Skip navigation

Stanford University

Stanford Microfluidics Laboratory

Isoelectric Focusing for Multi-dimensional Separations in Microfludic Devices

Conventional implementations of multi-dimensional separations (e.g., slab-gel and coupled column formats) aid in accurate identification and quantification of specific proteins present in complex samples.  In a multi-dimensional separation, independent separation mechanisms are employed sequentially, resulting in a total peak capacity that is approximately the product of the peak capacities of each respective separation dimension.  Such systems have the potential to resolve a multitude of species. 

Project Description

Researchers have recently coupled chromatographic techniques to electrophoresis on-chip.  In this work, we investigated on-chip coupling of IEF to zone electrophoresis.  Microchannels having an internal cross-section measuring  200 mm wide x 20 mm deep were fabricated in acrylic using a MEMS-based embossing tecnique.  Epi-fluorescence microscopy was used to characterize the spatial and temporal separation and sampling behaviors.

We chose to couple liquid phase IEF to free solution electrophoresis.  Ampholytes were used as the running buffer for both dimensions, while boundary condition control of the applied voltage and terminal buffer chemistries was used to govern the acting separation mechanisms. Low-dispersion electroosmotic flow mobilized focused species to junctions in a t-geometry.  Once species arrived at the junction, they were electrokinetically-sampled into the electrophoresis dimension.  Subsequently, species remaining in the IEF dimension were refocused and subjected to electrophoretic analysis.  This algorithm was repeated until all fluid volumes of interest from the first dimension were analyzed by the second dimension.

What is IEF?
IEF is electrophoresis in a stable pH gradient. This high-resolution method separates amphoteric molecules.  Under an applied electric field, molecules migrate to the pH corresponding to their isoelectric point. The pH gradient is produced by electrophoresis of ampholytes, heterogenous molecules synthesizd with a continuum of isoelectric points. Resolution is determined by the number of ampholytes and the evenness of the pI distribution.

What is an isoelectric point, pI?
pI is the pH at which a protein carries no net charge. Below the isoelectric point proteins carry a net positive charge; above, a net negative charge. Due to a prevalence of weakly acidic residues, almost all proteins are negatively charged at neutral pH.  At the pI, the mobility a protein in an electrofocusing system is zero (and therefore the point at which the protein will accumulate).

We have shown that on-chip IEF acts to “focus” an initially uniform sample into a tight volume at the isoelectric point (pI) resulting in enrichment factors of 80x (Figure 1).  Species focused quickly in the IEF dimension (< 60s) and can be rapidly sampled and analyzed by the second dimension (periods of 1 min). 

(Mouseover figure to begin movie)

Figure 1. Real-time focusing of three fluorescent species during IEF. Images are were taken 1 second apart, the channel is 400 microns wide, and an electric field of 500 V/cm was applied.

Two-dimensional “gel-like” plots were constructed from time-sequences of CCD images collected during the 2D separations (Figure 2). The gel-like plots were formatted to display intensity information so as to mimic a slab-gel result.  Based on the CCD images, the horizontal axis of the gel-like plots represents the relative location of the fluidic volumes during IEF.  The vertical axis of the gel-like plots corresponds to the axial spatial coordinates of the second dimension.  Thus, each vertical ‘lane’ of the 2D separation corresponds to successive CE separations at the same elapsed CE analysis time.

Figure 2.  Gel-like plot of results from a 2D on-chip separation of fluorescent species.

While retaining assay information content, miniaturization of multi-dimensional systems reduces necessary reagent and sample volumes, eliminates manual intervention (impacting reproducibility and system automation design), and significantly improves throughput.


1.) A.E. Herr, J.I. Molho, K.A. Drouvalakis, J.C. Mikkelsen, P.J. Utz, J.G. Santiago, T.W. Kenny. "On-Chip Coupling of Isoelectric Focusing and Free Solution Electrophoresis for Multidimensional Separations", Analytical Chemistry, 75(5), pp. 1180-1187, 2003.

2.) A.E. Herr, J.C. Mikkelsen, J.G. Santiago, T.W. Kenny, "Two-Dimensional Chip-Based Protein Analysis using Coupled Isoelectric Focusing and Capillary Electrophoresis", Proceedings of the Sold-State Sensor, Actuator, and Microsystems Workshop, pp. 366-7, June 2-6, 2002.

3.) A.E. Herr, J.I. Molho, R. Bharadwaj, J.G. Santiago, T.W. Kenny, D.A. Borkholder, M.A. Northrup. "Miniaturized Isoelectric Focusing (uIEF) as a Component of a Multi-Dimensional Microfluidic System", Proceedings of the Micro Total Analysis Systems Symposium, Monterey, California, pp. 51-53, October 21-25 2001.