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

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 (50   mM imidazole/HCl pH   =   8 as leading electrolyte and 100   mM imidazole/12   mM HEPES pH   =   8 as terminating electrolyte) separation of both proteins was achieved in less than 4   min with peak resolution of 1.5. The LODs were 55   nM and 380   nM, 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.