Use of external electrolysis to improve electrophoretic separations of sugars on glass microfluidic devices

In electrophoresis, applying high voltage on electrolytes with high conductivity and ionic strength can lead to a variation in terms of migration times due to the electrolysis phenomena. This effect can compromise the separation reproducibility and repeatability. Herein, we propose the adaptation of a scheme to minimize the electrolysis in microfluidic systems. The proof-of-concept was tested on the separation of five sugars, including sucrose (SUC), Lactose (LAC) galactose (GAL), glucose (GLU) and Fructose (FRU), using a high pH electrolyte to promote the ionization of sugars. A commercial microchip electrophoresis system model ER455 Quad MCE System ( eDAQ pty, Austrália) and a glass microchip (T85100C4D, Micronit, Netherlands) with 90 mm long was employed to perform the separations. Separated electrolysis system was made by applying the voltage in four 1.5 mL polypropylene tubes connected to microfluidic platform reservoirs by a saline bridge made with Tygon tubes (0.4 mm i.d.). Firstly, an electrolyte composed by NaOH (75 mmol/L) was used to promote the separation of SUC, GLU and FRU (Fig. 1A). A variation on migration time and co-migration between FRU and GLU were observed. In the 7th run, it was observed that SUC peak disappeared indicating that it was neutral and co-migrating with EOF marker (water). Trisodium phosphate (15mmol/L) was added to insert a buffering effect and increase the ionic strength, improving the separation between FRU and GLU. The improvement on the performance allowed the addition and separation of LAC and GAL to the previous mixture (Fig. 1B). However, the problem remained unaltered. To minimize the electrolysis, the separated electrolysis system was implemented reaching now the complete separation of the five sugars and improved stability and repeatability (Fig. 1C). With the external reservoirs, the variation in terms of migration times was reduced from 4% to less than 1%. Also, the separation efficiency was increased from c.a. 14,000-29,000 plates/m to ca. of 45,000-70,000 plates/m for the five sugars. In summary, we propose for the first time the electrophoretic separation of five sugars in a microfluidic system. This was achieved due to the use of additional reservoirs for external electrolysis that ensured stability on migration times and resolution working with a high conductive and ionic strength electrolyte. Moreover, external electrolysis provided enhanced separation efficiency.