Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

Abstract

A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA-EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.

Figure 1

The representative chromatograms for separation of proteins on (a) column 7 and (b) column 10. Conditions: mobile phase A: 98% ACN with 0.1% TFA in water; mobile phase B: 2% ACN with 0.1% TFA in water; linear gradient program, 16% A at 0 min, 40% A at 0.5 min; injection volume, 100 nL; flow rate, 1.1 μL/min; detection, 214 nm. Solutes: (1) ribonuclease A; (2) cytochrome c; (3) myoglobin. Each analytes was injected at concentration of 0.3 mg/mL in water.

Figure 2

The regression coefficients plots for proteins separation. A: Average resolution (Rs_(avg)); B: Analysis time (t_R).

Figure 3

The 2-D contour plots obtained for (A) Rs_avg and (B) t_R of proteins as a function of significant factors. %AAUA, %water and %1-propanol are the X1-, X2-, and X3-axes, respectively, with EDMA fixed at 18.5% and 1-propanol fixed at 62%.

Figure 4

Chromatograms of protein separations using (a) fast separation column OF-1 and (b) optimized high resolution column OH-1. Other conditions are the same as Figure 1. The inset table describes the differences between predicted values and experimental values.

Figure 5

Protein digest separation using the (a) optimized fast separation column OF-1 and (b) high resolution column OH-1. Conditions: mobile phase A: 98% ACN with 0.1% TFA in water; mobile phase B: 2% ACN with 0.1% TFA in water; linear gradient program, 16% A at 0 min, 20% A at 10 min, 50% A at 15 min, 80% A at 20 min; injection volume, 100 nL; flow rate, 1.1 μL/min; detection, 214 nm. Sample, 1.0 mg/mL myoglobin tryptic digest in water.

Figure 6

Scanning electron micographs of monolith columns. A: column 7; B: column 10; C: column OF-1. D: column OH-1. Detailed information of the polymerization mixture composition for the monolith is described in Table 2 and section 3.2.

Figure 7

Plots of the volumetric flow rate of mobile phase against the applied pressure. The composition of column 7, 10, OF-1 and OH-1 are described in Table 2 and results section. Mobile phase, 60% ACN in water. All data were collected on μ-HPLC system. The inset plot shows the expanded trend obtained on column 10 and column OH-1.