Experimental evidence for zwitterions

If I have a diprotic acid that is +1 positively charged in its fully protonated state, I can figure out the apparent equilibrium constants by titration with base. The net charge will be neutral after losing one proton, and -1 after losing the second proton.


Examples would be the hydronium ion or the amino acid glycine at a pH < 2.


How could you determine whether the single-protonated state is a zwitterion (glycine in neutral aqueous solution) or a molecule without charged functional groups (glycine in neutral polar aprotic solvent, or water at neutral pH)?


Is it necessary to do an experiment, or is there a simple rule to figure out whether a zwitterion is expected?




That is a very difficult problem worth a PhD project in physical chemistry. Intially, I thought one could try capillary electrophoresis at different pHs and if the analyte travels with the electrosmotic flow marker at a certain pH, one can say a zwitterion has existed (or a neutral form) by inference. However, you query is about *distinguishing* the neutral state and the zwitterionic state. One has to think about spectroscopic techniques that rely on dipole moments. Since you are interested in aqueous phase, gas phase microwave spectroscopy of amino-acids is out of question (there are papers on it). Since zwitterion ions can possess large dipole moments as compared to their neutral counterparts, two remaining ones are infrared and photoelectron spectroscopy.


Zwitterion formation in hydrated amino acid, dipole bound anions: How many water molecules are required? J. Chem. Phys. 119, 10696 (2003); <https://doi.org/10.1063/1.1620501>



> 
> We utilize the facts that zwitterions possess very large dipole
> moments, and that excess electrons can bind to strong dipole fields to
> form dipole bound anions, which in turn display distinctive and
> recognizible photoelectron spectral signatures. The appearance of
> dipole-bound photoelectron spectra of hydrated amino acid anions,
> beginning at a given hydration number, thus signals the onset of
> greatly enhanced dipole moments there and, **by implication, of
> zwitterion formation**.
> 
> 
> 


IR would be finiky with water, but there are reports that water bound to zwitterions has a different vibrational frequency


Stability and IR Spectroscopy of Zwitterionic Form of β-Alanine in Water Clusters, J. Phys. Chem. B 2019, 123, 20, 4392–4399, <https://doi.org/10.1021/acs.jpcb.9b00654>



> 
> We perform an experimental and computational study on the number of
> water molecules needed for zwitterion formation of β-alanine. Our
> density functional theory investigation reveals that a minimum of five
> water molecules are required to form and stabilize the zwitterion. A
> characteristic connecting water molecule located between the COO– and
> NH3+ groups is found to enhance the stability. This water molecule is
> also involved in a characteristic infrared active vibration at ≈1560
> cm–1, which is slightly shifted with the number of surrounding water
> molecules and is located in a spectral region outside of water
> vibrations.
> 
> 
> 


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