Incorporation of carbon nanotubes in porous polymer monolithic capillary columns to enhance the chromatographic separation of small molecules

Abstract

Multiwalled carbon nanotubes have been entrapped in monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary columns to afford stationary phases with enhanced liquid chromatographic performance for small molecules in the reversed phase. While the column with no nanotubes exhibited an efficiency of only 1800 plates/m, addition of a small amount of nanotubes to the polymerization mixture increased the efficiency to over 15,000 and 35,000 plates/m at flow rates of 1 and 0.15 μL/min, respectively. Alternatively, the native glycidyl methacrylate-based monolith was functionalized with ammonia and, then, shortened carbon nanotubes, bearing carboxyl functionalities, were attached to the pore surface through the aid of electrostatic interactions with the amine functionalities. Reducing the pore size of the monolith enhanced the column efficiency for the retained analyte, benzene, to 30,000 plates/m at a flow rate of 0.25 μL/min. Addition of tetrahydrofuran to the typical aqueous acetonitrile eluents improved the peak shape and increased the column efficiency to 44,000 plates/m calculated for the retained benzene peak.

Introduction

Since the inception of rigid organic polymer monolithic columns in the early 1990s [1], their use as chromatographic separation media has continued to grow. The popularity of monolithic columns is fueled by their high permeability, which enables excellent performance in the fast separation of large molecules such as peptides, proteins, nucleic acids, and synthetic polymers at high flow velocities using gradient elution [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The high speed achieved in these separations results from the rapid convective mass transport in the large through pores – the only pores that are present in the unmodified monoliths. In this instance, the lack of small pores in the monolithic structure avoids the normally slow diffusional mass transport. However, this comes at the cost of surface area, thus making the monoliths unsuitable for the separation of small molecules in an isocratic mode due to the absence of the numerous interaction sites required for sufficient sample loading capacity. In our initial experiments, we found that a poor efficiency of only 18,000 plates/m for benzene could be achieved with the first generation of monolithic poly(styrene-co-divinylbenzene) columns [3]. Since the original development of rigid monoliths, several groups have attempted to increase the column efficiency for small molecules. For example, a recent optimization of the polymerization conditions for methacrylate-based monoliths has afforded capillary columns with 35,000–50,000 plates/m for retained benzene [14], [15], [16], [17], [18]. Other groups have sought alternative processes such as the polymerization of a single crosslinker [19], [20], [21], [22], the termination of the polymerization reaction at an early stage [23], [24], [25], and the use of polymerizations at high temperature [26], [27], [28]. We have recently introduced a new modification reaction, hypercrosslinking, which enables a significant increase in the efficiency of monolithic columns [29]. While this reaction works well with monoliths prepared from styrene, chloromethylstyrene, and divinylbenzene, it is not readily applied to methacrylate-based monolithic columns. Even as all these methods have led to porous polymer monolithic columns with efficiencies exceeding those of our early columns [3], the preparation of highly efficient polymer-based monolithic columns for the isocratic separation of small molecules that perform as well as their silica-based monolithic counterparts [30], [31] remains a challenge.

Due to unique characteristics of nanoparticles, such as their large surface-to-volume ratio and their properties that differ from those of corresponding bulk materials, the use of nanomaterials in separation science is growing rapidly [32], [33], [34]. For example, nanostructures, such as polymer latex nanoparticles, fullerene derivatives, metal oxides, and carbon nanotubes have been used for the modifications of separation media for application in gas and liquid chromatography, capillary electrophoresis, and electrochromatography [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]. In the field of polymer monoliths, methacrylate columns with attached functionalized polymer nanoparticles were introduced first and these columns were used for the separation of saccharides [44] and in ion chromatography [45], [46], [47], [48]. Monoliths with pores coated with gold nanoparticles have recently been prepared [49], [50], [51] and used for the pre-concentration of thiol containing peptides and the separation of proteins [49], [50], while monoliths with embedded hydroxyapatite nano-needles proved useful in the extraction of phosphorylated peptides from complex protein digests [52]. Li et al. entrapped carbon nanotubes into a poly(chloromethylstyrene-co-ethylene dimethacrylate) monolith to afford capillary columns for HPLC and capillary electrochromatography [53].

Thus, nanostructures hold a great potential for achieving efficient separations of small molecules. This article demonstrates the use of carbon nanotubes entrapped within or attached to the pore surface of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths in order to improve the performance of the monolithic capillary columns in the isocratic separation of small molecules.