Instrumental requirements for nanoscale liquid chromatography.

Nanoscale liquid chromatography (nano-LC), with packed columns of typically 75 μm i.d. × 15 cm length, packed with C18, 5 μm of stationary phase, and optimal flow rates of 180 nL/min, can be considered as a miniaturized 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 calculated values and the values obtained using the down-scaling factor (∼3800) has been observed. This factor was applied to all system components, including flow rate, injection and detection volumes, and connecting capillaries. Down-scaling of a conventional HPLC system to a nano-LC system is easy to realize in practice and involves using a microflow processor for nanoflow delivery (50-500 nL/min), a longitudinal nanoflow cell (≤3 nL), a microinjection valve (≤ 20 nL), low-dispersion connecting tubing, and zero dead volume connections. Excellent retention time reproducibility was measured with RSD values of ±0.1% for isocratic and ±0.2% for gradient elution. Plates counts of more than 100 000/m indicate the excellent performance of the entire nano-LC system. With minimal detectable 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 valuable and/or minute proteinaceous samples. Coupling nano-LC with concomitant mass spectrometry using nanoscale ion spray or electrospray interfaces looks very promising and is obviously the next step for future work.