Chromatography Forum

Hi,
I have two queries regarding HESI MS injections which I am seeking assistance for.

what is an appropriate background solvent when running a HESI MS loop injection of a small mass compound (300-1500da). The compound is soluble in MeOH .

I have an unknown peak causing contamination at 550. Any suggessions what this could be?? I have tried different solvents- MeOH, ACN from different groups and have this peak at 10^7 ion count when i inject the solvents.

thanks
p

http://www.lc-ms.nl/contaminants.htm

indicates m/z 550 is from rubber tip of disposable syringe plunger.

A good general "mobile phase" for loop injections is water/acetonitrile (1:1, v/v) containing 0.1% formic or trifluoroacetic acid. If this particular analyte is soluble in MeOH, but not MeCN, then water/MeOH instead of water/MeCN.

What is the "H" in HESI ??

I was wondering too: http://www.thermoscientific.de/eThermo/ ... e_6667.pdf

I guess my ABSciex API3200 has always had a Heated ESI probe, is it really a new idea?

As for the contamination coming from the rubber tips of disposable syringes, you really don't realize just how much contamination you can get from things like this until you start doing LC/MS, or how much contamination is present in your Ultra Pure solvents

http://www.lc-ms.nl/contaminants.htm

indicates m/z 550 is from rubber tip of disposable syringe plunger.

A good general "mobile phase" for loop injections is water/acetonitrile (1:1, v/v) containing 0.1% formic or trifluoroacetic acid. If this particular analyte is soluble in MeOH, but not MeCN, then water/MeOH instead of water/MeCN.

What is the "H" in HESI ??

Thanks for your help. The H in HESI is heated!. Would be the same mobile phase be used for both positive and negative ion mode?

Could the sample (dissolved in MeOH) be run in 100% MeCN? I was instructed to run it in 100% ACN, but from my understanding the sample has to be ionised in solution using a volatile buffer.

Thanks heaps for your input.
paddy

You can certainly mix solvents (methanol with MeCN etc) as long as they are fully miscible; organic solvents have lower surface tension than water and generally will improve sensitivity.

As above, 0.1% formic acid willassist protonation

You can certainly mix solvents (methanol with MeCN etc) as long as they are fully miscible; organic solvents have lower surface tension than water and generally will improve sensitivity.

As above, 0.1% formic acid willassist protonation

Thanks for you help.
paddy

Hi again,

Would I need to change the mobile phase (cont 0.1% formic acid) when I go from positive to the negative ionisation mode?

Also is the formic acid (0.1%) in both the organic and aqueous bottles or only in the aqueous?

thanks
paddy

(1) I'm lazy, and add the formic acid only to the aqueous bit, not to the organic. But I'm lazy...

(2) No, you don't need to change the solvent to work in negative mode. It's true that formic acid should protonate, so it should favour positive ions, and given a weak acid analyte, should favour the neutral species, but electrospray is an electrochemical process, and it will ionise things at a pH where they really shouldn't be ionised (for example, although sugars don't ionise particularly well, they do give a signal in positive mode at neutral pH, and sugars aren't noted as particularly strong bases! The amount of protonated sugar in a neutral solution should be veerrrrry small).

In fact, since electrospray is electrochemical, introducing a lot of salt can affect ionisation efficiency because you are changing the conductivity of the solution in the spray tip. I use this as a feeble hand-wavy excuse for the fact that sometimes when I've tried to change pH to improve sensitivity, I've actually decreased sensitivity.

Mostly I add the formic acid in hopes it will improve my chromatography rather than for the benefit of the mass spec.

(3) Yes, if your sample dissolves nicely in acetonitrile there's nothing stopping you from injecting in pure acetonitrile; it'll probably work. For a long time this worried me intensely, as I couldn't see how an acid/base electrochemical process can work in a solvent that doesn't do anything acid-basey. I still don't really understand.

(1) I'm lazy, and add the formic acid only to the aqueous bit, not to the organic. But I'm lazy...

(2) No, you don't need to change the solvent to work in negative mode. It's true that formic acid should protonate, so it should favour positive ions, and given a weak acid analyte, should favour the neutral species, but electrospray is an electrochemical process, and it will ionise things at a pH where they really shouldn't be ionised (for example, although sugars don't ionise particularly well, they do give a signal in positive mode at neutral pH, and sugars aren't noted as particularly strong bases! The amount of protonated sugar in a neutral solution should be veerrrrry small).

In fact, since electrospray is electrochemical, introducing a lot of salt can affect ionisation efficiency because you are changing the conductivity of the solution in the spray tip. I use this as a feeble hand-wavy excuse for the fact that sometimes when I've tried to change pH to improve sensitivity, I've actually decreased sensitivity.

Mostly I add the formic acid in hopes it will improve my chromatography rather than for the benefit of the mass spec.

(3) Yes, if your sample dissolves nicely in acetonitrile there's nothing stopping you from injecting in pure acetonitrile; it'll probably work. For a long time this worried me intensely, as I couldn't see how an acid/base electrochemical process can work in a solvent that doesn't do anything acid-basey. I still don't really understand.

I will usually add the Formic Acid to both so I don't dilute it when using a gradient elution, but it doesn't seem to make that much difference unless you are eluting in 100% organic.

I think the reason you still get ionization from ESI is that the probe is imparting quite a charge to the liquid, and as the liquid droplet evaporates that charge becomes intensely concentrated and as the last of the solvent evaporates that charge is transferred to the analyte molecules that were contained within the droplet. The electrostatic charge then causes the ionization to take place even if the analyte would not normally ionize in solution. That is why neutrals can be analyzed by ESI, not just Acid and Base compounds.

That is just how I read one explanation somewhere, so don't take it as absolute truth

(1) I'm lazy, and add the formic acid only to the aqueous bit, not to the organic. But I'm lazy...

(2) No, you don't need to change the solvent to work in negative mode. It's true that formic acid should protonate, so it should favour positive ions, and given a weak acid analyte, should favour the neutral species, but electrospray is an electrochemical process, and it will ionise things at a pH where they really shouldn't be ionised (for example, although sugars don't ionise particularly well, they do give a signal in positive mode at neutral pH, and sugars aren't noted as particularly strong bases! The amount of protonated sugar in a neutral solution should be veerrrrry small).

In fact, since electrospray is electrochemical, introducing a lot of salt can affect ionisation efficiency because you are changing the conductivity of the solution in the spray tip. I use this as a feeble hand-wavy excuse for the fact that sometimes when I've tried to change pH to improve sensitivity, I've actually decreased sensitivity.

Mostly I add the formic acid in hopes it will improve my chromatography rather than for the benefit of the mass spec.

(3) Yes, if your sample dissolves nicely in acetonitrile there's nothing stopping you from injecting in pure acetonitrile; it'll probably work. For a long time this worried me intensely, as I couldn't see how an acid/base electrochemical process can work in a solvent that doesn't do anything acid-basey. I still don't really understand.

I will usually add the Formic Acid to both so I don't dilute it when using a gradient elution, but it doesn't seem to make that much difference unless you are eluting in 100% organic.

I think the reason you still get ionization from ESI is that the probe is imparting quite a charge to the liquid, and as the liquid droplet evaporates that charge becomes intensely concentrated and as the last of the solvent evaporates that charge is transferred to the analyte molecules that were contained within the droplet. The electrostatic charge then causes the ionization to take place even if the analyte would not normally ionize in solution. That is why neutrals can be analyzed by ESI, not just Acid and Base compounds.

That is just how I read one explanation somewhere, so don't take it as absolute truth

Thank you both. That sounds about what I am getting. I have been running samples (basic and neutral) in 100% MeCN (as instructed to me) as Mobile phase and still getting results.

I had to run a sample containing small amount of DMSO and now cant get rid of it. I tried washing it with MeCN/water 50:50 for a couple of hrs but it still persists. Is there any way to get rid of it?

hi,

I have a new problem today (its just not working for me this week!). Phthalate contamination peak at 391 up to 3 x 10^7 ion count. I know how i got it, while rinsing the bottle before transferring fresh mobile phase, I capped it and shook the bottle containing MeOH.. silly and costly mistake...

Any one has any idea how I can get rid off it from the HESI MS??

Phthalates: they will gradually disappear. Unless they're seriously stopping you from getting any decent results, I'd just change to fresh solvent, run the instrument a lot, and wait for them to fade down to an acceptable level. No mass-spec-aware person will criticise you too heavily for a spectrum with 391 in it (in fact Orbitrap people often use it as an unofficial lock-mass).

DMSO. Horrible stuff. It hangs around because it's so non-volatile. I don't mind standards that came from a DMSO solution, but recommend that the original solution is kept nice and concentrated so it can be diluted drastically to produce working standards with not too much DMSO. My only suggestion for getting rid of it is to run the instrument for a bit with a nice high LC flow to wash anything through, and try as high a source-temperature as you dare to get as much to evaporate as you can. Again, it will gradually disappear, but it tends to make in-source non-covalent cluster ions and hang around irritating.

Phthalates: they will gradually disappear. Unless they're seriously stopping you from getting any decent results, I'd just change to fresh solvent, run the instrument a lot, and wait for them to fade down to an acceptable level. No mass-spec-aware person will criticise you too heavily for a spectrum with 391 in it (in fact Orbitrap people often use it as an unofficial lock-mass).

DMSO. Horrible stuff. It hangs around because it's so non-volatile. I don't mind standards that came from a DMSO solution, but recommend that the original solution is kept nice and concentrated so it can be diluted drastically to produce working standards with not too much DMSO. My only suggestion for getting rid of it is to run the instrument for a bit with a nice high LC flow to wash anything through, and try as high a source-temperature as you dare to get as much to evaporate as you can. Again, it will gradually disappear, but it tends to make in-source non-covalent cluster ions and hang around irritating.

Thanks..will do

hi,

I have a new problem today (its just not working for me this week!). Phthalate contamination peak at 391 up to 3 x 10^7 ion count. I know how i got it, while rinsing the bottle before transferring fresh mobile phase, I capped it and shook the bottle containing MeOH.. silly and costly mistake...

Any one has any idea how I can get rid off it from the HESI MS??

Best way to clean the mobile phase bottles for LCMS is using a muffle furnace at about 550C. It will burn out all the organics in a few hours and you have a nice shiny clean bottle after. Just make sure you are using Pyrex, Kimax or Schott bottles so they don't soften at those temps.