The Ultimate 3000 system consists of a temperature controlled autosampler, heated column compartment, variable wavelength detector and an in-line degasser. The system uses biocompatible PEEK fluidics limiting sample loss through the system. In addition to the PEEK tubing, used fussed silica capillaries with 20mm internal diameter are also employed. The configuration allows for low solvent volumes and sample dispersion. The temperature controlled autosampler allows the analysis of temperature labile components and retards the artifactual modification of peptides prior to analysis. As the samples are linked to a nano-LC system, small volumes (0.5 mL) can reproducibly injected. In the current configuration a maximum of 20mL can be injected. The temperature controlled column compartment houses a cation exchange column for 2 dimensional analyses, a C18 reverse phase trap column and a 75mm internal diameter by 15 cm nano-LC column. The small diameter couple to 3 mm particles results in a very efficient column. This configuration allows for the analysis of small amounts of sample optimizing the number of analyses that can be performed from the total sample amount. The temperature control of the column increases the reproducibility of the system by limiting environmental factors that influances separation. The variable wavelength detector allows for the monitoring of the separation irrespective of the system being coupled to a mass spectrometer. If the injected sample is intended for mass spectrometric analysis the outflow from the column is mixed in-line with a suitable MALDI matrix and fraction collected directly onto a MALDI target plate.