Devices and methods for the sensitive detection and quantitation of biomolecules

A method for separating and analyzing one or more intact proteins comprising:

• introducing a sample comprising the one or more intact proteins into an inlet of a chromatographic separation device that comprises
• (a) sorbent particles formed from an organic polymer formed from one or more monofunctional or multifunctional aromatic monomers and having width ranging from 0.5 to 100 m and
• (b) the chromatographic separation device comprising the inlet, an outlet and an internal volume between the inlet and the outlet, the internal volume being filled with a volume of the sorbent particles, wherein the internal volume has a length ranging from 2 to 50 mm and a width ranging from 50 m to 10 mm, wherein a direction of fluid flow from the inlet to the outlet is along the length of the volume of the sorbent particles, and wherein a ratio of the length to the width of the volume of the sorbent particles ranges from 10:1 to 2:1,
• thereby loading the one or more intact proteins onto the sorbent particles;
• and introducing an acidic mobile phase comprising formic acid, acetic acid and/or a fluorinated carboxylic acid into the inlet of the chromatographic separation device in a volume sufficient to elute at least a portion of the loaded one or more intact proteins from the sorbent particles in an eluent stream from the outlet of the chromatographic separation device;
• and
• analyzing the eluent stream with a mass spectrometer, wherein the mass spectrometer is calibrated with a lockspray standard electrosprayed under conditions which produce a lockmass ion between 2,000 and 4,000 m/z.