A novel imidazolium-based organic–silica hybrid monolith for per aqueous capillary electrochromatography

Abstract

A new imidazolium-based organic–silica hybrid monolith by binding the imidazole-1-acetic acid onto the surface of monolithic silica was prepared through a simple route. The obtained monolith exhibited per aqueous capillary electrochromatography (PACEC) characteristics in water-rich mobile phases. A typical per aqueous chromatography behavior was observed for the test solutes (an increase in H₂O content resulted in an increase in retention). The effects of water content, pH and ion strength of mobile phase on the retention of test compounds in highly aqueous eluents were investigated in detail. PACEC in itself is a green chromatography mode that makes green capillary electrochromatography possible. Various hydrophilic compounds including amino acids were baseline separated with enhanced resolution of the obtained monolith under PACEC mode. It is evidenced that solutes were retained predominantly by hydrophobic interactions under PACEC conditions. Therefore, PACEC has the potential to replace hydrophilic interaction chromatography and meanwhile it is complementary to the reverse-phase chromatography.

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1. Introduction

Green chemistry is a concept falling into the scope of sustainable development.¹ Green analytical chemistry focuses on minimizing the amount of waste associated with either sample preparation or analysis, which has gained rising attention and acceptance in recent years.² Analysis and determination of organic compounds in various matrices is usually performed by using chromatographic techniques. It is thus very important that these methods have negligible environmental impacts. Owing to its unique characteristics, acetonitrile (ACN) is by far the preferred organic solvent used in chromatographic analysis. However, in the framework of green chromatography and the current shortage of ACN, most of the efforts have focused on overall reduction in the amount of solvent used. An attractive solution is to develop new reduction of ACN-consuming separation modes, in which water-rich eluents were used. Per aqueous chromatography is such an alternative chromatographic mode. Per aqueous liquid chromatography (PALC), which was also called as reversed hydrophilic interaction liquid chromatography (HILIC), was named by Sandra et al.³ Under PALC mode, mobile phases contain a high percentage of water to separate polar compounds, which has aroused wide concern as a green LC mode in recent years.^3–8 However, there is no report about the application of per aqueous chromatography mode in capillary electrochromatography (PACEC) yet.

Monolithic columns can be described as integrated continuous porous separation media for separation science. Nowadays, monolithic columns have proved to be good alternatives to the particle-packed columns due to the simplicity of their in situ preparation method, ready control of their porous properties and surface chemistries as well as their unique structure and exceptional characteristics.^9–15 On the basis of the nature of the constituting materials, the monolithic columns can be mainly classified into organic polymer-based,^16–18 inorganic silica-based^19,20 and organic–silica hybrid monolithic columns.^21–23 The majority of organic polymer-based monolithic columns are prepared by in situ polymerization reactions, while the silica-based monolithic columns are prepared through the sol–gel process. It can be easily found that the problems of both the swelling of organic monoliths in some solvents and the tedious fabrication procedures of the silica-based monoliths always limit their applications and encumber the researchers.²⁴ However, organic–silica hybrid monolithic columns can overcome the above shortcomings and are receiving more and more attention since they possess the advantages of easy fabrication, wide pH range, good mechanical stability and high permeability.^25–29

Various chemical modifications of organic–silica hybrid monoliths can be applied in various separation modes.³⁰ To date, most of publications dealing with monolithic columns still focus on reversed-phase separation represented by octadecyl (C18) column³¹ due to its great resolving ability for nonpolar analytes. In recent years, monolithic organic–silica hybrid stationary phases modified with phenyl,³² cyano,³³ and amino³⁴ groups have also been studied. These columns offer additional retention mechanisms to obtain the desired chromatographic characteristics since they introduce additional molecular interactions such as hydrophilic, hydrophobic, hydrogen bonding, π–π and ionic interactions. Compared with those common used modifiers, imidazole-1-acetic acid is a water-soluble functional monomer, enabling the surface modification of the monoliths to be simple, economical and environmentally friendly. In addition, the particular characteristics and property of imidazole-1-acetic acid also inspired us to introduce it into the surface of monolithic silica since its imidazolium can be responsible for hydrophobic, π–π and ionic interactions. Meanwhile, the carboxylic group can provide hydrogen bonding or ionic interactions in its dissociated form. Therefore, the imidazole-1-acetic seems to be an attractive modifier to extend the application of organic–silica hybrid monoliths for separation of different types of compounds.

In the present study, we illustrate the facile preparation of a new imidazolium-based organic–silica hybrid monolith by binding the imidazole-1-acetic acid onto the monolithic silica surface. The optimized monolithic column was successfully used for the CEC separation of a wide variety of hydrophilic test mixtures (amides, organic acids, nucleosides and amino acids) under per aqueous chromatographic conditions. Under PACEC mode, highly aqueous eluents (90–100% H₂O) were mainly studied, which not only contributes to address the problem associated with hazardous solvents, but also makes green CEC possible. In addition, the influences of different chromatographic conditions including water content, pH and buffer salt concentration on retention behavior have also been investigated.

2. Experimental

2.1. Chemicals and materials

Tetramethoxysilane (TMOS) (99%) and γ-choloropropyl-trimethoxysilane (CPTMS) (98%) were purchased from Shanghai Bore Chemical reagent Co. (Shanghai, China). Polyethylene glycol (PEG, M_n = 10 000) was obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Imidazole-1-acetic acid (98%) was purchased from Energy Chemical (Shanghai, China). Fused silica capillaries (Hebei Ruifeng Instrumental Company, China) of 33.5 cm (25 cm to detector) length (i.d. = 75 μm) were used. HPLC-grade ACN from Merck (Darmstadt, Germany) was used for the preparation of mobile phases. All other reagents used in the experiment were of analytical grade. All solutions were passed through 0.45 μm filter prior to use.

2.2. Column preparation

2.2.1. Pretreatment of capillary. A bare capillary was preconditioned prior to use by flushing with methanol for 20 min, H₂O for 20 min, 1 M NaOH for 1.5 h and kept for a night, and then flushed with H₂O for 20 min, 1 M HCl for 2 h, H₂O for another 20 min in sequence until the pH value of the outlet solution was 7.0, and finally, rinsed with methanol for 20 min. Then the pretreated capillary was dried by purging nitrogen gas at 120 °C for further use.

2.2.2. Preparation of organic–silica hybrid monolith. The preparation conditions of the organic–silica hybrid monolithic column are similar to that reported in our previous work.²² Briefly, 240 mg of PEG was dissolved in 2.5 ml of 0.01 M acetic acid in a glass vial, and then 0.9 ml of TMOS and 0.3 ml of CPTMS were respectively added dropwise. The solution was stirred at 0 °C for 4 h until a homogenous solution was obtained. The precondensation mixture was sonicated for 3 min at 0 °C, and then introduced into the pretreated capillary to an appropriate length. Afterwards, the capillary was placed in an incubator at 55 °C for 12 h with both ends sealed to complete the condensation reaction. The resulting chloropropyl modified (CP–silica) hybrid monolith was then flushed with H₂O and methanol to remove the PEG and other residuals. Finally, the obtained CP–silica hybrid monolithic column was rinsed with 0.01 M ammonium hydroxide and kept at 120 °C for 3 h, followed by washing with H₂O.

2.2.3. Preparation of imidazole-1-acetic acid-based hybrid monolith. An aqueous solution containing a large excess of imidazole-1-acetic acid (100 mg ml⁻¹) was pumped through the CP–silica hybrid monolithic column for 20 min. Then the column was heated at 70 °C for 24 h with both ends sealed in an incubator. Finally, the imidazole-1-acetic-modified silica (IAS) hybrid monolithic column was flushed with H₂O to remove the unreacted residue. A scheme detailing the column preparation is shown in Fig. 1. Since no organic solvent is involved in the process, the preparation route is simple and economic.

Fig. 1 Scheme detailing preparation of the IAS hybrid monolithic column.

2.3. Instrumentation and methods

Separations were performed on an Agilent CE system (Agilent Technologies, Germany) equipped with a diode array detector at a constant temperature of 25 °C. An Agilent CE Chemstation (Rev. A 09.03) was used for the data analysis and instrumental control. The columns were heated in an incubator (Binder BD, Germany). pH values of the mobile phases were measured using a Sartourius PB-10 pH meter (Beijing, China). Prior to CEC, the prepared column was placed in a CE cartridge with desired length and preconditioned with mobile phase for at least 20 min with an HPLC pump. After that, the cartridge was installed in the CE instrument, and the column was equilibrated by applying a voltage of 10 kV until stable current and baseline were achieved. NaH₂PO₄ solution (100 mM), which was used as the phosphate buffer, was adjusted with NaOH and HCl to different pH values. The mobile phases were composed of ACN with various concentrations of phosphate buffer. Working solutions were prepared by dissolving sample in the mobile phase at suitable concentrations. Separations were performed at 25 °C at different voltages (stated in the figure captions). Prior to use, the mobile phases were degassed ultrasonically for 15 min and filtered. Samples were injected by flushing for 0.01 min. The detection window was fabricated by burning out a 2–3 mm segment in the empty section of capillary at the edge of continuous bed, which is located as close to the monolithic matrix as possible. The monolithic column was then cut to an effective length of 25 cm with a total length of 33.5 cm before use. Thiourea was selected as EOF marker. The electroosmotic mobility (EOF) and retention factor (k) was calculated by the following equations: μ_EOF = L_eL_t/(Vt₀) and k = (t_R − t₀)/t₀, respectively. Where L_e and L_t are the effective length and total length of column, respectively. V is the applied voltage, t_R and t₀ represent the retention time of the analytes and the thiourea in the study, respectively.

Scanning electronic microscopic (SEM) images were obtained by a JSM-5601LV and a JSM-6701F scanning electron microscope (JEOL, Japan). The nitrogen content of the IAS monolithic matrix was determined by elemental analysis performed on an Elementar Vario EL cube (Hanau, Germany). The specific surface area (BET) of the IAS monolithic matrix was determined on an ASAP 2010 Accelerated Surface Area and Porosimetry System (Micromeritics, USA).

3. Results and discussion

3.1. Characterization of the IAS hybrid monolithic column

The morphology of the IAS hybrid monolithic column was characterized by SEM. As shown in Fig. 2, a uniform monolithic matrix with macro-porous structure was observed and the IAS hybrid monolithic matrix was well attached to the inner wall of the capillary. The quantitative evaluation of imidazole-1-acetic acid on the surface of the matrix was also made. From the nitrogen content of the monolith, the average content of the bonded imidazole-1-acetic acid on the surface of the silica skeleton was calculated as 0.76 μmol m⁻². The calculation formula of the surface coverage is as follows: coverage of imidazolium groups (μmol m⁻²) = (N% × 10⁴)/(28 × S), where N% represents the percentage of nitrogen as determined by elemental analysis (1.21%), S is the specific surface area of the IAS hybrid monolith (570.61 m² g⁻¹).

Fig. 2 SEM images of the IAS hybrid monolithic column. Magnification: (A) 1200×, (B) 2000×, (C) 3000× and (D) 5000×.

3.2. EOF characteristic

EOF is a basic driving force that plays a key role in CEC separation. Knowing the characteristics of the EOF of a monolithic column will be of great use to select appropriate separation conditions, and further, to understand the separation behavior.³⁵ The magnitude of EOF can be determined by the net surface charge density of all of the chargeable groups, including imidazolium groups, carboxylic groups and residual silanol groups. Accordingly, the direction of EOF is determined by the sign of the net surface charge. Fig. 3(A) shows the change of EOF as a function of the mobile phase pH on the IAS hybrid monolith. A reversed anodic EOF was observed in the pH range of 3.0–6.0, and the absolute magnitude of EOF decreased as the pH increased. At lower pH, the dissociation of carboxylic acids and residual silanol groups was suppressed and the net surface charge of the monolithic column was determined by the imidazolium groups. Thus, the column exhibited anodic EOF under such pH conditions. While increasing the mobile phase pH, the dissociation of carboxylic acids and residual silanol groups became stronger and the ionization of imidazolium groups became weaker; thus, the absolute magnitude of anodic EOF decreased. When the pH value was near 6.5, the net surface charges of the stationary phase were near zero; accordingly, no obvious EOF could be observed. As the pH value further increased, at a pH above 7.0, the direction of the EOF was changed from anode to cathode, and the cathodic EOF increased with increasing the mobile phase pH, indicating the net negative charges of the monolith which can be attributed to the fact that the dissociation of carboxylic acids and residual silanol groups increased and the ionization of imidazolium groups was partially suppressed. These results show that the EOF on the obtained hybrid monolith is pH-dependent and can be controlled easily by adjusting the mobile phase pH. Meanwhile, it is worth noting that larger EOF could be generated on the IAS monolithic column at lower pH relative to our previously reported N-methylimidazole-bonded monolith.²²

Fig. 3 (A) Influence of mobile phase pH on the EOF of the IAS hybrid monolithic column. Conditions: 10 mM NaH₂PO₄ buffer at different pH values; applied voltage, ±20 kV; detection wavelength, 214 nm. EOF marker, thiourea. (B) Effect of ACN content on the EOF velocity of the IAS hybrid monolithic column. Conditions: ACN–H₂O (ACN 0–20, v/v), NaH₂PO₄ (10 mM, pH 3.0); applied voltage, −20 kV; detection wavelength, 214 nm. (C) Effect of salt concentration on the EOF velocity of the IAS hybrid monolithic column. Conditions: NaH₂PO₄ (2.5–12.5 mM, pH 3.0); applied voltage, −20 kV; detection wavelength, 214 nm.

The influence of ACN content on the EOF was also investigated. As shown in Fig. 3(B), with the increase of ACN content from 0% to 20% in the solution (10 mM NaH₂PO₄ buffer, pH 3.0), the EOF velocity decreased from 2.10 to 1.56 mm s⁻¹. The decrease of EOF observed with the higher content of ACN might be caused by the change in the viscosity and zeta potential. Besides, the influence of phosphate buffer concentration was also tested and shown in Fig. 3(C). The EOF velocity decreased from 2.84 to 2.08 mm s⁻¹ in the buffer range of 2.5–12.5 mM. The reduced EOF was due to the decrease of thickness of electrical double-layer and zeta potential in a high salt concentration buffer.

3.3. Mechanical stability and column reproducibility

The mechanical stability of the obtained hybrid monolith was examined by connecting the column to a HPLC pump and the backpressure was measured by pumping the mobile phase (ACN–H₂O = 50/50, v/v). The backpressure linearly (R² = 0.999) increased from 0.8 to 1.8 MPa with the flow rate increasing from 0.001 to 0.011 ml min⁻¹, indicating that the hybrid monolith possessed good mechanical stability. The permeability of the monolithic capillary column was evaluated by the Darcy's Law: B₀ = FηL/πr²ΔP,³⁶ where F is the flow rate of mobile phase, η is the viscosity of mobile phase (ACN–H₂O = 50 : 50, η = 0.81 cP), L is the effective length of column, r is the inner radius of the column and ΔP is the pressure drop of the column, the permeability of this hybrid monolith was calculated with ACN–H₂O = 50 : 50 as the mobile phase and gained at 4.83 × 10⁻¹² m², indicating the good permeability of the obtained hybrid monolith.

The reproducibility of column production was assessed through measuring the percent relative standard deviations (RSD) of the EOF. The column to column (n = 3) reproducibility was acceptable with RSD of 5.2% for EOF, as well as RSD of 2.9% for retention time. The run-to-run (n = 5) and day-to-day (n = 3) reproducibility were calculated on a single capillary monolithic column, and the satisfactory RSDs for EOF and retention time were 1.6%, 0.86%, and 2.3%, 1.3%, respectively. These results indicated that the hybrid-silica monolithic columns were of good reproducibility and the preparation of the IAS hybrid monolith was feasible. Besides, the endurance of the hybrid–silica monolith was also investigated by measuring the migration of the EOF maker (thiourea) over a period of 2 months. The result showed that the migration time of thiourea changed only slightly from 1.967 to 1.978 min (after 300 runs) (relative standard deviation, RSD, was 0.64%). It confirms again that the obtained hybrid monolith is very stable.

3.4. Column efficiency

The column efficiency of the IAS hybrid monolithic column was evaluated in CEC by changing the applied voltage from −2 to −23 kV. Linear flow velocity is calculated by μ_EOF = L_d/t_m, where L_d is the length of the column from the inlet to the detection window, t_m is the migration time of EOF marker. Plate height (H) is calculated by H = L_d/N, where N is the theoretical plate number. The relationship between the flow velocity and the plate height of thiourea was demonstrated in Fig. 4. The lowest plate height of ∼8.5 μm was obtained, corresponding to column efficiency (theoretical plates, N) of ∼118 000 N/m. It can be observed that flat Van Deemter curves were obtained when the linear velocity of the mobile phase was larger than 1.5 mm s⁻¹, which was one of the typical behaviors of monolithic-type stationary phases.³⁵ The result suggests that rapid separation can be achieved with a minor loss in separation efficiency on this hybrid monolith.

Fig. 4 Dependence of the plate height of thiourea on the linear velocity of mobile phase by the IAS hybrid monolithic column. Conditions: mobile phase, 10 mM NaH₂PO₄ buffer at pH 3.0; applied voltage from −2 kV to −23 kV; detection wavelength, 214 nm.

3.5. Retention properties under the PACEC mode

To investigate the PACEC properties of the new column, four polar hydrophilic compounds (acrylamide, DMF, N,N-dimethyl acetamide and caprolactam) were selected as test probes. The retention factor of four test probes were plotted against the volume fraction of water in the eluent was shown in Fig. 5(A). As shown in Fig. 5(A), above 65% H₂O the retention factors were observed to be proportional to the water content in the eluent, which exhibited a typical PACEC behavior on the new hybrid monolith. The retention time of all the test compounds slightly increased with increasing H₂O% from 65 to 90%, and then dramatically increased as the H₂O% increased above 90%. It showed that the retention of the test probes was extremely sensitive to very small variation of ACN content in water-rich eluents. This result was consistent with the previous reported literature.³⁷ The reason might be that the surfaces of the new stationary phase were significantly saturated with water and ACN when the H₂O content was 65–90% in mobile phase, resulting in a slight increase of the retention factors of compounds. However, within the H₂O concentration range of 90–100%, the accumulation of water and ACN onto the new stationary phase is maximal as illustrated from their respective excess of adsorption.⁸ The change in the composition of the adsorbed eluent multilayer onto the new stationary phase is also maximal and drastic composition change could be caused by small variation of ACN content. Therefore, the retention factors were strongly sensitive to little change of mobile phase in the bulk water concentration.^5,7

Fig. 5 Effect of (A) water content in mobile phase on retention, (B) mobile phase pH on retention, (C) ionic strength of mobile phase on retention on the IAS hybrid monolithic column. Conditions: (A) 10 mM NaH₂PO₄ at pH 3.0 with different water contents; (B) 10 mM NaH₂PO₄ containing 4% ACN at different pH; (C) various salt concentrations containing 4% ACN at pH 3.0. Applied voltage, −15 kV; detection wavelength, 214 nm.

The column efficiency were about 172 500, 79 100, 54 300 and 30 200 N/m for acrylamide, DMF, DMA and caprolactam in water–ACN (96/4, v/v) mobile phase, respectively.

3.5.1. Effect of mobile phase pH on retention. As is well to known, mobile phase pH plays an important role in retention and selectivity in CEC separation. Fig. 5(B) shows the effect of the mobile phase pH on the PACEC retention by changing the pH at 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 while keeping the concentration of phosphate buffer constant at 10 mM. It was observed that the retention factors of all the test compounds slightly increased in the pH range from 2.5 to 5.0. The possible reason was that stronger dissociation of carboxylic acids and residual silanol groups would occur as the pH increased, and then the stationary phase acquired more negative charge, which generated ion-exchange effect with the positively charged basic compounds, thus leading to a slight increase in retention.

3.5.2. Effect of ionic strength of mobile phase on retention. The effect of salt concentration on the retention of the four compounds under the PACEC mode with 96% water in the mobile phase was also investigated by varying the concentration of phosphate buffer from 2.5 to 15 mM. As shown in Fig. 5(C), the retention factors exhibited a slightly increasing tendency with an increase in salt concentration for all test probes. It could be attributed to the increase in hydrophobic interactions. As previously reported, on silica with water-rich mobile phases solute retention resembles that of conventional reversed-phase systems (hydrophobic retention).⁵ Higher salt concentration could drive more solvated salt ions into the water layer, leading to the more ACN composition of the adsorbed eluent multilayer onto the new monolithic silica surface. Thus, an increase in both hydrophobic interactions and retention of the solutes would be observed.

3.6. Application

To demonstrate the retention characteristics and separation selectivity of the new hybrid monolith under PACEC mode, some hydrophilic compounds that were difficult to retain and separate by reversed-phase CEC were selected as the target analytes. Mixtures of ACN and water (H₂O% > 90%) were used as the mobile phases and low concentration of buffers (10 mM) were added to the eluents.

3.6.1. Separation of amides. CEC separation of a mixture of five amides is shown in Fig. 6(A) under the optimal conditions with 10 mM NaH₂PO₄ buffer (pH 3.0)–ACN (96/4 v/v). Very small change of ACN concentrations has a drastic influence on retention, which is also typical for HILIC and here for PACEC (clearly seen in Fig. 6(B)). It is well known that better peak shapes for cationic solutes can be achieved in RP separations at low pH, due to the suppression of the ionization of silanol groups.³⁸ The effect of mobile phase pH on the retention behavior of amides in PACEC (4% ACN) was investigated at various pH. The complete set of data from these experiments is summarized in Table 1. As can be seen, under PACEC mode the retention of amides increased with the increase of mobile phase pH. In general, the ion-exchange interaction between the basic compounds and the stationary phase is expected to derive from the extent of ionization of both surface silanols and the solute. Since in our case all the solutes remain completely ionized at both pHs, the increased retention at higher pH can be attributed to the increase in fraction of surface silanols in the ionized state. In addition to retention time effects, the increase in the buffer pH resulted in the decrease in the column efficiency for all solutes under PACEC mode. Moreover, as the retention increased, the peak tailing considerably increased at both pHs. Such behavior may be ascribed to the increased sample overloading effects which occur more readily at higher k values.³⁹ Based on the above discussion, it can be concluded that multiple effects of hydrophobic and ion-exchange interactions were involved in the separation of amides on the IAS hybrid monolithic column.

Fig. 6 Electrochromatogram of five amides on the IAS hybrid monolithic column. Conditions: (A) 10 mM NaH₂PO₄ buffer containing 4% v/v ACN at pH 3.0; (B) 10 mM NaH₂PO₄ buffer containing various ACN content at pH 3.0. Applied voltage, −15 kV; detection wavelength, 214 nm. Peak identification: (1) formamide; (2) acrylamide; (3) DMF; (4) N,N-dimethyl acetamide; (5) caprolactam.

Table 1 Retention factors (k) and column efficiencies (N/m) for amides in PACEC (4% ACN) at various pH

Solute	pH 3.0	pH 3.5	pH 4.0	pH 4.5	pH 5.0
	k, N/m	k, N/m	k, N/m	k, N/m	k, N/m
Acrylamide	0.29, 159 600	0.31, 147 700	0.35, 12 900	0.38, 116 700	0.45, 82 800
DMF	0.76, 77 500	0.82, 76 700	0.92, 73 400	1.01, 62 800	1.18, 58 500
DMA	1.32, 49 300	1.42, 46 600	1.60, 44 500	1.75, 42 500	2.06, 36 400
Caprolactam	2.76, 25 400	2.98, 24 000	3.33, 23 900	3.65, 21 800	4.34, 21 500

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3.6.2. Separation of organic acids. To make a further examination of the separation capability of the hybrid monolith, a mixture of organic acids including sulfanilic acid, p-hydroxybenzoic, p-aminobenzoic acid and benzoic acid was selected as test compounds, which cannot be separated in their undissociated form on the CP–silica hybrid monolith. Fig. 7(A) displays the separation electrochromatogram of the four organic acids. The analytes are baseline separated under the optimal conditions with 6% ACN, 10 mM NaH₂PO₄ (pH 3.0). And it is easy to find that the elution order of these compounds was in accordance with the increase of their hydrophobicities. The effect of the amount of ACN in the mobile phases on the retention factors of compounds was also investigated, and the result exhibited a decreasing tendency with an increase of ACN content (data not shown). These results revealed that the retention of organic acids is mainly due to hydrophobic interaction, which was in agreement with the separation mechanism under PALC mode.

Fig. 7 (A) Electrochromatogram of four organic acids on the IAS hybrid monolithic column. Conditions: 10 mM NaH₂PO₄ buffer containing 6% v/v ACN at pH 3.0, applied voltage, −20 kV; detection wavelength, 254 nm. Peak identification: (0) thiourea; (1) sulfanilic acid; (2) p-hydroxybenzoic; (3) p-aminobenzoic acid; (4) benzoic acid. (B) Electrochromatogram of four nucleosides and nucleotide bases on the IAS hybrid monolithic column. Conditions: 10 mM NaH₂PO₄ buffer at pH 4.0, applied voltage, −15 kV; detection wavelength, 214 nm. Peak identification: (1) uridine; (2) inosine; (3) thymidine; (4) hypoxanthine. Electrochromatograms of three amino acids on a bare fused silica capillary under CZE mode (C) and on the IAS hybrid monolithic column (D). Conditions: (C) 20 mM NaH₂PO₄ at pH 11.0; applied voltage, +15 kV; (D) 10 mM NaH₂PO₄ buffer containing 4% v/v ACN at pH 3.0; applied voltage, −20 kV. Detection wavelength, 214 nm. Peak identification: (1) L-tryptophan; (2) D,L-phenylalanine; (3) L-tyrosine.

3.6.3. Separation of nucleosides and nucleotide bases. Nucleotides are essential components in DNA and RNA and play important roles in all living species. The analysis of nucleosides is often used to illustrate the features of HILIC. Such separation can also be successfully achieved in PACEC by using 100% water-based mobile phases. In this separation process, all possible interactions may take place and the separation can be fine-tuned through pH and ionic strength. As shown in Fig. 7(B), four nucleosides and nucleotide bases were separated on the hybrid monolith under PACEC conditions with 10 mM NaH₂PO₄ buffer (pH 4.0). It could be seen that all the components were well resolved and the nucleosides displayed strong retention on the new column. The hypoxanthine was eluted last. It was reasonable to assume that surface carboxylic groups on the new stationary phase might interact with the basic hypoxanthine, thus leading to stronger retention and greater tailing for hypoxanthine. Separation of the four nucleosides and nucleotide bases cannot be achieved either on a bare capillary under capillary zone electrophoresis (CZE) mode or on a CP–silica hybrid monolithic column under the same conditions.

3.6.4. Separation of amino acids. A bare capillary and an IAS hybrid monolithic column were respectively used to analyze a mixture of three common amino acids with benzene ring (L-tryptophan, D,L-phenylalanine, and L-tyrosine). As seen in Fig. 7(C), three amino acids can be basically resolved under extreme conditions (pH 11.0) on a bare capillary under CZE mode. The elution order was L-tryptophan (symmetry = 2.41) < D,L-phenylalanine (symmetry = 2.87) < L-tyrosine (symmetry = 6.71), which was most likely related to the electrophoretic mobility of different amino acids. While, under the optimal CEC conditions: 10 mM NaH₂PO₄–ACN (96 : 4 v/v), pH 3.0, a mixture of three amino acids were also baseline separated on the IAS hybrid monolithic column. Fig. 7(D) clearly shows that the elution order of the amino acids was L-tyrosine (symmetry = 1.03) < D,L-phenylalanine (symmetry = 0.92) < L-tryptophan (symmetry = 0.88). Compared with the separation on a bare capillary, the elution order of the amino acids was changed, which can be illustrated by the complexity of the different mechanisms taking place in the separation. Besides, baseline separation can be achieved under mild conditions with moderate pH, and CEC separation on the hybrid monolith gave enhanced resolution of three amino acids (better peak symmetry).

4. Conclusion

In this work, a new imidazolium-based organic–silica hybrid monolith was prepared, characterized and evaluated. The obtained hybrid monolith exhibited PACEC properties, which could provide similar retention as hydrophilic interaction CEC for polar and hydrophilic compounds. The features of PACEC on the obtained monolith were illustrated through investigating the effects of water content, pH and ion strength of mobile phase on the retention of test compounds. A wide variety of test mixtures, including amides, organic acids, nucleosides and amino acids, were respectively separated under the PACEC mode. In the framework of green chromatography and the current shortage of ACN, PACEC could be a suitable chromatography mode as the complementarity of reversed-phase chromatography and the replacement of hydrophilic interaction chromatography, which is highly consuming in ACN. All in all, per aqueous chromatography was first applied in CEC for the separation of various hydrophilic compounds and it has proved to be an excellent and attractive chromatography mode. Therefore, ongoing studies on the application of per aqueous chromatography mode in both HPLC and CEC for various practical applications have good prospects and will be further explored in the near future.

Acknowledgements

The authors express their thanks to the support of the National Nature Science Foundation of China (no. 21175143, 21105107 and 20905072), and the National Science & Technology Major Project of China (no. 2011ZX05010 and 2011ZX05011).

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