On-chip single column transient isotachophoresis with free zone electrophoresis for preconcentration and separation of α-lactalbumin and β-lactoglobulin

Abstract

Isotachophoresis (ITP) coupled to zone electrophoresis (ZE), either free zone electrophoresis (FZE) or gel electrophoresis (GE), carried out mainly in capillaries and, although less frequently, also in microchips, is a powerful preconcentration and separation technique which has been successfully used for the study of many low molecular-weight analytes. However, this analytical technique has been scarcely applied for proteins separation. In this work, an on-chip transient ITP coupled to free zone electrophoresis (t-ITP-MFZE) mode with LIF detection is developed for the preconcentration and separation of the proteins α-lactalbumin and β-lactoglobulin.Firstly, for LIF detection, the proteins were off-chip fluorescently labeled with the fluorogenic reagent Chromeo P503. Then, several separation parameters in t-ITP-MFZE mode such as leading electrolyte, terminating electrolyte, separation voltage, and injection time were optimized to achieve the maximum sensitivity while maintaining an adequate resolution between α-lactalbumin and β-lactoglobulin in a single column configuration t-ITP. Using the optimized electrolytes (50mM imidazole/HCl pH=8 as leading electrolyte and 100mM imidazole/12mM HEPES pH=8 as terminating electrolyte) separation of both proteins was achieved in less than 4min with peak resolution of 1.5. The LODs were 55nM and 380nM, for α-lactalbumin and β-lactoglobulin, respectively, which are adequate for some food allergenicity studies.Finally, comparison of the optimized t-ITP-MFZE method to the equivalent MFZE method, carried out also in microchips but without the isotachophoretic preconcentration step, provided preconcentration indexes for both proteins around 10.