Molecular weight determination is performed by High Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS). Depending on the sample complexity, sampling is either performed by flow-injection-analysis (FIA) or by preceding chromatographic separation via HPLC.
The analyte is dissolve in an appropriate solvent containing a volatile acidic additive (e.g. formic acid). This mixture is infused into the electron spray ion source of the mass spectrometer. Here, droplets are formed which contain charged, quasi molecular ions. These droplets are further dried by a heated stream of nitrogen. The remaining, fine mist of particulates is transferred into the ion optics of the mass spectrometer and accelerated towards the analyser part. We employ a time-of-flight (TOF) analyser where the charged analyte is transferred into a drift-tube. Determination of the mass to charge ratio (m/z), thus the weight, is derived from the ions flight time.
The polarity of the ion optics can be switched between positive and negative ion mode, allowing for the detection of either positively or negatively charged entities. Routinely, the positive ion mode is engaged and allows for the detection of most analytes.
The charge state of the analyte is dependent upon the presence of hetero atomic moieties where protonation by the acidic additive might occur. Peptide which are comprised of several peptide bonds are usually found to carry more than one charge. This leads to charge to mass ratios corresponding to a fraction of the intact molecular weight. These multiply charged ions are deconvoluted and used to verify the singly charged, quasi molecular ion of the analyte.