Highlight October 2007: Free-flow electrophoresis separation methods

Free-flow electrophoresis separation methods have been developed and investigated for around 50 years and have been applied to many types of analytes for various biomedical applications. In contrast to their large-scale counterparts microfluidic FFE devices offer new possibilities due to the very rapid separations within several seconds or below and the requirement for sample volumes in the micro liter range. Eventually, these microfluidic FFE systems might find application in lab-on-a-chip devices for real-time monitoring and separation applications.

A new microfluidic free-flow isoelectric focusing glass chip for separation of proteins has been developed. Microfluidic free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. Several chip improvements led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. A linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. 7 isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 seconds and an electrical field of 20 Vmm^-1. The theoretical minimum difference in isoelectric point is delta(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. The performance of this microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.

figure 3 - chip photograph copy

Photographs of a fabricated µ-FFIEF chip: (a) the chip size is 20 mm x 20 mm with. The separation chamber has a volume of 0.3 µl (b) Membrane close-up.

Free-Flow Isoelectric focusing of 7 fluorescent IEF markers: (a) Photograph shows the device, when no voltage was applied. (b) When 150 V (I=50 µA) were applied, the markers (pI 4, 5.1, 6.2, 7.2, 8.1, 9 and 10.3) fully separated within less than two seconds.

A detailed description can be found in the full paper published in Analytical Chemistry September 2007: http://dx.doi.org/10.1021/ac071419b

For more information please contact Dietrich Kohlheyer: d.kohlheyer@utwente.nl