Preparation, characterization, and application of a porous poly (TAIC-co-MMA-co-EDMA) monolithic column for small molecules of aromatics and Chinese herbal medicine notoginseng by HPLC

Highlights

• Triallyl isocyanurate and methyl methacrylate were adopted to prepare a polymer monolith.

• The monolith showed high efficiency for aromatic small molecules and the column efficiency was up to 31,080 plates m⁻¹.

• Isomer of p-Hydroquinone, m-Hydroquinone, and a-Hydroquinone were achived baseline separation by homemade monolith.

• It could be used in active ingredients of Chinese herbal medicine notoginseng by HPLC.

• A promising separation method for small molecules in actual sample.

Abstract

A novel porous monolithic column was synthesized via in situ free-radical polymerization within a stainless steel column (50 × 4.6 mm i.d.) by using triallyl isocyanurate and methyl methacrylate as co-monomers, ethylene glycol dimethacrylate as a crosslinking agent, 1,4-butanediol, 1- dodecanol, and N, N-dimethylformamide as tri-porogens, and azobisisobutyronitrile as an initiator. The optimized monolithic columns were investigated by scanning electron microscopy, a nitrogen absorption-desorption apparatus, and mercury intrusion porosimetry. The effect of mobile phase composition and temperature on chromatographic behavior was studied in detail as well. The results showed that homemade monolithic columns not only have relative uniform porosity structure, low back pressure, and high thermal stability, but also show high column efficiency, the theoretical plates number of the new polymer monolithic column exceeded 31,000 plates m⁻¹. Good resolution was achieved for HPLC separation of small molecules, including polycyclic aromatic hydrocarbons, isomers of phenols, and the Chinese herbal medicine notoginseng. Methodological validation of six drugs in notoginseng show that correlation coefficients of the linear regressions were in the range of 0.9989–0.9997; the value of relative standard deviation for precision were in the range of 1.37–4.21% for intra-day and 0.34–5.31% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 100.57–102.51%, 95.18–102.68%, 101.61–102.82%, 100.94–102.40%, 100.99–103.15% and 99.49–103.91% for the six drugs in order, respectively. Taking aromatic hydrocarbons as samples, the relative standard deviation values of the run-to-run (n = 5) and column-to-column (n = 5) based on peak area were < 2.01% and 2.39%, and two RSDs based on retention time were < 2.56% and 2.23%, respectively. The result showed that the polymer monolithic column has good separation ability and high permeability for small molecular substances.

Introduction

The medicinal value of traditional Chinese medicine has received more and more attention. How to separate effective medicinal ingredients from traditional Chinese medicine has become a hot work in the field of analysis. For example, Panax notoginseng is a species of ginseng in the Araliaceae, which has the effect of promoting blood circulation, relieving phlegm, and phlegm and blood stasis [1]. Notoginseng contains a variety of ginsenoside active ingredients, such as ginsenosides R_g1 and R_b1 and notoginsenoside R₁. Notoginsenoside R₁ distends blood vessels, inhibits platelet aggregation, is hypolipidemic, and has other pharmacologic effects. Ginsenosides R_b1 and R_g1 have a good effect on learning and memory function in addition to ginsenoside R_e and ginsenoside R_d. C18 column was generally used for the separation of notoginseng, but such column had the disadvantages of long analysis time and large solvent consumption. In recent years, the monolithic column had often been used as a main means of separation of traditional Chinese medicine [2], [3], [4], [5]. Monolithic columns were prepared by in situ radical polymerization as the stationary phase of high performance liquid chromatography (HPLC) for several decades [6], [7], [8], [9], [10]. Because monolithic columns prepared by this method have the advantages of low back pressure, fast mass transfer, high sample loading, and ease of preparation, in situ radical polymerization technology has become the most commonly used method for preparing monoliths [11], [12], [13]. At the same time, because of the low mesopore volume and irregularity of the structure [14], [15], poor column efficiencies appeared in this reaction. Several methods have been used to overcome the deficiencies, such as using different polymerization methods [16], [17], [18], applying new crosslinkers [19], and adding nanomaterials [20], [21]. In situ radical polymerization technology is also a good way to improve the efficiency of the column by reducing the mass transfer distance. The same materials were used to make different columns in 4.6-mm steel columns and 150-μm capillaries, and the efficiency of capillary columns (1.25 × 10⁵ plates m⁻¹) was 15 times higher than steel columns (8.32 × 10³ plates m⁻¹) [18]; however, low capacity and poor reproducibility becomes the most prominent disadvantage of the capillary monolithic column. To obtain high sample loading and high stability, people focused more attention on the 4.6 mm i.d. steel column. In addition, to improve the homogeneity of the structure and enhance column efficiency, combined monomers have become a common method [22], [23], [24], [25].

Trially isocyanurate (TAIC) is one of the polyfunctional monomers and has a thermally stable triazine ring and three allyl groups, and thus has many advantages, such as thermal stability, mechanical stability, hydrolysis resistance, and resistance to drying [21], [22]. TAIC is widely used as a crosslinking agent for biodegradable polymers to improve thermal stabilities and mechanical properties [26], [27]. With application of TAIC in the chromatographic field, Sun et al. [28]. had further convinced the potential use of TAIC in the development of novel hydrophilic polymer monoliths. Lin et al. [29] also contributed outstanding work in the application of TAIC, with the highest column efficiency reaching 2.66 × 10⁵ plates m⁻¹, but that work was implemented in capillary columns, so the injection volume was limited. Due to the stable chemical properties, methyl methacrylate was often used for the synthesis of organic polymers. In this study, a novel TAIC-co-methyl methacrylate (MMA) polymeric monolith was prepared in the 50 mm × 4.6 mm stainless-steel column for the separation of small molecules, including anilines, polycyclic aromatic hydrocarbons, and isomers of phenols compounds. Furthermore, the optimized TAIC-co-MMA-co-EDMA monolithic column was assessed by separating six main drugs from the Chinese herbal medicine notoginseng. Furthermore, the methodological validation showed that presented monolithic column exhibited good precision, good recovery and good repeatability. The novel monolithic column could become a potential tool as the stationary phase for analyzing small molecules based on its easily preparation, short analysis time and high efficiency.