Itaconic Acid-Based Organic-Polymer Monolithic Column for Hydrophilic Capillary Electrochromatography and Its Application in Pharmaceutical Analysis

Abstract

Itaconic acid is an excellent hydrophilic monomer owing to the dicarboxylic group possessing strong polarity. This study reports on the preparation of a new organic-polymer monolithic column poly(itaconic acid-co-3-(acryloyloxy)-2-hydroxypropyl methacrylate) (poly(IA-co-AHM)) featuring excellent hydrophilic chromatography ability and its application in pharmaceutical analysis. The monolithic column was successfully synthesized by using the monomer itaconic acid and the cross-linker AHM through an in situ copolymerization method. Optical microscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) were employed for the characterization of the poly(IA-co-AHM) monolithic column, and all of these demonstrated that the prepared itaconic acid-based monolithic column exhibited satisfactory permeability and a homogeneous porous structure. Owing to the carboxylic groups of itaconic acid, a cathodic electroosmotic flow (EOF) was generated on the itaconic acid-based monolithic column among the pH ranges of the mobile phase from 4.0 to 9.0. Depending on the powerful hydrophilic interactions, different kinds of polar substances, including thioureas, nucleoside drugs, sulfonamides, and polypeptides, were separated efficiently by the itaconic acid-based monoliths poly(IA-co-AHM). The separations of polar compounds were successfully realized, even at a lower level of 50% acetonitrile content on this monolithic column. The highest column efficiencies corresponding to N,N'-dimethylthiourea and idoxuridine were 102 720 and 124 267 N/m, respectively. The poly(IA-co-AHM) monolithic column displayed excellent repeatability, whose relative standard deviations (RSDs) of the retention time and peak area were both lower than 5.0%. All experimental results demonstrated that the new itaconic acid-functionalized monolithic column was greatly appropriate to separate the polar compounds under the HILIC mode.

Figure 1

Scheme for the preparation of a poly(IA-co-AHM) monolithic column by in situ copolymerization.

Figure 2

Optical microscope images of the monolithic column, 400×.

Figure 3

Separation of thioureas on the poly(IA-co-AHM) monolithic column (Columns 2 and Column 3). Experimental conditions: mobile phase, 60% acetonitrile in 10 mM, pH 7.0 phosphate buffer; applied voltage: 20 kV; electrokinetic injection, 5 kV × 5 s; detection wavelength, 214 nm. Peaks: 1, toluene; 2, N,N′-dimethylthiourea; 3, N′-dimethylthiourea; 4, thiourea.

Figure 4

SEM images of the stationary phases of Column 2 (A, 750×; B, 3000×; C, 10 000×).

Figure 5

(A) Relationship between the retention factor (k) and acetonitrile content on the poly(IA-co-AHM) monolithic column to separate thioureas with toluene as an unretained marker. Experimental conditions: mobile phase, different acetonitrile contents in 10 mM, pH 7.0 phosphate buffer; other conditions are the same as in Figure 2. (B) Separation of nucleoside drugs. Experimental conditions: mobile phase, 60% acetonitrile in 10 mM, pH 8.0 phosphate buffer; applied voltage, 25 kV; electrokinetic injection, 10 kV × 5 s; detection wavelength, 214 nm. Peaks: 1, toluene; 2, idoxuridine; 3, 5-methyluridine; 4, cytarabine; 5, azacitidine; 6, cyclocytidine. (C) Separation of sulfonamides. Experimental conditions: mobile phase, 50% acetonitrile in 15 mM, pH 8.0 phosphate buffer; applied voltage, 20 kV; electrokinetic injection, 5 kV × 5 s; detection wavelength, 256 nm. Peaks: 1, toluene; 2, sulfamethoxazole; 3, sulfamethazine; 4, sulfamerazine; 5, sulfadiazine. (D) Separation of polypeptides. Experimental conditions: mobile phase, 50% acetonitrile in 20 mM, pH 6.0 phosphate buffer; applied voltage, 20 kV; electrokinetic injection, 5 kV × 10 s; detection wavelength, 214 nm. Peaks: 1, toluene; 2, Leu–Leu; 3, Leu–Gly; 4, Ala–Tyr.