Analytical Chemistry, 2005: Microchip HPLC of peptides and proteins
Citation: Reichmuth DS, Shepodd TJ, Kirby BJ. Microchip HPLC of peptides and proteins, Analytical Chemistry, 77:2997-3000. doi pdf
Abstract: Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.
Figures:
- Micrograph of injector valve and beginning of separation media. Inset is scanning electron micrograph of separation media polymerized inside 150-µm-i.d. glass capillary.
- Linear dependency of peak area on injection duration. Rhodamine 560 injections were performed at 450 psi; buffer flow was constant at 300 psi. Error bars are standard deviation, with at minimum nine replicates at each injection duration.
- Repeated 470-pL injections of a peptide mixture. Isocratic separation using 30% ACN with 0.1% TFA in 5 mM phosphate buffer (pH 2.2) at 300 psi.
- Repeated 6400pL, 750-ms injections of a protein mixture. Isocratic separation was performed using 24% ACN + 0.16% HFBA in 5 mM phosphate buffer (pH 2.0) at 300 psi. Peak identities and retention factors: a, free dye; b, insulin (3.2); c, anti-biotin (6.0); d, R-lactalbumin (9.1).
- Four-port valve allowing rapid sample changes. Sample is changed by flushing at low pressure when the sample waste port is open. Sample is injected by closing sample waste port and pressurizing the sample line.
- Rapid sample switching performed without sample carryover. Injections at 50-s spacings, alternating between a sample containing rhodamine 560 and a blank sample of run buffer.