The preparation of a novel organic–inorganic hybrid monolithic column with sonication-assist and its application

Abstract

A novel hydroxyl functionalized organic–inorganic hybrid monolithic column for high performance liquid chromatography (HPLC) was synthesized viafree radical copolymerization with sonocation-assist, which could shorten the time of the sol–gel process. Vinyltrimethoxysilane (VTMS) was used as the monomer and vinyl ester resin was used as both the monomer and crosslinker. The conditions of preparation were investigated and the characteristics of the hybrid column were studied by SEM and Fourier transform infrared spectroscopy. The obtained column showed high permeability and low backpressure. The column was used to separate lysozyme from egg white in a short time (6 min) by HPLC, and benzene and its homologs were separated by the hybrid column. In addition, influences on the elution of lysozyme, such as the pH value and the buffer concentration, were studied. Additionally, the lysozyme separated by the hybrid column showed high biological activity, which was assayed by the method of turbidimetry.

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1 Introduction

Monolithic columns can be described as integrated continuous porous separation media for separation science. In the past decade, monolithic column have drawn our attention and applied widely in chromatographic separation. There are many advantages that monolithic columns possess, including easy preparation, versatility in surface modification, great permeability, and good peak capacity. These unique merits have made monolithic columns widely used in analytical separation science.^1,2

Basically, monolithic materials are divided into two groups: organic polymer and inorganic polymer. Organic polymer-based monolithic columns possess many advantages, such as good stability to pH. But there are also disadvantages, such as poor mechanical stability, which results in a short lifetime, and undesirable retention reproducibility in some cases.^3,4 Additionally, lack of commercially available polar monomers and the limited solubility of very polar monomers have influenced the development of hydrophilic polymer-monolithic columns. As a result, reports on hydrophilic polymer-based monolithic columns are still very limited. In contrast, the inorganic silica-based monolithic columns demonstrate good solvent resistance and high mechanical stability. Nevertheless, silica-based columns suffer from the disadvantage of poor pH resistance and are time consuming to prepare. This is because certain steps (e.g. the drying and cladding of the rods) are difficult to overcome in academic laboratories and it took us several days to fabricate silica-based columns.^5–8

In order to overcome the disadvantages of inorganic and organic monolithic columns, the organic–inorganic hybrid monolithic columns have been developed via a sol–gel process and have attracted great attention since the organic functional moieties can be incorporated into the inorganic silica monolithic matrixes to combine the merits of inorganic and organic monoliths, such as good mechanical stability and solvent resistance, and overcome the disadvantages of both inorganic and organic monolithic columns.^7–11 Yan et al. have prepared an octyl-functionalized hybrid silica monolithic column using tetraethoxysilane (TEOS) and n-octyltriethoxysilane (C8-TEOS) as the monomers and the octyl-functionalized hybrid silica monolithic column was successfully used in electrochromatography, where polycyclic aromatic hydrocarbons (PAHs) and phenols were successfully separated with high column efficiency of up to 180 000 plates m⁻¹.¹²

Other approaches have been attempted to prepare the hybrid columns and the performance of the hybrid columns have been investigated.¹³ Zou et al. reported an approach (one pot process) in which the hydrolyzed alkoxysilanes of tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) as precursors were copolymerized with the organic monomer of allyldimethyldodecylammonium bromide (ADDAB) for synthesis of a hybrid monolith. The column was also applied in the analysis of tryptic digests of bovine serum albumin (BSA) and mouse liver extract by micro-liquid chromatography tandem mass spectrometry (μLC-MS/MS), demonstrating its potential in proteome analysis.⁸ There are also other kinds of hybrid monolithic columns. For example, metals can be used as the inorganic moiety, as reported in other articles.¹⁴ Bai et al. used vinyl ester resin as the monomer, sodium bisulfite both as organic adjunct and coadunate initiator to prepare a strong cation hybrid column, which was used to separate lysozyme (Lys) from egg white.¹⁵

Hou et al. have prepared an organic–inorganic hybrid silica monolith based immobilized titanium ion affinity chromatography column. Using such a hybrid silica monolithic Ti⁴⁺–IMAC column, phosphopeptides were effectively isolated from the digest mixture of α-casein and BSA with a molar ratio as low as 1 : 200, illustrating its superior selectivity.¹⁶ Xu et al. used glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA) and 2-mercaptoethanol to fabricate a thiol-modified monolithic column, then in situreduction of chloroauric acid within the column was used to form gold nanoparticles attached to the surface of the pores of the monolith. The monolithic column provided a means to selectively retain cysteine-containing peptides. Application of the gold-modified hybrid monolith in tandem with a packed C18 capillary column was demonstrated with baseline separation of six peptides (three cysteine-containing peptides).¹⁷ However, the column was not directly used in analysis and the effect of column was enriching peptides. Additionally the preparation process of the column was complicated and would take us at least two days to fabricate the columns. Yang et al. used the method of radical polymerization of the continuous phase of oil in water high-internal-phase emulsions to fabricate porous poly(vinyl ester) resin monolithic supports, and it could be used to separate immunoglobulin from human plasma and chicken egg yolk.¹⁸ However, the preparation method of the column was complicated and it was difficult to control the formation of column.

In our work, an organic–inorganic hybrid monolithic column with hydroxyl functional group was developed with sonication-assist and it could be used to separate lysozyme from egg white, and benzene from its homologs. With the use of sonication, the time of sol–gel process was shortened from 12 h to 20 min. The characteristics of the hybrid column and factors influencing elution were investigated. The results showed that the column exhibited good mechanical strength and pH resistance. Moreover, the lysozyme separated from egg white by the hybrid column possessed high biological activity after being assayed by the method of turbidimetry.

2 Experiments

2.1 Chemicals

Vinyl ester resin was synthesized from bisphenol A diglycidyl ether (BADE). Lysozyme was obtained from Sigma Chemical Co. (St Louis, MO, USA). Dodecyl alcohol was obtained from China Medicament Co. Ltd. (Beijing, China). Polyethylene glycol (PEG, MW = 10 000) was purchased from Sigma Chemical Co. (St Louis, MO, USA). 2, 2′-Azobisisobutyronitrile (AIBN) was purchased from Shanghai Chemical Plant (Shanghai, China). Vinyltrimethoxysilane (VTMS) was purchased from Guibao Co. Ltd. (Hangzhou, China). HPLC-grade methanol was used for the preparation of mobile phases. All of these chemicals were analytical reagent grade. Triplex distilled water was used for all experiments.

A 1100 system from Agilent Technologies (Shanghai, China) was applied to chromatographic studies. The HPLC system consisted of a quaternary pump with an online vacuum degasser, an autosampler with variable injection capacity from 0.1 to 100 μL and a UV detector. Chromatographic separation of Lys was carried out on the polymeric monolithic column (50 mm × 4.6 mm i.d.). All sample solutions injected into the chromatographic system were filtered through a Millipore membrane (0.45 μm) to remove particles. Scanning electron microscopy was purchased by Hitachi High Technologies, (S-4300, Japan).

2.2 Preparation of the organic–inorganic hybrid monolith

2.2.1 Synthesis of the vinyl ester resin. The synthetic procedure of the vinyl ester resin was followed as described previously.¹⁹ 20 mL of 1,4-dioxane, 0.4 g of tetrabutyl ammonium bromide, and 20 g of BADE were put into a three-necked flask and heated. 8.6 mL of methacrylic acid was added drop-wise once the temperature reached 80 °C. The temperature was then increased to 90 °C and kept at that temperature for 4.5 h, after which time the vinyl ester resin had been synthesized. The schematic synthesis of the vinyl ester resin is illustrated in Fig. 1.

Fig. 1 The synthesis process of vinyl ester resin.

2.2.2 Fabrication of hybrid column. VTMS (0.6 mL), PEG (10 000 MW, 0.01 g) and acetic acid (0.05 M, 0.1 mL) were mixed in a polytetrafluoroethylene tube, and then sonicated to form a homogeneous solution at 40 °C for 20 min. After that, 1 mL of dodecyl alcohol, 0.8 mL of vinyl ester resin and 2 mg of AIBN were added into the homogeneous solution with 10 min sonication. The solution was then injected into the stainless-steel tube of a 50 mm × 4.6mm i.d. chromatographic column, both ends sealed with rubber and the column was submerged in a water bath at 60 °C for 12 h for polymerization. After that, the column was rinsed by methanol to flush out the residual material for further use.

2.3 Chromatographic characters of the monolith

2.3.1 Selection of mobile phase. A series of different salt concentrations (NaH₂PO₄, 0.005, 0.01, 0.02, 0.05, 0.1, and 0.2 mol L⁻¹) and pH values (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) were investigated in order to study the effects of pH value and concentration of mobile phase on the elution of Lys.

2.3.2 Separation of Lys. Chicken egg white was separated from fresh eggs and diluted to 50% (v/v) with phosphate buffer (0.05 mol L⁻¹, pH 7.0). The diluted egg white was centrifuged (12 000 rpm) at 4 °C for 15 min. The supernatant fluid was used as the Lys source. The chromatographic separation was performed using a gradient which was: water for the first 2.5 min and 0.02 mol L⁻¹Na₂HPO₄ (pH = 4.0) for the next 10 min. The chromatography of separation is shown in Fig. 6.

2.4 Assay binding capacity of hybrid column

The method of frontal analysis was carried out to determine the dynamic binding capacity of hybrid monolithic column for Lys, with 3 mg mL⁻¹ Lys in the mobile phase of 0.02 mol L⁻¹Na₂HPO₄ aqueous solution (pH = 4).

2.5 The determination of Lys activity

The biological activity of lysozyme separated on the hybrid column was measured by a turbidimetric assay. The main procedures were followed from the article reported previously.^20,21 Firstly, the lysozyme separated by the hybrid column was lyophilized, then dissolved in 0.05 mol L⁻¹phosphate (pH = 6.2). Secondly, a Micrococcus lysodeikticus suspension (0.05 mol L⁻¹phosphate, pH = 6.2) was poured into Lys solution and mixed to form a homogeneous solution. Thirdly, the homogeneous solution was placed on a rocking bed (25 °C, 50 rpm, 1 min), and the decline of the absorbance measured at 450 nm every 15 s was detected. All samples were assayed in triplicate.

3 Results and discussions

3.1 Characterizations of the hybrid monolithic column

3.1.1 Preparation of hybrid monolithic column. The purity of the vinyl ester resin is controlled by its physicochemical properties, such as viscosity and acid value. Its viscosity is 0.4–0.5 Pa s⁻¹, and the acid value is 15.0–16.0 mg KOH g⁻¹, and so the vinyl ester resin synthesized is very stable. Moreover, the unreacted reagents in the synthesis of the vinyl ester resin will be washed out by methanol during the washing stage, so do not affect the preparation of hybrid monolith. The formation of the hybrid monolithic column involves two major reactions: the polycondensation of VTMS, and the copolymerization of vinyl ester resin and the polycondensated product of VTMS at temperatures of 40 and 60 °C, respectively. In our study, different conditions were investigated to assess the factors affecting formation of the hybrid column. Table 1 shows the conditions of the prepared hybrid columns.

Table 1 The conditions of preparation of the hybrid column

No.	PEG (g)	0.05 M HAc (mL)	Vinyl ester resin (mL)	Dodecyl alcohol (mL)	VTMS (mL)	AIBN (g)	Polycondensation temperature (°C)	Copolymerization temperature (°C)
C1	0.01	0.1	0.9	1	0.6	0.01	40	60
C2	0.01	0.1	0.8	1	0.6	0.01	40	60
C3	0.01	0.1	0.7	1	0.6	0.01	40	60
C4	0.01	0.1	0.8	0.9	0.6	0.01	40	60
C5	0.01	0.1	0.8	0.8	0.6	0.01	40	60
C6	0.01	0.1	0.8	1	0.6	0.01	50	60
C7	0.01	0.1	0.8	1	0.6	0.01	30	60
C8	0.02	0.1	0.8	1	0.6	0.01	40	60
C9	0.005	0.1	0.8	1	0.6	0.01	40	60
C10	0.02	0.1	0.8	1	0.7	0.01	40	60
C11	0.02	0.1	0.8	1	0.5	0.01	40	60

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From Table 1, we learned that if the ratio of vinyl ester resin was increased, the permeability of the column would be decreased and the backpressure would increase because the vinyl ester resin was not only the monomer but also the cross linking agent. Dodecyl alcohol was the porogenic solvent, and a low content of it could result in a drop in permeability; that is, the backpressure of the column would increase. HAc aqueous solution was used as the catalyzer of polycondensation in our studies. Generally, the polycondensation reaction comprises of two stepwise reactions: hydrolysis and condensation of metal alkoxide. Si–O–C bonds of VTMS were hydrolyzed and formed Si–OH in the presence of HAc aqueous solution, then Si–OH condensed and formed the polycondensation product Si–O–Si in the presence of PEG, which induced the separation of phases. The polycondensation of silicone oxide is usually performed at 40 °C for 12 h.^8,12 The copolymerization of monomers is performed at 60 °C for 12 or 24 h. With the assistance of sonication, the process of sol–gel accelerates, the time of the sol–gel process will decrease and the total time of preparation of the hybrid column will be shortened. Compared with the ordinary method of sol–gel process at 40 °C for 12 h without sonication-assist, there is no obvious change in the hybrid monolithic column prepared with sonication-assist. As a result, the use of sonication decreases the total time of preparation of the hybrid column.

In our work, the polycondensation was performed at a relatively low temperature (30, 40 or 50 °C), and the copolymerization was performed at 60 °C, as usually adopted for the preparation of organic monolithic columns with AIBN as the initiator. The results showed that the temperature of 30 °C was not appropriate for the polycondensation of VTMS, because the column of C7 had a high permeability. When the temperature is low, the reaction of polycondensation is not complete. However, when the temperature was increased to 50 °C, the column of C6 showed bad permeability and high backpressure, so 40 °C was chosen as the polycondensation temperature. The content of PEG and VTMS also affected the permeability of the hybrid monolithic column. With an increase of PEG, column C8 showed low permeability and high backpressure. With a decrease of PEG, column C9 showed low backpressure with high permeability. That was because PEG induced the separation of phases in the sol–gel process. The phenomenon of PEG in the formation of hybrid column was similar to the article reported previously.²² In our research, the content of VTMS was also considered. The polycondensation product of VTMS was not only the monomer but also the cross linker in the formation of the hybrid column. Therefore, an increase in the VTMS in the composition led to high backpressure and low permeability (C10). The backpressure became low with a decrease in VTMS in the composition of hybrid column (C11). As a result, the conditions of C2 gave the best preparation conditions for the column.

3.1.2 IR spectrum of the hybrid column. The IR spectrum of the hybrid monolithic column (C2) is shown in Fig. 2, which was taken by Fourier-transfer IR spectroscopy. The peaks at 1040 cm⁻¹ and 1146 cm⁻¹ were the absorption of the Si–O bond and Si–C bond. The absorption at 3440 cm⁻¹ was caused by the presence of –OH. From Fig. 2 we know that the copolymerization of VTMS and vinyl ester resin had taken place with the initiator AIBN.

Fig. 2 The IR spectrum of the hybrid column.

3.1.3 Permeability of the hybrid column. The SEM images of the column (C2) are shown in Fig. 3. Fig. 3 shows that there are many through-pores in the hybrid column and the specific surface of the column is too large, which indicated the backpressure might be low. So we measured the backpressure of the column in different flow rates with HPLC (Fig. 4) and the backpressure had a good linear relationship with flow rate. When the flow rate was raised to 3 mL min⁻¹, the backpressure was only 37 bars, which means the prepared hybrid column had high porosity. Additionally, the mechanical stability of the obtained hybrid monolithic column was examined by connecting the column (50 mm × 4.6 mm i.d) to an HPLC pump (Agilent) with the flow rate ranging from 0.1 to 3 mL min⁻¹ (mobile phase, water). The measured backpressure linearly (R = 0.999) increased as the flow rate was increased. This indicated that the hybrid monolith possessed good mechanical stability under the flow rate of 3 mL min⁻¹.

Fig. 3 Scanning electron microscopy of the hybrid column.

Fig. 4 The relationship of flow rate and backpressure (chromatographic conditions: the prepared hybrid monolith C2, 50 mm × 4.6 mm i.d.; mobile phase: water.).

The pore diameter distribution of C2 was characterized by mercury intrusion porosimetry and is shown in Fig. 5. From the results, it was determined that the general pore volume, average pore diameter and interval porosity were 1.327 mL g⁻¹, 0.74 μm and 65.38%, respectively.

Fig. 5 Pore size distribution profiles for the monolith by mercury intrusion porosimetry.

3.2 Separation of Lys from egg white

Fig. 6 shows the chromatogram and two peaks were observed. Lys was separated from egg white within 6 min was found to be the second peak. In previous studies, there were many methods which were used for the separation of Lys. Bai¹⁵ used the –SO₃H hybrid column to separate Lys from egg white in 10 min. But the hybrid column we prepared had an advantage: the time of separation of Lys was shorter than that reported previously; it only took us 6 min to separate the Lys from egg white. What is more, the hybrid column had a good stability in the experiment. The RSD of the retention peak area of the Lys in different times and column-to-column were 0.81% (n = 5) and 2.51% (n = 5), respectively.

Fig. 6 The figure of separation of Lys from egg white. HPLC conditions: the prepared hybrid monolith C2, 50 mm × 4.6 mm i.d; a) mobile phase: water for the first 2.5 min and 0.02 mol L⁻¹NaH₂PO₄ (pH = 4.0) for 10 min; b) mobile phase: water.

3.3 Effect of pH and buffer concentration on the elution of Lys

The effects of pH and the concentration of the mobile phase were studied in our research, and Fig. 7 and 8 show the results. When the mobile phase was water, Lys was not eluted from the hybrid column (Fig. 6), so we chose aqueous phosphate solution as the elution agent. As the salt concentration increased, the protein bound to the column desorbed.²³Fig. 7 showed that the concentration of buffer salt had an effect on the elution of Lys. The elution effect increased with the increase of salt concentration and the greatest elution effect on Lys was at 0.02 mol L⁻¹ aqueous phosphate solution. It is not difficult to explain the phenomenon. The stability of a protein is related to the size of mass point, electric charge and hydration layer, and electrolyte destroys the structure of electric double layer. So the stability of Lys solution was degraded and the Lys precipitated. As a result, the Lys was eluted by aqueous phosphate solution and 0.02 mol L⁻¹NaH₂PO₄ which possessed the greatest ability of elution was chosen as the mobile phase. Fig. 8 revealed the effect of pH on the elution of Lys and it showed that the ability of elution decreased with an increase in pH value; that is, the Lys was retained on the hybrid monolith. The phenomenon can be explained as follows: because the isoelectric point (pI) of Lys is close to 11.0, when pH < pI, Lys is positively charged. When the value of pH increases in the range of 4 to 10, the positive charge on Lys decreases. There are lots of hydroxyl group on the stationary phase, which leads to positively charged stationary phase. So the repulsion of positive charges which exists between the monolith with hydroxyl functional groups and the Lys will reduce with the increase of pH in the range of 4 to 7. In contrast, when pH value of the mobile phase is higher than 7, the hydroxyl group on the monolith will be hydrolyzed, and the stationary phase is negative charged. Therefore, there will be an electrostatic attraction between the stationary phase and Lys, and the electrostatic attraction will be enhanced with the increase of pH in the range of 7 to 10 and the ability of elute will decrease. As a result, 0.02 mol L⁻¹NaH₂PO₄ (pH = 4) was selected as the elution mobile phase.

Fig. 7 Effect of buffer salt concentration on the elution of Lys. HPLC conditions: the prepared hybrid monolith C2, 50 mm × 4.6 mm i.d.; sample: Lys, 1 mg mL⁻¹; volume: 1.0 μL. Mobile phases (NaH₂PO₄ aqueous solution): a, 0.005 mol L⁻¹; b, 0.01 mol L⁻¹; c, 0.02 mol L⁻¹; d, 0.05 mol L⁻¹; e, 0.1 mol L⁻¹; f, 0.2 mol L⁻¹.

Fig. 8 The pH effect on the elution of Lys. HPLC conditions: the prepared hybrid monolith C2, 50 mm × 4.6 mm i.d.; sample: Lys, 1 mg mL⁻¹; volume: 5.0 μL. Mobile phase: 0.02 mol L⁻¹buffer phosphate with the pH as follows: 1. pH = 4.0; 2. pH = 5.0; 3. pH = 6.0; 4. pH = 7.0; 5. pH = 8.0; 6. pH = 9.0; 7. pH = 10.0.

3.4 Separation of benzene and its homologs from a mixture

The monolith was also used to separate benzene and its homologs from a mixture with the mobile phase methanol/water (80/20, v/v). The chromatogram was shown in Fig. 9. The peaks were benzene, toluene and ethyl benzene, in that order. The order of the peaks can be explained by the theory of hydrophobic interaction. The stationary phase of the column is composed of two parts, one is the inorganic moiety and the other is the resin moiety, which provides sufficient hydrophobic properties (phenyl groups), allowing the hybrid column to be used in the separation of organic small molecules.

Fig. 9 Chromatogram of benzene and its homologs. HPLC conditions: the prepared hybrid monolith C2, 50 mm × 4.6 mm i.d.; samples: benzene, toluene and ethyl benzene, 1μg mL⁻¹; volume: 5.0 μL. Mobile phase: methanol/water (80/20, v/v).

3.5 The dynamic binding capacity of the hybrid monolith for Lys

Using the method described in section 2.4, the dynamic binding capacity of the hybrid monolith for Lys is 0.18 mg g⁻¹.

3.6 Biological activity assay of Lys

One unit of enzyme activity is defined as the reduction of the absorbance at 450 nm by 0.001 min⁻¹ under the conditions (25 °C, pH = 6.2) employed. Specific activity is defined in terms of units of activity per milligram of protein (U mg⁻¹). The results of the biological activity of Lys are shown in Table 2 and the specific activity of Lys was 14572 ± 140 U mg⁻¹. The biological activity we assayed is constant with the result reported previously,²³ indicating that the Lys separated by the hybrid column was not deactivated and possessed high biological activity, meaning it can be used in food and medicine industries.

Table 2 The relationship of concentration and specific activity of Lys (ΔA: The decrease of absorbance in 1 min.)

Concentration (μg mL^−1)	5	6.25	7.5	10.0	12.5
ΔA	0.0760	0.09686	0.1110	0.1521	0.1856
Specific activity (U mg^−1)	15 200 ± 125	15 500 ± 150	14 800 ± 140	15 210 ± 120	14 850 ± 165
Average specific activity (U mg^−1)			14 572 ± 140

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4 Conclusions

A novel organic–inorganic hybrid column with hydroxyl functional groups was prepared from VTMS and vinyl ester resin. The column showed high permeability, low backpressure, strong pH resistance and strong mechanical stability. The results demonstrated the hybrid column could be used as the solid phase of HPLC and used to separate lysozyme from egg white in a short time; benzene and its homologs were also successfully separated. Additionally, the Lys separated by the hybrid column possesses high biological activity. Therefore, such hybrid columns with hydroxyl functional groups could be used in the separation of proteins and organic molecules by HPLC.

Acknowledgements

We are grateful for financial support from the National Natural Science Foundation of China (21175031), Hebei Province Programs for Science and Technology Development (No. 11966411D) and the Education Department of Hebei Province (No. ZD2010234).

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