Mobile phase / LCQTOF - question

[no_peak]

I am creating a screening method on LC-QTOF-MS.
Since I don't know what chemicals to expect, I was recommended a mobile phase:
A: Water/MeOH (95/5, v/v) + 5 mM ammonium formate + 0.1% formic acid;

B: MeOH/Water (95/5) + 5 mM ammonium formate + 0.1 % formic acid

Gradient (A/B): 95/5 --30min--> 5/95

I am not 100% convinced about the organic phase with the addition of salt. In particular, I feel that the column pressure at a flow rate of 0.3 mL/min (100x2.1x1., which is about 500 bar, is too high.
Do you use a phase with this composition?

[millerk8561]

Wouldn't the point of having mixed mobile phases (5% either direction) be to enable doing a 0-100% programmed gradient without hitting 100% composition on either end?

[TylerSmith123]

Hi no_peak,

The amount of salt you're adding is so small that I wouldn't expect any precipitation when you're formulating your MP. Additionally, adding that salt will have virtually no affect on your pressure unless it's precipitating onto the column/out, so it is not the buffer's fault for your high pressures. Your high pressure is mostly likely a product of your method, mobile phase (potentially change to ACN for lower pressure), and especially column. As for Miller's comment, he's correct. The formulation of the MP is intended not to reach 100% org because that may cause precipitation of your buffers and subsequent errors/pressure increases.

TS

[Gaetan Glauser]

Previous comments gave correct explanations. You asked if we use such mobile phases (so implicitely if we use different ones): for screening/untargeted LC-high resolution MS, I usually start with only solvents and formic acid. I use ACN instead of MeOH (sharper peak shapes, lower backpressure). You will find this combination in a majority of papers in the literature.

[no_peak]

Previous comments gave correct explanations. You asked if we use such mobile phases (so implicitely if we use different ones): for screening/untargeted LC-high resolution MS, I usually start with only solvents and formic acid. I use ACN instead of MeOH (sharper peak shapes, lower backpressure). You will find this combination in a majority of papers in the literature.

thank you for your reply.
Of course, ACN seems to be a better solution.
But in my opinion, I don't like the fact that at a concentration of about 100% ACN began to disappear reference masses - I also found such a note in the guide from the manufacturer.

[TylerSmith123]

Hi No_peak,

What did you mean by this comment: "But in my opinion, I don't like the fact that at a concentration of about 100% ACN began to disappear reference masses - I also found such a note in the guide from the manufacturer" ?

What reference masses are you talking about? Additionally, for the screening method, do you have any information on the molecules of analysis? It seems strange that you would have bough a column, mobile phases, etc, without any background knowledge on your sample. Of course it could be completely proprietary information in which case I will not push more, but if you're even a little more specific with your analytes you'll receive better help from the forum's users.

[trozen]

no_peak,

My favorite is 0.1% FA in water / 0.1% FA in methanol (5/95 to 95/5). See if your column can tolerate heating to 60°C to decrease the backpressure, or try one of superficially porous columns such as Waters Cortecs (2.7 µm).

Note that in general you should expect lower sensitivity with water/ACN mobile phase; addition of salts also decreases method robustness to some extent and may cause ionization suppression (though this is very application specific).

Lastly, 30 min run time is an overkill for such a column. I would recommend 15 min maximum unless you have challenging isomer pairs.

[lmh]

Acetonitrile versus methanol: methanol will give the higher back-pressure especially in the middle of the gradient as 50% methanol is more viscous than either methanol or water. But there are a few things that ionise in methanol but do not in acetonitrile. Some things run sharper in acetonitrile. There is no perfect solvent.

Reference masses: I'm guessing the original poster means that they're running a Q-ToF that uses a second sprayer to introduce lock-masses, which Agilent call reference masses. Ionisation of the reference mass in an Agilent Q-ToF often depends on the solvent spray from the main sprayer, so it can disappear at the extremes of a gradient if the solvents aren't helping the reference mass sprayer do its job. You might be able to get the reference masses to work better at the extremes if you add ammonium formate to your reference mass recipe, but I don't know...

And yes, I'm also too lazy to bother with the ammonium bit and tend to run water + formic acid versus acetonitrile (probably without the formic acid). I probably miss some compounds.

With 1.7u particles in a 100mm column, your back-pressure seems quite believable to me. If the column is rated to 500 bar and the pump is happy at 500bar, I wouldn't worry!