components on the HPLC Good point. The pumps operate by volume, not by weight. That said, I hate pump mixing as it can make each HPLC slightly different.

Peter