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A novel surface-confined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography夽 Lizhen Qiao, Shuangyuan Wang, Hua Li, Yuanhong Shan, Abo Dou, Xianzhe Shi ∗ , 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
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Article history: Received 2 June 2014 Received in revised form 10 July 2014 Accepted 28 July 2014 Available online xxx Keywords: Glucaminium-based ionic liquid Hydrophilic interaction chromatography Anion-exchange Stationary phase
a b s t r a c t Glucaminium-based ionic liquids are a new class of recently developed ionic liquids and prepared by functionalizing the amine group of N-methyl-d-glucamine, which renders them good hydrophilicity due to the presence of the glucose structure and charged quaternary ammonium group. In the present study, a glucaminium-based ionic liquid N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized and subsequently bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The obtained stationary phase was characterized by elemental analysis and infrared spectroscopy, and then packed as a HPLC column. A mixture of five nucleosides was used to characterize the separation performance of the obtained column under HILIC mode and the column efficiency was determined with cytidine as the test solute, reaching 80,000 plates/m. Then, the retention behavior was evaluated by investigating the effect of various chromatographic factors on retention of different types of solutes, and the results revealed that the developed surface-confined glucaminium-based ionic liquid stationary phase exhibited a hydrophilic interaction/anion-exchange mixed-mode retention mechanism. Finally, two mixtures of nucleotides and flavonoids were separated on the glucaminium-based ionic liquid column, respectively under hydrophilic interaction and hydrophilic interaction/anion-exchange mixedmode chromatography. In conclusion, the multimodal retention capabilities of the glucaminium-based ionic liquid column could offer a wider range of retention behavior and flexible selectivity toward polar and hydrophilic compounds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hydrophilic interaction chromatography (HILIC) is playing an increasingly important role in separating polar and hydrophilic compounds in recent years with some unique advantages over conventional techniques, such as the good solubility of polar samples in aqueous mobile phase and the compatibility with mass spectrometry (MS), and currently it has been applied in various fields. In order to obtain a wider range of selectivity to various polarity of compounds and better performance, the development of HILIC stationary phases has always been a hot research field. Surface modification to silica materials with different polar
夽 Invited paper for the Honor Issue of Professor Peichang Lu’s 90th birthday. ∗ 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).
functional groups is key to provide different retention behavior and has gained great progress. Commercially available HILIC stationary phases mainly include bare silica, chemically bonded diol, amino and amide silica, and ZIC-HILIC columns, and several researchers also developed other HILIC stationary phases through covalently bonding hydrophilic molecules to the surface of silica materials, such as cyclofructans [1–3], zwitterionic functionalities [4–6] and so on. Besides the new bonded phases, introduction of multiple retention modes into one stationary phase was another strategy to expand the selectivity, i.e. mixed-mode stationary phases, and the combination of more than one retention modes could offer flexible and adjustable selectivity. Under HILIC mode, the hydrophilic-partition, adsorption mechanism and electrostatic interactions might coexist between the stationary phase and solutes and further lead to chromatographic retention. Moreover, charged groups in bonded functionalities could provide the possibility of ion-exchange with oppositely charged solutes while
http://dx.doi.org/10.1016/j.chroma.2014.07.096 0021-9673/© 2014 Elsevier B.V. All rights reserved.
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improving hydrophilicity, and thus a HILIC/ion-change mixedmode chromatographic system is obtained. It was reported that HILIC/ion-exchange showed many advantages (such as tunable selectivity, more efficient retention and better separation) for the application of peptides [7], small molecular drugs [8], and proteins [9]. Recently some new HILIC stationary phases were developed and exhibited HILIC/ion-change mixed-mode retention behavior. For example, a glutathione-based mixed-mode HILIC/cation-exchange stationary phase was prepared and successfully used to separate neutral fructosan with high degree of polymerization, basic chitooligosaccharides and strongly acidic carrageenan oligosaccharides [10]. In the previous work [11] we developed a new type of surface-confined dicationic ionic liquids stationary phases, which exhibited HILIC/anion-exchange mixed-mode retention mechanism. Ionic liquids (ILs) are an important class of non-molecular solvents possessing many outstanding properties such as high thermal stability, negligible vapor pressure and intrinisic conductivity, and have been applied in many fields. In addition, ILs possess the potential to undergo multiple solvation interactions and can be tailored to obtain desired properties by introducing appropriate functional groups or combining different cationic and anionic moieties. These beneficial characteristics have made ILs attractive for analytical chemistry applications as extraction phases, dissolution solvents, separation media, and electrochemical sensing systems, etc. [12,13]. In HPLC, ILs were applied in two ways, as mobile phase additives to mask the adverse effect of residual silanol groups [14–16] or being immobilized to the surface of supporting materials as stationary phases [17,18]. Qiu et al. developed a number of imidazolium ionic liquid-based stationary phases [19–23], and found that ILs of different structures might exhibit reversed phase, ion-exchange or HILIC retention mechanism. Apart from the conventional monocationic ILs, our group recently prepared several imidazolium dicationic ILs-based stationary phases exhibiting considerably high column efficiency under HILIC mode [11]. All the studies indicated that ILs-modified HPLC columns could retain a wide range of solutes through multimodal retention mechanism such as electrostatic, ionic, – and dipole interactions [17,18,24], and are enjoying increasing popularity as a promising class of HPLC stationary phases. Depending on the cationic moieties, ILs can be divided into imidazolium, ammonium, phosphonium, pyridinium and guanidinium-based ILs. However, currently only imidazolium and pyridinium ILs are used as bonded selectors for HPLC stationary phases. Glucaminium-based ILs were a new kind of ILs recently developed to remove or extract boron-species, improve the separation of hydrophilic boron compounds under RPLC by using the complexion behavior with borate [25–27]. Moreover, glucaminium-based ILs presented good extraction performances to alkyl halides and aromatics [28], deoxyribonucleic acid (DNA) [29], indicating they could provide the ability to function with solutes by multiple interactions. Glucaminium-based ILs are prepared from N-methyl-d-glucamine through the functionalization of the amine group with halogen substituted compounds [30], and the presence of the glucose structure renders the IL molecule good hydrophilicity. These properties make glucaminium-based ILs possess great potential as attractive selectors for HILIC stationary phase, and therefore in the present study the chromatographic performance of glucaminium-based IL modified silica was studied as HILIC stationary phase. A new glucaminium-based IL with allyl groups was synthesized and grafted to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The glucaminium-based IL bonded silica was packed as a HPLC column after characterization and a mixture of five nucleosides was separated on the obtained column to evaluate the selectivity under HILIC mode. The effect of chromatographic factors (including salt
concentration in mobile phase, water content in mobile phase and the mobile phase pH) on retention was investigated by using a series of solutes to reveal the retention mechanism. Finally, the glucaminium-based IL column was used to separate two mixtures of nucleotides and flavonoids. 2. Experimental 2.1. Materials and instruments Allyl bromide, N-methyl-d-glucamine, (3-mercaptopropyl)trimethoxysilane, sodium carbonate, ammonium formate (NH4 FA), formic acid and involved nucleotides were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2,2 -Azobis(2methylpropionitrile) (AIBN) was obtained from J&K Scientific (Beijing, China). Toluene was refluxed to remove water in the presence of sodium before use. Acetonitrile (ACN) of HPLC grade (Merck, Germany) and Milli-Q water (resistivity of 18 M cm−1 ) were used to prepare the mobile phase. The supporting porous silica with 5 m spherical diameter, 10 nm pore size and 310 m2 g−1 surface area was purchased from Fuji Silysia Chemical (Kasugai, Japan). Nucleotides from Sigma–Aldrich included thymidine 5 -monophosphate disodium salt hydrate (TMP), uridine 5 monophosphate disodium salt (UMP), inosine 5 -monophosphate (IMP), adenosine 5 -monophosphate monohydrate (AMP), cytidine 5 -monophosphate (CMP), guanosine 5 -monophosphate disodium salt hydrate (GMP), uridine 5 -diphosphate disodium salt hydrate (UDP), adenosine 5 -diphosphate disodium salt (ADP), cytidine 5 -diphosphate sodium salt hydrate (CDP), guanosine 5 -diphosphate sodium salt (GDP), inosine 5 -diphosphate sodium salt (IDP), thymidine 5 -diphosphate sodium salt (TDP), thymidine 3 :5 -cyclic monophosphate sodium salt (cTMP), inosine 3 :5 cyclic monophosphate sodium salt (cIMP), adenosine 3 :5 -cyclic monophosphate sodium salt monohydrate (3 :5 -cAMP), guanosine 3 :5 -cyclic monophosphate (cGMP), adenosine 2 :3 -cyclic monophosphate (2 :3 -cAMP), cytidine 2 :3 -cyclic monophosphate monosodium salt (cCMP), adenosine 5 -triphosphate disodium salt hydrate (ATP). Involved flavonoids included daidzin, chrysin, prunetin, naringenin, 2 -hydroxychalcone, diosmetin, phlorizindihydrate, naringin, daidzein, genistin, apigenin, (±)dihydrokaempferol, vitexin, hesperetin, phloretin and hesperidin, mainly from several reagent suppliers such as Sigma–Aldrich and J&K Scientific. The elemental analysis was performed on a Vario EL III elemental analyzer (Elementar, Germany). The measurement of infrared spectrum to the bonded silica materials and 1 H NMR (nuclear magnetic resonance) spectrum of the synthesized IL were conducted respectively on a FT-IR (Fourier-transform infrared) system Spectrum GX (PerkinElmer, USA) and a Bruker ultrashieldTM Plus 400 NMR spectrometer (Bruker, Switzerland). The chromatographic evaluation to the stationary phase was performed on an Agilent 1290 UPLC system equipped with a diode array detector (DAD). 2.2. Preparation of the glucaminium-based IL bonded silica materials First, N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized according to Refs. [25,26] with some modification. 2.5 g of N-methyl-d-glucamine was dissolved in 50 mL of methanol, and then 0.8 g of sodium carbonate was added to the solution under stirring at 45 ◦ C. After being stirred for 30 min, 9.7 g of allyl bromide dissolved in 10 mL of methanol was slowly added dropwise to the reaction solution within one hour, and the reaction was conducted at 45 ◦ C for 98 h. The reaction mixture was evaporated under reduced pressure at 50 ◦ C and then cooled when about
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10 mL of mixture remained. The bottom solid was removed, and methanol was added to the mixture. The procedures of evaporation and cooling were repeated three times in order to remove sodium bromide and the remaining sodium carbonate. The collected product N,N-diallyl-N-methyl-d-glucaminium bromide was dried under vacuum and characterized by 1 H NMR, 13 C NMR and electrospray ionization-mass spectrometry (ESI-MS), as shown in Fig. S1. Then, the obtained glucaminium-based IL was bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry [31] in methanol with AIBN as a catalyst. First, the 3-mercaptopropyl modified silica materials were prepared as follows. (3-Mercaptopropyl)trimethoxysilane was added slowly to the suspension of silica materials in anhydrous toluene, and the reaction mixture was refluxed under argon for 24 h, followed by centrifuging and washing. Then, glucaminium-based IL was added to the suspension of 3-mercaptopropyl modified silica in methanol and refluxed in the presence of AIBN for 38 h. After being washed with methanol and dried, the glucaminium-based IL bonded silica materials were confirmed by IR spectroscopy as shown in Fig. S2, and the surface coverage was calculated from the elemental analysis results. 2.3. Column packing and chromatographic evaluation A HPLC column of 3.0 mm × 150 mm was prepared by a slurry packing method. The glucaminium-based IL bonded silica materials were suspended in methanol and slurry-packed into a stainless steel column of 150 mm × 3.0 mm i.d. under a constant pressure of 60 MPa with methanol as the pushing solvent. The nucleotides mixture consisted of 50 g/mL cyclic nucleotides and nucleotide monophosphates, 100 g/mL nucleotide diphosphates. The standard solution of adenosine nucleotides mixture included 50 g/mL AMP, 140 g/mL ADP and 200 g/mL ATP. The concentration of standard solutions was 20 g/mL for nucleosides, 10–30 g/mL for acidic solutes and 20–40 g/mL for flavonoids. The injection volume was 2–5 L and all the injections were repeated at least twice. The dead time t0 was measured with toluene as the test solute. In the investigation of effect of water content in mobile phase on retention, the mobile phase consisted of an aqueous phase with 10 mM ammonium formate (NH4 FA) and an organic phase ACN/H2 O (95:5, v/v) containing 10 mM NH4 FA. The effect of salt concentration on retention was evaluated with a mobile phase of ACN/H2 O (85/15, v/v) containing the corresponding concentration of NH4 FA (5 mM, 10 mM, 15 mM, 20 mM, 25 mM). For the effect of mobile phase pH, the pH of the aqueous phase containing 100 mM NH4 FA was adjusted with formic acid to various pH values (4.2, 5.6, 6.4), and pure ACN with the same volume ratio of formic acid was used as the organic phase. In the whole experiment for the effect of chromatographic factors on retention, the flow rate was 0.4 mL/min and the column temperature was maintained at 30 ◦ C. All the used test solutes were listed in Fig. S3 with their structures and pKa values. 3. Results and discussion 3.1. Characterization of the glucaminium-based IL stationary phase The synthesized glucaminium-based IL N,N-diallyl-N-methyld-glucaminium bromide was confirmed by 1 H NMR (Fig. S1a), 13 C NMR (Fig. S1b) and ESI-MS (m/z = 276.18 for [M+H]+ ) (Fig. S1c), and was bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry, as shown in Fig. 1.
Fig. 1. Preparation scheme of the glucaminium-based IL and its bonded silica materials.
The obtained glucaminium-based IL bonded silica materials were characterized by IR spectroscopy and elemental analysis. As can be seen from the IR spectrum in Fig. S2, stretching vibration of CH3 at ∼2950 cm−1 was stronger in glucaminium-based IL silica (SiO2 -glu IL) than in 3-mercaptopropyl modified silica (SiO2 SH), though C H stretching vibration at 2800–3000 cm−1 was present in the both types of materials compared with unmodified silica (SiO2 ). Furthermore, the presence of the peak at ∼1400 cm−1 in glucaminium-based IL silica was due to the bending vibration of the increased C H bonds. The elemental analysis results indicated that the carbon loading of the 3-mercaptopropyl modified silica was 3.09%, while the glucaminium-based IL bonded silica possessed a carbon loading of 8.78% and a nitrogen loading of 0.70%. Calculated from the nitrogen loading content, the coverage of the glucaminium-based IL molecule on the surface of supporting silica materials was 2.18 mol/m2 . The column packed from the prepared glucaminium-based IL bonded silica exhibited reasonable retention and good separation selectivity to nucleosides with satisfactory symmetry under HILIC conditions as shown in Fig. 2. With cytidine as the test solute, the column efficiency was near 70,000 plates/m when the retention factor k was 4.67 under the mobile phase condition of 100 mM NH4 FA/ACN (15/85, v/v), and the column efficiency can reach nearly 80,000 plates/m with the k value of 9.22 under 100 mM NH4 FA/ACN (10/90, v/v).
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r2
s
Anisic acid Benzoic acid 3-Chlorobenzoic acid p-Nitrobenzoic acid 3,5-Dinitrobenzoic acid 4-Hydroxybenzoic acid Hippuric acid
0.9987 0.9981 0.9994 0.9994 0.9968 0.9918 0.9954
0.714 0.736 0.843 0.835 0.970 0.613 0.732
r, correlation coefficient.
Fig. 2. Separation chromatogram of a nucleoside mixture on the obtained glucaminium-based IL column. Separation conditions: mobile phase 100 mM NH4 FA/ACN (15/85, v/v), flow rate at 0.4 mL/min, DAD at 254 nm, column temperature at 30 ◦ C. Peaks: (1) 3-methyluridine, (2) 5-methyluridine, (3) 1methylguanosine, (4) inosine, (5) cytidine.
3.2. Effect of chromatographic factors on retention behavior The effect of some chromatographic factors including salt concentration, water content in mobile phase and the mobile phase pH on retention behavior was investigated on the glucaminium-based IL column by using a series of test solutes as listed in Fig. S3. 3.2.1. Salt concentration in mobile phase For basic and weak basic compounds such as melamine, nucleobases or nucleosides, the retention was slightly enhanced with the increase of salt concentration (Fig. 3a). For charged stationary phases, electrostatic interactions existed between the stationary phase and solutes besides hydrophilic interaction, and the combination has been reported as electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) [32]. On the one hand, the increase of salt concentration would suppress the electrostatic interactions (mainly repulsion here) and further enhance the retention. On the other hand, hydrophilic retention was based on the partition of solutes between the water enriched layer on the surface of stationary phase and mobile phase of high organic solvent, and higher salt concentration would make more ions preferentially enter the water enriched layer, which potentially resulted in larger volume of the water enriched layer and stronger retention of solutes [33,34]. For acidic compounds (mostly substituted benzoic acids), the retention exhibited obvious decrease when the salt concentration increased, which might originate from anion-exchange with the
positive charge on the surface of the glucaminium-based IL stationary phase. In an ion-exchange system, the retention factor k of a solute vs. the concentration of counterions in the mobile phase can be described by the following equation [35]: log k = −s log [Conc.] + constant
(1)
where [Conc.] is the salt concentration and s is the slope, a constant usually found to be close to the ratio of the charge of the solute/couterion charge, which is 1 in the study. The retention factors of these acidic solutes and the corresponding [Conc.] were plotted and fitted to Eq. (1), and the results were shown in Fig. 3b and Table 1. The plots exhibited good linearity and all the correlation coefficients were more than 0.99, which indicated that these solutes were retained on the glucaminium-based IL column through anion-exchange. However, the slope s was below 1 with some deviation from the theoretical value. This can be explained that the retention mechanism was not pure anionexchange and hydrophilic mechanism also contributed to the retention, i.e. a mixed-mode retention mechanism. Moreover, the value of s increased with the decrease of pKa values, indicating that anion-exchange exhibited greater contribution to the retention of more acidic compounds. Similarly, in reversed-phase mixed ion-exchange chromatographic system, it was concluded that the retention was dominated by hydrophobic interactions when s was close to zero and by ion exchange when s close to one [36,37]. 3.2.2. Water content in mobile phase The changes of retention with the water content in the mobile phase were investigated within the water volume fraction of 50–95%, and the data were plotted as retention factors vs. water volume fractions in Fig. 4. Basic and weak basic solutes displayed a continuous decreased retention when the water content in mobile phase increased, acting as steep decrease from 5% to 20% of water volume fraction, then exhibiting weak retention and slight decrease
Fig. 3. Change of retention with salt concentration in mobile phase. (a) Retention factors vs. salt concentration for basic and weak basic solutes and (b) log k vs. log [Conc.] for acidic solutes. Mobile phase: H2 O/ACN (15/85, v/v) with different concentrations of NH4 FA, other conditions were the same as Fig. 2.
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Fig. 4. Change of retention with water volume fraction in mobile phase. (a) Basic and weak basic solutes and (b) for acidic solutes. Mobile phases with different volume fractions of ACN and H2 O containing 10 mM NH4 FA, other conditions were the same as Fig. 2.
over 20% of water volume fraction. This retention mode was typical HILIC behavior, mainly from the change of partitioning between predominantly organic mobile phase and water enriched layer on the surface of stationary phase [38], although the retention was a result of hydrophilic-partition mechanism, adsorption mechanism and possible electrostatic interactions [39–44]. On the whole, the acidic solutes presented weak retention at low water volume fractions and the retention was enhanced when water content increased, exhibiting different change trends from the above basic solutes. Specifically, the retention decreased at the initial low water volume fractions and then increased for acids with pKa values below 4, or continually increased through the studied water volume fractions for acids with pKa values over 4. The situation of hippuric acid was an exception, and it presented strong retention at low water volume fractions and then experienced an obvious decrease at higher water volume fractions. Although the retention began to increase before reaching the highest water fraction investigated, it never rose to the same value as at the initial low water volume fraction any more. These acidic solutes (substituted benzoic acids) were retained on the
glucaminium-based IL column through HILIC mechanism and anion-exchange as shown above. Under high ACN contents, the dissociation of acids and anion-exchange were severely suppressed, but the hydrophilic interaction was strong and dominated the retention. With the increase of water volume fraction, the HILIC retention became weaker. However, the suppression was also weakened and anion-exchange contributed more significantly to the retention of acids. The hydrophilic interactions were weaker for acids with smaller pKa values as they were prone to exist in molecular forms and possessed weaker hydrophilicity due to the suppression of dissociation. Therefore there was no decrease in retention even at low water volume fractions for acidic solutes with pKa values over 4. 3.2.3. Mobile phase pH Two test mixtures (nucleosides and substituted benzoic acids) were used to investigate the effect of the mobile phase pH on retention and their separation performances under three different pH values were given in Fig. 5. The retention of nucleosides presented no obvious change under various mobile phase pH values, only
Fig. 5. Separation performances of mixtures of nucleosides (a) and substituted acids (b) under three different mobile phase pH values (4.2, 5.6, 6.4). Separation condition: mobile phase 100 mM NH4 FA/ACN 15/85 (v/v) for (a) and 10/90 (v/v) for (b), the pH of the aqueous phase was adjusted with formic acid to various pH values, and a same volume of formic acid was added to the organic phase. Other conditions were the same as Fig. 2. Peaks: (1) 3-methyluridine, (2) 5-methyluridine, (3) 1-methylguanosine, (4) Inosine, (5) Cytidine. (1 ) Phenol, (2 ) anisic acid, (3 ) benzoic acid, (4 ) 4-hydroxybenzoic acid, (5 ) 3-chlorobenzoic acid, (6 ) 3-hydroxybenzoic acid, (7 ) 3,5-dinitrobenzoic acid, (8 ) p-nitrobenzoic acid.
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slight increase at pH 5.6 and 6.4 compared with at pH 4.2. The pH variation would not change the charge state and density of the stationary phase molecules, and the nucleosides were neutral and unaffected by the pH difference. However, free silanol groups (pKa 9.5) were deprotonated and negatively charged under pH 7, and the charged amount would decrease with pH decreasing as a result of protonation, which would decrease the electrostatic attraction between the silanol groups and nucleosides and further resulted in their retention slightly weaker at the lowest pH at 4.2. As for the benzoic acids, the retention was greatly influenced by the mobile phase pH, mostly exhibiting an obvious increase when pH changed from 4.2 to 6.4. These acidic solutes might exist in molecular forms or anionic forms, and could be retained through HILIC mechanism and anion-exchange. Both of the retention behaviors were associated with the ionization and dissociation degree of acids, which would be greatly affected by the pH variation. Under the lower pH 4.2, benzoic acid existed at low ionization state, exhibiting weaker retention. When the pH increased, the ionization was enhanced and resulted in more molecules in ionic forms. For these acidic solutes, their ionized forms were retained stronger than their neutral forms under HILIC conditions [42]. Furthermore, the ionic forms could be retained through anionexchange, which displayed a greater contribution to the increased retention under higher pH values. These benzoic acids presented different retention and change degrees depending on the pKa value, the type of functional groups and the substituted position, which resulted in different elution orders under various pH values. For 3,5-dinitrobenzoic acid with the minimum pKa value, the retention exhibited no change as the ionization remained constant within the studied pH range. The hydroxyl group substituted benzoic acids exhibited relatively stronger retention than other acids under higher pH values, especially 3-hydroxybenzoic acid. The reduction of protonation with increased pH favored the formation of hydrogen bond and would further enhance the retention. It would be easier for 4-hydroxybenzoic acid to form intermolecular hydrogen bond than 3-hydroxybenzoic acid, which decreased the opportunity to interact with the stationary phase through hydrogen bond and resulted in its weaker retention. The investigation into the effect of various chromatographic factors on retention behavior indicated that the developed glucaminium-based IL stationary phase presented a mixed-mode retention mechanism with hydrophilic interaction and anionexchange.
Fig. 6. Separation chromatogram of sixteen flavonoids on the glucaminium-based IL column under HILIC mode. Separation conditions: mobile phases were 100 mM NH4 FA (A) and ACN (B), with a gradient elution of 95%B maintained 6 min, linearly increased to 70%B from 6 to 12 min, maintained at 70%B from 12 to 17 min, to 95%B from 17 to 20 min, equilibration at 95%B to 35 min; flow rate at 0.4 mL/min; column temperature at 30 ◦ C; DAD at 283 nm. Peaks: (1) 2 -hydroxychalcone, (2) prunetin, (3) chrysin, (4) hesperetin, (5) naringenin, (6) diosmetin, (7) daidzein, (8) apigenin, (9) (±)-dihydrokaempferol, (10) phloretin, (11) genistin, (12) daidzin, (13) phlorizindihydrate, (14) hesperidin, (15) naringin, (16) vitexin.
3.3. Separation performance Flavonoids are a large family of phenolic compounds widely present in plants, and possess significant biological functions, such as pharmaceutical effect and absorbing radicals as antioxidants [45,46]. The separation and analysis of flavonoids are commonly carried out by using RPLC, but in recent years some other techniques were used to improve resolution, including HILIC [47]. A mixture of sixteen flavonoids was separated on the prepared glucaminiumbased IL column, and the chromatogram was given in Fig. 6. These flavonoids existed mainly in neutral forms under the experimental conditions (mobile phase pH about 6.4) as the pKa values of flavonoids range from 7 to 11 [48], and therefore they were retained on the glucaminium-based IL column through HILIC mechanism without a significant contribution from anion-exchange. It can be seen that the basic flavonoids (peaks 1–10 in Fig. 6) displayed relatively weak retention under HILIC mode, and this was because that there were hydrophobic aromatic rings in the structures and therefore they did not possess sufficient hydrophilicity. However, flavonoid glycosides (peaks 11–16 in Fig. 6) were strongly retained due to the introduction of hydrophilic glycoside group. The prepared glucaminium-based IL column was also used to separate a nucleotides mixture, including cyclic nucleotides,
Fig. 7. Separation chromatograms for a mixture of cyclic nucleotides, nucleoside monophosphates and nucleoside diphosphates (a) and a mixture of adenosine mono/di/triphosphates (b). Separation conditions for (a): mobile phases were 200 mM NH4 FA with 0.1% FA (A) and ACN with 0.1% FA (B), with a gradient elution of 85%B maintained 4 min, increased to 70% B 4–7 min, 70%B maintained 7–9 min, to 60%B 9–12 min, 60%B maintained 12–15 min, to 40%B 15–17 min, maintained at 40%B; flow rate at 0.8 mL/min; column temperature at 30 ◦ C; DAD at 254 nm. Separation conditions for (b): mobile phases were 200 mM NH4 FA with 0.1% FA (A) and ACN with 0.1% FA (B), with an isocratic elution at 30%B, flow rate at 0.8 mL/min; column temperature at 30 ◦ C; DAD at 254 nm. Peaks for (a): (1) cTMP, (2) 3 :5 -cAMP, (3) 2 :3 -cAMP, (4) cIMP, (5) cGMP, (6) cCMP; (7) TMP, (8) UMP, (9) IMP, (10) AMP, (11) CMP, (12) GMP; (13) TDP, (14) UDP, (15) IDP, (16) CDP, (17) ADP, (18) GDP. Peaks for (b): (1 ) AMP, (2 ) ADP, (3 ) ATP.
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nucleoside monophosphates and nucleoside diphosphates, and a mixture of adenosine mono/di/tri-phosphates. As shown in Fig. 7, most nucleotides were separated well in order of cyclic nucleotides, nucleoside monophosphates and nucleoside diphosphates under the mixed-mode retention mechanism of HILIC and anion-exchange. Within the three types, the elution order was mainly based on the hydrophilicity of the nucleoside part of nucleotides. For the adenosine nucleotides mixture, AMP, ADP and ATP were eluted with high resolution, but the peak shape became more tailing with the increase of the phosphate group due to the strong anion-exchange with the glucaminium-based IL column. Although the peak shape was not satisfactory, it was suggested that HILIC stationary phases mixed with weak anion-exchange would exhibit good separation ability toward a wide range of nucleotides with improved peak shapes. 4. Conclusions A surface-confined glucaminium-based IL stationary phase was successfully prepared by bonding N,N-diallyl-N-methyl-dglucaminium bromide to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The obtained glucaminium-based IL column possessed a hydrophilic glucose structure and positively charged quaternary ammonium group immobilized onto silica, which provided the possibility to interact with solutes by hydrophilic interaction or anion-exchange. The glucaminium-based IL column presented good separation performances toward neutral polar solutes such as nucleosides with a column efficiency of 80,000 plates/m with cytidine as the test solute under HILIC mode. The retention behavior was evaluated by investigating the effect of different chromatographic factors on retention using various types of solutes, which indicated that the glucaminium-based IL column exhibited a mixed-mode HILIC/anion-exchange retention mechanism to negatively charged solutes, such as substituted benzoic acids. The multimodal retention capabilities could offer a wider range of retention behavior and flexible selectivity, and the developed glucaminium-based IL stationary phase displayed reasonable retention and good separation efficiency toward mixtures of flavonoids and nucleotides under HILIC and mixed-mode HILIC/anion-exchange chromatography, respectively. Acknowledgements The study was supported by the National Basic Research Program (Nos. 2012CB720801, 2012CB517506) from the State Ministry of Science and Technology of China, the foundations (Nos. 21375011 and 21205114) and the creative research group project (No. 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.2014.07.096. References [1] P. Kozlik, V. Simova, K. Kalikova, Z. Bosakova, D.W. Armstrong, E. Tesarova, Effect of silica gel modification with cyclofructans on properties of hydrophilic interaction liquid chromatography stationary phases, J. Chromatogr. A 1257 (2012) 58–65. [2] N.L.T. Padivitage, D.W. Armstrong, Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography, J. Sep. Sci. 34 (2011) 1636–1647.
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A novel surface-confined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography夽 Lizhen Qiao, Shuangyuan Wang, Hua Li, Yuanhong Shan, Abo Dou, Xianzhe Shi ∗ , 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 2 June 2014 Received in revised form 10 July 2014 Accepted 28 July 2014 Available online xxx Keywords: Glucaminium-based ionic liquid Hydrophilic interaction chromatography Anion-exchange Stationary phase
a b s t r a c t Glucaminium-based ionic liquids are a new class of recently developed ionic liquids and prepared by functionalizing the amine group of N-methyl-d-glucamine, which renders them good hydrophilicity due to the presence of the glucose structure and charged quaternary ammonium group. In the present study, a glucaminium-based ionic liquid N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized and subsequently bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The obtained stationary phase was characterized by elemental analysis and infrared spectroscopy, and then packed as a HPLC column. A mixture of five nucleosides was used to characterize the separation performance of the obtained column under HILIC mode and the column efficiency was determined with cytidine as the test solute, reaching 80,000 plates/m. Then, the retention behavior was evaluated by investigating the effect of various chromatographic factors on retention of different types of solutes, and the results revealed that the developed surface-confined glucaminium-based ionic liquid stationary phase exhibited a hydrophilic interaction/anion-exchange mixed-mode retention mechanism. Finally, two mixtures of nucleotides and flavonoids were separated on the glucaminium-based ionic liquid column, respectively under hydrophilic interaction and hydrophilic interaction/anion-exchange mixedmode chromatography. In conclusion, the multimodal retention capabilities of the glucaminium-based ionic liquid column could offer a wider range of retention behavior and flexible selectivity toward polar and hydrophilic compounds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hydrophilic interaction chromatography (HILIC) is playing an increasingly important role in separating polar and hydrophilic compounds in recent years with some unique advantages over conventional techniques, such as the good solubility of polar samples in aqueous mobile phase and the compatibility with mass spectrometry (MS), and currently it has been applied in various fields. In order to obtain a wider range of selectivity to various polarity of compounds and better performance, the development of HILIC stationary phases has always been a hot research field. Surface modification to silica materials with different polar
夽 Invited paper for the Honor Issue of Professor Peichang Lu’s 90th birthday. ∗ 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).
functional groups is key to provide different retention behavior and has gained great progress. Commercially available HILIC stationary phases mainly include bare silica, chemically bonded diol, amino and amide silica, and ZIC-HILIC columns, and several researchers also developed other HILIC stationary phases through covalently bonding hydrophilic molecules to the surface of silica materials, such as cyclofructans [1–3], zwitterionic functionalities [4–6] and so on. Besides the new bonded phases, introduction of multiple retention modes into one stationary phase was another strategy to expand the selectivity, i.e. mixed-mode stationary phases, and the combination of more than one retention modes could offer flexible and adjustable selectivity. Under HILIC mode, the hydrophilic-partition, adsorption mechanism and electrostatic interactions might coexist between the stationary phase and solutes and further lead to chromatographic retention. Moreover, charged groups in bonded functionalities could provide the possibility of ion-exchange with oppositely charged solutes while
http://dx.doi.org/10.1016/j.chroma.2014.07.096 0021-9673/© 2014 Elsevier B.V. All rights reserved.
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improving hydrophilicity, and thus a HILIC/ion-change mixedmode chromatographic system is obtained. It was reported that HILIC/ion-exchange showed many advantages (such as tunable selectivity, more efficient retention and better separation) for the application of peptides [7], small molecular drugs [8], and proteins [9]. Recently some new HILIC stationary phases were developed and exhibited HILIC/ion-change mixed-mode retention behavior. For example, a glutathione-based mixed-mode HILIC/cation-exchange stationary phase was prepared and successfully used to separate neutral fructosan with high degree of polymerization, basic chitooligosaccharides and strongly acidic carrageenan oligosaccharides [10]. In the previous work [11] we developed a new type of surface-confined dicationic ionic liquids stationary phases, which exhibited HILIC/anion-exchange mixed-mode retention mechanism. Ionic liquids (ILs) are an important class of non-molecular solvents possessing many outstanding properties such as high thermal stability, negligible vapor pressure and intrinisic conductivity, and have been applied in many fields. In addition, ILs possess the potential to undergo multiple solvation interactions and can be tailored to obtain desired properties by introducing appropriate functional groups or combining different cationic and anionic moieties. These beneficial characteristics have made ILs attractive for analytical chemistry applications as extraction phases, dissolution solvents, separation media, and electrochemical sensing systems, etc. [12,13]. In HPLC, ILs were applied in two ways, as mobile phase additives to mask the adverse effect of residual silanol groups [14–16] or being immobilized to the surface of supporting materials as stationary phases [17,18]. Qiu et al. developed a number of imidazolium ionic liquid-based stationary phases [19–23], and found that ILs of different structures might exhibit reversed phase, ion-exchange or HILIC retention mechanism. Apart from the conventional monocationic ILs, our group recently prepared several imidazolium dicationic ILs-based stationary phases exhibiting considerably high column efficiency under HILIC mode [11]. All the studies indicated that ILs-modified HPLC columns could retain a wide range of solutes through multimodal retention mechanism such as electrostatic, ionic, – and dipole interactions [17,18,24], and are enjoying increasing popularity as a promising class of HPLC stationary phases. Depending on the cationic moieties, ILs can be divided into imidazolium, ammonium, phosphonium, pyridinium and guanidinium-based ILs. However, currently only imidazolium and pyridinium ILs are used as bonded selectors for HPLC stationary phases. Glucaminium-based ILs were a new kind of ILs recently developed to remove or extract boron-species, improve the separation of hydrophilic boron compounds under RPLC by using the complexion behavior with borate [25–27]. Moreover, glucaminium-based ILs presented good extraction performances to alkyl halides and aromatics [28], deoxyribonucleic acid (DNA) [29], indicating they could provide the ability to function with solutes by multiple interactions. Glucaminium-based ILs are prepared from N-methyl-d-glucamine through the functionalization of the amine group with halogen substituted compounds [30], and the presence of the glucose structure renders the IL molecule good hydrophilicity. These properties make glucaminium-based ILs possess great potential as attractive selectors for HILIC stationary phase, and therefore in the present study the chromatographic performance of glucaminium-based IL modified silica was studied as HILIC stationary phase. A new glucaminium-based IL with allyl groups was synthesized and grafted to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The glucaminium-based IL bonded silica was packed as a HPLC column after characterization and a mixture of five nucleosides was separated on the obtained column to evaluate the selectivity under HILIC mode. The effect of chromatographic factors (including salt
concentration in mobile phase, water content in mobile phase and the mobile phase pH) on retention was investigated by using a series of solutes to reveal the retention mechanism. Finally, the glucaminium-based IL column was used to separate two mixtures of nucleotides and flavonoids. 2. Experimental 2.1. Materials and instruments Allyl bromide, N-methyl-d-glucamine, (3-mercaptopropyl)trimethoxysilane, sodium carbonate, ammonium formate (NH4 FA), formic acid and involved nucleotides were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2,2 -Azobis(2methylpropionitrile) (AIBN) was obtained from J&K Scientific (Beijing, China). Toluene was refluxed to remove water in the presence of sodium before use. Acetonitrile (ACN) of HPLC grade (Merck, Germany) and Milli-Q water (resistivity of 18 M cm−1 ) were used to prepare the mobile phase. The supporting porous silica with 5 m spherical diameter, 10 nm pore size and 310 m2 g−1 surface area was purchased from Fuji Silysia Chemical (Kasugai, Japan). Nucleotides from Sigma–Aldrich included thymidine 5 -monophosphate disodium salt hydrate (TMP), uridine 5 monophosphate disodium salt (UMP), inosine 5 -monophosphate (IMP), adenosine 5 -monophosphate monohydrate (AMP), cytidine 5 -monophosphate (CMP), guanosine 5 -monophosphate disodium salt hydrate (GMP), uridine 5 -diphosphate disodium salt hydrate (UDP), adenosine 5 -diphosphate disodium salt (ADP), cytidine 5 -diphosphate sodium salt hydrate (CDP), guanosine 5 -diphosphate sodium salt (GDP), inosine 5 -diphosphate sodium salt (IDP), thymidine 5 -diphosphate sodium salt (TDP), thymidine 3 :5 -cyclic monophosphate sodium salt (cTMP), inosine 3 :5 cyclic monophosphate sodium salt (cIMP), adenosine 3 :5 -cyclic monophosphate sodium salt monohydrate (3 :5 -cAMP), guanosine 3 :5 -cyclic monophosphate (cGMP), adenosine 2 :3 -cyclic monophosphate (2 :3 -cAMP), cytidine 2 :3 -cyclic monophosphate monosodium salt (cCMP), adenosine 5 -triphosphate disodium salt hydrate (ATP). Involved flavonoids included daidzin, chrysin, prunetin, naringenin, 2 -hydroxychalcone, diosmetin, phlorizindihydrate, naringin, daidzein, genistin, apigenin, (±)dihydrokaempferol, vitexin, hesperetin, phloretin and hesperidin, mainly from several reagent suppliers such as Sigma–Aldrich and J&K Scientific. The elemental analysis was performed on a Vario EL III elemental analyzer (Elementar, Germany). The measurement of infrared spectrum to the bonded silica materials and 1 H NMR (nuclear magnetic resonance) spectrum of the synthesized IL were conducted respectively on a FT-IR (Fourier-transform infrared) system Spectrum GX (PerkinElmer, USA) and a Bruker ultrashieldTM Plus 400 NMR spectrometer (Bruker, Switzerland). The chromatographic evaluation to the stationary phase was performed on an Agilent 1290 UPLC system equipped with a diode array detector (DAD). 2.2. Preparation of the glucaminium-based IL bonded silica materials First, N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized according to Refs. [25,26] with some modification. 2.5 g of N-methyl-d-glucamine was dissolved in 50 mL of methanol, and then 0.8 g of sodium carbonate was added to the solution under stirring at 45 ◦ C. After being stirred for 30 min, 9.7 g of allyl bromide dissolved in 10 mL of methanol was slowly added dropwise to the reaction solution within one hour, and the reaction was conducted at 45 ◦ C for 98 h. The reaction mixture was evaporated under reduced pressure at 50 ◦ C and then cooled when about
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10 mL of mixture remained. The bottom solid was removed, and methanol was added to the mixture. The procedures of evaporation and cooling were repeated three times in order to remove sodium bromide and the remaining sodium carbonate. The collected product N,N-diallyl-N-methyl-d-glucaminium bromide was dried under vacuum and characterized by 1 H NMR, 13 C NMR and electrospray ionization-mass spectrometry (ESI-MS), as shown in Fig. S1. Then, the obtained glucaminium-based IL was bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry [31] in methanol with AIBN as a catalyst. First, the 3-mercaptopropyl modified silica materials were prepared as follows. (3-Mercaptopropyl)trimethoxysilane was added slowly to the suspension of silica materials in anhydrous toluene, and the reaction mixture was refluxed under argon for 24 h, followed by centrifuging and washing. Then, glucaminium-based IL was added to the suspension of 3-mercaptopropyl modified silica in methanol and refluxed in the presence of AIBN for 38 h. After being washed with methanol and dried, the glucaminium-based IL bonded silica materials were confirmed by IR spectroscopy as shown in Fig. S2, and the surface coverage was calculated from the elemental analysis results. 2.3. Column packing and chromatographic evaluation A HPLC column of 3.0 mm × 150 mm was prepared by a slurry packing method. The glucaminium-based IL bonded silica materials were suspended in methanol and slurry-packed into a stainless steel column of 150 mm × 3.0 mm i.d. under a constant pressure of 60 MPa with methanol as the pushing solvent. The nucleotides mixture consisted of 50 g/mL cyclic nucleotides and nucleotide monophosphates, 100 g/mL nucleotide diphosphates. The standard solution of adenosine nucleotides mixture included 50 g/mL AMP, 140 g/mL ADP and 200 g/mL ATP. The concentration of standard solutions was 20 g/mL for nucleosides, 10–30 g/mL for acidic solutes and 20–40 g/mL for flavonoids. The injection volume was 2–5 L and all the injections were repeated at least twice. The dead time t0 was measured with toluene as the test solute. In the investigation of effect of water content in mobile phase on retention, the mobile phase consisted of an aqueous phase with 10 mM ammonium formate (NH4 FA) and an organic phase ACN/H2 O (95:5, v/v) containing 10 mM NH4 FA. The effect of salt concentration on retention was evaluated with a mobile phase of ACN/H2 O (85/15, v/v) containing the corresponding concentration of NH4 FA (5 mM, 10 mM, 15 mM, 20 mM, 25 mM). For the effect of mobile phase pH, the pH of the aqueous phase containing 100 mM NH4 FA was adjusted with formic acid to various pH values (4.2, 5.6, 6.4), and pure ACN with the same volume ratio of formic acid was used as the organic phase. In the whole experiment for the effect of chromatographic factors on retention, the flow rate was 0.4 mL/min and the column temperature was maintained at 30 ◦ C. All the used test solutes were listed in Fig. S3 with their structures and pKa values. 3. Results and discussion 3.1. Characterization of the glucaminium-based IL stationary phase The synthesized glucaminium-based IL N,N-diallyl-N-methyld-glucaminium bromide was confirmed by 1 H NMR (Fig. S1a), 13 C NMR (Fig. S1b) and ESI-MS (m/z = 276.18 for [M+H]+ ) (Fig. S1c), and was bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry, as shown in Fig. 1.
Fig. 1. Preparation scheme of the glucaminium-based IL and its bonded silica materials.
The obtained glucaminium-based IL bonded silica materials were characterized by IR spectroscopy and elemental analysis. As can be seen from the IR spectrum in Fig. S2, stretching vibration of CH3 at ∼2950 cm−1 was stronger in glucaminium-based IL silica (SiO2 -glu IL) than in 3-mercaptopropyl modified silica (SiO2 SH), though C H stretching vibration at 2800–3000 cm−1 was present in the both types of materials compared with unmodified silica (SiO2 ). Furthermore, the presence of the peak at ∼1400 cm−1 in glucaminium-based IL silica was due to the bending vibration of the increased C H bonds. The elemental analysis results indicated that the carbon loading of the 3-mercaptopropyl modified silica was 3.09%, while the glucaminium-based IL bonded silica possessed a carbon loading of 8.78% and a nitrogen loading of 0.70%. Calculated from the nitrogen loading content, the coverage of the glucaminium-based IL molecule on the surface of supporting silica materials was 2.18 mol/m2 . The column packed from the prepared glucaminium-based IL bonded silica exhibited reasonable retention and good separation selectivity to nucleosides with satisfactory symmetry under HILIC conditions as shown in Fig. 2. With cytidine as the test solute, the column efficiency was near 70,000 plates/m when the retention factor k was 4.67 under the mobile phase condition of 100 mM NH4 FA/ACN (15/85, v/v), and the column efficiency can reach nearly 80,000 plates/m with the k value of 9.22 under 100 mM NH4 FA/ACN (10/90, v/v).
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r2
s
Anisic acid Benzoic acid 3-Chlorobenzoic acid p-Nitrobenzoic acid 3,5-Dinitrobenzoic acid 4-Hydroxybenzoic acid Hippuric acid
0.9987 0.9981 0.9994 0.9994 0.9968 0.9918 0.9954
0.714 0.736 0.843 0.835 0.970 0.613 0.732
r, correlation coefficient.
Fig. 2. Separation chromatogram of a nucleoside mixture on the obtained glucaminium-based IL column. Separation conditions: mobile phase 100 mM NH4 FA/ACN (15/85, v/v), flow rate at 0.4 mL/min, DAD at 254 nm, column temperature at 30 ◦ C. Peaks: (1) 3-methyluridine, (2) 5-methyluridine, (3) 1methylguanosine, (4) inosine, (5) cytidine.
3.2. Effect of chromatographic factors on retention behavior The effect of some chromatographic factors including salt concentration, water content in mobile phase and the mobile phase pH on retention behavior was investigated on the glucaminium-based IL column by using a series of test solutes as listed in Fig. S3. 3.2.1. Salt concentration in mobile phase For basic and weak basic compounds such as melamine, nucleobases or nucleosides, the retention was slightly enhanced with the increase of salt concentration (Fig. 3a). For charged stationary phases, electrostatic interactions existed between the stationary phase and solutes besides hydrophilic interaction, and the combination has been reported as electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) [32]. On the one hand, the increase of salt concentration would suppress the electrostatic interactions (mainly repulsion here) and further enhance the retention. On the other hand, hydrophilic retention was based on the partition of solutes between the water enriched layer on the surface of stationary phase and mobile phase of high organic solvent, and higher salt concentration would make more ions preferentially enter the water enriched layer, which potentially resulted in larger volume of the water enriched layer and stronger retention of solutes [33,34]. For acidic compounds (mostly substituted benzoic acids), the retention exhibited obvious decrease when the salt concentration increased, which might originate from anion-exchange with the
positive charge on the surface of the glucaminium-based IL stationary phase. In an ion-exchange system, the retention factor k of a solute vs. the concentration of counterions in the mobile phase can be described by the following equation [35]: log k = −s log [Conc.] + constant
(1)
where [Conc.] is the salt concentration and s is the slope, a constant usually found to be close to the ratio of the charge of the solute/couterion charge, which is 1 in the study. The retention factors of these acidic solutes and the corresponding [Conc.] were plotted and fitted to Eq. (1), and the results were shown in Fig. 3b and Table 1. The plots exhibited good linearity and all the correlation coefficients were more than 0.99, which indicated that these solutes were retained on the glucaminium-based IL column through anion-exchange. However, the slope s was below 1 with some deviation from the theoretical value. This can be explained that the retention mechanism was not pure anionexchange and hydrophilic mechanism also contributed to the retention, i.e. a mixed-mode retention mechanism. Moreover, the value of s increased with the decrease of pKa values, indicating that anion-exchange exhibited greater contribution to the retention of more acidic compounds. Similarly, in reversed-phase mixed ion-exchange chromatographic system, it was concluded that the retention was dominated by hydrophobic interactions when s was close to zero and by ion exchange when s close to one [36,37]. 3.2.2. Water content in mobile phase The changes of retention with the water content in the mobile phase were investigated within the water volume fraction of 50–95%, and the data were plotted as retention factors vs. water volume fractions in Fig. 4. Basic and weak basic solutes displayed a continuous decreased retention when the water content in mobile phase increased, acting as steep decrease from 5% to 20% of water volume fraction, then exhibiting weak retention and slight decrease
Fig. 3. Change of retention with salt concentration in mobile phase. (a) Retention factors vs. salt concentration for basic and weak basic solutes and (b) log k vs. log [Conc.] for acidic solutes. Mobile phase: H2 O/ACN (15/85, v/v) with different concentrations of NH4 FA, other conditions were the same as Fig. 2.
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Fig. 4. Change of retention with water volume fraction in mobile phase. (a) Basic and weak basic solutes and (b) for acidic solutes. Mobile phases with different volume fractions of ACN and H2 O containing 10 mM NH4 FA, other conditions were the same as Fig. 2.
over 20% of water volume fraction. This retention mode was typical HILIC behavior, mainly from the change of partitioning between predominantly organic mobile phase and water enriched layer on the surface of stationary phase [38], although the retention was a result of hydrophilic-partition mechanism, adsorption mechanism and possible electrostatic interactions [39–44]. On the whole, the acidic solutes presented weak retention at low water volume fractions and the retention was enhanced when water content increased, exhibiting different change trends from the above basic solutes. Specifically, the retention decreased at the initial low water volume fractions and then increased for acids with pKa values below 4, or continually increased through the studied water volume fractions for acids with pKa values over 4. The situation of hippuric acid was an exception, and it presented strong retention at low water volume fractions and then experienced an obvious decrease at higher water volume fractions. Although the retention began to increase before reaching the highest water fraction investigated, it never rose to the same value as at the initial low water volume fraction any more. These acidic solutes (substituted benzoic acids) were retained on the
glucaminium-based IL column through HILIC mechanism and anion-exchange as shown above. Under high ACN contents, the dissociation of acids and anion-exchange were severely suppressed, but the hydrophilic interaction was strong and dominated the retention. With the increase of water volume fraction, the HILIC retention became weaker. However, the suppression was also weakened and anion-exchange contributed more significantly to the retention of acids. The hydrophilic interactions were weaker for acids with smaller pKa values as they were prone to exist in molecular forms and possessed weaker hydrophilicity due to the suppression of dissociation. Therefore there was no decrease in retention even at low water volume fractions for acidic solutes with pKa values over 4. 3.2.3. Mobile phase pH Two test mixtures (nucleosides and substituted benzoic acids) were used to investigate the effect of the mobile phase pH on retention and their separation performances under three different pH values were given in Fig. 5. The retention of nucleosides presented no obvious change under various mobile phase pH values, only
Fig. 5. Separation performances of mixtures of nucleosides (a) and substituted acids (b) under three different mobile phase pH values (4.2, 5.6, 6.4). Separation condition: mobile phase 100 mM NH4 FA/ACN 15/85 (v/v) for (a) and 10/90 (v/v) for (b), the pH of the aqueous phase was adjusted with formic acid to various pH values, and a same volume of formic acid was added to the organic phase. Other conditions were the same as Fig. 2. Peaks: (1) 3-methyluridine, (2) 5-methyluridine, (3) 1-methylguanosine, (4) Inosine, (5) Cytidine. (1 ) Phenol, (2 ) anisic acid, (3 ) benzoic acid, (4 ) 4-hydroxybenzoic acid, (5 ) 3-chlorobenzoic acid, (6 ) 3-hydroxybenzoic acid, (7 ) 3,5-dinitrobenzoic acid, (8 ) p-nitrobenzoic acid.
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slight increase at pH 5.6 and 6.4 compared with at pH 4.2. The pH variation would not change the charge state and density of the stationary phase molecules, and the nucleosides were neutral and unaffected by the pH difference. However, free silanol groups (pKa 9.5) were deprotonated and negatively charged under pH 7, and the charged amount would decrease with pH decreasing as a result of protonation, which would decrease the electrostatic attraction between the silanol groups and nucleosides and further resulted in their retention slightly weaker at the lowest pH at 4.2. As for the benzoic acids, the retention was greatly influenced by the mobile phase pH, mostly exhibiting an obvious increase when pH changed from 4.2 to 6.4. These acidic solutes might exist in molecular forms or anionic forms, and could be retained through HILIC mechanism and anion-exchange. Both of the retention behaviors were associated with the ionization and dissociation degree of acids, which would be greatly affected by the pH variation. Under the lower pH 4.2, benzoic acid existed at low ionization state, exhibiting weaker retention. When the pH increased, the ionization was enhanced and resulted in more molecules in ionic forms. For these acidic solutes, their ionized forms were retained stronger than their neutral forms under HILIC conditions [42]. Furthermore, the ionic forms could be retained through anionexchange, which displayed a greater contribution to the increased retention under higher pH values. These benzoic acids presented different retention and change degrees depending on the pKa value, the type of functional groups and the substituted position, which resulted in different elution orders under various pH values. For 3,5-dinitrobenzoic acid with the minimum pKa value, the retention exhibited no change as the ionization remained constant within the studied pH range. The hydroxyl group substituted benzoic acids exhibited relatively stronger retention than other acids under higher pH values, especially 3-hydroxybenzoic acid. The reduction of protonation with increased pH favored the formation of hydrogen bond and would further enhance the retention. It would be easier for 4-hydroxybenzoic acid to form intermolecular hydrogen bond than 3-hydroxybenzoic acid, which decreased the opportunity to interact with the stationary phase through hydrogen bond and resulted in its weaker retention. The investigation into the effect of various chromatographic factors on retention behavior indicated that the developed glucaminium-based IL stationary phase presented a mixed-mode retention mechanism with hydrophilic interaction and anionexchange.
Fig. 6. Separation chromatogram of sixteen flavonoids on the glucaminium-based IL column under HILIC mode. Separation conditions: mobile phases were 100 mM NH4 FA (A) and ACN (B), with a gradient elution of 95%B maintained 6 min, linearly increased to 70%B from 6 to 12 min, maintained at 70%B from 12 to 17 min, to 95%B from 17 to 20 min, equilibration at 95%B to 35 min; flow rate at 0.4 mL/min; column temperature at 30 ◦ C; DAD at 283 nm. Peaks: (1) 2 -hydroxychalcone, (2) prunetin, (3) chrysin, (4) hesperetin, (5) naringenin, (6) diosmetin, (7) daidzein, (8) apigenin, (9) (±)-dihydrokaempferol, (10) phloretin, (11) genistin, (12) daidzin, (13) phlorizindihydrate, (14) hesperidin, (15) naringin, (16) vitexin.
3.3. Separation performance Flavonoids are a large family of phenolic compounds widely present in plants, and possess significant biological functions, such as pharmaceutical effect and absorbing radicals as antioxidants [45,46]. The separation and analysis of flavonoids are commonly carried out by using RPLC, but in recent years some other techniques were used to improve resolution, including HILIC [47]. A mixture of sixteen flavonoids was separated on the prepared glucaminiumbased IL column, and the chromatogram was given in Fig. 6. These flavonoids existed mainly in neutral forms under the experimental conditions (mobile phase pH about 6.4) as the pKa values of flavonoids range from 7 to 11 [48], and therefore they were retained on the glucaminium-based IL column through HILIC mechanism without a significant contribution from anion-exchange. It can be seen that the basic flavonoids (peaks 1–10 in Fig. 6) displayed relatively weak retention under HILIC mode, and this was because that there were hydrophobic aromatic rings in the structures and therefore they did not possess sufficient hydrophilicity. However, flavonoid glycosides (peaks 11–16 in Fig. 6) were strongly retained due to the introduction of hydrophilic glycoside group. The prepared glucaminium-based IL column was also used to separate a nucleotides mixture, including cyclic nucleotides,
Fig. 7. Separation chromatograms for a mixture of cyclic nucleotides, nucleoside monophosphates and nucleoside diphosphates (a) and a mixture of adenosine mono/di/triphosphates (b). Separation conditions for (a): mobile phases were 200 mM NH4 FA with 0.1% FA (A) and ACN with 0.1% FA (B), with a gradient elution of 85%B maintained 4 min, increased to 70% B 4–7 min, 70%B maintained 7–9 min, to 60%B 9–12 min, 60%B maintained 12–15 min, to 40%B 15–17 min, maintained at 40%B; flow rate at 0.8 mL/min; column temperature at 30 ◦ C; DAD at 254 nm. Separation conditions for (b): mobile phases were 200 mM NH4 FA with 0.1% FA (A) and ACN with 0.1% FA (B), with an isocratic elution at 30%B, flow rate at 0.8 mL/min; column temperature at 30 ◦ C; DAD at 254 nm. Peaks for (a): (1) cTMP, (2) 3 :5 -cAMP, (3) 2 :3 -cAMP, (4) cIMP, (5) cGMP, (6) cCMP; (7) TMP, (8) UMP, (9) IMP, (10) AMP, (11) CMP, (12) GMP; (13) TDP, (14) UDP, (15) IDP, (16) CDP, (17) ADP, (18) GDP. Peaks for (b): (1 ) AMP, (2 ) ADP, (3 ) ATP.
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nucleoside monophosphates and nucleoside diphosphates, and a mixture of adenosine mono/di/tri-phosphates. As shown in Fig. 7, most nucleotides were separated well in order of cyclic nucleotides, nucleoside monophosphates and nucleoside diphosphates under the mixed-mode retention mechanism of HILIC and anion-exchange. Within the three types, the elution order was mainly based on the hydrophilicity of the nucleoside part of nucleotides. For the adenosine nucleotides mixture, AMP, ADP and ATP were eluted with high resolution, but the peak shape became more tailing with the increase of the phosphate group due to the strong anion-exchange with the glucaminium-based IL column. Although the peak shape was not satisfactory, it was suggested that HILIC stationary phases mixed with weak anion-exchange would exhibit good separation ability toward a wide range of nucleotides with improved peak shapes. 4. Conclusions A surface-confined glucaminium-based IL stationary phase was successfully prepared by bonding N,N-diallyl-N-methyl-dglucaminium bromide to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The obtained glucaminium-based IL column possessed a hydrophilic glucose structure and positively charged quaternary ammonium group immobilized onto silica, which provided the possibility to interact with solutes by hydrophilic interaction or anion-exchange. The glucaminium-based IL column presented good separation performances toward neutral polar solutes such as nucleosides with a column efficiency of 80,000 plates/m with cytidine as the test solute under HILIC mode. The retention behavior was evaluated by investigating the effect of different chromatographic factors on retention using various types of solutes, which indicated that the glucaminium-based IL column exhibited a mixed-mode HILIC/anion-exchange retention mechanism to negatively charged solutes, such as substituted benzoic acids. The multimodal retention capabilities could offer a wider range of retention behavior and flexible selectivity, and the developed glucaminium-based IL stationary phase displayed reasonable retention and good separation efficiency toward mixtures of flavonoids and nucleotides under HILIC and mixed-mode HILIC/anion-exchange chromatography, respectively. Acknowledgements The study was supported by the National Basic Research Program (Nos. 2012CB720801, 2012CB517506) from the State Ministry of Science and Technology of China, the foundations (Nos. 21375011 and 21205114) and the creative research group project (No. 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.2014.07.096. References [1] P. Kozlik, V. Simova, K. Kalikova, Z. Bosakova, D.W. Armstrong, E. Tesarova, Effect of silica gel modification with cyclofructans on properties of hydrophilic interaction liquid chromatography stationary phases, J. Chromatogr. A 1257 (2012) 58–65. [2] N.L.T. Padivitage, D.W. Armstrong, Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography, J. Sep. Sci. 34 (2011) 1636–1647.
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