University of Southampton


School of Chemistry - Mass Spectrometry


Interpretation of spectra - Atmospheric Pressure Ionisation (API)

Electrospray - Positive Ion

Method of choice for molecules that possess a basic site, i.e. a position to protonate, or preformed cations e.g. quaternary ammonium compounds.

Ions observed:
(M + H)+ => (M+ 1)
(M + NH4)+ => (M + 18)
(M + Na)+ => (M + 23)
(M + K)+ => (M + 39)
M+ => for quaternary ammonium species - very intense signal.

If you see two ions separated by 5 amu then it suggests you have (M + NH4)+ and (M + Na)+. Look for accompanying (M + H)+ and (M + K)+. Similarly if you suspect (M + Na)+ look for (M + K)+ at 16 Da higher than (M + Na)+ etc.

If you see ions at much higher mass than you expect check for multimers e.g. (2M + H)+, (2M + NH4)+, (2M + Na)+, (3M + H)+ etc. Generally multimers are observed if the sample is too concentrated, though some molecules 'self assemble' to give these higher mass species. (You can get 'mixed assemblies') Acetonitrile and methanol adducts (+ 41 & +32 Da respectively) are also sometimes observed.

Electrospray - Negative Ion

For molecules that possess an acidic site i.e. a position to deprotonate.

(M - H)- => (M - 1)-

Deprotonation can be aided by addition of a trace of base, NH4OH, TEA, TPA. Formic acid and TFA adducts are often observed:
(M + HCOOH - H)- => (M + 45)-
(M + TFA - H)- => (M + 113)-

Multiply Charged Molecules (Electrospray Only)

Electrospray can produce ions of the type (M+nH)n+ or (M-nH)- This capability allows molecules of mass greater than the mass range of the spectrometer to be analysed since the mass to charge ratio brings the peaks onto the range of the mass analyser.

For the molecules that produce these multiple charge envelopes, acquire the data in continuum format. This allows the data to be post processed to provide a molecular ion on a real mass scale. This also simplifies determination of the charge states.

N.B. Addition of ~ 0.1 to 1% HCOOH (or TFA) for positive ion or 0.1 to 1% NH4OH, TEA (TPA excellent for oligonucleotides) for -ve ion will aid ionisation and is necessary to multiply charged species.


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