Effect of capillary cross-section geometry and size on the separation of proteins in gradient mode using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns

Abstract

Porous polymer monoliths have been prepared in capillaries with circular or square cross-sections and lateral dimensions of 50, 75, 100 microm as well as in a rectangular 38 microm x 95 microm capillary. These capillaries have been used to determine the effect of the size and shape of their cross-section on the porous and hydrodynamic properties of poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths. The capillaries were studied by scanning electron microscopy and evaluated for their permeability to flow and their performance in the liquid chromatographic separation of a protein mixture comprising ribonuclease A, cytochrome c, myoglobin, and ovalbumin using a linear gradient of acetonitrile in the mobile phase. No differences resulting from channel geometry were found for the various capillary columns. These results demonstrate that standard capillaries with circular geometry are a good and affordable alternative conduit for modeling the processes carried out in microfluidic chips with a variety of geometries.

Fig. 1

Experimental setup used in this study. Zero dead volume connections were used for installation of the conduits.

Fig. 2

Effect of flow rate on back pressure generated in conduits containing porous poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths prepared from the same polymerization mixture and differing in shape and size. Conditions: Column length 15 cm, mobile phase 0.1 %v/v formic acid solution in 50/50 v/v acetonitrile-water.

Fig. 3

Scanning electron micrographs of the cross section of fused silica capillaries with different size and shape containing porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith.

Fig. 3

Scanning electron micrographs of the cross section of fused silica capillaries with different size and shape containing porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith.

Fig. 3

Scanning electron micrographs of the cross section of fused silica capillaries with different size and shape containing porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith.

Fig. 4

Separation of ribonuclease A, cytochrome c, myoglobin, and ovalbumin using poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic columns in cylindrical and square conduits with indicated lateral length dimensions. Conditions: Column length 15 cm, mobile phase A 0.1 %v/v formic acid solution in water, B 0.1 %v/v formic acid solution in acetonitrile, gradient 0–80 % B in A in 5 min, flow rate 1.6 μL/min, superficial velocities in the respective conduits are shown in chromatograms.

Fig. 5

Effect of superficial flow velocity on elution volume of ribonuclease A (■), cytochrome c (●), myoglobin (▲), and ovalbumin (▼) using poly(butyl methacrylate-co-ethylene dimethacrylate)monolith located in a capillary with 38×95 μm rectangular and 75 μm I.D. circular cross section. Conditions: Column length 15 cm, mobile phase A 0.1 %v/v formic acid solution in water, B 0.1 %v/v formic acid solution in acetonitrile, gradient 0–80 % B in A with varying times according to a constant gradient volume of 8 μl.

Fig. 6

Effect of flow rate on the separation of ribonuclease A, cytochrome c, myoglobin, and ovalbumin (elution order) using poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic column in the 38×95 μm rectangular (a,b) and 75 μm I.D. circular capillary (c,d). Conditions: Column length 15 cm, mobile phase A: 0.1 %v/v formic acid solution in water, B: 0.1 %v/v formic acid solution in acetonitrile, gradient 0–80 % B in A in 5 min (a,c) and 11.4 min (b,d); flow rate 1.6 μL/min (a,c) and 0.7 μL/min (b,d); superficial velocities in the conduits are shown in chromatograms.

Fig. 7

Effect of flow velocity on resolution of pairs ribonuclease A - cytochrome c (closed symbols) and myoglobin ovalbumin (open symbols) using a constant gradient volume of 8 μL by adjusting the gradient time. Conditions: Column length 15 cm, mobile phase A: 0.1 %v/v formic acid solution in water, B: 0.1 %v/v formic acid solution in acetonitrile, gradient 0–80 % B in A; Sizes of the conduits are shown in figure.