Chromatography Forum

Hello to all,

Can anybody explain the ionisation technique of Zwitter ion, which ionisation is preferred, and in which mode(Positive or Negative).
What happens actually when zwitter ions are ionised?

Dear Ramasarma,

The ionisation of zwitter ion depends on the pH.
What is your pH?, and what is your zwitter ion?

good luck
Safwat H. Ali

If this is for quantitation, I recommend you try to optimize the signal for your analyte in both positive and negative modes. Follow that with injection of standards under the HPLC conditions you intend to use. If one mode is dramatically better than the other at this point, go with that mode. If performance is similar in both, prepare some matrix spikes and evaluate based on signal-to-noise, interference, and matrix suppression or enhancement.

safwatali,

My compound is 5-amino salcilycacid, Pka of that compound is 2-5.

How the pH will effect the ionisation of zwitter ion? Have you done any zwitter ions? Let me know regarding this issues.

Thankyou MG,

I have done exactly same as you guided, my system is micromass quattro ultima, i am getting in positive aswellas in negative mode same response. But the response is very low it is around one lac, for one microgram aqueous analyte. Is there any way to improve the response?

Ramasarma, you may have already done the following, but if not it will help:

Infuse a solvent standard of the compound into your ion source, run a SIM experiment on your compound's M+H or M-H ion, and ramp any up-front lens voltage that affects the fragmentation of your analyte. I am not familiar with Micromass instruments so I don't know the specific names for these lenses.

Also change your ion source temperature, gas flows, and electrospray voltage (needle voltage) and find optimum values, but keep in mind that you must infuse at the flow rate you intend to use for LC, for these optimums to be meaningful. If you cannot infuse at high flows, optimize the temperatures, gas flows, and needle voltage by using flow-injection instead.

Once you have optimized signal for the M+H or M-H ion, you can move on to MS/MS. While infusing your standard, perform a product ion scan and ramp your collision energy (in Q2). Pick several of the most intense product ions you see. Set-up an MRM experiment with those product ions and repeat the ramping of collision energy. Then you will have the optimum collision energy for each transition. Once you have these, you can also optimize any other parameters affecting MS/MS fragmentation (such as collision gas pressure). At this point, you can run your LC/MS/MS experiment with matrix spike, to see which transition suffers the least from matrix background or interference. Loss of water (18) and loss of CO2 (44) will probably be too noisy to be useful, and I usually eliminate them immediately.