Preparation and evaluation of surface-bonded tricationic ionic liquid silica as stationary phases for high-performance liquid chromatography

Journal of Chromatography A, 1396 (2015) 62–71

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Preparation and evaluation of surface-bonded tricationic ionic liquid silica as stationary phases for high-performance liquid chromatography Lizhen Qiao, Xianzhe Shi ∗ , Xin Lu, Guowang Xu ∗∗ Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

a r t i c l e

i n f o

Article history: Received 21 January 2015 Received in revised form 30 March 2015 Accepted 30 March 2015 Available online 4 April 2015 Keywords: Tricationic ionic liquid, Stationary phase Reversed-phase liquid chromatography, Hydrophilic interaction chromatography

a b s t r a c t Two tricationic ionic liquids were prepared and then bonded onto the surface of supporting silica materials through “thiol-ene” click chemistry as new stationary phases for high-performance liquid chromatography. The obtained columns of tricationic ionic liquids were evaluated respectively in the reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode, and possess ideal column efficiency of 80,000 plates/m in the RPLC mode with naphthalene as the test solute. The tricationic ionic liquid stationary phases exhibit good hydrophobic and shape selectivity to hydrophobic compounds, and RPLC retention behavior with multiple interactions. In the HILIC mode, the retention and selectivity were evaluated through the efficient separation of nucleosides and bases as well as flavonoids, and the typical HILIC retention behavior was demonstrated by investigating retention changes of hydrophilic solutes with water volume fraction in mobile phase. The results show that the tricationic ionic liquid columns possess great prospect for applications in analysis of hydrophobic and hydrophilic samples. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Stationary phases play an important role in the applications of high-performance liquid chromatography (HPLC), and the development of new stationary phases has always been one of the most important subjects, especially for mixed-mode and HILIC stationary phases driven by the need to obtain better selectivity for a wider range of compounds [1]. Introduction of mixed-mode retention mechanism into conventional RPLC could improve the resolution of some complex samples, and HILIC could retain hydrophilic solutes while solving the solubility in mobile phase and maintaining the compatibility with mass spectrometry. Surface modification to supporting silica with different selectors is one significant strategy to obtain novel stationary phases, and ionic liquids (ILs) have attracted increasing interest as a class of stationary phase selectors in recent years. ILs are a special class of molten salts composed of organic cations and organic or inorganic anions, and they possess a series of unique properties, including negligible vapor pressure, and multiple

∗ Corresponding author. Tel.: +86 411 84379757; fax: +86 411 84379559. ∗∗ Corresponding author. Tel.: +86 411 84379530; fax: +86 411 84379530. E-mail addresses: [email protected] (X. Shi), [email protected] (G. Xu). http://dx.doi.org/10.1016/j.chroma.2015.03.081 0021-9673/© 2015 Elsevier B.V. All rights reserved.

solvation interactions. In particular, ILs can be tuned with desired physicochemical properties to satisfy specific demands by introducing certain functional groups or varying combinations of cations and anions. Based on these outstanding features, ILs have been a hot research topic drawing great attention and have been applied in many areas, such as electrochemistry [2,3] and separation fields [4]. Immobilizing ILs on supported materials can expand the scope of applications and improve efficiency in some cases. Although it is generally accepted that ILs would lose the liquid state once immobilized on supported materials especially solid substrate, the unique properties of ILs can be retained while also combining the advantages of supporting materials [5,6]. Surface-confined or supported ILs have been an important studied branch with great attention for surface modification, catalyst [7], solid-phase extraction [8] and HPLC stationary phases [9]. HPLC stationary phases were obtained by bonding ILs onto the surface of silica through covalent linkage, and there have been a lot of involved studies mainly based on imidazolium ILs, also including pyridinium [10], quinolium [11] and guanidinium-based ILs [12]. Surface-confined or supported ILs present great potential as candidates for HPLC stationary phases, and the investigation into chromatographic retention mechanism and linear solvation energy relationship (LSER) method indicates that surface-confined IL stationary phases could undergo multimodal interactions with

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solutes, such as ␲–␲ and dipolar interactions, hydrogen bonding, electrostatic forces and ion-exchange [5,13–16]. The mixed-mode retention mechanism provides beneficial performances in chromatographic separation, from RPLC, ion-exchange to HILIC and mixed-mode chromatography. For example, some IL modified silica stationary phases exhibit better selectivity to certain isomeric solutes with high ␲ electron density because of the multiple interactions including ␲–␲ and dipolar interactions, which is different from conventional stationary phases (such as C18) simply based on hydrophobic mechanism [17–21]. Moreover, ILs based stationary phases were also evaluated for HILIC and mixed-mode chromatography and present good separation efficiency and a wide range of selectivity as promising candidates [12,22–24]. So far, most of reported studies have been focused on traditional monocationic ILs. Compared with conventional monocationic ILs, multifunctional ILs (mainly dicationic ILs and tricationic ILs) can possess a broader range of physicochemical properties by tuning the cationic and anionic moieties or varying their combinations [25–27]. Especially, dicationic ILs have received a lot of attention with some beneficial features for applications in dye-sensitized solar cells [28,29], as solvents for organic reactions [30], as stationary phases of capillary gas chromatography (CGC) with exceptionally good thermal stability [31]. Typical tricationic ILs are composed of three positively charged moieties anchored to a central core and coordinating anions, and enable greater possibility for a variety of properties. As CGC stationary phases, tricationic ILs possess unique selectivity and present considerable promising prospect [32]. Besides, immobilizing free multiple cationic ILs on supporting materials would offer special advantages. In supported multiple cationic ILs for catalyst, bisimidazolium and tri-imidazolium moieties contribute to formation of prominent three-dimensional catalytic environment providing more catalytic active sites, and further result in higher catalytic efficiency and recyclability [33–35]. Besides, a bi-guanidinium IL bridged polysilsesquioxane membrane presents good fuel cell performance with impressive hydroxide conductivity and decent alkali-stability [36]. In our previous study, dicationic imidazolium IL bonded silica exhibited appropriate retention and good separation efficiency as HILIC stationary phases, but their monocationic analogues did not possess effective hydrophilic retention behavior [23]. Similarly, tricationic ILs should have potential as HPLC selectors, and the increase of cations and the special arrangement would bring unique chromatographic behavior with different solvation properties. However, there is no related research available about surface-confined tricationic ILs as HPLC stationary phases. In the present work, we evaluated the chromatographic performance of the tricationic IL modified silica as HPLC stationary phases. Two tricationic imidazolium ILs were synthesized and then bonded to the surface of porous silica modified with 3-mercaptopropyl through “thiol-ene” click chemistry, and the obtained modified silica materials were packed as HPLC columns. Two columns from each surface-confined tricationic IL were separately prepared to evaluate the chromatographic behavior in the RPLC and HILIC modes. Four hydrophobic mixtures were used to characterize the RPLC selectivity and separation performance, and the mixed-mode retention mechanism was investigated by correlating retention factors with hydrophobic nature and compared with a commercial C8 column. The HILIC retention and selectivity were evaluated by separating typical hydrophilic solutes nucleosides and bases, and retention changes with water content in mobile phase were investigated to reveal the retention mechanism. Besides, a mixture of 21 flavonoids was separated on the prepared tricationic IL columns in the HILIC mode followed by the injection of extracted soybean flavonoids. All the results indicate that the surface-confined tricationic imidazolium ILs possess mixed-mode retention behavior

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with multiple interactions, and present great potential as both RPLC and HILIC stationary phases. 2. Experimental 2.1. Materials and instrumental Bis(trifluoromethane)sulfonimide lithium salt (LiNTf2 ), 2,4,6tris(bromomethyl)mesitylene, 1,3,5-tris(bromomethyl)benzene, 1-vinylimidazole, (3-mercaptopropyl)trimethoxysilane and ammonium formate (NH4 FA) were purchased from Sigma-Aldrich (St. Louis, Mo, USA). 2,2 -Azobis(2-methylpropionitrile) (AIBN) was obtained from J&K Scientific (Beijing, China). Porous silica spheres with 5 ␮m diameter, 10 nm pore size and 310 m2 g−1 surface area were used as the stationary phase matrix and were from Fuji Silysia Chemical (Kasugai, Japan). Flavonoids daidzin, formononetin, genistein, daidzein, apigenin and phloretin were from J&K Scientific (Beijing, China); chrysin, naringenin and catechin were from Acros Organics (Geel, Belgium); prunetin, taxifolin, phlorizindihydrate, naringin, genistin, (±)-dihydrokaempferol, vitexin, hesperetin and hesperidin were from Sigma-Aldrich (St. Louis, Mo, USA); 2 -hydroxychalcone was from Alfa Aesar (Tianjin, China); diosmetinand pelargonidin chloride were from Chromadex (Irvine, USA). Anhydrous toluene was obtained by refluxing analytically pure toluene in the presence of sodium under argon. The mobile phases were prepared from acetonitrile (ACN) of HPLC grade (Merck, Germany) and Milli-Q water (resistivity of 18 M cm−1 ). The surface coverage was determined by elemental analysis on an Elementar Vario EL III elemental analyzer (Hanau, Germany). The infrared spectra and 1 H NMR (nuclear magnetic resonance) spectra were measured respectively on a FT-IR (Fourier-transform infrared) system of PerkinElmer Spectrum GX Spectrometer (Waltham, MA, USA) and a Bruker ultrashieldTM Plus 400 NMR spectrometer (Karlsruhe, Germany). An Agilent 1290 UPLC system equipped with a diode array detector (DAD) was used to evaluate the chromatographic performance of the prepared stationary phases. A RPLC Ascentis® Express C8 column (150 mm × 2.1 mm, 2.7 ␮m, carbon coverage of 3.7 ␮mol/m2 , Supelco) was used in the RPLC behavior evaluation to compare with the prepared tricationic IL columns, and the used silica particle possesses a 1.7 ␮m solid fused core and 0.5 ␮m porous shell with 9 nm pore size and 150 m2 g−1 surface area. 2.2. Preparation of the tricationic ionic liquid-bonded silica materials The structures of the two tricationic ILs used as the stationary phases molecules immobilized onto the surface of silica materials are shown in Fig. 1. First, the tricationic bromides were prepared according to the literature [26] with some modification. One mole equivalents of 2,4,6-tris(bromomethyl)mesitylene or 1,3,5-tris(bromomethyl)benzene and six to nine equivalents of 1-vinylimidazole were refluxed in isopropanol for eight days under argon, and then the solvent was removed by rotary evaporation under reduced pressure. The obtained products were washed with ethyl acetate several times and the tricationic bromides were collected as white solids. Through the method, 1,3,5-{tris(3-vinylimidazolium)methyl}mesitylene trisbromide and 1,3,5-{tris(3-vinylimidazolium)methyl}benzene trisbromide were prepared. Then, the tricationic ILs with NTf2 − counteranion as shown in Fig. 1 were obtained by anion exchange. LiNTf2 of five moles equivalent to the tricationic bromide was dissolved in water and added into the aqueous solution of the bromide salt under stirring.

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Fig. 1. The structures of tricationic ILs (Me3 Benz(VMIm)3 -NTf2 and Benz(VMIm)3 NTf2 ) bonded to the surface of supporting silica materials.

After 48 h of stirring under room temperature, dichloromethane was added and there were separated layers in the reaction solution. The top water layer was removed and the remaining solution was washed with water several times. The solvent was removed under reduced pressure and the tricationic IL with NTf2 − counteranion was collected. Through the same procedures, two tricationic ILs 1,3,5-{tris(3-vinylimidazolium)methyl}mesitylene tris(bis(trifluoromethane)-sulfonimide) (Me3 Benz(VMIm)3 -NTf2 ) and 1,3,5-{tris(3-vinylimidazolium)methyl}benzene tris(bis(trifluoromethane)sulfonimide) (Benz(VMIm)3 -NTf2 ) were obtained as stationary phase selectors to be bonded to the surface of silica. The obtained tricationic ILs with NTf2 − counteranion were bonded to the surface of porous spherical silica. Firstly, 3mercaptopropyl modified silica was prepared by refluxing the suspension of silica materials in anhydrous toluene with (3mercaptopropyl)trimethoxysilane under argon for 24 h. Secondly, the tricationic IL was dissolved in methanol and added to the suspension of 3-mercaptopropyl modified silica in methanol. The reaction mixture was stirred in the presence of AIBN at 60 ◦ C under argon for 26 h, and the tricationic IL-bonded silica was obtained by centrifugation and washing with methanol. Through the method, two tricationic ILs surface-confined silica stationary phases SilMe3 Benz(VMIm)3 -NTf2 and Sil-Benz(VMIm)3 -NTf2 were obtained. 2.3. Column packing and chromatographic evaluation The two types of tricationic IL-bonded silica materials were respectively packed as HPLC columns of 150 mm × 3.0 mm i.d. by a slurry packing method. The bonded silica materials were suspended in methanol and the suspension was slurry packed into stainless steel columns under a constant pressure of 60 MPa with methanol as the pushing solvent. For RPLC evaluation, pure acetonitrile and water were used as the mobile phase. The concentration of hydrophobic test solutes in standard solutions was different depending on the detector response, and the injection volume was 1–3 ␮L. The alkyl benzene mixture included seven normal alkyl benzenes from benzene to hexylbenzene, and the concentrations were between 0.2 and 0.35 ␮L/mL. The polycyclic aromatic hydrocarbon (PAH) mixture consisted of uracil, nitrobenzene, naphthalene, biphenyl, fluorine, phenanthrene and pyrene with the concentration ranging from 10

to 100 ␮g/mL. The Tanaka test mixture consisted of uracil, amylbenzene, butylbenzene, triphenylene, o-terphenyl, phenol and benzylamine with the concentration in the range of 5–80 ␮g/mL or 0.5–0.8 ␮L/mL. A test mixture of geometric PAH isomers included o-terphenyl, m-terphenyl, p-terphenyl and triphenylene, and the concentrations respectively were 70, 30, 170 and 20 ␮g/mL. For HILIC evaluation, the mobile phase consisted of ACN/H2 O (95/5, v/v) containing 10 mM NH4 FA and water containing 10 mM NH4 FA. The concentrations of standard solutions were 20 ␮g/mL for nucleosides, 20 ␮g/mL for bases and 20–40 ␮g/mL for flavonoids, and the injection volumes for the three mixtures, respectively, were 5, 2 and 3 ␮L. Throughout the experiments, the flow rate was 0.4 mL/min (0.2 mL/min for the commercial C8 column) and the column temperature was kept at 30 ◦ C. The dead time t0 was determined with toluene or uracil as the test solute under HILIC conditions (ACN/H2 O, 90/10, v/v) and RPLC conditions (ACN/H2 O, 50/50, v/v), respectively. All the injections were repeated at least three times. Soybean flavonoids were extracted by adding 1.5 mL CH3 OH/H2 O (80:20, v/v) into a 2 mL Eppendorf tube containing 50 mg of soybean powder, followed by vortex-mixing for 30 s, sonication for 10 min and centrifugation at 14,000 × g, 4 ◦ C for 20 min. Then, 1 mL supernatant was collected into a new Eppendorf tube and freeze-dried in a Labconco Freezone 4.5 centrifuge concentrator. Then, the residue was redissolved using 250 ␮L CH3 OH/H2 O (80:20, v/v) for injection. 3. Results and discussion 3.1. Characterization of the tricationic IL stationary phases The synthesized tricationic bromides were characterized by 1 H NMR in deuterated water and dimethyl sulphoxide (DMSO) and the spectra are shown in Figure S1. The preparation of tricationic ILs with NTf2 − counteranion was based on a metathesis reaction. In order to ensure the completeness of anion exchange from Br− to NTf2 − and the absence of bromide impurities or possible salts, an excess of LiNTf2 was used and the top washing water layer was tested using silver nitrate until there was no visible precipitate. The obtained NTf2 − tricationic ILs were bonded to the surface of supporting silica materials modified with 3-mercaptopropyl through “thiol-ene” click chemistry, and the obtained materials were characterized by FT-IR spectroscopy and elemental analysis (C, H, N). The IR spectra in Figure S2 show that both 3-mercaptopropyl modified silica (–SH SiO2 ) and tricationic IL-bonded silica possess –C–H stretching vibration peaks at ∼2800–3000 cm−1 . Besides, there are =C–H stretching vibration peaks above 3000 cm−1 , peaks at ∼1500–1600 cm−1 from imidazole ring and benzene ring and characteristic peaks of anion NTf2 − at ∼1200, ∼1350 and ∼600 cm−1 in IR spectra of the tricationic IL-bonded silica materials. The elemental analysis data are given as loading percentages of elements (C, H, N) by weight. The 3mercaptopropyl modified silica (C% 3.58, N% 0, H% 0.91) possess a surface coverage density of 3.5 ␮mol/m2 calculated from carbon loading. The surface coverage density of Sil-Me3 Benz(VMIm)3 -NTf2 (C% 9.62, N% 2.22, H% 1.22) and Sil-Benz(VMIm)3 -NTf2 (C% 9.21, N% 2.24, H% 1.10) respectively are 0.73 and 0.74 ␮mol/m2 from nitrogen loading values. 3.2. RPLC evaluation 3.2.1. Chromatographic performance in the RPLC mode A normal alkyl benzene mixture and a PAH mixture were used to investigate the RPLC chromatographic performance of the prepared tricationic IL stationary phases. As shown in Fig. 2, the

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Fig. 2. Separation chromatograms of normal alkylbenzenes (a) and PAHs (b) on two tricationic IL columns and a C8 column. Peaks: 1, benzene; 2, toluene; 3, ethylbenzene; 4, propylbenzene; 5, butylbenzene; 6, amylbenzene; 7, hexylbenzene; 1 , uracil; 2 , nitrobenzene; 3 , naphthalene; 4 , biphenyl; 5 , fluorine; 6 , phenanthrene; 7 , pyrene. Chromatographic conditions: an isocratic elution of ACN/H2 O (50/50, v/v), flow rate of 0.4 mL/min for tricationic IL columns and ACN/H2 O (60/40, v/v), 0.2 mL/min for C8 column, column temperature of 30 ◦ C and DAD at 254 nm.

tricationic IL columns exhibit effective retention and selectivity to these hydrophobic solutes with symmetrical peak shape. With naphthalene as the test solute, the column efficiency is 80,000 plates/m for Sil-Me3 Benz(VMIm)3 -NTf2 column and 73,000 plates/m for Sil-Benz(VMIm)3 -NTf2 column, respectively with retention times of 5.44 and 5.68 min at a mobile phase of ACN/H2 O (50/50, v/v), flow rate of 0.4 mL/min and column temperature of 30 ◦ C. The two mixtures were separated on a commercial C8 column and the results are also shown in Fig. 2. It can be seen that the tricationic IL columns possess lower hydrophobicity than the commercial C8 column since a stronger eluting mobile phase was used for C8 column. Specifically, the C8 stationary phase presents better methylene selectivity, and the tricationic IL stationary phases exhibit more efficient retention for most PAHs. 3.2.2. Investigation into retention mechanism of the tricationic IL stationary phases The retention changes of some hydrophobic solutes with the ACN content in mobile phase were investigated in the range of 40–70% ACN volume fractions with an interval of 5%. Figure S3 describes the relationship between retention factors and corresponding ACN volume fractions, and displays a typical

RPLC retention mode, the retention decreases when the ACN content increased. Besides, the positive correlation of log k and log P (octanol–water partition coefficient used to represent the hydrophobicity of a solute) in Fig. 3 also demonstrates the RPLC retention mode of hydrophobic mechanism. Meanwhile, the plot of log k versus log P on the commercial C8 column is given in Fig. 3. It can be observed that the linear curves of PAHs and alkylbenzenes possess the nearly same slopes for the C8 column and the plot of alkylbenzenes is above the plot of PAHs. On the tricationic IL columns, the curve for PAHs presents a larger slope than that for alkylbenzes, and the retention factors of PAHs are significantly stronger than those of alkylbenzenes at the larger log P values. Conventional C8 stationary phases retain solutes mainly based on hydrophobic interaction mechanism, but the retention on surface-confined IL stationary phases was from multiple interactions. The tricationic IL stationary phases have great ability to undergo ␲–␲ and dipolar interactions due to the presence of core benzene ring and three terminal imidazole rings. These interactions would enhance the retention of solutes with higher electron density and greater conjugated aromatic system such as PAHs. The separation of Tanaka test mixture also demonstrates the retention difference between C8 stationary phase and the tricationic IL

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Fig. 3. The correlation plots of log k versus log P for two tricationic IL columns and a C8 column. The chromatographic conditions were the same as in Fig. 2. Black lines with squares were based on normal alkylbenzenes benzene, ethylbenzene, butylbenzene, hexylbenzene, octylbenzene, decylbenzene and dodecylbenzene, and red lines with circular points were based on PAHs, being benzene, naphthalene, anthracene, tetracene and penracene in ascending order of hydrophobicity (For interpretation of the color information in this figure legend, the reader is referred to the web version of the article.).

stationary phases. As shown in Fig. 4, butylbenzene, amylbenzene and o-terphenyl, triphenylene exhibit different elution orders on the tricationic IL columns and C8 column. Specifically, o-terphenyl and triphenylene exhibit reversed elution order, and they elute later than butylbenzene and amylbenzene on tricationic IL columns but elute between butylbenzene and amylbenzene on C8 column. 3.2.3. Selectivity of the tricationic IL stationary phases to isomeric PAHs A test mixture of PAH geometrical isomers including oterphenyl, m-terphenyl, p-terphenyl and triphenylene was used to evaluate the shape selectivity of the prepared tricationic IL columns, and the separation chromatograms together with that on C8 column are shown in Fig. 5. Compared with C8 stationary phase, the two tricationic IL columns present enhanced selectivity to these PAH isomers, and display reversed elution orders for the three positional isomeric terphenyls and triphenylene. As mentioned above, the retention on C8 stationary phase mainly originates from hydrophobic mechanism, but the tricationic IL stationary phases can undergo multiple interactions with solutes. On the one hand, the lower molecular rigidity of o-terphenyl, mterphenyl and p-terphenyl than triphenylene favors twisting and bending, and further getting access to interact with stationary phase molecules through hydrophobic mechanism. On the other hand, four conjugated aromatic rings in the structure of triphenylene render it higher electron density, and provide stronger ␲-␲ and dipolar interactions with tricationic IL stationary phases. The two tricationic IL columns exhibit the same elution orders and similar separation capability to the isomeric PAH mixture except obvious differentiation in retention to triphenylene. In the structure of Me3 Benz(VMIm)3 -NTf2 , three substituted methyl groups in the tricationic core benzene ring bring greater structural rigidity, which is favorable to the interactions between the stationary phase molecules and rigid triphenylene and further contribute to the later elution than that on more flexible Benz(VMIm)3 -NTf2 molecule based column. 3.3. HILIC retention behavior 3.3.1. Separation performance in the HILIC mode Buffer salt is commonly used in the HILIC mode in order to improve the peak shape and obtain better separation performance, and in the HILIC evaluation ammonium formate was added in the mobile phase. However, the counteranions of the tricationic IL columns might have been exchanged after HILIC application [17,23], which would have an effect on the retention of hydrophobic solutes in the RPLC mode usually without buffer salt in mobile

phase. Therefore, the evaluation of RPLC and HILIC retention was performed separately using two same columns. Two mixtures of nucleosides and bases were separated on the two tricationic IL columns, and the chromatograms are given in Fig. 6. It can be seen that both the two columns exhibit good selectivity and efficient hydrophilic retention to these typical hydrophilic solutes. Moreover, slightly different selectivity and some reversed elution orders are displayed although the stationary phases are similar. For example, nucleosides 4 (N6 -methyladenosine) and 5 (5-methyluridine) coeluting on Sil-Me3 Benz(VMIm)3 -NTf2 column are successfully separated on Sil-Benz(VMIm)3 -NTf2 column. Sil-Benz(VMIm)3 -NTf2 column shows no selectivity to nucleosides 7 (uridine) and 8 (adenosine), 9 (7-methylguanosine) and 10 (1-methylguanosine), but they get effective separation on Sil-Me3 Benz(VMIm)3 -NTf2 column. Besides, reversed elution order such as bases 4 (cytosine) and 5 (adenine) was observed on the two tested columns. In terms of hydrophilic retention, the two columns did not exhibit significant difference, although the separation performance seemed better on Sil-Me3 Benz(VMIm)3 -NTf2 column, such as successful separation of bases 7 (xanthine) and 8 (guanine), and better selectivity to nucleosides 9–14. The different separation performances on the two columns mainly resulted from the slight difference in the structures of the two tricationic ILs, which would bring steric interactions into the separation process. Structurally, Sil-Me3 Benz(VMIm)3 -NTf2 column presents greater steric interactions, and might have better selectivity to solutes with steric difference in molecular structure. 3.3.2. Retention mechanism in the HILIC mode The HILIC retention behavior of the tricationic IL stationary phases was evaluated by investigating the retention changes of hydrophilic solutes (including nucleosides and bases) with the content of water in mobile phase, and the results are expressed as trend curves of retention factors versus water volume fractions in Figure S4. It can be seen that the HILIC retention exhibits a decrease when the water volume fraction increases on the two tricationic IL columns, indicating typical HILIC retention behavior, which has been explained from molecular dynamics point through the partitioning mechanism between the ACN-rich mobile phase and a water layer on the surface of the stationary phase [37]. Moreover, the fitting results of the retention factors at different water volume fractions based on two equations respectively describing partitioning mechanism and adsorption mechanism are listed in Table S1, and the retention does not follow the partitioning equation exclusively. Present studies have drawn a widely accepted conclusion that the HILIC retention is a mixed-mode

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Fig. 4. The separation of a Tanaka test mixture on two tricationic IL columns and a C8 column. Peaks: 1, benzylamine; 2, uracil; 3, phenol; 4, butylbenzene; 5, amylbenzene; 6, o-terphenyl; 7, triphenylene. The chromatographic conditions were the same as in Fig. 2.

process including hydrophilic partitioning, adsorption-like mechanisms and other possible interactions [38–40]. The retention change resulting from interactions other than partitioning mechanism is remarkable and might lead to fitting better to the adsorption equation.

3.3.3. Selectivity and retention to flavonoids in the HILIC mode Flavonoids are widespread secondary plant metabolites with great structural diversity. They possess high therapeutic/pharmaceutical significance, and the determination has been of considerable interest for decades. In the field of

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Fig. 5. Separation selectivity of the tricationic IL columns and a C8 column to four isomeric PAHs. Peaks: 1, o-terphenyl; 2, m-terphenyl; 3, p-terphenyl; 4, triphenylene. The chromatographic conditions were the same as in Fig. 2.

chromatography, RPLC is the most used technique to separate flavonoids and further conduct characterization and determination. Moreover, in recent years some other techniques have been applied in separation of flavonoids in order to improve throughput and resolution, including multi-dimensional LC [41] and HILIC [12,42]. In fact, flavonoids often exist in the form of glycosides by combining sugar substituents, which render them sufficiently hydrophilic to be retained appropriately on HILIC columns. However, in our previous study to separate a flavonoids mixture in the HILIC mode [12], the chromatographic peaks are divided into two separate groups, one group consisting of weakly retained basic flavonoids and the other group being hydrophilic flavonoid glycosides. The chromatograms of a mixture of 21 flavonoids on the two tricationic IL columns in the HILIC mode are shown in Fig. 7, and it can be seen that the tricationic IL columns could effectively retain and separate flavonoids with peaks uniformly distributed through the HILIC gradient. Flavonoids exist in neutral molecular forms under the present chromatographic conditions with a mobile phase of pH

6 as their pKa values range between 7 and 11 [43], and thereby they are mainly retained through hydrophilic partitioning mechanism and other mixed-mode interactions between solutes and tricationic ILs without ionic exchange. Compared with reported HILIC columns used to separate flavonoids, tricationic ILs possess stronger hydrogen bonding, dipolar, ␲–␲, electrostatic and possible steric interactions. These interactions are significant for enhanced retention of basic flavonoid structures, and the flavonoids are not eluted in strict order of hydrophilicity. Therefore, the tricationic IL columns not only display selectivity and effective retention to hydrophilic flavonoid glycosides but also to basic flavonoids without hydrophilic substituents. After the separation of flavonoids standards, extracted soybean flavonoids were injected and the chromatograms are given in Fig. 7 with good separation efficiency. All the results indicate that the tricationic IL columns could improve selectivity and retention to a wide range of flavonoids in the HILIC mode, and have the potential to be applied in the resolution of complex samples.

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Fig. 6. Separation chromatograms of nucleosides (a) and bases (b) on tricationic IL columns in the HILIC mode. Peaks for (a): 1, thymidine; 2, 5 -deoxy-5 (methylthio)adenosine; 3, 1-methyladenosine; 4, N6 -methyladenosine; 5, 5-methyluridine; 6, 5-methylcytosine; 7, uridine; 8, adenosine; 9, 7-methylguanosine; 10, 1-methylguanosine; 11, pseudouridine; 12, inosine; 13, cytidine; 14, 2 -deoxyguanosine; 15, guanosine. Peaks for (b): 1 , thymine; 2 , uracil; 3 , 1-methylxanthine; 4 , cytosine; 5 , adenine; 6 , hypoxanthine; 7 , xanthine; 8 , guanine. Chromatographic conditions: a gradient elution consisted of mobile phase A water containing 10 mM NH4 FA and mobile phase B ACN/H2 O (95/5, v/v) containing 10 mM NH4 FA, 10% A maintained 6 min, then increased to 30% A within 6–10 min and kept at 30% A, column temperature 30 ◦ C, flow rate 0.4 mL/min, DAD 254 nm.

Fig. 7. Separation of a flavonoids mixture (a) and extracted soybean flavonoids (b) on tricationic IL columns in the HILIC mode. Peaks for (a): 1, 2 -hydroxychalcone; 2, prunetin; 3, formononetin; 4, chrysin; 5, hesperetin; 6, naringenin; 7, genistein; 8, daidzein; 9, diosmetin; 10, (±)-dihydrokaempferol; 11, apigenin; 12, phloretin; 13, pelargonidin chloride; 14, genistin; 15, hesperidin; 16, daidzin; 17, naringin; 18, taxifolin; 19, phlorizindihydrate; 20, catechin; 21, vitexin. Chromatographic conditions: gradient elution consisted of mobile phase A water containing 10 mM NH4 FA and mobile phase B ACN/H2 O (95/5, v/v) containing 10 mM NH4 FA, 5% A maintained 6 min, then increased to 30% A within 6–12 min and kept at 30% A for the flavonoids mixture, 5% A maintained 6 min, then increased to 40% A within 6–14 min and kept at 40% A for extracted soybean flavonoids, column temperature 30 ◦ C, flow rate 0.4 mL/min, DAD 254 nm.

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4. Conclusions The prepared tricationic IL stationary phases present appropriate retention to hydrophobic solutes in the RPLC mode and the retention decreases with increase of ACN content in mobile phase. Compared with a commercial C8 column, the tricationic IL columns possess weaker hydrophobicity, but exhibit exceptionally good selectivity to isomeric PAHs due to multiple interactions including ␲–␲ and dipolar interactions. Moreover, PAHs, especially PAHs with great electron density, possess different retention on the two kinds of tricationic IL stationary phases, which could be contributed to the different structural rigidity. The evaluation of chromatographic behavior in the HILIC mode indicates that the tricationic IL stationary phases also present typical HILIC retention mechanism and could effectively retain hydrophilic solutes (nucleosides and bases) with good selectivity. Besides, the tricationic IL columns exhibited more efficient retention and better selectivity to flavonoid standards than reported HILIC columns, and show great prospect for application in analysis of complex samples. In conclusion, the tricationic IL stationary phases possess mixed-mode retention mechanism based on multiple interactions and exhibit enhanced selectivity to isomeric PAHs and flavonoids respectively in the RPLC and HILIC modes. Acknowledgement The study was funded by the foundations (21275141, 21375011 and 21435006) and the creative research group project (21321064) from the National Natural Science Foundation of China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2015.03.081. References [1] T.L. Chester, Recent developments in high-performance liquid chromatography stationary phases, Anal. Chem. 85 (2013) 579–589. [2] D.S. Silvester, Recent advances in the use of ionic liquids for electrochemical sensing, Analyst 136 (2011) 4871–4882. [3] M. Opallo, A. Lesniewski, A review on electrodes modified with ionic liquids, J. Electroanal. Chem. 656 (2011) 2–16. [4] T.D. Ho, C. Zhang, L.W. Hantao, J.L. Anderson, Ionic liquids in analytical chemistry: fundamentals, advances, and perspectives, Anal. Chem. 86 (2014) 262–285. [5] V. Pino, A.M. Afonso, Surface-bonded ionic liquid stationary phases in highperformance liquid chromatography—a review, Anal. Chim. Acta 714 (2012) 20–37. [6] B. Xin, J. Hao, Imidazolium-based ionic liquids grafted on solid surfaces, Chem. Soc. Rev. 43 (2014) 7171–7187. [7] R. Skoda-Foeldes, The use of supported acidic ionic liquids in organic synthesis, Molecules 19 (2014) 8840–8884. [8] H. Yu, T.D. Ho, J.L. Anderson, Ionic liquid and polymeric ionic liquid coatings in solid-phase microextraction, TrAC Trends Anal. Chem. 45 (2013) 219–232. [9] M. Zhang, X. Liang, S. Jiang, H. Qiu, Preparation and applications of surfaceconfined ionic-liquid stationary phases for liquid chromatography, TrAC Trends Anal. Chem. 53 (2014) 60–72. [10] D.S. Van Meter, O.D. Stuart, A.B. Carle, A.M. Stalcup, Characterization of a novel pyridinium bromide surface confined ionic liquid stationary phase for highperformance liquid chromatography under normal phase conditions via linear solvation energy relationships, J. Chromatogr. A 1191 (2008) 67–71. [11] M. Sun, J. Feng, C. Luo, X. Liu, S. Jiang, Quinolinium ionic liquid-modified silica as a novel stationary phase for high-performance liquid chromatography, Anal. Bioanal. Chem. 406 (2014) 2651–2658. [12] L. Qiao, S. Wang, H. Li, Y. Shan, A. Dou, X. Shi, G. Xu, A novel surfaceconfined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography, J. Chromatogr. A 1360 (2014) 240–247. [13] B.J. VanMiddlesworth, A.M. Stalcup, Characterization of surface confined ionic liquid stationary phases: Impact of cation revisited, J. Chromatogr. A 1364 (2014) 171–182.

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