An improved method for tracking and reducing the void volume in nano HPLC-MS with micro trapping columns

Tandem mass spectrometry coupled to HPLC is the state of the art technique in proteomic research. Here we describe a highly sensitive nano liquid chromatography system (nano HPLC) for analysis of protein digests. Using preconcentration in a column-switching set-up, we were able to inject large sample volumes (less than or equal to250 muL) without significant loss of sensitivity. The major problem with this type of preconcentration. is usually the occurrence of void volumes. In order to diagnose void volumes a simple and easy test was developed by which the UV trace and the pressure profile in the separation column were monitored. Part by part replacement of connection tubing restored a void volume-free system. A major pre-requisite for handling samples in the femtomol range was found to be the use of protein/peptide-saturated columns tryptic digests of cytochrome C were injected directly onto the reversed-phase nano separation column (75 mum inner diameter) and the separation results were compared with chromatograms obtained from separations using column switching. By using column switching we were able to inject large sample volumes in a short time period without losing resolution.