Switching from ESI + to -

[shemesh199]

Quick question, I have a method setup on ESI +, I am interested to investigate ESI - , so when switching from ESI+ to ESI- does the original mobile phase need to be changed, or can that be kept the same, and what will happen to signal intensity. My Mobile phase conditions are
A) 10mM Aqueous Ammonium Acetate pH 3.0, and B) ACN.

Since the parent ion will presumably change to MW-1, is a quick infusion necessary with a MS2 full scan, or would a product ion scan of MW-1 be optimal as a quicker approach to method development.

Is signal intensity typical lost for negative mode? I know that most methods are using ESI+ , what is the key advantage of using + over -.

I have some strange analyte behavior in + mode so I am searching - mode to see if the trend follows.

Thanks Kindly

[Jetjamnong]

Negative ion analysis,Analytes that are rather acidic are generally analyzed in negative ion mode. Thesample molecule (acid) loses a proton and transfers it to a base (pH >7) in solution andbecomes negatively charged. Therefore, for high sensitivity negative ion analysis, it isimportant to have a base in solution. Ammonia and other volatile bases yield bestresults.
For negative ionization, analytes with functional groups that deprotonate readily, such
as carboxylic or sulfonic acids, show the best sensitivity. Analytes that are polar but
contain no acid groups show less sensitivity.
Charge exchange is another mechanism that can occur in negative ion mode. It results
in an [M] ion instead of an [M – H] ion.

[shemesh199]

Thanks for the reply. Can my original mobile phase that I used for ESI + be used for ESI - , or must I change composition?

[Jetjamnong]

Yes, you can use the same composition of this mobile phase in positive and negative mode. by using a base mobile phase would provide you a good results with negative ion mode.
Try to run some compoud(s) that can ionized well on positive and negative ion mode with the same mobile phase and then compare the intensity ratios, such as common drug, Paracetamol and Ibuprofen.

[Camisotro]

Quick question, I have a method setup on ESI +, I am interested to investigate ESI - , so when switching from ESI+ to ESI- does the original mobile phase need to be changed, or can that be kept the same, and what will happen to signal intensity. My Mobile phase conditions are
A) 10mM Aqueous Ammonium Acetate pH 3.0, and B) ACN.

Since the parent ion will presumably change to MW-1, is a quick infusion necessary with a MS2 full scan, or would a product ion scan of MW-1 be optimal as a quicker approach to method development.

Is signal intensity typical lost for negative mode? I know that most methods are using ESI+ , what is the key advantage of using + over -.

I have some strange analyte behavior in + mode so I am searching - mode to see if the trend follows.

Thanks Kindly

Funny things can happen in the electrospray ionization mechanism, and "wrong-way-round" ionization has been widely reported - you can have an acidic mobile phase and still get [M - H]- negative ions. If you don't see it when you try at pH 3, by all means try again around pH 7.

In positive mode the acidity in the highly charged spray is significantly greater than the original mobile phase. I would thus assume the opposite can happen in negative mode.

I think there are just a lot more analytes that respond well in positive mode, and even ones that respond better in negative are often still highly visible in positive. In the charged spray environment, many carbonyl groups can be easily protonated that would not normally do so in solution. But in negative mode it's still a challenge to pry off a not-very-acidic proton. Of course in negative mode there are also adducts that are sometimes the best visible ion.

[Kenn]

Quick question, I have a method setup on ESI +, I am interested to investigate ESI - , so when switching from ESI+ to ESI- does the original mobile phase need to be changed, or can that be kept the same, and what will happen to signal intensity. My Mobile phase conditions are
A) 10mM Aqueous Ammonium Acetate pH 3.0, and B) ACN.

Since the parent ion will presumably change to MW-1, is a quick infusion necessary with a MS2 full scan, or would a product ion scan of MW-1 be optimal as a quicker approach to method development.

Is signal intensity typical lost for negative mode? I know that most methods are using ESI+ , what is the key advantage of using + over -.

I have some strange analyte behavior in + mode so I am searching - mode to see if the trend follows.

Thanks Kindly

If the original mobile phase works for both independently it will work fine, what you need is some separation. Some may say you can do it with less but I'd want at least and preferably more than a minute between peaks looked at in different modes. The sensitivity depends on your ion but you can bank on losing some of it. You play it too close and get a retention shift for your positive ion and it pops out after a switch to negative or visa versa and that data is gone, you get nothing. I would prefer to have two methods one positive and one negative and tell the instrument to change methods and run the second one after the first. If there are any slight differences in your gradient that maximizes one peak versus the other you can do it. I would include at least a half dozen priming injections in the method with the new mode before submitting the definitive sequence.

[shemesh199]

Hi Kenn,

Interestingly I did keep the same mobile phase conditions, A) 10mM Aqueous Ammonium Acetate pH 3.0, and B) ACN. When I switched over to Negative mode with a MS2 Full Scan covering the range, I did not find my molecular ion, and seemed to have lost all sensitivity. I am guessing the mobile phase needs to be changed.

More interestingly it seems that the pKa of my compound is 3.2. I am not sure why the previous authors have adjusted pH to 3.0, my understanding was to shy away from a mobile phase pH near your pKa because 50% of the analyte will be uncharged.

However, when I switched to 5.0 I remember losing signal?

What do you recommend.

Many Thanks

[Kenn]

To clarify, are you looking at only one compound or transition and wondering whether to run it in positive or negative mode? I thought you were switching modes inside your method to look at 2 different ions, one positive and one negative. If that's not the case and your compound ionizes in either mode then yes, infuse it and look at it in both for your answer.

[shemesh199]

This is my compound.

[shemesh199]

Actually, this problem is a bit complicated, I have not been obtaining very linear curves under positive mode,no matter what is done, that is why I would like to investigate the negative mode, to see if the peak abundances for a standard curve have the same trend. This compound is a bit troublesome, as it is a larger molecule.

[Kenn]

Hi Kenn,

Interestingly I did keep the same mobile phase conditions, A) 10mM Aqueous Ammonium Acetate pH 3.0, and B) ACN. When I switched over to Negative mode with a MS2 Full Scan covering the range, I did not find my molecular ion, and seemed to have lost all sensitivity. I am guessing the mobile phase needs to be changed.

Or I'm guessing it doesn't ionize in negative mode? As far as running in scan mode somebody else may be able to help you a little better, like 99.9% of the work I do is MRM. I infuse and scan to find my parent and products and go straight to MRM.

More interestingly it seems that the pKa of my compound is 3.2. I am not sure why the previous authors have adjusted pH to 3.0, my understanding was to shy away from a mobile phase pH near your pKa because 50% of the analyte will be uncharged.

I would certainly look at the compound without the use of the buffer to see if it is imperative. I have eliminated a buffer from an analysis more times than I can count. What does it look like with A channel being 0.1% formic and B with ACN?

What do you recommend.

I'm not sure what it is you are trying to do so this is difficult. If you are trying to analyze a compound and it's sensitivity is best in positive mode why are you interested in negative.

[Kenn]

Actually, this problem is a bit complicated, I have not been obtaining very linear curves under positive mode,no matter what is done, that is why I would like to investigate the negative mode, to see if the peak abundances for a standard curve have the same trend. This compound is a bit troublesome, as it is a larger molecule.

I would try and stay in positive mode and fix the reason your curves are not linear. I don't think your compound is naturally doing that. Can you simplify your method for the sake of achieving linearity and then add to it later and see where it comes apart? There are so many other factors involved that I could be here all night. You see a sodium adduct in Q1 when you infuse it as well as your parent?

If you can see it in negative mode I would not be at all surprised if you do see that same trend.

[shemesh199]

The ionization for this compound is pretty interesting. I am not sure what exactly is going on in the ionization source. It seems that the ionization is variable from injection to injection, I have spent weeks on the method but has some issues, most all other compounds work perfectly with exceptional linearity. I am thinking one of the reasons has to do with my reconstitution technique, after spiking the standards I dry down and reconstitute in pure ACN, as opposed to starting mobile phase conditions 60:40, 10 mM aqueous ammonium acetate with the pH adjusted to 3.0 using formic acid, and ACN.

This is performed because ICG has some instability in water, and usually split peaks are observed if reconc is in starting conditions. I am not so sure if something is precipitating out in the injector. Another thing is this compound requires very high fragmentor voltage, outside of the normal range for the instrument Agilent 6410 QQQ ~220V and high CE ~38.

Here you can see exactly what is going on, I have attached a picture with the calibration and peak abundances, additionally this is definately a method issue because the instrument is in perfect shape and other assays run beautifully.

Many Thanks

[Kenn]

The peaks are fronting a little, are you overloading the column. What does the peak look like if you put it in 50:50:0.1 MEOH:H2O:Formic?

Either shoot less or reduce concentration. I would not shoot 100% ACN in an isocratic method of approx 50% aqueous unless I absolutely had to and it would be small injection volume like 10 µL. I have often gone to methanol aqueous sample composition to stabilize a compound in conjunction with ACN mobile phase and it works fine. I'm not sure why you are drying your standards. If you prepare a set at roughly 1/10the the concentration I see in your chromatogram in the 50:50:0.1 I mentioned what does that peak look like then? You look like you have plenty of sensitivity. Are you certain you need that buffered mobile phase? What is your column and flow?

[shemesh199]

Interesting idea, I am not sure that will work since I have tried to reconstitute in 60:40 , buffer with ACN, but I will definately give it a shot! Usually when there is water present I have observed some peak splitting, some previous authors published an article stating they had to reconc in pure ACN due to decomposition in water. Maybe using MeOH as the organic modifier will have better results, as well as an increased pH, so far I have been using a buffer at pH 3.0. I will try some alterate approach. I will keep you posted and many thanks.