Monolithic porous polymer stationary phases in polyimide chips for the fast high-performance liquid chromatography separation of proteins and peptides

Abstract

Poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in monolithic format have been prepared by thermally initiated free radical polymerization within polyimide chips featuring channels having a cross-section of 200micromx200microm and a length of 6.8cm. These chips were then used for the separation of a mixture of proteins including ribonuclease A, myoglobin, cytochrome c, and ovalbumin, as well as peptides. The separations were monitored by UV adsorption. Both the monolithic phases based on methacrylate and on styrene chemistries enabled the rapid baseline separation of most of the test mixtures. Best performance was achieved with the styrenic monolith leading to fast baseline separation of all four proteins in less than 2.5min. The in situ monolith preparation process affords microfluidic devices exhibiting good batch-to-batch and injection-to-injection repeatability.

Fig. 1

Schematic layout of microfluidic polyimide chip with integrated LC monolith column and exit ports for UV absorbance detection. The wide line shows the LC separation column. The thin line shows the return channel. These channels are connected through the off-chip UV detection cell (cross section 120 × 120 μm; length 6 mm). The UV cell itself is not shown to simplify the scheme.

Fig. 2

SEM images of the cross section of the separation channel in a polyimide HPLC chip filled with poly(lauryl methacrylate-co-ethylene dimethacrylate) (left) and poly(styrene-co-divinylbenzene) monolith (right).

Fig. 3

Optical micrographs of a part of an empty polyimide chip (A), a chip with the injection channel empty and the separation channel filled with the monolith (B), and a chip with both injection and separation channels containing the monolith (C).

Fig. 4

On-chip separations of ribonuclease A, cytochrome c, myoglobin, ovalbumin (order of elution) using the monolithic poly(lauryl methacrylate-co-ethylene dimethacrylate) (A) and poly(styrene-co-divinylbenzene) (B) stationary phases at different flow rates: (a) 4, (b) 3, (c) 2, and (d) 1 μL/min. Gradient: 0-60% of acetonitrile in water (0.05% v/v of formic acid) in 10 min. Concentration of proteins in sample solution: 0.2 and 0.1 mg/mL for poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) monoliths, respectively. Injection volume 40 nL. Detection: UV at 210 nm.

Fig. 5

On-chip separations of ribonuclease A, cytochrome c, myoglobin, ovalbumin (order of elution) using the monolithic (A) poly(lauryl methacrylate-co-ethylene dimethacrylate) and (B) poly(styrene-co-divinylbenzene) stationary phases. Gradient of the mobile phase: 0-60% of acetonitrile in water (0.05% v/v of formic acid) in (a) 30, (b) 20, (c) 10, and (d) 5 min, and 0-100% of acetonitrile in water (0.05 % v/v of formic acid) in (e) 2 min. Concentration of proteins in sample solution: 0.1 mg/mL. Injection volume 40 nL. Flow rate: 4 μL/min. Detection: UV at 210 nm.

Fig. 6

On-chip separation of a mixture of peptides Tyr-Gly-Gly, [Ala²]-methionine-enkephalin, LHRH, alytesin, and somatostatin (elution order) (A) and of a tryptic digest of cytochrome c (B) using the monolithic poly(styrene-co-divinylbenzene) stationary phase. Gradient: 0-60% of acetonitrile in water (0.05% v/v of formic acid) in 7 min (A) and 10 min (B). Concentration of peptides in sample solution: 0.1 mg/mL. Injection volume 40 nL. Flow rate: 4 μL/min. Detection: UV at 210 nm.

Fig. 7

Pressure drop in the polyimide chip normalized by viscosity of the solvent as a function of flow rate of acetonitrile, methanol, and water through channel containing poly(lauryl methacrylate-co-ethylene dimethacrylate) (A) and poly(styrene-co-divinylbenzene) monolith (B).

Fig. 8

Overlaid chromatograms obtained by 10 successive gradient separations of ribonuclease A, cytochrome c, myoglobin, ovalbumin (order of elution) using chips containing both monolithic poly(styrene-co-divinylbenzene) (A) and poly(lauryl methacrylate-co-ethylene dimethacrylate) (B) stationary phases. Gradient: 0-60% of acetonitrile in water (0.05% v/v of formic acid) for 10 min. Concentration of proteins in sample solution: 0.2 and 0.1 mg/mL for poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) monoliths, respectively. Injection volume 40 nL. Flow rate: 4 μL/min. Detection: UV at 210 nm.