Mechanistic Interpretation of the Dependence of Charge State Distributions on Analyte Concentrations in Electrospray Ionization Mass Spectrometry

Abstract

The effect of analyte concentrations upon charge state distributions in electrospray ionization mass spectrometry (ESMS) was investigated using four compounds which showed doubly charged and singly charged positive ions (gramicidin S, 4,4'-bipiperidine), multiply charged negative ions (Trypan Blue), or doubly charged and singly charged negative ions (eosin Y) in ES mass spectra. For each of the above compounds, the charge associated with analyte molecules was calculated to increase in solution with increasing concentration, yet the analyte charge state distributions in ES mass spectra were observed to monotonically shift toward lower values. In order to rationalize these observations, we introduce the ratio N/N o , the ratio of the number of excess charges on all droplets produced by electrospray (N) to the total number of analyte molecules in all droplets (N o ). For each compound, the N/N o value decreases with increasing analyte concentration. The lower N/N o value is proposed to be an underlying factor critical to explaining the desorption of a higher proportion of gas-phase ions of lower charge state at elevated analyte concentrations. We propose that the lower N/N o value is indicative of a decreased efficiency of analyte charging and an increased level of ion pairing of charged analyte molecules with available counterions. Furthermore, in comparing N/N o values and observed analyte charge state distributions as functions of increasing analyte concentration, the decreasing N/N o ratio predicts a much greater extent of shifting of analyte charge states toward lower values than was observed. This implies that the actual degree of ion pairing in droplets was considerably alleviated as compared to that indicated by the calculated N/N o values. This finding conforms to a description of the ES process wherein uneven fission of droplets occurs at the Rayleigh limit, thereby generating offspring droplets of higher charge to mass ratio, hence an augmented N/N o value.