A benzenesulfonic acid-modified organic polymer monolithic column with reversed-phase/hydrophilic bifunctional selectivity for capillary electrochromatography

Abstract

Here, a styrene-based polymer monolithic column poly(VBS-co-TAT-co-AHM) with reversed-phase/hydrophilic interaction liquid chromatography (RPLC/HILIC) bifunctional separation mode was successfully prepared for capillary electrochromatography by the in situ polymerization of sodium p-styrene sulfonate (VBS) with cross-linkers 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AHM) and 1,3,5-triacryloylhexahydro-1,3,5-triazine (TAT). The preparation conditions of the monolith were optimized. The morphology and formation of the poly(VBS-co-TAT-co-AHM) monolith were confirmed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). The separation performances of the monolith were evaluated systematically. It should be noted that the incorporation of VBS functional monomer can provide π−π interactions, hydrophilic interactions, and ion-exchange interactions. Hence, the prepared poly(VBS-co-TAT-co-AHM) monolith can achieve efficient separation of thiourea compounds, benzene series, phenol compounds, aniline compounds and sulfonamides in RPLC or HILIC separation mode. The largest theoretical plate number for N,N′-dimethylthiourea reached 1.7 × 10⁵ plates/m. In addition, the poly(VBS-co-TAT-co-AHM) monolithic column showed excellent reproducibility and stability. This novel monolithic column has great application value and potential in capillary electrochromatography (CEC).

Graphical abstract

Highlights

• •A novel functional monomer VBS was explored as monolithic column stationary phase.
• •The prepared column showed excellent separation performances toward various compounds.
• •The maximum column efficiency can be up to 170,000 plates/m.
• •Satisfactory reproducibility and stability were obtained.

1. Introduction

Capillary electrochromatography (CEC) is a separation technique which combines the high separation efficiency of capillary electrophoresis (CE) [1] with high selectivity of high performance liquid chromatography (HPLC) [[2], [3], [4]]. CEC has the advantages of being time-saving, less reagents consumption, and good compatibility with mass spectrometer [5], and thus has been used in many fields, such as food [6], medical [7,8] and environmental analysis [9,10]. Among the various types of CEC columns, monolithic columns have gained great attention because of their excellent chemical and physical stability, high phase ratios, continuous porous bed, and good separation selectivity [[11], [12], [13]]. Presently, monolithic columns can be classified into organic polymer monoliths, silicon-based monoliths [[14], [15], [16]], and organic silica hybrid monoliths [17,18]. Silicon-based monolithic columns are mainly fabricated directly in capillary tubes, and subsequently surfaces are modified by silanization reaction to fix the stationary phase with the interactive moieties [14]. The monolithic silica column has strong mechanical stability, good tolerance to organic solvents, and is suitable for the separation of various substances. However, some problems include narrow pH application range, and being easy to dry and crack and easy to be destroyed under high pH conditions. Compared with silicon-based monoliths, organic polymer monolithic column has the advantages of simple preparation method, good acid-base tolerance, diverse surface chemical properties of stationary phases, adjustable pore structure, fast mass transfer rate, good separation selectivity, and high separation efficiency [12,[19], [20], [21], [22]]. It is a kind of widely studied monolithic column.

Recently, styrene-based materials have been widely used in capillary monolithic columns [23]. Studies showed that styrene compounds have good cross-linking degrees in the preparation process and good stability in the wide pH range [24,25]. Up to now, some styrene or ethylvinylbenzene polymers such as poly(styrene-divinylbenzene) (PS-DVB) have been applied to CEC [26,27] and reversed-phase (RP) [28] or ion exchange chromatography [29] mode HPLC. Schmitt et al. [29] reported the resins, prepared by carboxylation of highly crosslinked monodisperse PS-DVB, were used to develop mixed-acidic cation-exchange (MCX) columns with both strong cation-exchange (SCX) and weak cation-exchange (WCX) acidic functional groups for the separation of amino acids. Obviously, these styrene-based materials with high hydrophobic property can exhibit satisfactory separation performance toward hydrophobic analytes. However, their application in hydrophilic interaction liquid chromatography (HILIC) mode for the separation of highly polar substances is limited. Therefore, if styrene is used in HILIC, it will be useful to introduce a functional group that can provide sufficient hydrophilicity and effectively shield the surface of hydrophobic aromatic groups [30,31].

In this work, sodium p-styrene sulfonate (VBS) was selected as a functional monomer. The benzene ring groups in VBS can provide π−π conjugation and hydrophobic interaction which benefit the separation of hydrophobic compounds in RPLC mode. In addition, the combination of sulfonic acid groups and hydrophilic crosslinking agents can enhance their hydrophilicity; thus the separation of polar compounds can be achieved in HILIC mode. As a proof-of-concept, the separation performance of the poly(VBS-co-TAT-co-AHM) monolithic column was evaluated by separating different polar analytes in RPLC/HILIC modes. The prepared columns showed exceptional separation performance toward thiourea compounds, alkylbenzenes, phenol compounds, aniline compounds, and sulfonamides. And the obtained largest number of theoretical plates could reach 1.7 × 10⁵ plates/m (for N,N′-dimethylthiourea). This VBS-based monolithic column provides a new route for the separation of different polar analytes.

2. Experimental

2.1. Reagents and instrumentation

VBS and 3,5-dimethylaniline were obtained from Mackin Biochenical Co., Ltd. (Shanghai, China). 3-(trimethoxysilyl)propylmethacrylate (γ-MAPS), 1-dodecanol, ethylbenzene, n-propylbenzene, n-butylbenzene, and 2,6-diethylaniline were bought from Aladdin Reagent Factory (Shanghai, China). N-methylthiourea and sulfaguanidine were obtained from Energy Chemical (Shanghai, China). N,N′-dimethylthiourea was purchased from J&K Company (Beijing, China). 4-methylaniline was bought from Shanghai Jinshanting New Chemical Reagent Factory (Shanghai, China). Ammonium persulfate ((NH4)₂S₂O₈), N,N-dimethylformamide (DMF), toluene, thiourea, phenol, pyrocatechol, phloroglucinol, sodium hydroxide, diethylene glycol, and hydrochloric acid were procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AHM) and 1,3,5-triacryloylhexahydro-1,3,5-triazine (TAT) were bought from TCI (Shanghai, China). Sulfadiazine and sulfamethazine sodium salt were bought from Tianjin Xien Biochemical Technology Co., Ltd. (Tianjin, China). Acetonitrile and methanol were got from Tedia (Cincinnati, Ohio, USA). Fused silica capillary (100 μm i.d., 365 μm o.d.) was obtained from Ruifeng Chromatographic Devices (Yongnian, Hebei, China). Other reagents of analytical grade used in experiments were from commercial sources.

All our experimental results were from Agilent 7100 CE system (Waldbronn, Germany) which was equipped with an auto-sampler, a temperature-controlled column compartment and a diode array detector. The data acquisition was completed by a chromatographic workstation. The Fourier transform infrared spectroscopy (FT-IR) was scanned and recorded on a Thermo Nexus 470 FT-IR system (Waltham, MA, USA). The morphology of the monolithic columns was characterized by a Carl Zeiss Ultra Plus Field Emission scanning electron microscope (FESEM; Oberkochen, Germany) at an accelerating voltage of 5.0 kV. Deionized water was purified by a Milli-Q system (Waltham, MA, USA). A precision mechanical syringe pump (LSP04-1A, Longer Pump Company, Baoding, China) was used to inject the corresponding solution into the capillary. The backpressure of monolithic columns was got from a Shimadzu LC-20AD NANO pump (Tokyo, Japan).

2.2. Preparation of poly(VBS-co-TAT-co-AHM) monolithic column

First, the fused-sillica capillary was washed with 1.0 M NaOH (1 h), H₂O (30 min), 1.0 M HCl (1 h), H₂O (30 min), and methanol (1 h), sequentially. And then it was dried by N₂ stream. The capillary pretreatment process was completed. After a solution of methanol and γ-MAPS (1:1, V/V) was injected into the preconditioned capillary and both ends were sealed up, the capillary was immersed in a water bath at 45 °C for 12 h to modify γ-MAPS. After removing it from the water bath, the capillary was flushed with methanol to remove the residuals and dried by N₂ stream for further use. The procedure of the monolithic column preparation is shown in Fig. 1. Monomers (VBS, AHM, TAT) and porogens (diethylene glycol, 1-dodecanol, DMF) were weighed according to the mass fraction shown in Table 1, mixed with 2.4 mg (NH₄)₂S₂O₈, and then sonicated to form a homogeneous copolymerization solution. The total mass of the copolymer solution was 918.4 mg. Each compound’s mass fraction in copolymerization solution can be easily calculated via mass fraction in monomer/porogen and the ratio of monomers and porogens (e.g., mass fraction of VBS in No. 1 column = 25% × 10% = 2.5%, so VBS in copolymerization solution in No. 1 column = (918.4–2.4) mg × 2.5% = 22.9 mg). After cutting the capillary modified by γ-MAPS into corresponding lengths, a certain length of copolymerization solution was injected into the capillary. In order to complete the combination of polymer and capillary, the capillary was immersed in a water bath at 80 °C for 24 h with both ends sealed. Finally, the obtained poly(VBS-co-TAT-co-AHM) columns were rinsed by methanol overnight. At the edge of the monolithic continuous bed, a detection window (about 3 mm) was made for the instrumentation experiment. The total length of the obtained poly(VBS-co-TAT-co-AHM) monolithic column was 38.0 cm and the effective length was 27.0 cm.

Fig. 1.

Procedure for the preparation of the poly(VBS-co-TAT-co-AHM) monolithic column. VBS: sodium p-styrene sulfonate; AHM: 3-(acryloyloxy)-2-hydroxypropyl methacrylate; TAT: 1,3,5-triacryloylhexahydro-1,3,5-triazine.

Table 1.

Compositions of the polymerization mixtures for the poly(VBS-co-TAT-co-AHM) monoliths.

Column NO.	Monomers/porogens (%, m/m)	Monomers			Porogens			Permeability (10−13, m2)	Homogeneity
		VBS (%, m/m)	AHM (%, m/m)	TAT (%, m/m)	Diethylene glycol (%, m/m)	1-dodecanol (%, m/m)	DMF (%, m/m)
1	10:90	25	67.5	7.5	40	50	10	4.05	Poor
2	15:85	15	76.5	8.5	40	50	10	3.21	Better
3	15:85	25	67.5	7.5	40	50	10	3.45	Better
4	15:85	35	58.5	6.5	40	50	10	2.23	Best
5	20:80	25	67.5	7.5	40	50	10	–	Best
6	15:85	25	71.3	3.7	40	50	10	3.16	Better
7	15:85	25	67.5	7.5	40	50	10	3.45	Better
8	15:85	25	63.8	11.2	40	50	10	3.27	Better

The permeability was characterized by backpressure. Backpressure was obtained with methanol as the mobile phase at 0.3 μL/min; the length of the capillary was kept at 38 cm (monolithic length 27 cm).

VBS: sodium p-styrene sulfonate; AHM: 3-(acryloyloxy)-2-hydroxypropyl methacrylate; TAT: 1,3,5-triacryloylhexahydro-1,3,5-triazine; DMF: N,N-dimethylformamide.

2.3. Sample solutions and CEC experiment

Standard solutions for all analytes were made by dissolving the substance in methanol. The concentrations of toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, p-toluidine, 3,5-dimethylaniline, and 2,6-diethylaniline standard solutions were 4 mg/mL. The standard solutions of thiourea, N-methylthiourea, and N,N′-dimethylthiourea were 1 mg/mL, and the standard solutions of phenol, pyrocatechol, and phloroglucinol were 5 mg/mL.

The mobile phase consisted of acetate buffer and appropriate contents of acetonitrile. The acetate buffer was prepared in five gradients of concentration from 5 to 25 mM, and its pH values ranged from 3.0 to 10.0 by adding CH₃COOH solution or NH₄OH solution (CH₃COOH–CH₃COONH₄ for pH 3.0–6.0, NH₄OH–CH₃COONH₄ for pH 7.0–9.0). All the solutions were stored at 4 °C for further use.

3. Results and discussion

3.1. Optimization of poly(VBS-co-TAT-co-AHM) monolithic column

In the process of column preparation, permeability and homogeneity are significant factors that determine the column efficiency and stability of the whole separation, which are closely related to the proportion of functional monomers, crosslinkers and porogens in polymer solution. Good permeability and homogeneity are the prerequisites for the application of the newly-formed monolithic columns. Therefore, crosslinkers, monomers and porogens are investigated to achieve integrated optimization.

A crosslinker is a substance that binds multiple molecules to form a chain or mesh structure through the chemical bonds. The proportion of crosslinking agents greatly affects the polymerization reaction on capillary wall, so it was adjusted first to reach better permeability and homogeneity in monolithic column. Throughout the experiment, when the ratio of monomers to porogens was 10:90 (m/m), the homogeneity was too poor to meet the requirement of electrochromatographic experiment. Therefore, TAT, with three cross-linkable sites, was added to adjust the column morphology. It could show better dimensional stability and increase the cross-linking density of the polymer [32].

Although porogens do not participate in the polymerization crosslinking reaction, it separates the copolymers to form the porous structure to affect the permeability directly, so the effect of the ratio of monomers on porogens was the following sectors to be investigated under three proportions of 10:90, 15:85, and 20:80 (m/m). As shown in Table 1, when the ratio of porogens decreased from 90% to 85%, the homogeneity of the column improved while permeability declined. Homogeneity continued to increase as porogens were reduced to 80%, but the back pressure rose sharply due to the reduction of the through-pores in the capillary, causing a total block for mobile phase. Combined with the discussion above, the column prepared in 85% of the porogens behaved best in comprehensive performance.

Among the substances that act as monomers, the ratio of functional monomers to cross-linking agents should also be considered when the ratio of monomers to porogens fixed to be 15:85. According to Table 1, when the ratio of VBS was 15% and 25%, there was no significant difference in overall permeability and homogeneity. When the weight percentage of VBS was added to 35%, although the homogeneity was improved to a certain extent, the column permeability was greatly reduced. So CEC experiments were used to further investigate which monolithic columns with a VBS ratio of 15% or 25% (No. 2 and No. 3 columns in Table 1) had better separation efficiency. In RP mode, the analytes comprising thiourea, toluene, ethylbenzene, propylbenzene, and butylbenzene were separated as shown in Fig. 2A. Obviously, the resolution of No. 3 column is greater than that of No. 2 column. While in the hydrophilic mode, the separation of toluene, thiourea, N-methylthiourea, and N,N′-dimethylthiourea is shown in Fig. 2B. The separation performance of No. 3 column was better than that of No. 2 column as well with smaller tailing factor attributed to stronger hydrophobic interactions. Therefore, 25% proved to be the best ratio of VBS.

Fig. 2.

The chromatographic performance of No. 2 and No. 3 monolithic columns in Table 1 with different ratios of copolymerization solution. Experimental conditions: (A) Mobile phase, 50% methanol in pH 9.0 20 mM NH₄AC buffer; applied voltage, +15 kV; electrokinetic injection, +5 kV × 5 s; detection wavelength, 200 nm. Peaks: 1, thiourea; 2, toluene; 3, ethylbenzene; 4, propylbenzene; and 5, butylbenzene. (B) Mobile phase, 90% methanol in pH 9.0 20 mM NH₄AC buffer; applied voltage, +15 kV; electrokinetic injection, +5 kV × 5 s; detection wavelength, 254 nm. Peaks: 1, toluene; 2, N,N′-dimethylthiourea; 3, N-methylthiourea; and 4, thiourea.

When the composition of monomers is further optimized, the ratio of the two cross-linking agents should be adjusted to change the polarity of the monolithic columns. According to Table 1, columns were characterized by scanning electron microscopy (SEM), as shown in Fig. 3 for No. 6, No. 7, and No. 8 columns, and as shown in Fig. S1 for No. 1 to No. 5 columns. It can be seen that the spherical clusters of No. 7 column (Fig. 3E) were significantly best aggregated and uniformed, possessing thorough-pores channel and small spherical units which could provide effective surface area for specific analytes to selectively bind. Therefore, the optimal ratio for establishing the overall column monomer was VBS (%, m/m):AHM (%, m/m):TAT (%, m/m) = 25:67.5:7.5.

Fig. 3.

Scanning electron microscope (SEM) of No. 6, No. 7, and No. 8 columns in Table 1. (A) No. 6 column; (B) No. 7 column; (C) No. 8 column; (D) No. 6 column; (E) No. 7 column; (F) No. 8 column.

3.2. Characterization of poly(VBS-co-TAT-co-AHM) monolithic column

SEM was used to observe the morphology of the monolithic column. As shown in Fig. 3, the polymer formed into spherical units and aggregated into clusters, the spherical units were small and uniformly arranged, and pore channels were formed between different spherical clusters, while the polymer was concentrated in the capillary.

FT-IR was used to characterize the formation of poly(VBS-co-TAT-co-AHM) monolith As shown in Fig. S2, the absorption peak at 3,360 cm⁻¹ was related to the stretching vibration of the hydroxyl formed by hydrolysed VBS, while the absorption at 1,123 and 1,055 cm⁻¹ was consistent with the characteristic absorption of the sulfonic acid group. The above characteristic absorption peaks proved the successful modification of the functional monomer VBS on the capillary, verifying the successful construction of the poly (VBS-co-TAT-co-AHM) monolithic column.

3.3. Evaluation of electroosmotic flow (EOF)

In CEC, EOF is the main driving force of mobile phase. In the poly (VBS-co-TAT-co-AHM) monolithic column, the sulfonic acid groups in stationary phase can generate an EOF from anode to cathode. The calculation of electroosmotic flow can be derived from the following formula: EOF = (L_e × L_t)/(V × t₀). L_e and L_t are the effective length and total length of the monolithic column, respectively. V is the applied voltage, and t₀ is the migration time of EOF marker toluene [33]. Here, the influence of the pH value, the concentration of acetate buffer and acetonitrile content on EOF were investigated under both hydrophilic and reversed phase conditions, where thiourea and toluene were respectively to be the unretained marker.

As shown in Figs. S3A−C for RP mode, as the pH increased in the range of pH 6.0–10.0, the EOF increased as well. But the EOF dropped mildly when the concentration of acetate buffer and acetonitrile content increased from 5 to 25 mM and 35% to 60%, respectively. As shown in Figs. S3D−F for HILIC mode, EOF increased along with the increase of pH value. Meanwhile, as the concentration of acetate buffer increased from 5 to 25 mM, and acetonitrile content increased from 75% to 95%, EOF dropped obviously. With the increase of pH, more sulfonic acid groups in stationary phase were dissociated, leading to an increase of electrical double layer and Zeta potential, triggering the enhancement in EOF. As the concentration of buffer increased, the ion concentration and repulsive force increased, causing a thinner electric double layer and a smaller Zeta potential, resulting in a decrease in EOF. While the content of organic phase increased, the conductivity in mobile phase gradually decreased, and the cooperation of εr/η of acetonitrile and the net negatively charged ionizable silanol groups resulted in a reduction in EOF [3,12].

3.4. HILIC and RP chromatographic performance

The poly (VBS-co-TAT-co-AHM) monolithic column constructed forward mainly possesses three kinds of forces to achieve the selective separation of analytes, namely, π−π conjugation, hydrophilic interaction, and cation exchange interaction. Therefore, both HILIC and RP separation modes were introduced to study the chromatographic performance of the poly (VBS-co-TAT-co-AHM) monolithic column.

3.4.1. HILIC chromatographic performance

The sulfonic acid group contained in functional monomer VBS has a certain polarity and can provide hydrophilic interaction and ion exchange interaction when dissociating at a specific pH value. Under the condition of high organic phase, thiourea substances such as thiourea (logP 0.285), N-methylthiourea (logP 0.700), and N,N′-dimethylthiourea (logP 1.020) were selected as analytes to evaluate the hydrophilicity of the column. As shown in Fig. S4. The elution order of these thiourea substances was consistent with their polarity from low to high. The baseline separation was achieved within 9 min when the mobile phase was made up of 80% acetonitrile and 20% acetate buffer (20 mM, pH 8.0). When the acetonitrile content became greater than 75%, the non-polar compound toluene was basically not retained. Therefore, it can be used as unretained marker to research the relationship between acetonitrile content and retention factor (k) of these three test compounds. The formula: k =(t_r − t₀)/t₀ was used to calculate the retention factor, where t_r and t₀ denote the retention time of thiourea substances and toluene, respectively. In Fig. S5, while the acetonitrile content increased from 75% to 95%, the retention factor of the three polar substances increased significantly, proving that the separation of polar substances by poly (VBS-co-TAT-co-AHM) monolithic column was a typical hydrophilic chromatographic separation mechanism.

3.4.2. RP chromatographic performance

The functional monomer VBS can provide π−π conjugation and hydrophobic interaction to achieve efficient separation of compounds with low polarity when organic phase ratio is relatively low. Thiourea was used as no-retention marker, whereas toluene (logP 2.72), ethylbenzene (logP 3.23), propylbenzene (logP 3.73), and butylbenzene (logP 4.24) were chosen as target analytes to evaluate the performance of the column in RP chromatography. As shown in Fig. S6, the four analytes eluted in the order of toluene, ethylbenzene, propylbenzene, and butylbenzene, showing a typical RP characteristic. The baseline separation was obtained within 17 min in the mobile phase of 35% acetonitrile in the acetate buffer (20 mM, pH 8.0). Fig. S7 shows the relationship between retention factor (k) and acetonitrile content. With the increase of acetonitrile content from 20% to 45%, the retention factor of analytes decreased, demonstrating that the separation of the four aromatic substances was a typical RP separation mechanism.

3.5. Reproducibility and stability of the monolithic column

The reproducibility and stability of poly(VBS-co-TAT-co-AHM) monolithic columns were evaluated. Using the method in Section 2.2 and the optimal ratio in Section 3.1, different batches of poly(VBS-co-TAT-co-AHM) monolithic columns were prepared. There were three columns in each batch, and a total of three batches were prepared. Under the same experimental conditions, reproducibility and stability of poly(VBS-co-TAT-co-AHM) monolith were tested. In Table 2, the relative standard deviations (RSDs) between columns were calculated by the retention time of four benzene compounds. The RSDs of intra-day (n = 5), inter-day (n = 5), column-to-column (n = 3), and batch-to-batch (n = 3) were in the range of 1.27%–1.55%, 1.04%–3.15%, 2.26%–3.44%, and 3.86%–5.88%, respectively. Besides, after 60 consecutive runs in CEC mode, the chromatographic separation performance of the monolithic column did not change significantly. The results proved that the poly(VBS-co-TAT-co-AHM) monolithic columns were of great reproducibility and stability.

Table 2.

Reproducibility and stability of the monolithic column.

Analytes	Retention time (%RSD)
	Intra-day (n = 5)	Inter-day (n = 5)	Column-to-column (n = 3)	Batch-to-batch (n = 3)
Toluene	1.36	1.04	2.41	3.86
Ethylbenzene	1.27	2.09	2.26	5.32
n-propylbenzene	1.46	2.52	2.35	5.45
n-butylbenzene	1.55	3.15	3.44	5.88

RSD: relative standard deviation.

Experimental conditions: mobile phase, 70% acetonitrile in pH 8.0 20 mM ammonium acetate buffer; applied voltage, +15.0 kV; electric injection, +5 kV × 3 s; detection wavelength, 254 nm.

3.6. Application of monolithic column

3.6.1. Separation of phenolic substances

The prepared poly(VBS-co-TAT-co-AHM) monolithic column was applied to separate phenolic substances. Three phenolic substances consisting of phloroglucinol (logP 0.004), hydroquinone (logP 0.620) and phenol (logP 1.540) were used as analytes to test the columns’ separation performance. The electrochromatographic separation results are exhibited in Fig. 4. The analytes were eluted according to the polarity as the sequence: phenol < hydroquinone < phloroglucinol, and baseline separation of three phenolic compounds was achieved within 11.5 min.

Fig. 4.

Separation of phenol, hydroquinone, and phloroglucinol. Experimental conditions: mobile phase, 80% acetonitrile in pH 9.0 10 mM ammonium acetate buffer; applied voltage, +15.0 kV; electric injection, +5 kV × 3 s; detection wavelength, 200 nm. Peaks: 1. phenol; 2. pyrocatechol; 3. phloroglucinol.

3.6.2. Separation of alkaline substances

Three aniline substances, namely, 4-methylaniline (logP 1.533), 3,5-dimethylaniline (logP 1.972) and 2,6-diethylaniline (logP 2.650), were successfully separated on poly(VBS-co-TAT-co-AHM) monolithic column. The result is shown in Fig. 5. The poly(VBS-co-TAT-co-AHM) monolithic column showed good separation performance toward the above aniline compounds. In terms of the N,N′-dimethylthiourea, the largest theoretical plate number reached 1.7 × 10⁵ plates/m. The successful separation of these three aniline substances was mainly based on the hydrophilic interaction and the electrostatic interaction.

Fig. 5.

Separation of three aniline substances. Experimental conditions: mobile phase, 80% acetonitrile in pH 8.0 20 mM ammonium acetate buffer; applied voltage, +15.0kV; electric injection, +5 kV × 3 s; detection wavelength, 254 nm. Peaks: 1. 2,6-diethylaniline; 2. 3,5-dimethylaniline; 3. 4-methylaniline.

3.6.3. Separation of sulfa drugs

Sulfonamides, an important kind of compounds with broad antibacterial spectrums and stable properties, have been used clinically for nearly 50 years. Hence, the development of efficient methods to determine sulfonamides is of great significance. Here, three sulfa drugs (sulfadiazine, sulfamethazine and sulfaguanidine) were used to evaluate the separation ability of the prepared monolithic column. In Fig. 6, when the chromatographic condition was that the volume ratio of acetonitrile to acetate buffer (20 mM, pH 7) was 90:10, baseline separation for three sulfa drugs with high resolution was achieved. The elution sequence was as follows: sulfamethazine < sulfadiazine < sulfaguanidine, consistent with the order of the hydrophilicity increase. The above results revealed that poly(VBS-co-TAT-co-AHM) monolithic column exhibited good separation selectively toward sulfonamides due to hydrophilic interaction and π−π interaction.

Fig. 6.

Separation of sulfadimidine, sulfadiazine, and sulfaguanidine. Experimental conditions: mobile phase, 90% acetonitrile in pH 7.0 20 mM ammonium acetate buffer; applied voltage, +15.0 kV; electric injection, +5 kV × 3 s; detection wavelength, 254 nm. Peaks: 1. sulfadimidine; 2. sulfadiazine; 3. sulfaguanidine.

4. Conclusions

In summary, VBS was first used as a functional monomer in monolithic column. Hence, a poly(VBS-co-TAT-co-AHM) monolithic column with RPLC/HILIC bifunctional separation mode was developed and applied in CEC successfully. Thanks to the π−π interaction and hydrophobic sites provided by benzene rings and the hydrophilic interaction and ion exchange interaction sites endowed by benesulfonic groups, the obtained poly(VBS-co-TAT-co-AHM) monolithic column could effectively separate a variety of compounds with different polarities in RPLC or HILIC chromatographic mode. In addition, the poly(VBS-co-TAT-co-AHM) monolithic column showed great reproducibility and stability. One monomer can bring multifunctional separation mode. This monolithic column has great application potential in the separation field.

CRediT author statement

Yikun Liu: Conceptualization, Methodology; Ning He: Investigation, Data Curation, Writing - Original draft preparation; Yingfang Lu: Investigation, Visualization; Weiqiang Li: Investigation, Writing - Original draft preparation; Xin He: Writing - Original draft preparation; Zhentao Li: Writing - Reviewing & Editing; Zilin Chen: Supervision, Funding acquisition, Project administration, Writing - Reviewing & Editing.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: