Postcolumn renewal of sensor surfaces for high-performance liquid chromatography-surface plasmon resonance detection

The combination of high-performance liquid chromatography (HPLC) with surface plasmon resonance (SPR) for continuous separation and label-free detection of protein samples is described. The detection was realized by electrostatic adsorption of proteins bearing positive and negative charges onto chemically modified SPR sensors in two separate SPR channels. One SPR channel is coated with carboxymethylated dextran which facilitates the detection of positively charged proteins, whereas the other, devoted to the monitoring of negatively charged proteins, is covered with ethylenediamine molecules attached onto a dextran surface. Renewal of the sensor surface in the channels can be accomplished by introducing regeneration solutions through two six-port valves. The coupled technique (HPLC-SPR) was assessed for its analytical figures of merit and applied to the quantification of lysozyme in human milk samples. Unlike the SPR detection of bulk solution refractive index changes during chromatographic peak elutions, the highest sensitivity of SPR is retained in this work since the measurement is performed at the SPR sensor surface where the evanescent field is the strongest. The renewable SPR detection of continuous separations is reproducible and versatile and does not require the separated proteins to contain chromophores or to be prelabeled with a tag (e.g., a redox-active or fluorescent molecule). Such generality makes SPR complementary to other types of chromatographic detectors.