Effects of separation length and voltage on isoelectric focusing in a plastic microfluidic device.

This paper describes the investigation on the effects of separation length and voltage on IEF in a plastic microfluidic device. A LIF, whole-channel imaging detection (WCID) system was developed to monitor proteins while they were moving under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers (hydroxyethylcellulose and hydroxypropylcellulose). We found that the IEF separation resolution is essentially independent of separation length when the same voltage is applied, which agrees with the theory. This result supports the notion that IEF in a microfabricated device leads to more rapid analysis without sacrificing the resolving power. A higher separation voltage also brought about more rapid analysis and superior separation resolution. IEF of two proteins (green fluorescence protein and R-phycoerythrin) was achieved in 1.5 min when 500 V was applied across a 1.9-cm channel. We found that a linear relationship exists between the focusing time and the inverse of the electrical field strength. In addition, we confirmed the phenomenon in which the pH gradient was compressed to the middle of a channel, and we found that the relative amount of the gradient compression decreased with the channel length.