Microchannel Isoelectric Focusing (IEF): Elucidating the Separation Performance Impacts of Chemical Mobilization

Introduced in the 1960’s, isoelectric focusing (IEF) remains a powerful electrophoresis technique. In particular, IEF finds a dominant role in modern day analyses of proteins by isoelectric point (pI), making the assay critical for detection of post-translational modifications and in 2D separations. Capillary IEF enables rapid separations, but pH zones must be mobilized after IEF if a sensitive single-point detector is used (i.e., photomultiplier tube) [1]. Chemical mobilization is the technique of choice, as this mechanism leads to less shear-flow broadening (compared to pressure driven mobilization) and is independent of surface chemistry. Intriguingly, despite prior chemical mobilization optimization efforts and recent numerical simulations, our understanding of chemical mobilization and its implications on analytical performance remain limited [2]. Previous studies have tested conditions for mobilization, but are incomplete regarding the quantitative performance assessment and detailed mechanistic descriptions connecting performance with theory [3, 4]. Given this gap in the current literature, in this as of yet not reported study we: 1) detail experimental disagreements with previously proposed theoretical descriptions, 2) highlight areas where there is incomplete theoretical understanding, and 3) provide quantification performance metrics for different mobilization schemes.

We measure the effect of mobilization solution pH, the differences between ionic and zwitterionic mobilization, and the impact of diffusion as a source of zone broadening.  To this end, we define quantitative metrics and utilize microchannel IEF to assess the performance of different typically used mobilization schemes. Leveraging the planar format of glass microfluidic chips that allows easy interfacing with microscopy equipment, we compare the location of the pH gradient using fluorescent pH markers before and during mobilization. Performing our separations in the microfluidic format enables rapid and automated separations, thus enabling multiple conditions to be quickly tested with technical replicates with minimal experimental variation. Additionally, we polymerize cross-linked polyacrylamide gels within the device channels to remove the effects of electroosmotic flow and be able to isolate distortions stemming from mobilization. 

From our results, we quantify performance of the different tested mobilization schemes including the efficiency of mobilization across the entire pH range, the distortion of the focused zones resulting from zone broadening, and changes in pH gradient linearity. Our results will inform future assay design for IEF and 2D separations as well as help elucidate the physical mechanisms for the varying performance of different mobilization schemes.

REFERENCES

[1] Hjertén, S. & Zhu M. D., J. Chromatogr. (1985), 346, 265-270.

[2] Thormann, W. & Kilár, F., Electrophoresis (2013), 34, 716-724.

[3] Hjertén, S., Liao, J. L.,Yao, K., J. Chromatogr. A (1987), 387, 127-138.

[4] Zhu, M. D., Rodriguez, R., Wehr, T., J. Chromatogr. (1991), 559, 479-488.