INSTRUMENTAL REQUIREMENTS FOR NANOSCALE LIQUID-CHROMATOGRAPHY

Nanoscale liquid chromatography (nano-LC), with packed columns of typi cally 75 mu m i.d. x 15 cm length, packed with C18, 5 pm of stationary phase, and optimal now rates of 180 nL/min, can be considered as a mi niaturized version of conventional HPLC, Using the down-scaling factor , which corresponds to the ratio of the column diameter in square, (d( conv)/d(micro))(2), excellent agreement between the theoretically calc ulated values and the values obtained using the down-scaling factor (s imilar to 3800) has been observed. This factor was applied to all syst em components, including now rate, injection and detection volumes, an d connecting capillaries, Down-scaling of a conventional HPLC system t o a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitud inal nanoflow cell (less than or equal to 3 nL), a microinjection valv e (less than or equal to 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibilit y was measured with RSD values of +/-0.1% for isocratic and +/-0.2% fo r gradient elution. Plates counts of more than 100 000/m indicate the excellent performance of the entire nano-LC system, With minimal detec table amounts of proteins in the low femtomole and subfemtomole ranges (e.g., 520 amol for bovine serum albumin), high mass sensitivity was found, making nano-LC attractive for the microcharacterization of valu able and/or minute proteinaceous samples. Coupling nano-LC with concom itant mass spectrometry using nanoscale ion spray or electrospray inte rfaces looks very promising and is obviously the next step for future work.