Preparation of rosin-based polymer microspheres as a stationary phase in high-performance liquid chromatography to separate polycyclic aromatic hydrocarbons and alkaloids

2.1 Experimental principles

Figure 1 illustrates the principles and schematic of the preparation of rosin-based polymer microspheres. Methyl methacrylate (MMA), ethyleneglycol dimethacrylate (EGDMA) cross-linked with AAEGA effectively to form a macromolecule with network structure, and the three-ring phenanthrene skeleton structure with high rigidity in rosin was introduced into the polymer chain through free radical polymerization by using azodiisobutyronitrile (AIBN) as initiator. The AAEGA was provided by the Guangxi Key Laboratory of Forest Products Chemistry and Engineering (Nanning, China).

Figure 1

Principles of the synthesis of rosin-based polymer microspheres.

2.2 Preparation of rosin-based polymer microspheres

(i) MMA (10 mmol), EGDMA (4 mmol), AAEGA (10 mmol), chloroform (30 mL), cetanol, polypropylene glycol, and AIBN were mixed together and ultrasonicated for 10 min as the oil phase (dispersion phase). The amounts of cetanol, polypropylene glycol, and AIBN were 5%, 2%, and 0.3% of the total mass of MAA, EGDMA, and AAEGA, respectively. (ii) Second, 40 g of the dispersion phase was added into the aqueous phase (400 mL) containing 2% (w/w) polyvinyl alcohol (PVA) in deionized (DI) water. (iii) Crude oil-in-water emulsions were poured into the membrane emulsification setup (FM0210/500M, Senhui company, Beijing, China). The emulsions were homogenized by squeezing them through a membrane (SPG) with an average pore size of 8 μm under the pressure of 1.0 bar and collected using a beaker. (iv) The emulsions (droplets) were heated to 75°C and maintained for 2 h, raised to 80°C for 2 h, and aged at 95°C for 2 h. The copolymers were then treated through sedimentation and centrifugation. (v) Finally, porogens, unreacted monomers and assistant agents were removed through Soxhlet extraction with acetone, ethyl acetate, and ethyl alcohol, respectively. The final products (microspheres) were obtained after washing with DI water for removing PVA.

2.3 Characterization

Scanning electron microscopy (SEM; SUPPRA 55 Sapphire, Carl Zeiss, Germany) was conducted to investigate the sample’s morphology. Particle sizes and distributions of microspheres were measured by laser granulometry (Mastersizer 3000, Malvern, UK). Specific surface area and porosity were examined through N₂ adsorption experiments by using a surface area and pore size analyzer (ASAP 2020, Micromeritics, Atlanta, USA) at 77 K. Surface areas were calculated by the Brunauer–Emmett–Teller (BET) equations. Pore volume, average pore size, and pore size distribution were calculated using the Barrett–Joyner–Halenda equations. The thermal stabilities of the microspheres were characterized by thermogravimetric analysis (TGA; STA449F3, Netzsch-Gerätebau GmbH, Germany). The samples (2–4 mg) were placed in a standard crucible (Al₂O₃) and heated under nitrogen from 30°C to 800°C at a rate of 10°C min⁻¹ to obtain thermogravimetric and differential thermogravimetric curves.

2.4 Chromatographic evaluation of polymers

The copolymers were packed into a stainless-steel chromatographic column (250 mm × 4.6 mm i.d.) with the wet method of packing column by using a slurry packer (CP–24, Scientific Systems company, Science Park Road State College, PA, USA) under the pressure of 3250 psi with chloroform–methanol (2.6:1, v/v) as the slurry solvent. The prepared rosin-based polymer column was connected to an HPLC (LC-6AD, Shimadzu, Japan) system and flushed with 4 L of methanol at 0.1 mL/min.

Chromatographic experiments were performed on an HPLC (LC-6AD, Shimadzu, Japan) system with a UV detector (Shimadzu, Japan). Unless otherwise stated, separations were conducted at 30°C using methanol as mobile phase at a flow rate of 0.3 mL/min. In brief, 20 μL of each sample was injected, and the wavelength of the UV detector was set at 254 nm. R_S, the resolution, is expressed as follows:

Where t_R1 and t_R2 is the is the retention time of Peak 1 and Peak 2, respectively, and W₁ and W₂ is the peak width of Peak 1 and Peak 2, respectively.

The mixed solution of PAHs was prepared, in which 1 L of solution (methanol as the solvent) contains benzene, naphthalene, fluorine, pyrene, and benzo(e) pyrene at 0.05, 0.005, 2.5×10^-4, 2.5×10^-3, and 2.5×10^-3 mol, respectively. The mixed solution of THO and CAF (CPT and HCPT) was prepared in methanol with concentrations of 1.25×10^-4 mol L^-1.

3.1 Characterization of rosin-based polymer microspheres

In Figure 2a, microspheres with a narrow pore distribution exhibit unimodal particle size distribution profiles ranging from 1 μm to 10 μm, which meets the requirement for particle sizes of the liquid chromatographic stationary phase. The thermal weight loss of the polymer microspheres was characterized by TGA, and the results are presented in Figure 2b The decomposition temperature for polymer microspheres was about 280°C. The temperature at the highest decomposition rates was about 422°C, and decomposition was completed at about 480°C. Thus, TGA demonstrated that the microspheres possessed good thermal stability. Figure 2c shows that the microspheres displayed typical-IV isotherms with well-defined H₁ hysteresis loops, implying a uniform mesoporous structure (Figure 2d) containing cylindricalpores. The BET surface area, pore volume, average pore diameter, and average pore size of the microspheres are 67.4723 m²/g, 0.2026 cm³/g, and 6.8192 nm, respectively. The SEM micrographs of the microspheres magnified to 1 and 5 million times are shown in Figures 2e and 2f As shown in Figure 2e, the rosin-based polymers are regular spheres with a rough and porous surface, and the pores in the microspheres have formed a 3D network (Figure 2f)

Figure 2

Characterization of microspheres. (a) Particle size distribution of microspheres. (b) Weight loss of microspheres. (c) N₂ adsorption–desorption isotherm of microspheres. (d) Pore size distribution of microspheres. (e) SEM image of microspheres which was magnified to one million times. (f) SEM image of microspheres which was magnified to five millions times.

3.2 Chromatographic characterization of polymers

Figure 3a shows that five PAHs were well separated in the rosin-based polymer column with the resolution (Rs) (the two components which elute closest to each other) of 6.088 ± 0.006, 5.759 ± 0.017, 7.019 ± 0.020, and 8.584 ± 0.063. Figure 3b shows a linear relationshipbetween the carbon numbers of PAHs and the ln k, which followed the equation of the carbon number rule:

Figure 3

(a) Separation of mixed samples of PAHs in the chromatographic column. (b) The carbon number rule plots obtained by plotting ln k and n_c. (c) Separation of mixed samples of THO and CAF in the chromatographic column. (d) Separation of mixed samples of CPT and HCPT in the chromatographic column.

Where k is the retention factor, and n_c is the homologue carbon atom number (18). This linear fitting equation is as follows:

Basing on the equation and Figure 3a, we can discover information as follows: (1) According to the equation, we can infer the retention value of other PHAs. (2) All of the five PAHs can enter into the hole interior of polymers; otherwise, the relationship shows an fold line (19). (3) When the sample enters the hole interior, the mass transfer resistance of the molecules changes according to molecular weights. The diffusion rate quickens when the molecular weight decreases (20). (4) The rosin-based polymer microspheres can be used in the separation of organic compounds with similar structure but different molecular sizes.

As shown in Figure 3c, THO and CAF obtained a good separation (Rs = 4.6 ± 0.005). The structure of the CAF has one more methyl group than THO (Figure 4), which increases the mass transfer resistance, leading to the prolonged retention time. Moreover, the N-H bonds of the THO can form hydrogen with the methanol, which also helps the separation.

Figure 4

Structures of CPT, HCPT, THO and CAF.

As shown in Figure 3d, the Rs of CPT and HCPT is 2.245 ± 0.049. CPT and HCPT belong to polycyclic alkaloid; hence, their retention times were longer than those of THO and CAF. The molecular structure of HCPT is different than that of CPT due to a hydroxyl (Figure 4), and the extra hydroxyl increased the polarity of HCPT. When usingmethyl alcohol as the mobile phase, the influence of the polarity of the sample molecules plays a major role in the chromatographic separation. As a result, the retention time of HCPT was shorter than that of CPT.