Simplified gradient generator for micro- and nano-liquid chromatography

We have developed a simple device to generate gradient elution profiles using split tubing array (STAR) units for micro- and nano-HPLC. This gradient device consists of a delivery pump, a splitter, tubes in parallel, and a relatively large-volume mixing chamber. In the mixing chamber, an initially filled water-rich solvent is adjusted to an organic-rich solvent by delivery of appropriate components through the split paths, in which the flow rate and residence time are controlled by the sizes of the tubes employed. A program was developed to describe the output gradient profiles, and the predicted gradient profiles were highly consistent with the observed ones. A linear gradient generated by this STAR system was successfully applied to micro-HPLC systems for separation of digested peptides derived from serum albumin with sufficient reproducibility. Further miniaturization of STAR systems for nano-HPLC coupled with tandem mass spectrometry was accomplished to separate digested peptides from serum albumin with a reproducibility of retention times of better than 0.2%, and the obtained spectra from the well-separated chromatographic peaks allowed protein identification with high confidence by protein database searching. We believe that this simple and reproducible gradient system using an isocratic pump will be especially advantageous for nanoLC–MS, instead of flow-controlled gradient pumps.