Simple automated liquid chromatographic system for splitless nano column gradient separations

A simple splitless gradient liquid chromatographic system for micro and nano column separations has been assembled and tested. It consists of an OEM programmable syringe pump equipped with a glass microsyringe and ten-port selector valve. Gradient of mobile phase was created in the syringe barrel due to turbulent mixing. Capability of suggested system to create various gradient profiles was verified using 50-μl, 100-μl, and 250-μl glass syringes. Acetone, thiourea, and uracil were tested as gradient markers and finally, uracil was proved to be an excellent way of water–acetonitrile gradient tracing. It was found that up to 80% of the total syringe volume is available as a linear gradient section. In context to micro and nano column chromatography, the best results were obtained using the 100-μl syringe. Separations were performed on the capillary monolithic column Chromolith CapRod RP-18e (150 mm × 0.1 mm) and system performance was evaluated using a test mixture of six alkylphenones as well as tryptic digest of bovine serum albumin. Results proved that suggested system is able to create uniform gradients with high repeatability of retention times of test solutes (RSD < 0.3%). Repeatability of injection of sample volumes in the range of 0.1–3 μl was evaluated using on-column preconcentration technique which means that sample was diluted in mobile phase of low eluting strength. Repeatability of the peak areas was measured and statistically evaluated (RSD < 5%).

► We assembled a simple splitless gradient nano liquid chromatographic system. ► Capability of automated system to create various gradient profiles was verified. ► System was tested by mixture of alkylphenones and by BSA tryptic digest. ► A high repeatability of retention times (RSD ∼ 0.3%) was obtained. ► A sample volumes from 0.1 to 3 μl could be injected with RSD < 5%.