Incorporating high-pressure electroosmotic pump and a nano-flow gradient generator into a miniaturized liquid chromatographic system for peptide analysis

• We demonstrate the feasibility of a low-cost high-pressure electroosmotic pump for gradient HPLC separation.
• We develop a means for isolating a gradient eluent from electroosmotic pump solution, ensuring a stable pump rate.
• We develop a means for producing a nanogradient eluent for capillary chromatographic separation.
• We develop a total-capillary chromatographic system.
• We couple the total-capillary chromatographic system with an MS for protein and peptide analysis.

We integrate a high-pressure electroosmotic pump (EOP), a nanoflow gradient generator, and a capillary column into a miniaturized liquid chromatographic system that can be directly coupled with a mass spectrometer for proteomic analysis. We have recently developed a low-cost high-pressure EOP capable of generating pressure of tens of thousands psi, ideal for uses in miniaturized HPLC. The pump worked smoothly when it was used for isocratic elutions. When it was used for gradient elutions, generating reproducible gradient profiles was challenging; because the pump rate fluctuated when the pump was used to pump high-content organic solvents. This presents an issue for separating proteins/peptides since high-content organic solvents are often utilized. In this work, we solve this problem by incorporating our high-pressure EOP with a nano-flow gradient generator so that the EOP needs only to pump an aqueous solution. With this combination, we develop a capillary-based nano-HPLC system capable of performing nano-flow gradient elution; the pump rate is stable, and the gradient profiles are reproducible and can be conveniently tuned. To demonstrate its utility, we couple it with either a UV absorbance detector or a mass spectrometer for peptide separations.

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