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Fabrication of an ionic liquid-based macroporous polymer monolithic column via atom transfer radical polymerization for the separation of small molecules
Talanta 149 (2016) 62–68
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Fabrication of an ionic liquid-based macroporous polymer monolithic column via atom transfer radical polymerization for the separation of small molecules Hang Zhang a,b,c, Ligai Bai a,b,c,n, Zhen Wei a,b,c, Sha Liu a,b,c, Haiyan Liu a,b,c,n, Hongyuan Yan a,b,c a b c
Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Baoding 071002, China College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Baoding 071002, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 4 November 2015 Accepted 11 November 2015 Available online 14 November 2015
A polymer monolithic column was prepared in a stainless steel column (50 4.6 mm i.d.) via atom transfer radical polymerization technique using triallyl isocyanurate and ionic liquid (1-allyl-3-methylimidazolium chloride) as co-monomers, ethylene dimethacrylate as cross linking agent, polyethylene glycol 200, 1,4-butanediol, and N, N- dimethylformamide as porogen system, CCl4 as initiator, and FeCl2 as catalyst. The optimized polymer columns were characterized by scanning electron microscope, nitrogen adsorption-desorption instrument, mercury intrusion porosimetry, infrared spectrometer, and thermogravimetric analysis technique. Respectively, all of these factors above could illustrate that the optimized columns had relative uniform macroporous structure and high thermal stability. A series of basic and acidic small molecules, isomers, and homologues were used to evaluate the performance of these monoliths and enhanced column efficiency was obtained. & 2015 Elsevier B.V. All rights reserved.
Keywords: Monolithic polymer column Ionic liquids (ILs) High performance liquid chromatography (HPLC) small molecules
1. Introduction Over the past two decades, compared to the silica-based columns, there has been a strong interest in polymer monolithic columns[1,2] due to their advantages of time-saving preparation, enhanced permeability, reduced mass transfer resistance, and better pH tolerance [3–5]. Polymer monoliths have been successfully used for the separation of large biomolecules, such as proteins, oligonucleotides, and peptides [6–11]. In contrast, it is unfavorable for the chromatographic performance of polymer monolithic columns for the separation of small molecules as a result of low mesopore volume and inhomogeneity of the structure [12,13]. Several methods have been proposed to solve this problem, such as applying a single cross-linker [14], using various living-control radical polymerization [15–17], adding nanostructures [18,19], and introducing click chemistry reaction [9,20,21]�which mainly focused on the improvement of the performance of capillary monoliths. To some extent, all these methods led to varying degrees of improvement of column efficiency. n Corresponding authors at: College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China. E-mail addresses: [email protected] (L. Bai), [email protected] (H. Liu).
http://dx.doi.org/10.1016/j.talanta.2015.11.028 0039-9140/& 2015 Elsevier B.V. All rights reserved.
The highest efficiency of obtained monoliths exceeded 100,000 plates/m for capillary liquid chromatography (CLC). However, there are still many difficulties to overcome. Many of click chemistry reactions are easily to tolerate conditions that will be damaging for processes currently used for the preparation of monoliths with some reactive chemistries [5]. As for nanostructure substance, because of its poor dispersion in the polymer matrix, it is suitable to be attached on the pore surface of the well-defined monolith in case of being buried and inaccessible, which is more time-consuming and complex operation compared to one-pot polymerization [5,22]. In addition, in terms of capillary monolith column, it also brings difficulties to the process of analysis for the too low capacity and poor reproducibility. For routine monolithic column (50 4.6 mm i.d.), there is not any prominent progress in recent time. The attempts mainly concentrated on the variation of monomers, cross-linkers, and porogen solvent mixture, the adjustment of the polymerization conditions (including temperature, time, and polymerization technique) [23], but these methods have not improved the applicability of the polymer monoliths to the desired degree. In order to develop a uniform and stable system with simple optimization of polymerization conditions, especially focus on improving the homogeneity of the structure and enhancing column efficiency, the green solvent ionic liquid (IL) as co monomer was proposed in the present work.
H. Zhang et al. / Talanta 149 (2016) 62–68
Ionic liquids (ILs) are molten salts containing relatively asymmetric organic cations and inorganic or organic anions, whose physical and chemical properties are convenient to be changed by controlling the cations and anions [24]. Furthermore, contributed to the properties of high thermal stability, low volatility, good adjustability, high electrolytic conductivity, and miscibility [25], IL has been widely applied in chromatography [26–28] and aroused considerable scientific interest in the fabrication of monolithic column. IL-based monolithic columns [29,30] have been successfully utilized to separate a variety of analytes especially complex biological samples by CEC., These success mainly benefited from the property of good electrolytic conductivity of IL to generate a stable reversed EOF in a wide pH range for the CEC mode. However, it didn't help improve the morphology and enhance the separation of small molecules in HPLC. Our group [31,32] employed ILs as function monomer for the preparation of polymer monolithic columns via in situ free radical polymerization. The obtained monolithic columns, as the station phase for reversed-phase liquid chromatography (RPLC), exhibited good separation performance for small molecules. It demonstrated that the addition of IL made the structure more uniform with larger surface area. Furthermore, the conditions of polymerization could be optimized to get better mechanical and thermal stability, greater performance improvement and higher repeatability. The combination of the unique advantages of porous polymer monoliths, and the specific features of IL might present a promising improvement in the performance of monoliths for HPLC. Herein, IL (1-allyl-3-methylimidazolium chloride, AMIM þ Cl ) and a high concentration of crosslinking monomer triallyl isocyanurate (TAIC) build up a co-monomers system, with ethylene dimethacrylate (EDMA) as single cross-linker. The atom transfer radical polymerization (ATRP) technique was introduced to prepare the monolith. After the optimization of porogen system and polymerization conditions, a series process of characterization were carried out respectively including scanning electron microscopy, infrared spectrometer, nitrogen adsorption/desorption measurement, mercury intrusion porosimetry and thermal gravimetric analysis. Besides, the separation ability of these columns was estimated comprehensively by separating a series of basic and acidic small molecules, isomers and homologues by HPLC.
2. Experimental 2.1. Materials and methods Triallyl isocyanurate (TAIC) was purchased from Shanghai Huangguan Chemical company (Shanghai, China). Ethylene dimethacrylate (EDMA) was obtained from Maya-Reagent (Zhejiang, China). Polyethylene glycol 200 (PEG-200), 1,4-butanediol, N, Ndimethylformamide (DMF), CCl4, and FeCl2 were supplied by Tianjin Guangfu Fine Institute of Chemistry (Tianjin, China). Meanwhile, 2,2-Azobisisobutyronitrile(AIBN), HPLC-grade methanol (MeOH), acetonitrile (ACN), and KBr were produced by Kermel Chemical Reagent Factory (Tianjin, China). The analytes were provided by the National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, China). Besides, ultrapure water was used for all experiments and all medias were filtered through a 0.45 μm membrane before use. 2.2. Instruments All chromatographic experiments were performed on an 1100 system from Agilent Technologies (USA). Scanning electron microscopy (SEM) images of the monoliths were carried out on Hitachi S-3400 SEM instrument (Hitachi High Technologies, Japan).
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The Fourier transform infrared spectroscopy (FT–IR) spectra were recorded on an FTIR–8400S IR apparatus in the region of 400–4000 cm 1 (Shimadzu, Japan). The pore type was determined by the nitrogen adsorption/desorption measurment on a TriStar II 3020 instrument (Micromeritics, USA) while pore size measurement was performed on an AutoPore IV 9500 instrument (Micromeritics, USA ). Thermal gravimetric analysis (TGA) was carried out on a Setsys 16/18 instrument (Setaram, Caluire, France). Molecular weight data for ILs was obtained by MSD Trap XCT instrument with electrospray ionization (ESI) source which was bought from Agilent Technologies (USA). 2.3. Preparation and optimization of the poly(IL-co-TAIC-co-EDMA) monoliths 2.3.1. Synthesis of the IL (AMIM þ Cl ) Allyl chloride (9.95 g, 0.13 mol) was added dropwise to 1–methylimidazole (8.21 g, 0.1 mol). The mixture was heated at 55 °C under stirring for 11 h. When phase separation occurred, the brown viscous liquid was washed with ether (60 mL for three times). Then the product, IL (1-allyl-3-methylimidazolium chloride, AMIM þ Cl ), was filtered and dried in a vacuum oven until constant weigh (9.39 g, yield: 59.16%). Thus, the obtained IL was assayed by mass spectrometer. The reaction equation was shown in Scheme1(a). 2.3.2. Preparation of the poly(IL-co-TAIC-co-EDMA) monoliths The processes of preparation columns were as follows: IL and TAIC were used as co-monomers, EDMA as cross linking agent, 1,4butanediol, PEG200, and DMF as tri-porogens, CCl4 as initiator, FeCl2 as catalyst. All the reagents were put into a tube followed mixing and degassed by sonication for 15 min, and then the mixed solution was poured into a stainless-steel column (50 4.6 mm i. d.). The column was sealed and allowed to react in a water bath for 24 h. The resulting monolithic column was washed online with methanol by an HPLC pump to remove the porogens. The polymerization scheme was illustrated in Scheme 1(b). 2.3.3. Optimization of the poly(IL-co-TAIC-co-EDMA) monoliths In order to find the proper porogens then to improve the morphology and structure of the monolithic column, several candidates, such as propanol/1,4-butanediol, DMF/1,4-butanediol, 1,4-butanediol/PEG200, DMF/PEG200 and DMF/PEG200/1,4-butanediol were proposed according to their solubility and polarity, respectively. The pre-experiment showed that the ternary porogenic system DMF/PEG200/1,4-butanediol had prior effects on the property of the monolithic columns to that of single or double porogens. Table 1 listed the representative conditions and some of the obtained results, containing the effect of polymerization temperature on the property of monolithic column. Furthermore, compared to the IL-based monolith prepared via ATRP, columns H and I were prepared via in situ or without IL addition, respectively, with the details listed in Table 2. 2.4. HPLC procedures An Agilent 1100 system consisted of a quaternary pump with an online vacuum degasser, an autosampler with variable injection capacity from 0.1 to 100 μL, and UV detector. All sample solutions injected in the chromatographic system were filtered through a millipore membrane (0.45 μm) to remove particles and large aggregates. The chromatographic conditions were as follows: mobile phase: ACN/water; UV wave length: 254 nm; temperature: 25 °C; concentration of sample: 0.01 mol L 1; sample injection volume: 5.0 μL.
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Scheme1. The synthesis of polymer monolithic column. (a) Synthesis of IL(AMIM þ Cl ); (b) The polymerization scheme.
amount of DMF, according to columns C and D, which indicated that PEG200 and DMF acted as the good solvents in the porogen system for making microspores. However, the permeability increased following the increasing amount of 1,4-butanediol, according to columns A – C. Thus, 1,4-butanediol served as the poor solvent for making macrospores. Considering the mechanical ability and back pressure, component of porogens for column A was used in the following experiment. What's more, columns E – G listed in Table 1 were prepared to study the effect of preparation temperature on the property of the monolithic columns. Higher preparation temperature would not only improve the solubility of monomers, but also accelerate the reaction process, thus resulting in a decrease of permeability. Though higher permeability could be obtained at the lower temperature, according to columns E, it had soft property that was not
3. Results and discussion 3.1. Optimization of the monolithic column Results listed in Table 1 showed the ratio of the three porogens dramatically affected the permeability of the monoliths. With the increasing content of PEG200, the permeability significantly decreased from 9.68 to 3.22 ( 10 14 m2, according to columns A – C), calculated according to the Darcy's Law [33] of permeability. B0 ¼ FηL/ΔPπr2 where F was the flow rate of the mobile phase 3 1 (m s ), η was the dynamic viscosity of the mobile phase (Pa s), L was the length of column (m), ΔP was the back pressure of the column (Pa), and r was the inner radius of the column (m). MeOH was used as the mobile phase.( η ¼0.58 cP). The decreasing permeability was also as the result of increasing Table 1 Optimization of porogen solvent and temperature. Monolithic column label
A B C D E F G
Progenic solvent PEG200 (mL)
1,4-Butanediol (mL)
DMF (mL)
0.8 0.6 1.0 0.7 0.8 0.8 0.8
0.4 0.6 0.2 0.3 0.4 0.4 0.4
0.2 0.2 0.2 0.4 0.2 0.2 0.2
Back pressure (bar)
Polymerization temperature (°C)
Perameablity (10 14 m2)
5 3 9 12 2 – 14
70 70 70 70 60 50 80
5.81 9.68 3.22 2.42 14.5 – 2.06
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Table 2 Compositions of polymerization mixtures on monolithic column preparation. Monolithic column label
IL (mL) TAIC (mL)
EDMA (mL)
PEG200 (mL)
1,2-Propanediol (mL)
DMF (mL)
Fecl2 (g) CCl4 (mL)
AIBN (g) Back pressure (bar)
Perameablity (10 14 m2)
A H I
0.1 0.1 –
0.6 0.6 0.6
0.8 0.8 0.8
0.4 0.4 0.4
0.2 0.2 0.2
0.005 – 0.005
– 0.02 –
5.81 3.63 2.64
0.5 0.5 0.5
0.1 – 0.1
5 8 11
suitable for usage. Column F was allowed to react at 50 °C and there was no any change of the pre-polymerization solution after 24 h. Column A prepared at 70 °C was certified to have the proper property. 3.2. Characterizations of the monolithic columns 3.2.1. MS spectra of IL The MS spectra of IL were shown in Fig. S1 (in supplementary information (SI) file). ESI ionic source was used with positive mode in the MS experiment. The m/z 123.1 and 281.0 could be found in Fig. S1, which were in accordance with the molecular weight of AMIM þ and 2(AMIM þ )Cl (the isotope peak indicated the present of Cl), respectively. 3.2.2. FT-IR The FT-IR spectra of columns A and I were shown in Fig. 1. Compare to column I, the stretching bands at 1643 cm 1 and 1567 cm 1 were identified with the νC ¼ C and νC ¼ N in imidazole ring. The band at 3400 cm 1 was due to the νO–H, which was for the strong moisture absorption of AMIM þ Cl and the absorbed water easily forming hydrogen bond. These results confirmed the presence of AMIM þ Cl in column A. 3.2.3. Mechanical stability and permeability The relationship between linear velocity and back-pressure drop of column A was also measured with methanol, water, and ACN as the mobile phases, respectively. With the linear velocities increasing from 1.0 to 7.0 mm s 1, the curve obtained from the corresponding back-pressure showed excellent linear correlation (r 40.999) as shown in Fig. S2 in SI file. Furthermore, more than 100 injections were made to verify the stability of the column, the performance of the monolithic column was not reduced, which indicated good mechanical stability. In addition, it showed the good permeability as 5.81 10 14 m2 calculating with methanol as mobile phase.
Fig. 2. Chracterization of pore type and pore size. (a) Nitrogen adsorption–desorption isotherms; (b) Pore size distribution for column A by mercury intrusion porosimetry.
Fig. 1. The FT–IR spectrum of the monolithic columns.(a) the poly (ILs–co–TAIC– co–EDMA) monolithic column (column A); (b) the poly (TAIC-co-EDMA) monolithic column (column I).
3.2.4. Pore structure and pore size distribution Fig. 2(a, b) showed the type of the pores and the pore size distribution of column A, respectively. After drying in a vacuum, the pore structure of column A was examined by nitrogen adsorption/desorption measurements, and the results shown in Fig. 2(a) was adjusted to isotherm of type V with a small hysteresis loop, which indicated weak interaction between N2 and the material. The results showed that there was scarcely any micropore and mesopore in the material. The small hysteresis loop on the isotherm, type H3, was the result of the slit created by the lamellar accumulation of the particles. The large absorption at high pressure in the isotherm indicated that there were macropores in the material. The pore area detected by this
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method was 19.28 m2 g 1. So, the structure of the monolithic column with macroporous was measured by mercury intrusion porosimetry, and the total intrusion volume was 1.94 mL g 1, the porosity (through-pore) was 73.29%, the total pore area was 5.83 m2 g 1, and the mode pore diameter was 2520 nm, respectively, with the pore size distribution was shown in Fig. 2(b). The enlarged imagine for pore distribution from 0 to 100 nm in Fig. 2(b) showed that there were sparing micropores and mesopores, which certified the results from the isotherm of type V with a small hysteresis loop. The pore area detected by this method was 5.83 m2 g 1. So the total pore area should be the addition of 19.28 (pore area of micropores and mesopores) and 5.83 (pore area of macropores). That was 25.11 m2 g 1. 3.2.5. TGA TGA was used to investigate the thermal behavior and weight loss of monolithic column. For column A (as shown in Fig. S3 in SI file), the decomposition temperature was about 310 °C and the rate of decomposition was relatively low. Such a high heat resistance was good for organic monolithic column. 3.2.6. Morphology of the monolithic columns As presented in Table 2, columns A and H were prepared by ATRP and in situ free radical polymerization, respectively, and column I was prepared via ATRP without IL addition. The structures of the three columns were shown in Fig. 3 evaluated by the scanning electron microscopy (SEM). Image for column A had uniform and porous skeleton structure. Column H showed the morphology of an inhomogeneity structure and larger globule that would increase the eddy diffusion. The reason was that in situ free radical polymerization would lead to an increase of the dimensions of the globules as well as the macropore size of columns. ATRP technique could led to the fabrication of the uniform structure with a narrow molecular weight distribution. This was because that it could avoid the drawbacks of the typical free radical polymerization arguably listed as slow initiation, fast termination, and easy chain transfer. However, column I had lamellar particle accumulated pore structure with low permeability. So the pore structure of column A was preferred to use as the stationary phase of HPLC. To further compare the separation performance of the three columns in HPLC, four homologue compounds were used as test analytes. Comparing the chromatograms shown in Fig. 4, a baseline separation of the mixture was achieved with both columns A and H, while the column I exhibited poor resolution and low efficiency. It was the addition of IL that the resolution and column efficiency were improved in the separation of small molecules. Besides, the column A showed higher column efficiency than column H. Though both types of columns were prepared using the same monomers and porogens, they showed different retention characteristics. It should be attributed to the differences of initiation process, which confirmed the ATRP technique was more preferable than in situ radical polymerization. 3.3. Chromatographic performance The nitrogen adsorption/desorption measurement demonstrated low surface areas of column A, which might not contribute to the improvement of separation performance. The mercury intrusion porosimetry afforded porosity of column A at 73.29% and the total intrusion volume reached to 1.94 mL g 1, as shown in Section 3.2.4. The large pore volume would enable fast mass transfer as the convection is dominant during the separation process, at the same time, the thin framework would also accelerate the mass transfer by reducing the diffusion distance, which
Fig. 3. Scanning electron microscopy imagines of columns.
would lead to the decrease of the value of C in van Deemter equation and be beneficial to the separation of small molecules. Owing to the satisfactory mechanical stabilities, as well as the uniform and porous structure, column A was applied to separate a series of small molecules. As shown in Fig. 5(a), a reversed-phase
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Fig. 4. Separation of toluene and its homologues on the monoliths.Experimental conditions: monolith size: 50 mm 4.6 mm i.d.; mobile phase, ACN/water (v/v, 42:58); flow rate, 1.0 mL min 1; concentration of sample, 0.01 mol L 1; injection volume, 2.0 μL; detection wavelength, 254 nm. Analytes: (1) toluene (2) ethylbenzene (3) propylbenzene and (4) butylbenzene.
(RP) retention mechanism was illustrated when the mixture of seven acidic and basic compounds were separated with the mobile phase of ACN/water (40/60, v/v) according to their hydrophobicity in column A. The retention factors of these seven compounds decreased linearly with the increase of ACN content from 30% to 50% as shown in Fig. 5(b). It confirmed the typical RP chromatographic property of the monolithic station phase toward the hydrophobic solutes. To further evaluate the performance of the column in the separation of isomers, p-hydroquinone, m-hydroquinone, and a-hydroquinone were used as test analytes. As shown in Fig. 6, p-hydroquinone could be baseline separated from the other two compounds, but m-hydroquinone and a-hydroquinone couldn't be distinguished from each other because of the similar lgP value. These results illustrated a certain selectivity of plane structure. 3.4. The effect of velocity on the column efficiency of the monolithic column Plots of the plate height versus flow rate (benzene, 1-naphthylamine, o-hydroquinone, and propylbenzene) were used as test probes in Fig. S4 in SI file, which demonstrated that 1.0 mm s 1 (1.0 mL min 1 for column with 4.6 mm i.d.) was the best velocity for lowest plate height (highest column efficiency). 3.5. Repeatability Column repeatability was significant performance characteristics of monolithic columns that must be confirmed for their use in routine analysis. Column A was characterized by measuring the relative standard deviations (RSDs) of retention times and peak areas using phenol, benzene, p-aminoazobenzene as test compounds. The RSDs of run-to-run (n¼ 5) and column-to-column (n ¼5) based on retention times were less than 0.9% and 2.2%, respectively. The two RSDs based on peak areas were less than 1.9% and 2.3%, respectively.
4. Conclusions A facile monolithic column was fabricated by IL-co-TAIC-coEDMA system via ATRP technique. Contributed to the porous and
Fig. 5. Chromatographic behavior of column A for the separation of acid and basic compounds.(a) HPLC separation of acid and basic compounds. Experimental conditions: mobile phase, ACN/water (v/v, 40/60); flow rate, 1.0 mL min 1; concentration of sample, 0.01 mol L 1; injection volume, 5.0 μL; detection wavelength, 254 nm. Analytes: (1) benzotriazole, (2) phenol, (3) benzene (4) 1-naphthol (5) naphthalene, (6) p-aminoazobenzene, and (7) diphenylamine; (b) Relationship between ACN content in mobile phase and the retention factor (k) of compounds on column A.
uniform construction, the resulted monoliths exhibited good thermal and chemical stabilities and better chromatographic efficiency than traditional polymer monoliths prepared via in situ free radical polymerization. The combination of ILs and living-control radical polymerization technique benefited in enhancing the chromatographic performance of small molecules separation.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21175031, No. 21505030), Natural Science Foundation of Hebei Province (No. B2015201024, B2013201082), National Science Foundation of Hebei University (No. 2013-247), and the Post-graduate's Innovation Fund Project of Hebei University (No. X2015076).
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Fig.6. Chromatographic behaviors of separation of isomers.Conditions: mobile phase ACN/water (v/v, 35/65); flow rate, 0.8 mL min 1; concentration of sample, 0.01 mol L 1; injection volume, 5.0 μL; detection wavelength, 254 nm. Analytes: (1) p-Hydroquinone, (2) m-Hydroquinone and (3) a-Hydroquinone.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2015.11. 028.
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Talanta journal homepage: www.elsevier.com/locate/talanta
Fabrication of an ionic liquid-based macroporous polymer monolithic column via atom transfer radical polymerization for the separation of small molecules Hang Zhang a,b,c, Ligai Bai a,b,c,n, Zhen Wei a,b,c, Sha Liu a,b,c, Haiyan Liu a,b,c,n, Hongyuan Yan a,b,c a b c
Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Baoding 071002, China College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Baoding 071002, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 4 November 2015 Accepted 11 November 2015 Available online 14 November 2015
A polymer monolithic column was prepared in a stainless steel column (50 4.6 mm i.d.) via atom transfer radical polymerization technique using triallyl isocyanurate and ionic liquid (1-allyl-3-methylimidazolium chloride) as co-monomers, ethylene dimethacrylate as cross linking agent, polyethylene glycol 200, 1,4-butanediol, and N, N- dimethylformamide as porogen system, CCl4 as initiator, and FeCl2 as catalyst. The optimized polymer columns were characterized by scanning electron microscope, nitrogen adsorption-desorption instrument, mercury intrusion porosimetry, infrared spectrometer, and thermogravimetric analysis technique. Respectively, all of these factors above could illustrate that the optimized columns had relative uniform macroporous structure and high thermal stability. A series of basic and acidic small molecules, isomers, and homologues were used to evaluate the performance of these monoliths and enhanced column efficiency was obtained. & 2015 Elsevier B.V. All rights reserved.
Keywords: Monolithic polymer column Ionic liquids (ILs) High performance liquid chromatography (HPLC) small molecules
1. Introduction Over the past two decades, compared to the silica-based columns, there has been a strong interest in polymer monolithic columns[1,2] due to their advantages of time-saving preparation, enhanced permeability, reduced mass transfer resistance, and better pH tolerance [3–5]. Polymer monoliths have been successfully used for the separation of large biomolecules, such as proteins, oligonucleotides, and peptides [6–11]. In contrast, it is unfavorable for the chromatographic performance of polymer monolithic columns for the separation of small molecules as a result of low mesopore volume and inhomogeneity of the structure [12,13]. Several methods have been proposed to solve this problem, such as applying a single cross-linker [14], using various living-control radical polymerization [15–17], adding nanostructures [18,19], and introducing click chemistry reaction [9,20,21]�which mainly focused on the improvement of the performance of capillary monoliths. To some extent, all these methods led to varying degrees of improvement of column efficiency. n Corresponding authors at: College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China. E-mail addresses: [email protected] (L. Bai), [email protected] (H. Liu).
http://dx.doi.org/10.1016/j.talanta.2015.11.028 0039-9140/& 2015 Elsevier B.V. All rights reserved.
The highest efficiency of obtained monoliths exceeded 100,000 plates/m for capillary liquid chromatography (CLC). However, there are still many difficulties to overcome. Many of click chemistry reactions are easily to tolerate conditions that will be damaging for processes currently used for the preparation of monoliths with some reactive chemistries [5]. As for nanostructure substance, because of its poor dispersion in the polymer matrix, it is suitable to be attached on the pore surface of the well-defined monolith in case of being buried and inaccessible, which is more time-consuming and complex operation compared to one-pot polymerization [5,22]. In addition, in terms of capillary monolith column, it also brings difficulties to the process of analysis for the too low capacity and poor reproducibility. For routine monolithic column (50 4.6 mm i.d.), there is not any prominent progress in recent time. The attempts mainly concentrated on the variation of monomers, cross-linkers, and porogen solvent mixture, the adjustment of the polymerization conditions (including temperature, time, and polymerization technique) [23], but these methods have not improved the applicability of the polymer monoliths to the desired degree. In order to develop a uniform and stable system with simple optimization of polymerization conditions, especially focus on improving the homogeneity of the structure and enhancing column efficiency, the green solvent ionic liquid (IL) as co monomer was proposed in the present work.
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Ionic liquids (ILs) are molten salts containing relatively asymmetric organic cations and inorganic or organic anions, whose physical and chemical properties are convenient to be changed by controlling the cations and anions [24]. Furthermore, contributed to the properties of high thermal stability, low volatility, good adjustability, high electrolytic conductivity, and miscibility [25], IL has been widely applied in chromatography [26–28] and aroused considerable scientific interest in the fabrication of monolithic column. IL-based monolithic columns [29,30] have been successfully utilized to separate a variety of analytes especially complex biological samples by CEC., These success mainly benefited from the property of good electrolytic conductivity of IL to generate a stable reversed EOF in a wide pH range for the CEC mode. However, it didn't help improve the morphology and enhance the separation of small molecules in HPLC. Our group [31,32] employed ILs as function monomer for the preparation of polymer monolithic columns via in situ free radical polymerization. The obtained monolithic columns, as the station phase for reversed-phase liquid chromatography (RPLC), exhibited good separation performance for small molecules. It demonstrated that the addition of IL made the structure more uniform with larger surface area. Furthermore, the conditions of polymerization could be optimized to get better mechanical and thermal stability, greater performance improvement and higher repeatability. The combination of the unique advantages of porous polymer monoliths, and the specific features of IL might present a promising improvement in the performance of monoliths for HPLC. Herein, IL (1-allyl-3-methylimidazolium chloride, AMIM þ Cl ) and a high concentration of crosslinking monomer triallyl isocyanurate (TAIC) build up a co-monomers system, with ethylene dimethacrylate (EDMA) as single cross-linker. The atom transfer radical polymerization (ATRP) technique was introduced to prepare the monolith. After the optimization of porogen system and polymerization conditions, a series process of characterization were carried out respectively including scanning electron microscopy, infrared spectrometer, nitrogen adsorption/desorption measurement, mercury intrusion porosimetry and thermal gravimetric analysis. Besides, the separation ability of these columns was estimated comprehensively by separating a series of basic and acidic small molecules, isomers and homologues by HPLC.
2. Experimental 2.1. Materials and methods Triallyl isocyanurate (TAIC) was purchased from Shanghai Huangguan Chemical company (Shanghai, China). Ethylene dimethacrylate (EDMA) was obtained from Maya-Reagent (Zhejiang, China). Polyethylene glycol 200 (PEG-200), 1,4-butanediol, N, Ndimethylformamide (DMF), CCl4, and FeCl2 were supplied by Tianjin Guangfu Fine Institute of Chemistry (Tianjin, China). Meanwhile, 2,2-Azobisisobutyronitrile(AIBN), HPLC-grade methanol (MeOH), acetonitrile (ACN), and KBr were produced by Kermel Chemical Reagent Factory (Tianjin, China). The analytes were provided by the National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, China). Besides, ultrapure water was used for all experiments and all medias were filtered through a 0.45 μm membrane before use. 2.2. Instruments All chromatographic experiments were performed on an 1100 system from Agilent Technologies (USA). Scanning electron microscopy (SEM) images of the monoliths were carried out on Hitachi S-3400 SEM instrument (Hitachi High Technologies, Japan).
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The Fourier transform infrared spectroscopy (FT–IR) spectra were recorded on an FTIR–8400S IR apparatus in the region of 400–4000 cm 1 (Shimadzu, Japan). The pore type was determined by the nitrogen adsorption/desorption measurment on a TriStar II 3020 instrument (Micromeritics, USA) while pore size measurement was performed on an AutoPore IV 9500 instrument (Micromeritics, USA ). Thermal gravimetric analysis (TGA) was carried out on a Setsys 16/18 instrument (Setaram, Caluire, France). Molecular weight data for ILs was obtained by MSD Trap XCT instrument with electrospray ionization (ESI) source which was bought from Agilent Technologies (USA). 2.3. Preparation and optimization of the poly(IL-co-TAIC-co-EDMA) monoliths 2.3.1. Synthesis of the IL (AMIM þ Cl ) Allyl chloride (9.95 g, 0.13 mol) was added dropwise to 1–methylimidazole (8.21 g, 0.1 mol). The mixture was heated at 55 °C under stirring for 11 h. When phase separation occurred, the brown viscous liquid was washed with ether (60 mL for three times). Then the product, IL (1-allyl-3-methylimidazolium chloride, AMIM þ Cl ), was filtered and dried in a vacuum oven until constant weigh (9.39 g, yield: 59.16%). Thus, the obtained IL was assayed by mass spectrometer. The reaction equation was shown in Scheme1(a). 2.3.2. Preparation of the poly(IL-co-TAIC-co-EDMA) monoliths The processes of preparation columns were as follows: IL and TAIC were used as co-monomers, EDMA as cross linking agent, 1,4butanediol, PEG200, and DMF as tri-porogens, CCl4 as initiator, FeCl2 as catalyst. All the reagents were put into a tube followed mixing and degassed by sonication for 15 min, and then the mixed solution was poured into a stainless-steel column (50 4.6 mm i. d.). The column was sealed and allowed to react in a water bath for 24 h. The resulting monolithic column was washed online with methanol by an HPLC pump to remove the porogens. The polymerization scheme was illustrated in Scheme 1(b). 2.3.3. Optimization of the poly(IL-co-TAIC-co-EDMA) monoliths In order to find the proper porogens then to improve the morphology and structure of the monolithic column, several candidates, such as propanol/1,4-butanediol, DMF/1,4-butanediol, 1,4-butanediol/PEG200, DMF/PEG200 and DMF/PEG200/1,4-butanediol were proposed according to their solubility and polarity, respectively. The pre-experiment showed that the ternary porogenic system DMF/PEG200/1,4-butanediol had prior effects on the property of the monolithic columns to that of single or double porogens. Table 1 listed the representative conditions and some of the obtained results, containing the effect of polymerization temperature on the property of monolithic column. Furthermore, compared to the IL-based monolith prepared via ATRP, columns H and I were prepared via in situ or without IL addition, respectively, with the details listed in Table 2. 2.4. HPLC procedures An Agilent 1100 system consisted of a quaternary pump with an online vacuum degasser, an autosampler with variable injection capacity from 0.1 to 100 μL, and UV detector. All sample solutions injected in the chromatographic system were filtered through a millipore membrane (0.45 μm) to remove particles and large aggregates. The chromatographic conditions were as follows: mobile phase: ACN/water; UV wave length: 254 nm; temperature: 25 °C; concentration of sample: 0.01 mol L 1; sample injection volume: 5.0 μL.
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Scheme1. The synthesis of polymer monolithic column. (a) Synthesis of IL(AMIM þ Cl ); (b) The polymerization scheme.
amount of DMF, according to columns C and D, which indicated that PEG200 and DMF acted as the good solvents in the porogen system for making microspores. However, the permeability increased following the increasing amount of 1,4-butanediol, according to columns A – C. Thus, 1,4-butanediol served as the poor solvent for making macrospores. Considering the mechanical ability and back pressure, component of porogens for column A was used in the following experiment. What's more, columns E – G listed in Table 1 were prepared to study the effect of preparation temperature on the property of the monolithic columns. Higher preparation temperature would not only improve the solubility of monomers, but also accelerate the reaction process, thus resulting in a decrease of permeability. Though higher permeability could be obtained at the lower temperature, according to columns E, it had soft property that was not
3. Results and discussion 3.1. Optimization of the monolithic column Results listed in Table 1 showed the ratio of the three porogens dramatically affected the permeability of the monoliths. With the increasing content of PEG200, the permeability significantly decreased from 9.68 to 3.22 ( 10 14 m2, according to columns A – C), calculated according to the Darcy's Law [33] of permeability. B0 ¼ FηL/ΔPπr2 where F was the flow rate of the mobile phase 3 1 (m s ), η was the dynamic viscosity of the mobile phase (Pa s), L was the length of column (m), ΔP was the back pressure of the column (Pa), and r was the inner radius of the column (m). MeOH was used as the mobile phase.( η ¼0.58 cP). The decreasing permeability was also as the result of increasing Table 1 Optimization of porogen solvent and temperature. Monolithic column label
A B C D E F G
Progenic solvent PEG200 (mL)
1,4-Butanediol (mL)
DMF (mL)
0.8 0.6 1.0 0.7 0.8 0.8 0.8
0.4 0.6 0.2 0.3 0.4 0.4 0.4
0.2 0.2 0.2 0.4 0.2 0.2 0.2
Back pressure (bar)
Polymerization temperature (°C)
Perameablity (10 14 m2)
5 3 9 12 2 – 14
70 70 70 70 60 50 80
5.81 9.68 3.22 2.42 14.5 – 2.06
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Table 2 Compositions of polymerization mixtures on monolithic column preparation. Monolithic column label
IL (mL) TAIC (mL)
EDMA (mL)
PEG200 (mL)
1,2-Propanediol (mL)
DMF (mL)
Fecl2 (g) CCl4 (mL)
AIBN (g) Back pressure (bar)
Perameablity (10 14 m2)
A H I
0.1 0.1 –
0.6 0.6 0.6
0.8 0.8 0.8
0.4 0.4 0.4
0.2 0.2 0.2
0.005 – 0.005
– 0.02 –
5.81 3.63 2.64
0.5 0.5 0.5
0.1 – 0.1
5 8 11
suitable for usage. Column F was allowed to react at 50 °C and there was no any change of the pre-polymerization solution after 24 h. Column A prepared at 70 °C was certified to have the proper property. 3.2. Characterizations of the monolithic columns 3.2.1. MS spectra of IL The MS spectra of IL were shown in Fig. S1 (in supplementary information (SI) file). ESI ionic source was used with positive mode in the MS experiment. The m/z 123.1 and 281.0 could be found in Fig. S1, which were in accordance with the molecular weight of AMIM þ and 2(AMIM þ )Cl (the isotope peak indicated the present of Cl), respectively. 3.2.2. FT-IR The FT-IR spectra of columns A and I were shown in Fig. 1. Compare to column I, the stretching bands at 1643 cm 1 and 1567 cm 1 were identified with the νC ¼ C and νC ¼ N in imidazole ring. The band at 3400 cm 1 was due to the νO–H, which was for the strong moisture absorption of AMIM þ Cl and the absorbed water easily forming hydrogen bond. These results confirmed the presence of AMIM þ Cl in column A. 3.2.3. Mechanical stability and permeability The relationship between linear velocity and back-pressure drop of column A was also measured with methanol, water, and ACN as the mobile phases, respectively. With the linear velocities increasing from 1.0 to 7.0 mm s 1, the curve obtained from the corresponding back-pressure showed excellent linear correlation (r 40.999) as shown in Fig. S2 in SI file. Furthermore, more than 100 injections were made to verify the stability of the column, the performance of the monolithic column was not reduced, which indicated good mechanical stability. In addition, it showed the good permeability as 5.81 10 14 m2 calculating with methanol as mobile phase.
Fig. 2. Chracterization of pore type and pore size. (a) Nitrogen adsorption–desorption isotherms; (b) Pore size distribution for column A by mercury intrusion porosimetry.
Fig. 1. The FT–IR spectrum of the monolithic columns.(a) the poly (ILs–co–TAIC– co–EDMA) monolithic column (column A); (b) the poly (TAIC-co-EDMA) monolithic column (column I).
3.2.4. Pore structure and pore size distribution Fig. 2(a, b) showed the type of the pores and the pore size distribution of column A, respectively. After drying in a vacuum, the pore structure of column A was examined by nitrogen adsorption/desorption measurements, and the results shown in Fig. 2(a) was adjusted to isotherm of type V with a small hysteresis loop, which indicated weak interaction between N2 and the material. The results showed that there was scarcely any micropore and mesopore in the material. The small hysteresis loop on the isotherm, type H3, was the result of the slit created by the lamellar accumulation of the particles. The large absorption at high pressure in the isotherm indicated that there were macropores in the material. The pore area detected by this
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method was 19.28 m2 g 1. So, the structure of the monolithic column with macroporous was measured by mercury intrusion porosimetry, and the total intrusion volume was 1.94 mL g 1, the porosity (through-pore) was 73.29%, the total pore area was 5.83 m2 g 1, and the mode pore diameter was 2520 nm, respectively, with the pore size distribution was shown in Fig. 2(b). The enlarged imagine for pore distribution from 0 to 100 nm in Fig. 2(b) showed that there were sparing micropores and mesopores, which certified the results from the isotherm of type V with a small hysteresis loop. The pore area detected by this method was 5.83 m2 g 1. So the total pore area should be the addition of 19.28 (pore area of micropores and mesopores) and 5.83 (pore area of macropores). That was 25.11 m2 g 1. 3.2.5. TGA TGA was used to investigate the thermal behavior and weight loss of monolithic column. For column A (as shown in Fig. S3 in SI file), the decomposition temperature was about 310 °C and the rate of decomposition was relatively low. Such a high heat resistance was good for organic monolithic column. 3.2.6. Morphology of the monolithic columns As presented in Table 2, columns A and H were prepared by ATRP and in situ free radical polymerization, respectively, and column I was prepared via ATRP without IL addition. The structures of the three columns were shown in Fig. 3 evaluated by the scanning electron microscopy (SEM). Image for column A had uniform and porous skeleton structure. Column H showed the morphology of an inhomogeneity structure and larger globule that would increase the eddy diffusion. The reason was that in situ free radical polymerization would lead to an increase of the dimensions of the globules as well as the macropore size of columns. ATRP technique could led to the fabrication of the uniform structure with a narrow molecular weight distribution. This was because that it could avoid the drawbacks of the typical free radical polymerization arguably listed as slow initiation, fast termination, and easy chain transfer. However, column I had lamellar particle accumulated pore structure with low permeability. So the pore structure of column A was preferred to use as the stationary phase of HPLC. To further compare the separation performance of the three columns in HPLC, four homologue compounds were used as test analytes. Comparing the chromatograms shown in Fig. 4, a baseline separation of the mixture was achieved with both columns A and H, while the column I exhibited poor resolution and low efficiency. It was the addition of IL that the resolution and column efficiency were improved in the separation of small molecules. Besides, the column A showed higher column efficiency than column H. Though both types of columns were prepared using the same monomers and porogens, they showed different retention characteristics. It should be attributed to the differences of initiation process, which confirmed the ATRP technique was more preferable than in situ radical polymerization. 3.3. Chromatographic performance The nitrogen adsorption/desorption measurement demonstrated low surface areas of column A, which might not contribute to the improvement of separation performance. The mercury intrusion porosimetry afforded porosity of column A at 73.29% and the total intrusion volume reached to 1.94 mL g 1, as shown in Section 3.2.4. The large pore volume would enable fast mass transfer as the convection is dominant during the separation process, at the same time, the thin framework would also accelerate the mass transfer by reducing the diffusion distance, which
Fig. 3. Scanning electron microscopy imagines of columns.
would lead to the decrease of the value of C in van Deemter equation and be beneficial to the separation of small molecules. Owing to the satisfactory mechanical stabilities, as well as the uniform and porous structure, column A was applied to separate a series of small molecules. As shown in Fig. 5(a), a reversed-phase
H. Zhang et al. / Talanta 149 (2016) 62–68
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Fig. 4. Separation of toluene and its homologues on the monoliths.Experimental conditions: monolith size: 50 mm 4.6 mm i.d.; mobile phase, ACN/water (v/v, 42:58); flow rate, 1.0 mL min 1; concentration of sample, 0.01 mol L 1; injection volume, 2.0 μL; detection wavelength, 254 nm. Analytes: (1) toluene (2) ethylbenzene (3) propylbenzene and (4) butylbenzene.
(RP) retention mechanism was illustrated when the mixture of seven acidic and basic compounds were separated with the mobile phase of ACN/water (40/60, v/v) according to their hydrophobicity in column A. The retention factors of these seven compounds decreased linearly with the increase of ACN content from 30% to 50% as shown in Fig. 5(b). It confirmed the typical RP chromatographic property of the monolithic station phase toward the hydrophobic solutes. To further evaluate the performance of the column in the separation of isomers, p-hydroquinone, m-hydroquinone, and a-hydroquinone were used as test analytes. As shown in Fig. 6, p-hydroquinone could be baseline separated from the other two compounds, but m-hydroquinone and a-hydroquinone couldn't be distinguished from each other because of the similar lgP value. These results illustrated a certain selectivity of plane structure. 3.4. The effect of velocity on the column efficiency of the monolithic column Plots of the plate height versus flow rate (benzene, 1-naphthylamine, o-hydroquinone, and propylbenzene) were used as test probes in Fig. S4 in SI file, which demonstrated that 1.0 mm s 1 (1.0 mL min 1 for column with 4.6 mm i.d.) was the best velocity for lowest plate height (highest column efficiency). 3.5. Repeatability Column repeatability was significant performance characteristics of monolithic columns that must be confirmed for their use in routine analysis. Column A was characterized by measuring the relative standard deviations (RSDs) of retention times and peak areas using phenol, benzene, p-aminoazobenzene as test compounds. The RSDs of run-to-run (n¼ 5) and column-to-column (n ¼5) based on retention times were less than 0.9% and 2.2%, respectively. The two RSDs based on peak areas were less than 1.9% and 2.3%, respectively.
4. Conclusions A facile monolithic column was fabricated by IL-co-TAIC-coEDMA system via ATRP technique. Contributed to the porous and
Fig. 5. Chromatographic behavior of column A for the separation of acid and basic compounds.(a) HPLC separation of acid and basic compounds. Experimental conditions: mobile phase, ACN/water (v/v, 40/60); flow rate, 1.0 mL min 1; concentration of sample, 0.01 mol L 1; injection volume, 5.0 μL; detection wavelength, 254 nm. Analytes: (1) benzotriazole, (2) phenol, (3) benzene (4) 1-naphthol (5) naphthalene, (6) p-aminoazobenzene, and (7) diphenylamine; (b) Relationship between ACN content in mobile phase and the retention factor (k) of compounds on column A.
uniform construction, the resulted monoliths exhibited good thermal and chemical stabilities and better chromatographic efficiency than traditional polymer monoliths prepared via in situ free radical polymerization. The combination of ILs and living-control radical polymerization technique benefited in enhancing the chromatographic performance of small molecules separation.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21175031, No. 21505030), Natural Science Foundation of Hebei Province (No. B2015201024, B2013201082), National Science Foundation of Hebei University (No. 2013-247), and the Post-graduate's Innovation Fund Project of Hebei University (No. X2015076).
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Fig.6. Chromatographic behaviors of separation of isomers.Conditions: mobile phase ACN/water (v/v, 35/65); flow rate, 0.8 mL min 1; concentration of sample, 0.01 mol L 1; injection volume, 5.0 μL; detection wavelength, 254 nm. Analytes: (1) p-Hydroquinone, (2) m-Hydroquinone and (3) a-Hydroquinone.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2015.11. 028.
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