A stable and high-resolution isoelectric focusing capillary array device for micropreparative separation of proteins

A simple capillary array IEF device was developed for high resolution and micropreparative separation of trace amounts of proteins. Based on quasi-chip-scale manufacturing, the specific capillaries (600   μm i.d., 1200   μm o.d. and 20   mm length) were integrated with the miniaturized polymethyl-methacrylate electrode trays. Electroosmotic flow was suppressed effectively by modified cross-linked polyacrylamide coating, and instability of IEF was addressed using the designed concentration of electrolytes via moving reaction boundary theory. As a prototyping, the resolution, reproducibility, throughput, speed and linearity of pH gradient were systemically evaluated with model proteins. The results revealed the following advantages: (i) the reproducibility of array was assessed as RSD values of 0.95% (intra-day) and 2.88% (inter-day); (ii) IEF could be completed in 20   min with up to 400   V/cm electric field; (iii) high resolution separation of model proteins achieved in 20   mm length column; (iv) multi-units with 48 micro-columns can be easily integrated to obtain high throughput; and (v) good linearity of pH gradient ( R =0.9989). More importantly, utility of the device was tested by using hemoglobins sample from human red blood cell. HbA 0 and HbA 1c with only Δp I 0.03 have been successfully separated by the developed method.