LC/ESI-MS determination of proteins using conventional liquid chromatography and ultrasonically assisted electrospray.

Conventional unassisted electrospray ionization mass spectrometry (ESI-MS) has severe limitations as a liquid chromatography (LC) interface due to the few solvent compositions which can be electrosprayed without some type of assistance. LC mobile phases which have high flow rates (over 5 microL/min), high conductivity, or high surface tension are normally unsuitable for use with ESI. For this reason, an ultrasonic nebulizer has been developed which mechanically creates the fine spray of droplets needed for ESI and thus functions independently of the solvent composition. This device also operates at considerably higher liquid flow rates, up to several hundred microliters per minute. To characterize the system's performance, the ultrasonic nebulizer frequency, source electrode potentials, and drying and focusing gas flow rates were studied and optimized. Also, droplet size measurements were taken using a phase-Doppler anemometer, which showed consistent nebulizer performance up to a liquid flow rate of several hundred microliters per minute. Finally, the ultrasonic nebulizer was used for an LC application involving the separation of proteins on 1.0 and 2.1 mm i.d. columns. LC flow rates of up to 200 microL/min of mobile phases containing 0.1% trifluoroacetic acid could be easily nebulized. With the 1.0 mm i.d. column, as little as 32 fmol of cytochrome c could be detected in selected ion monitoring mode.