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ion exchange chromatography
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Preparation of two ionic liquid bonded stationary phases and comparative evaluation under mixed-mode of reversed phase/ hydrophilic interaction/ ion exchange chromatography Xiang Wang, Jingdong Peng ∗ , Huanjun Peng, Jun Chen, Hong Xian, Ranxi Ni, Shiyu Li, Dengying Long, Zhongying Zhang School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 9 April 2019 Received in revised form 9 July 2019 Accepted 14 July 2019 Available online xxx Keywords: Click chemistry Stationary phases Mixed-Mode Retention mechanism
a b s t r a c t The present work describes the preparation of two ionic liquid and carboxyl acid silane reagents via photo-initiated thiol-ene click chemistry that have been bonded to silica to afford two mixed-mode stationary phases (Sil-C4Im-C9Co and Sil-C9Im-C4Co). The two stationary phases provided satisfactory retention repeatability and efficiencies. The influence of acetonitrile content, salt concentration and pH of the mobile phase was investigated to clarify the retention properties of the prepared stationary phases. The results showed that the prepared Sil-C4Im-C9Co and Sil-C9Im-C4Co undergo multiple interactions with solutes under different chromatographic conditions. The retention mechanisms were further studied by the linear energy solvation relationship and Van’t Hoff plots. Finally, the stationary phases were employed to separate hydrophobic solutes (alkylbenzenes and polycyclic aromatic hydrocarbons) under reversed phase liquid chromatography (RPLC) mode, hydrophilic solutes (carboxylic acids, nucleosides and bases) under hydrophilic interaction liquid chromatography (HILIC) mode and inorganic anions under ion-exchange chromatography (IEC) mode, providing excellent performance and varying selectivity when compared with a commercial column. The bonding method in this work is feasible and the prepared stationary phases are promising when employed in RPLC/HILIC/IEC mixed-mode chromatography applications. © 2019 Published by Elsevier B.V.
1. Introduction Among the most employed separation modes for high performance liquid chromatography (HPLC), reversed phase liquid chromatography (RPLC), for its great performance in the separation of most organic compounds, still plays a dominating role [1]. Separations on RPLC are principally based on the varying hydrophobic interactions between solutes and stationary phases, with hydrophilic solutes being weakly retained and poorly separated. Hydrophilic interaction liquid chromatography (HILIC) has worked in tandem with RPLC and experienced a rapid development over the last few decades [2–4]. The interactions involved in HILIC appear to be more complex and may involve mixed partitioning and adsorption mechanisms [5,6]. With the increasing complexity of practical samples, ion-exchange chromatography (IEC) is
indispensable, especially for biological application [7,8]. Hence, the development of stationary phases for a wide range of applications remains a current topic of significant interest for chromatographic science. Mixed-mode chromatography (MMC) can achieve at least two separation modes on a single column [9]. MMC employs stationary phases modified by functional groups possessing different characteristics which provide multiple interactions with solutes. Numerous MMC stationary phases have been reported, including RPLC/HILIC [10,11], HILIC/IEC [12–14] and RPLC/HILIC/IEC [15–17]. A stationary phase modified with Lisoleucine and 4-phenylbutylamine has been reported to separate aromatic compounds of different polarities under RPLC mode and nucleotides/nucleosides under HILIC mode [10]. A 1,4-butanediol diglycidyl ether and dopamine grafted dendritic stationary phase
∗ Corresponding author at: School of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing, People’s Republic of China. E-mail address: [email protected] (J. Peng). https://doi.org/10.1016/j.chroma.2019.460372 0021-9673/© 2019 Published by Elsevier B.V.
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was synthesized, which demonstrated mixed RPLC, HILIC and IEC mechanisms, and was successful in separating alkylbenzenes, polycyclic aromatic hydrocarbons(PAHs), nucleosides and flavonoid, and some acidic and basic analytes under different modes [15]. A new column comprising graphene quantum dots bonded to silica showed satisfactory performances under RPLC, NPLC and HILIC modes, and a complex retention mechanism was observed under different modes [18]. Additionally, a stationary phase functionalized 2-methylimidazolium was used for HILIC and anion-exchange chromatography, and furthermore, this silica could be further modified to afford new stationary phases [19]. Ionic liquids (ILs) are a type of organic salt having low melting point, high thermal stability and negligible vapor pressure. ILs have been utilized in RPLC mobile phase to improve the retention of high polar solutes. As a result of the unique chemical and physical properties, ILs have been bonded to silica and employed as stationary phases for HPLC [20–23]. A series of ILs bearing different aliphatic chains were prepared and bonded to silica to determine their relative selectivity [24]. A monolithic column was prepared using pentafluorobenzyl imidazolium bromide ILs, and showed mixed mechanisms of hydrophobic interaction, − stacking, ion-exchange, electrostatic interaction and dipole–dipole interaction, enhancing better selectivity for halogenated compounds when compared with commercial C18 and pentafluorophenyl columns [25]. In this study, four silane reagents were synthesized via photoinitiated thiol-ene click chemistry and bonded to silica through one step bonded method. This may result in a higher yield of acylation and less undesirable interactions when compared with two step modification process [26]. Compared with existing IL based mixed mode stationary phases, we prepared two novel stationary phases in which both IL and carboxylic groups have been introduced. Furthermore, it has been investigated for the first time that how the performance of the column is influenced by the relative length of the IL and carboxylic silane reagents. The retention properties
were investigated as a function of the chromatographic conditions while the retention mechanisms were investigated by the linear solvation energy relationship and thermodynamic analysis. As for application, two prepared stationary phases were compared with a commercial column and used to separate hydrophobic alkylbenzenes and PAHs, hydrophilic carboxylic acids, nucleosides, bases and inorganic anions. 2. Experimental 2.1. Chemicals and materials The spherical porous silica (5 m particle size, 100 Å pore size, and 330 m2 g−1 surface area) was purchased from Fuji Silysia Chemical (Kasugai, Japan). 2, 2-Dimethoxy(DMPA), (3-Mercaptopropyl) 2-phenylacetophenone trimethoxysilane were purchased from Aladdin (Shanghai, China). Lithium Bis(trifluoromethanesulfonyl)imide (LiNTF2 ), 4bromobut-1-ene were obtained from Meryer Chemical Technology Co., Ltd. (Shanghai, China). 1-Methylimidazole, 9-bromonon-1ene, 9-decenoic acid and 4-pentenoic acid were purchased from Bide Pharmatech Ltd. (Shanghai, China). 2.2. Instrument and apparatus All chromatographic evaluations were performed using an Agilent 1100 series system with a G1311A quaternary pump, a G1379A on-line degasser, a G1316A column oven, a G1315B DAD detector and a 7725i manual injector with a sample loop of 20 L. The columns were packed using an RPL-ZD10 packing machine. The Ultimate C18 (150 × 4.6 mm, 5 m,) column was purchased from Welch materials, Inc., the APS-2 Hypersil (150 × 4.6 mm, 5 m,) column and Acclaim Mixed-Mode WAX (150 × 4.6 mm, 5 m) column were obtained from Thermo Fisher Scientific.
Fig. 1. Preparation route of Sil-C9Im-C4Co and Sil-C4Im-C9Co stationary phases.
Please cite this article in press as: X. Wang, et al., Preparation of two ionic liquid bonded stationary phases and comparative evaluation under mixed-mode of reversed phase/ hydrophilic interaction/ ion exchange chromatography, J. Chromatogr. A (xxxx), https://doi.org/10.1016/j.chroma.2019.460372
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C (%)
H (%)
N (%)
S (%)
Sil-C4Im-C9Co Sil-C9Im-C4Co
7.36 9.3
1.665 1.728
1.08 1.12
2.792 2.974
2.3. New stationary phases preparation 2.3.1. Preparation of four silane reagents The process of synthesizing four silane reagents is shown in Fig. 1 and described in detail in the supplementary information [27,28]. 2.3.2. One-step bonded silane reagents onto silica 6.0 g spherical silica was first activated by refluxing in 4 M HCl for 12 h, prior to being washed to pH 7 and dried under vacuum at 60 ◦ C overnight. 2.5 g activated silica was dispersed in 30 mL anhydrous dioxane, 2.0 mmol IL silane and 2.0 mmol carboxylic acid silane were added according to the configurations in Fig. 1. The mixture was refluxed under a N2 atmosphere for 24 h, and the obtained materials were washed by CH3 OH and H2 O respectively to remove unreacted reagents, dried under vacuum at 45 ◦ C overnight. The functionalized silica was slurry-packed into stainless steel columns (4.6 mm i.d. × 150 mm length). 2.4. Preparation of real sample-milk powder extract 1.0 g milk powder was dispersed in 10mLof formic acid solution (1%v/v), subjected to ultra-sonication for 30 min. Therefore, 40 mL of ACN was added to precipitate the protein, with the resulting suspension centrifuged at 10,000 r/min for 15 min, and the supernatant collected and filtered through a 0.22 m film for chromatographic analysis. 3. Results and discussion 3.1. Preparation and characterization of two stationary phases (Sil-C4Im-C9Co and Sil-C9Im-C4Co) Two stationary phases were modified using IL and carboxylic acid silane reagents having differing alkyl chain lengths. According to the structures, the stationary phases were named as SilC4Im-C9Co and Sil-C9Im-C4Co. The two stationary phases were characterized by element analysis, with the results presented in Table 1. The bonding densities of four silane reagents are
Fig. 2. Effect of ACN content on retention.
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calculated according to the carbon and nitrogen contents, provided that all the methoxyl groups have reacted or hydrolyzed. In Sil-C4Im-C9Co, short IL silane reagent cover the silica by the density of 0.257 mmol g−1 , long carboxylic acid silane reagent by 0.215 mmol g−1 , in Sil-C9Im-C4Co, long IL silane reagent by 0.267 mmol g−1 , short carboxylic acid silane reagent by 0.368 mmol g−1 . Lower bonding densities were found for long silane reagents on both two stationary phases, which may result from the steric hindrance effect. Furthermore, thermogravimetric analysis was used to prove the successful fabrication of the new stationary phases and to investigate their thermostability. As shown in Fig. S1, apart from the weight loss associated with absorbed water, a mass loss of >20% was observed in both Sil-C9Im-C4Co and Sil-C4Im-C9Co as the temperature was increased to 700 ◦ C, and both stationary phases demonstrated good stability ≤300 ◦ C. 3.2. Influence of the chromatographic conditions on retention The retention of solutes is greatly affected by chromatographic conditions employed, such as solvent composition, pH and salt concentration in the mobile phase, as these conditions determine the interactions between solutes and the selected stationary phase. Hence, investigation into the effects of chromatographic conditions on retention may elucidate the retention mechanism and properties of the stationary phase.
3.2.1. Effect of ACN content In this study, ACN content was varied from 95 to 25%, as shown in Fig. 2. With decreasing ACN content in the mobile phase, the hydrophilic solutes (cytidine and melamine) retention decreased obviously on Sil-C9Im-C4Co and Sil-C4Im-C9Co, displaying typical HILIC retention characteristics. Conversely, hydrophobic solutes (toluene and methylparaben) were more strongly retained when the ACN content decreased, exhibiting typical RPLC retention characteristics, more so for Sil-C9Im-C4Co. Interestingly, the retention of two carboxylic acids (benzoic acid and salicylic acid) showed a U-shaped curve when the ACN content changed, demonstrating a HILIC mode at an ACN content ≥85% and a RPLC mode at an ACN content <85%. In conclusion, both columns exhibit mixed-mode of HILIC and RPLC. Hydrophilic and hydrophobic interactions may form between solutes and synthesized stationary phases, and such interactions may vary in their dominance according to the solvent composition.
Mobile phase: 10 mM NH4FA, pH 5.5, flow rate:0.6 mL min−1 , column temperature: 25 ◦ C.
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Fig. 3. Effect of salt concentration on retention.
Mobile phase: ACN/NH4 FA buffer (90/10, v/v), flow rate: 0.6 mL min−1 , pH 5.5, column temperature: 25 ◦ C.
3.2.2. Effect of salt concentration An appropriate salt concentration is vital for HILIC separation, and herein, retention was studied as a function of varying the NH4 FA salt concentration from 0 to 20 mM. As shown in Fig. 3, with increasing of salt concentration, solutes of varying properties showed diverse retention trends. The retention of melamine increased, while the opposite trend was observed for methylparaben, which can be explained by the increased hydrophilic nature of the stationary phase as a result of the thickness of the absorbed water layer increasing on the stationary phases. Benzoic acid was strongly retained on both stationary phases in ˜ h), and experienced a sharp decrease the absence of the salt (1.5 when the salt concentration increased, especially in the range of 0–10 mM. The increase of salt concentration is thought to greatly suppress the ion-exchange interaction between the stationary phases and benzoic acid. Adenosine and cytidine show a slight decrease in retention when the salt concentration increased from 0 to 5 mM, and an increase in retention upon further increasing the salt concentration. The aforementioned two reasons may explain this observed trend, that is, variation of salt concentration may influence retention by two mechanisms, suppressing the
Fig. 4. Effect of pH on retention.
ion-exchange interaction and strengthening the hydrophilic partition. 3.2.3. Effect of pH To study the effect of pH on the retention properties of the prepared stationary phases, as shown in Fig. 4, the pH of the mobile phase was changed from 6.0 to 3.1. The retention of pyridine, melamine and nucleosides was little affected by pH, as the interactions between these solutes and the stationary phases were not changed. The observed retention changes of benzoic acid (pKa 4.12) and p-nitrobenzoic acid (pKa 3.42) showed opposite trends, since the pH affects the charge states of solutes and stationary phases. The degree of protonation for benzoic acid increases as pH decreased, therefore, the ion-exchange interactions and hydrophilic partitioning are greatly suppressed, resulting in a significant decrease in retention. For p-nitrobenzoic acid, as the pH decreased from 6.0 to 4.0, additional carboxyl and silanol groups on the stationary phases become protonated, while p-nitrobenzoic acid is not affected, hence, the weakened electrostatic repulsion resulted in stronger retention. As the pH is further decreased, protonation of
Mobil phase: ACN / 100 mM NH4 FA buffer solution (80/20, v/v), flow rate: 0.6 mL min−1 , column temperature: 30 ◦ C.
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Fig. 5. Column efficiency and retention repeatability of Sil-C4Im-C9Co and Sil-C9Im-C4Co. Mobile phase: ACN/100 mM NH4 FA (90/10, v/v), 0.6 mL min−1 , pH 5.5, UV 254 nm; (1) methylparaben (2) adenosine, (3) cytidine.
p-nitrobenzoic acid begins resulting in weakened retention, similar to benzoic acid. 3.3. Retention repeatability and efficiencies Satisfactory retention repeatability and efficiencies are a prerequisite for both qualitative and quantitative work. In this study, a mixture of methylparaben, adenosine and cytidine were injected and recorded, and repeated over 15 times. The relative standard deviation (RSD) for retention time, symmetry factors and plate numbers for each peak are shown in Fig. 5. Both columns presented satisfactory retention repeatability and column efficiencies with Sil-C9Im-C4Co outperforming Sil-C4Im-C9Co, which may relate to the bonding density and packing quality of the columns. 3.4. Investigation of the retention mechanism 3.4.1. Comparison of retention properties among different columns Various solutes were selected to research and compare the retention properties of different columns, including hydrophobic alkylbenzenes and PAHs, hydrophilic nucleosides and bases, carboxylic acids and amines. Typical RPLC stationary phase C18 and HILIC stationary phase APS-2 Hypersil were introduced to present further comparative information. The retention of hydrophobic alkylbenzenes and PAHs were not compared among the HILIC stationary phases because they are barely retained. As shown in Fig. 6, C18 and APS-2 Hypersil expectedly showed their opposing selectivity. In the presence of C18 column, hydrophobic alkylbenzenes and PAHs are more strongly retained when compared with the carboxylic acids and hydrophilic solutes, while the APS-2 Hypersil presented an opposite trend. C18 retains solutes mainly through hydrophobic interactions, whereas, APS2 Hypersil acts through hydrophilic partitioning. In comparisons between APS-2 Hypersil and the stationary phases prepared in this work, nucleosides and bases are evenly distributed on both sides of the first bisector, which means iso-elution, suggesting a similar hydrophilia but different selectivity. Furthermore, carboxylic acids and amines are more strongly retained on Sil-C4Im-C9Co and Sil-C9Im-C4Co, because of the presences of both anion-exchange sites (imidazole ring) and cation-exchange sites (carboxylic group) on the stationary phases. When compared Sil-C4Im-C9Co to
Sil-C9Im-C4Co, the points of nucleosides and bases located almost on the first bisector, however, the carboxylic acids are more strongly retained on Sil-C9Im-C4Co, while amines retention is more pronounced on Sil-C4Im-C9Co. This observation may be explained by the differences between their structures, since the imidazole ring is more accessible on Sil-C9Im-C4Co, and therefore, a stronger anion-exchange is expected than for Sil-C4Im-C9Co stationary phase. 3.4.2. Linear solvation energy relationship (LSER) LSER is a reliable method to investigate the retention mechanism and to compare the retention properties between various chromatographic systems [29–31], expressed as follows: log k = c + eE + sS + aA + bB + vV + d− D− + d+ D+ The capital letters are solute descriptors, c is a system constant, lowercase letters (e, s, a, b, v) are system descriptors that measure the magnitude of difference in the particular interactions between the stationary and mobile phases, corresponding to the excess polarizability contributions from n and electrons, dipolarity, hydrogen bond(HBD) donating ability, hydrogen bond accepting ability, and the hydrophobicity of a given chromatographic system respectively [32]. d− D− and d+ D+ were further introduced to describe the effect of ionization on retention [33]. The lowercase letters can be used to compare the selectivity between different stationary phases after setting the chromatographic condition [34]. In this study, the retentions of 35 solutes were measured under the same conditions as Fig. 6, LSER was fitted for four columns with the corresponding coefficients listed in Table 2. As expected, only the C18 column for RPLC possesses positive v, confirming the tendency to undergo hydrophobic interactions with solutes. Imidazole rings could interact with solutes through − and anion exchange interactions, therefore, positive e and d− were observed for both Sil-C4Im-C9Co and Sil-C9Im-C4Co. As the imidazole ring is more accessible in Sil-C9Im-C4Co, because of its longer alkyl chain, e and d- values are higher for Sil-C9Im-C4Co. Both the Sil-C4Im-C9Co and Sil-C9Im-C4Co stationary phases can provide additional polar groups than the C18 and APS-2 Hypersil columns, which promotes dipole–dipole interactions with the solutes, and therefore, the s coefficients is more positive and larger for Sil-C4Im-C9Co and SilC9Im-C4Co. As for hydrogen-bond interactions, coefficients a and b
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Fig. 6. Comparison of the retention selectivity of C18, APS-2 Hypersil, Sil-C9Im-C4Co and Sil-C4Im-C9Co. Mobile phase: ACN/100 mM NH4 FA (90/10, v/v), flow rate: 0.6 mL min−1 , column temperature: 25 ◦ C.
are negative for C18, because there are fewer hydrogen-bond acting sites present. Three hydrophilic stationary phases gave positive a and b of varying size, while two IL bonded stationary phases are more HBD basic (bigger a and smaller b) than APS-2 Hypersil. This may relate to the associated anion or absorbed water layer, because the mobile phase is less HBD basic than methylimidazolium Ntf2 -. 3.4.3. Thermodynamic analysis The Van’t Hoff plot is one of the most employed thermodynamic methods to investigate physicochemical process mechanisms [35,36], and changes in enthalpy and entropy can be calculated through the slope and intercept of the plot by the Van’t Hoff equation, expressed as: lnk = −
H S + + ln∅ RT R
R is the gas constant, ∅ represents the phase ratio (vs / vm ), H and S measure the change of enthalpy and entropy, respectively, of solutes transferring from the mobile phase to the stationary phase. As shown in Fig. 7, in accordance with previous works, increasing the temperature of the column from 10 to 50 ◦ C, the retention of solutes such as pyridine, melamine and benzoic acid showed continuous declination. The Van’t Hoff plots associated with additional solutes are presented in Fig. S2. Interestingly, p-nitrobenzoic acid showed an opposite trend, indicating that the retention mechanism may differ from other solutes. Apart from methylparaben, all solutes exhibit a Van’t Hoff plot with high correlation coefficients (R2 >0.97), therefore, no obvious
variations in the retention mechanism occurred in the temperature range studied. Furthermore, the H and S values of seven solutes were calculated, and are listed in Table 3, with different mechanisms observed for the solutes. Negative H and S values were observed for four solutes (cytidine, adenosine, pyridine and melamine), indicating an enthalpy-driven mechanism for their exothermic retention processes, the retention was assumed to be principally based on hydrophilic partition. Conversely, positive H and S values were observed for p-nitrobenzoic acid, indicating an entropy-driven process. A positive H value indicates a more energetically favorable system for p-nitrobenzoic acid to remain in the mobile phase. A large positive S suggest an increasing disorder of the system when p-nitrobenzoic acid moved into the stationary phase, appearing as an adsorption process. A positive S may result from two aspects when p-nitrobenzoic acid is adsorbed onto the stationary phase. First, the degrees of freedom decreased for p-nitrobenzoic acid, this is a process that increased the order of the system. Second, a portion of water molecules adsorbed on the surface of stationary phases are replaced by p-nitrobenzoic acid and moved into the mobile phase, which greatly increases the disorder of the system. Furthermore, according to the retention variation tendency for p-nitrobenzoic acid when the chromatographic conditions change, the retention of p-nitrobenzoic acid is considered to be principally based on ion-exchange interactions. A negative H and positive S were observed for the retention process of benzoic acid, indicating an enthalpy entropy dual driven mechanism, hence, a mixed hydrophilic partitioning and adsorption mechanism is considered. Lastly, the values of H and S for methylparaben are meaningless because of the poor correlation coefficients derived
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Table 2 LSER coefficients of C18, APS-2 Hypersil, Sil-C4Im-C9Co and Sil-C9Im-C4Co. Stationary phases
c
e
s
a
b
v
d−
d+
n
r2
C18 APS-2 Hypersil Sil-C4Im-C9Co Sil-C9Im-C4Co
−0.683 −0.609 −0.852 −1.077
0.226 0.043 0.374 0.601
−0.484 0.350 0.432 0.589
−0.444 0.327 0.648 0.940
−0.377 0.950 0.382 0.239
0.766 −1.445 −1.212 −1.542
−0.619 1.552 1.558 1.724
0.035 −0.242 −0.154 −0.135
35 35 35 35
0.892 0.980 0.973 0.952
Fig. 7. Chromatogram of solutes under different temperature. Mobile phase: ACN /100 mM NH4 FA (90/10, v/v), flow rate: 0.6 mL min−1 , UV 254 nm. (1) pyridine, (2) melamine, (3) benzoic acid, (4) p-nitrobenzoic acid. Table 3 H and S for the retention process of analytes on Sil-C4Im-C9C and Sil-C9Im-C4Co. Sil-C4Im-C9Co Analytes Methylparaben Cytidine Adenosine Pyridine Melamine Benzoic acid p-Nitrobenzoic acid
H (kJ/mol) −0.24 −9.27 −10.11 −9.79 −9.37 −4.82 4.72
from the Van’t Hoff plot, the relative contribution of each of the partition and adsorption mechanisms may change when the temperature increased. 3.5. Application 3.5.1. Application under HILIC mode First, the prepared Sil-C9Im-C4Co and Sil-C4Im-C9Co were employed to separate carboxylic acids, nucleoside and bases under HILIC mode, and a commercial Acclaim Mixed-Mode WAX was used for comparison. As shown in Fig. 8, separations of seven carboxylic acids were achieved on prepared stationary phases in a shorter time when compared with Acclaim Mixed-Mode WAX. And the selectivity are different for Sil-C9Im-C4Co (˛4,5 = 1.59, ˛5,6 = 1.55, ˛6,7 = 1.19), Sil-C4Im-C9Co (˛4,5 = 1.42, ˛5,6 = 1.55, ˛6,7 = 1.30) and Acclaim Mixed-Mode WAX (˛4,5 = 3.60, ˛5,6 = 1.03, ˛6,7 = 1.41). Six nucleosides and bases were approximately baseline separated on three stationary phases, but peak orders are different. Adenine is
Sil-C9Im-C4Co S (J/mol K) −2.44 −18.42 −16.85 −35.94 −19.24 6.22 39.09
H (kJ/mol) −8.38 −9.53 −10.92 −10.87 −9.67 −5.95 3.66
S(J/mol K) 29.26 −19.39 −20.01 −40.39 −21.07 2.04 36.50
more strongly retained on the prepared columns, and an approximate calculation of the interactions between adenine and the stationary phases was performed through the LSER coefficients obtained above. The results showed that dipole–dipole and − interactions were significantly stronger between adenine and the prepared stationary phases (data not shown). Hence, Sil-C9ImC4Co and Sil-C4Im-C9Co can provide comparable performance to Acclaim Mixed-Mode WAX under HILIC mode, however, with different selectivity. 3.5.2. Application under RPLC mode Alkylbenzenes and PAHs are the most common solutes used when researching the performances of stationary phases under RPLC mode. As shown in Fig. 9, Sil-C9Im-C4Co provide satisfactory performance in separating five alkylbenzenes and four PAHs. Sil-C4Im-C9Co showed weak hydrophobicity and fail to separate alkylbenzenes, however, four PAHs were separated. The difference between Sil-C9Im-C4Co and Sil-C4Im-C9Co under RPLC mode may
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Fig. 8. Separation of carboxylic acids, nucleoside and bases under HILIC mode. (a) ACN /66.6 mM NH4 FA buffer (80/20, v/v), pH 3.5, 0.6 mL min−1 , UV 240 nm; (b) ACN /66.6 mM NH4 FA buffer (87/13, v/v), pH 3.5, 0.6 mL min−1 , UV 240 nm; (1) ibuprofen, (2) p-aminobenzoic acid, (3) trans-cinnamic acid, (4) Benzoic acid, (5) p-trifluoromethyl benzoic acid, (6) p-nitrobenzoic acid, (7) o-nitrobenzoic acid, (8) Uracil, (9) uridine, (10) cytosine, (11) adenine, (12) cytosine, (13) guanosine.
Fig. 9. Separation of alkylbenzenes and PAHs under RPLC mode. Mobile phase: ACN/H2 O (40/60, v/v) for Sil-C9Im-C4Co and Sil-C4Im-C9Co, ACN/H2 O (50/50, v/v) for Acclaim Mixed-Mode WAX, flow rate: 0.6 mL min−1 , column temperature:25 ◦ C (1) benzene, (2) toluene, (3) ethylbenzene, (4) propyl benzene, (5) butyl benzene, (6) naphthalene, (7) phenanthrene, (8) pyrene.
result from the different hydrophobicity of the prepared silane reagents and discriminatory bonded densities. Acclaim MixedMode WAX performed better than prepared stationary phases under RPLC mode.
3.5.3. Application under IEC mode The evaluation of Sil-C9Im-C4Co and Sil-C4Im-C9Co indicated that they can retain solutes through strong ion-exchange interactions, therefore, in this section, these stationary phases were
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indicate that the stationary phases developed in this work could be used to detect melamine in dairy products. 4. Conclusions
Fig. 10. Separation of inorganic anions under IEC. Mobile phase: ACN/5 mM Na2 SO4 solution (10/90, v/v) for Sil-C9Im-C4Co and SilC4Im-C9Co, ACN/50 mM pH 6.0 Na3 PO4 solution (50/50, v/v) for Acclaim MixedMode WAX, 0.5 mL min−1 , UV 210 nm; (1) BrO3 − , (2) NO2 − , (3) NO3 − , (4) IO3 − , (5) SCN− .
In this work, two IL silane reagents and two carboxylic reagents possessing alkyl chains of varying length were prepared via photoinitiated thio-ene chemistry and bonded to silica to construct two novel stationary phases, Sil-C9Im-C4Co and Sil-C4Im-C9Co. Both two columns can retain solutes through hydrophobic, hydrophilic, − stacking and ion-exchanged interactions, which could be influenced by chromatographic conditions. Sil-C9Im-C4Co and Sil-C4Im-C9Co provide different selectivity when compared to commercial RPLC and HILIC stationary phases. The alkyl chain length of silane reagents can influence the selectivity of stationary phases, as anion-exchange interaction is stronger on Sil-C9ImC4Co than Sil-C4Im-C9Co. Thermodynamic analysis indicated that hydrophilic solutes retain on new stationary phases through partition mechanism while carboxyl acids through mixed mechanisms of partition and adsorption. Furthermore, the prepared stationary phases successfully separated hydrophobic solutes, hydrophilic solutes and inorganic anions under mixed mode of RPLC, HILIC and IEC. Declaration of competing interest None. Acknowledgements This work was financially supported by theNational Natural Science Foundation of China (no. 21277110). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 460372. References
Fig. 11. Chromatograms of milk powder extract on Sil-C9Im-C4Co and Sil-C4ImC9Co. Mobile phase, ACN/66.6 mM NH4 FA buffer (90/10, v/v); pH 4.1, flow-rate, 0.6 mL min−1 ; detection wavelength, 215 nm.
employed to separate inorganic anions under IEC mode. As shown in Fig. 10, five inorganic anions were well separated on the prepared columns, while the anions were weakly retained on Acclaim Mixed-Mode WAX and poorly separated. Hence, the prepared stationary phases can provide excellent performance when employed in ion-exchange chromatography. 3.5.4. Detection of melamine in milk powder extract Melamine is a heterocyclic compound with a high nitrogen content. As a result of the deficiencies of traditional methods to quantify protein content in dairy products, melamine may be illegally added to increase the detected protein content. However, accumulated melamine in the human body may cause severe kidney failure. Therefore, new stationary phases have been developed to detect melamine in milk powder extract. As shown in Fig. 11, melamine is adequately retained on Sil-C9Im-C4Co and Sil-C4ImC9Co and is somewhat unaffected by other compounds. The results
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Preparation of two ionic liquid bonded stationary phases and comparative evaluation under mixed-mode of reversed phase/ hydrophilic interaction/ ion exchange chromatography Xiang Wang, Jingdong Peng ∗ , Huanjun Peng, Jun Chen, Hong Xian, Ranxi Ni, Shiyu Li, Dengying Long, Zhongying Zhang School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 9 April 2019 Received in revised form 9 July 2019 Accepted 14 July 2019 Available online xxx Keywords: Click chemistry Stationary phases Mixed-Mode Retention mechanism
a b s t r a c t The present work describes the preparation of two ionic liquid and carboxyl acid silane reagents via photo-initiated thiol-ene click chemistry that have been bonded to silica to afford two mixed-mode stationary phases (Sil-C4Im-C9Co and Sil-C9Im-C4Co). The two stationary phases provided satisfactory retention repeatability and efficiencies. The influence of acetonitrile content, salt concentration and pH of the mobile phase was investigated to clarify the retention properties of the prepared stationary phases. The results showed that the prepared Sil-C4Im-C9Co and Sil-C9Im-C4Co undergo multiple interactions with solutes under different chromatographic conditions. The retention mechanisms were further studied by the linear energy solvation relationship and Van’t Hoff plots. Finally, the stationary phases were employed to separate hydrophobic solutes (alkylbenzenes and polycyclic aromatic hydrocarbons) under reversed phase liquid chromatography (RPLC) mode, hydrophilic solutes (carboxylic acids, nucleosides and bases) under hydrophilic interaction liquid chromatography (HILIC) mode and inorganic anions under ion-exchange chromatography (IEC) mode, providing excellent performance and varying selectivity when compared with a commercial column. The bonding method in this work is feasible and the prepared stationary phases are promising when employed in RPLC/HILIC/IEC mixed-mode chromatography applications. © 2019 Published by Elsevier B.V.
1. Introduction Among the most employed separation modes for high performance liquid chromatography (HPLC), reversed phase liquid chromatography (RPLC), for its great performance in the separation of most organic compounds, still plays a dominating role [1]. Separations on RPLC are principally based on the varying hydrophobic interactions between solutes and stationary phases, with hydrophilic solutes being weakly retained and poorly separated. Hydrophilic interaction liquid chromatography (HILIC) has worked in tandem with RPLC and experienced a rapid development over the last few decades [2–4]. The interactions involved in HILIC appear to be more complex and may involve mixed partitioning and adsorption mechanisms [5,6]. With the increasing complexity of practical samples, ion-exchange chromatography (IEC) is
indispensable, especially for biological application [7,8]. Hence, the development of stationary phases for a wide range of applications remains a current topic of significant interest for chromatographic science. Mixed-mode chromatography (MMC) can achieve at least two separation modes on a single column [9]. MMC employs stationary phases modified by functional groups possessing different characteristics which provide multiple interactions with solutes. Numerous MMC stationary phases have been reported, including RPLC/HILIC [10,11], HILIC/IEC [12–14] and RPLC/HILIC/IEC [15–17]. A stationary phase modified with Lisoleucine and 4-phenylbutylamine has been reported to separate aromatic compounds of different polarities under RPLC mode and nucleotides/nucleosides under HILIC mode [10]. A 1,4-butanediol diglycidyl ether and dopamine grafted dendritic stationary phase
∗ Corresponding author at: School of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing, People’s Republic of China. E-mail address: [email protected] (J. Peng). https://doi.org/10.1016/j.chroma.2019.460372 0021-9673/© 2019 Published by Elsevier B.V.
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was synthesized, which demonstrated mixed RPLC, HILIC and IEC mechanisms, and was successful in separating alkylbenzenes, polycyclic aromatic hydrocarbons(PAHs), nucleosides and flavonoid, and some acidic and basic analytes under different modes [15]. A new column comprising graphene quantum dots bonded to silica showed satisfactory performances under RPLC, NPLC and HILIC modes, and a complex retention mechanism was observed under different modes [18]. Additionally, a stationary phase functionalized 2-methylimidazolium was used for HILIC and anion-exchange chromatography, and furthermore, this silica could be further modified to afford new stationary phases [19]. Ionic liquids (ILs) are a type of organic salt having low melting point, high thermal stability and negligible vapor pressure. ILs have been utilized in RPLC mobile phase to improve the retention of high polar solutes. As a result of the unique chemical and physical properties, ILs have been bonded to silica and employed as stationary phases for HPLC [20–23]. A series of ILs bearing different aliphatic chains were prepared and bonded to silica to determine their relative selectivity [24]. A monolithic column was prepared using pentafluorobenzyl imidazolium bromide ILs, and showed mixed mechanisms of hydrophobic interaction, − stacking, ion-exchange, electrostatic interaction and dipole–dipole interaction, enhancing better selectivity for halogenated compounds when compared with commercial C18 and pentafluorophenyl columns [25]. In this study, four silane reagents were synthesized via photoinitiated thiol-ene click chemistry and bonded to silica through one step bonded method. This may result in a higher yield of acylation and less undesirable interactions when compared with two step modification process [26]. Compared with existing IL based mixed mode stationary phases, we prepared two novel stationary phases in which both IL and carboxylic groups have been introduced. Furthermore, it has been investigated for the first time that how the performance of the column is influenced by the relative length of the IL and carboxylic silane reagents. The retention properties
were investigated as a function of the chromatographic conditions while the retention mechanisms were investigated by the linear solvation energy relationship and thermodynamic analysis. As for application, two prepared stationary phases were compared with a commercial column and used to separate hydrophobic alkylbenzenes and PAHs, hydrophilic carboxylic acids, nucleosides, bases and inorganic anions. 2. Experimental 2.1. Chemicals and materials The spherical porous silica (5 m particle size, 100 Å pore size, and 330 m2 g−1 surface area) was purchased from Fuji Silysia Chemical (Kasugai, Japan). 2, 2-Dimethoxy(DMPA), (3-Mercaptopropyl) 2-phenylacetophenone trimethoxysilane were purchased from Aladdin (Shanghai, China). Lithium Bis(trifluoromethanesulfonyl)imide (LiNTF2 ), 4bromobut-1-ene were obtained from Meryer Chemical Technology Co., Ltd. (Shanghai, China). 1-Methylimidazole, 9-bromonon-1ene, 9-decenoic acid and 4-pentenoic acid were purchased from Bide Pharmatech Ltd. (Shanghai, China). 2.2. Instrument and apparatus All chromatographic evaluations were performed using an Agilent 1100 series system with a G1311A quaternary pump, a G1379A on-line degasser, a G1316A column oven, a G1315B DAD detector and a 7725i manual injector with a sample loop of 20 L. The columns were packed using an RPL-ZD10 packing machine. The Ultimate C18 (150 × 4.6 mm, 5 m,) column was purchased from Welch materials, Inc., the APS-2 Hypersil (150 × 4.6 mm, 5 m,) column and Acclaim Mixed-Mode WAX (150 × 4.6 mm, 5 m) column were obtained from Thermo Fisher Scientific.
Fig. 1. Preparation route of Sil-C9Im-C4Co and Sil-C4Im-C9Co stationary phases.
Please cite this article in press as: X. Wang, et al., Preparation of two ionic liquid bonded stationary phases and comparative evaluation under mixed-mode of reversed phase/ hydrophilic interaction/ ion exchange chromatography, J. Chromatogr. A (xxxx), https://doi.org/10.1016/j.chroma.2019.460372
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C (%)
H (%)
N (%)
S (%)
Sil-C4Im-C9Co Sil-C9Im-C4Co
7.36 9.3
1.665 1.728
1.08 1.12
2.792 2.974
2.3. New stationary phases preparation 2.3.1. Preparation of four silane reagents The process of synthesizing four silane reagents is shown in Fig. 1 and described in detail in the supplementary information [27,28]. 2.3.2. One-step bonded silane reagents onto silica 6.0 g spherical silica was first activated by refluxing in 4 M HCl for 12 h, prior to being washed to pH 7 and dried under vacuum at 60 ◦ C overnight. 2.5 g activated silica was dispersed in 30 mL anhydrous dioxane, 2.0 mmol IL silane and 2.0 mmol carboxylic acid silane were added according to the configurations in Fig. 1. The mixture was refluxed under a N2 atmosphere for 24 h, and the obtained materials were washed by CH3 OH and H2 O respectively to remove unreacted reagents, dried under vacuum at 45 ◦ C overnight. The functionalized silica was slurry-packed into stainless steel columns (4.6 mm i.d. × 150 mm length). 2.4. Preparation of real sample-milk powder extract 1.0 g milk powder was dispersed in 10mLof formic acid solution (1%v/v), subjected to ultra-sonication for 30 min. Therefore, 40 mL of ACN was added to precipitate the protein, with the resulting suspension centrifuged at 10,000 r/min for 15 min, and the supernatant collected and filtered through a 0.22 m film for chromatographic analysis. 3. Results and discussion 3.1. Preparation and characterization of two stationary phases (Sil-C4Im-C9Co and Sil-C9Im-C4Co) Two stationary phases were modified using IL and carboxylic acid silane reagents having differing alkyl chain lengths. According to the structures, the stationary phases were named as SilC4Im-C9Co and Sil-C9Im-C4Co. The two stationary phases were characterized by element analysis, with the results presented in Table 1. The bonding densities of four silane reagents are
Fig. 2. Effect of ACN content on retention.
3
calculated according to the carbon and nitrogen contents, provided that all the methoxyl groups have reacted or hydrolyzed. In Sil-C4Im-C9Co, short IL silane reagent cover the silica by the density of 0.257 mmol g−1 , long carboxylic acid silane reagent by 0.215 mmol g−1 , in Sil-C9Im-C4Co, long IL silane reagent by 0.267 mmol g−1 , short carboxylic acid silane reagent by 0.368 mmol g−1 . Lower bonding densities were found for long silane reagents on both two stationary phases, which may result from the steric hindrance effect. Furthermore, thermogravimetric analysis was used to prove the successful fabrication of the new stationary phases and to investigate their thermostability. As shown in Fig. S1, apart from the weight loss associated with absorbed water, a mass loss of >20% was observed in both Sil-C9Im-C4Co and Sil-C4Im-C9Co as the temperature was increased to 700 ◦ C, and both stationary phases demonstrated good stability ≤300 ◦ C. 3.2. Influence of the chromatographic conditions on retention The retention of solutes is greatly affected by chromatographic conditions employed, such as solvent composition, pH and salt concentration in the mobile phase, as these conditions determine the interactions between solutes and the selected stationary phase. Hence, investigation into the effects of chromatographic conditions on retention may elucidate the retention mechanism and properties of the stationary phase.
3.2.1. Effect of ACN content In this study, ACN content was varied from 95 to 25%, as shown in Fig. 2. With decreasing ACN content in the mobile phase, the hydrophilic solutes (cytidine and melamine) retention decreased obviously on Sil-C9Im-C4Co and Sil-C4Im-C9Co, displaying typical HILIC retention characteristics. Conversely, hydrophobic solutes (toluene and methylparaben) were more strongly retained when the ACN content decreased, exhibiting typical RPLC retention characteristics, more so for Sil-C9Im-C4Co. Interestingly, the retention of two carboxylic acids (benzoic acid and salicylic acid) showed a U-shaped curve when the ACN content changed, demonstrating a HILIC mode at an ACN content ≥85% and a RPLC mode at an ACN content <85%. In conclusion, both columns exhibit mixed-mode of HILIC and RPLC. Hydrophilic and hydrophobic interactions may form between solutes and synthesized stationary phases, and such interactions may vary in their dominance according to the solvent composition.
Mobile phase: 10 mM NH4FA, pH 5.5, flow rate:0.6 mL min−1 , column temperature: 25 ◦ C.
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Fig. 3. Effect of salt concentration on retention.
Mobile phase: ACN/NH4 FA buffer (90/10, v/v), flow rate: 0.6 mL min−1 , pH 5.5, column temperature: 25 ◦ C.
3.2.2. Effect of salt concentration An appropriate salt concentration is vital for HILIC separation, and herein, retention was studied as a function of varying the NH4 FA salt concentration from 0 to 20 mM. As shown in Fig. 3, with increasing of salt concentration, solutes of varying properties showed diverse retention trends. The retention of melamine increased, while the opposite trend was observed for methylparaben, which can be explained by the increased hydrophilic nature of the stationary phase as a result of the thickness of the absorbed water layer increasing on the stationary phases. Benzoic acid was strongly retained on both stationary phases in ˜ h), and experienced a sharp decrease the absence of the salt (1.5 when the salt concentration increased, especially in the range of 0–10 mM. The increase of salt concentration is thought to greatly suppress the ion-exchange interaction between the stationary phases and benzoic acid. Adenosine and cytidine show a slight decrease in retention when the salt concentration increased from 0 to 5 mM, and an increase in retention upon further increasing the salt concentration. The aforementioned two reasons may explain this observed trend, that is, variation of salt concentration may influence retention by two mechanisms, suppressing the
Fig. 4. Effect of pH on retention.
ion-exchange interaction and strengthening the hydrophilic partition. 3.2.3. Effect of pH To study the effect of pH on the retention properties of the prepared stationary phases, as shown in Fig. 4, the pH of the mobile phase was changed from 6.0 to 3.1. The retention of pyridine, melamine and nucleosides was little affected by pH, as the interactions between these solutes and the stationary phases were not changed. The observed retention changes of benzoic acid (pKa 4.12) and p-nitrobenzoic acid (pKa 3.42) showed opposite trends, since the pH affects the charge states of solutes and stationary phases. The degree of protonation for benzoic acid increases as pH decreased, therefore, the ion-exchange interactions and hydrophilic partitioning are greatly suppressed, resulting in a significant decrease in retention. For p-nitrobenzoic acid, as the pH decreased from 6.0 to 4.0, additional carboxyl and silanol groups on the stationary phases become protonated, while p-nitrobenzoic acid is not affected, hence, the weakened electrostatic repulsion resulted in stronger retention. As the pH is further decreased, protonation of
Mobil phase: ACN / 100 mM NH4 FA buffer solution (80/20, v/v), flow rate: 0.6 mL min−1 , column temperature: 30 ◦ C.
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Fig. 5. Column efficiency and retention repeatability of Sil-C4Im-C9Co and Sil-C9Im-C4Co. Mobile phase: ACN/100 mM NH4 FA (90/10, v/v), 0.6 mL min−1 , pH 5.5, UV 254 nm; (1) methylparaben (2) adenosine, (3) cytidine.
p-nitrobenzoic acid begins resulting in weakened retention, similar to benzoic acid. 3.3. Retention repeatability and efficiencies Satisfactory retention repeatability and efficiencies are a prerequisite for both qualitative and quantitative work. In this study, a mixture of methylparaben, adenosine and cytidine were injected and recorded, and repeated over 15 times. The relative standard deviation (RSD) for retention time, symmetry factors and plate numbers for each peak are shown in Fig. 5. Both columns presented satisfactory retention repeatability and column efficiencies with Sil-C9Im-C4Co outperforming Sil-C4Im-C9Co, which may relate to the bonding density and packing quality of the columns. 3.4. Investigation of the retention mechanism 3.4.1. Comparison of retention properties among different columns Various solutes were selected to research and compare the retention properties of different columns, including hydrophobic alkylbenzenes and PAHs, hydrophilic nucleosides and bases, carboxylic acids and amines. Typical RPLC stationary phase C18 and HILIC stationary phase APS-2 Hypersil were introduced to present further comparative information. The retention of hydrophobic alkylbenzenes and PAHs were not compared among the HILIC stationary phases because they are barely retained. As shown in Fig. 6, C18 and APS-2 Hypersil expectedly showed their opposing selectivity. In the presence of C18 column, hydrophobic alkylbenzenes and PAHs are more strongly retained when compared with the carboxylic acids and hydrophilic solutes, while the APS-2 Hypersil presented an opposite trend. C18 retains solutes mainly through hydrophobic interactions, whereas, APS2 Hypersil acts through hydrophilic partitioning. In comparisons between APS-2 Hypersil and the stationary phases prepared in this work, nucleosides and bases are evenly distributed on both sides of the first bisector, which means iso-elution, suggesting a similar hydrophilia but different selectivity. Furthermore, carboxylic acids and amines are more strongly retained on Sil-C4Im-C9Co and Sil-C9Im-C4Co, because of the presences of both anion-exchange sites (imidazole ring) and cation-exchange sites (carboxylic group) on the stationary phases. When compared Sil-C4Im-C9Co to
Sil-C9Im-C4Co, the points of nucleosides and bases located almost on the first bisector, however, the carboxylic acids are more strongly retained on Sil-C9Im-C4Co, while amines retention is more pronounced on Sil-C4Im-C9Co. This observation may be explained by the differences between their structures, since the imidazole ring is more accessible on Sil-C9Im-C4Co, and therefore, a stronger anion-exchange is expected than for Sil-C4Im-C9Co stationary phase. 3.4.2. Linear solvation energy relationship (LSER) LSER is a reliable method to investigate the retention mechanism and to compare the retention properties between various chromatographic systems [29–31], expressed as follows: log k = c + eE + sS + aA + bB + vV + d− D− + d+ D+ The capital letters are solute descriptors, c is a system constant, lowercase letters (e, s, a, b, v) are system descriptors that measure the magnitude of difference in the particular interactions between the stationary and mobile phases, corresponding to the excess polarizability contributions from n and electrons, dipolarity, hydrogen bond(HBD) donating ability, hydrogen bond accepting ability, and the hydrophobicity of a given chromatographic system respectively [32]. d− D− and d+ D+ were further introduced to describe the effect of ionization on retention [33]. The lowercase letters can be used to compare the selectivity between different stationary phases after setting the chromatographic condition [34]. In this study, the retentions of 35 solutes were measured under the same conditions as Fig. 6, LSER was fitted for four columns with the corresponding coefficients listed in Table 2. As expected, only the C18 column for RPLC possesses positive v, confirming the tendency to undergo hydrophobic interactions with solutes. Imidazole rings could interact with solutes through − and anion exchange interactions, therefore, positive e and d− were observed for both Sil-C4Im-C9Co and Sil-C9Im-C4Co. As the imidazole ring is more accessible in Sil-C9Im-C4Co, because of its longer alkyl chain, e and d- values are higher for Sil-C9Im-C4Co. Both the Sil-C4Im-C9Co and Sil-C9Im-C4Co stationary phases can provide additional polar groups than the C18 and APS-2 Hypersil columns, which promotes dipole–dipole interactions with the solutes, and therefore, the s coefficients is more positive and larger for Sil-C4Im-C9Co and SilC9Im-C4Co. As for hydrogen-bond interactions, coefficients a and b
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Fig. 6. Comparison of the retention selectivity of C18, APS-2 Hypersil, Sil-C9Im-C4Co and Sil-C4Im-C9Co. Mobile phase: ACN/100 mM NH4 FA (90/10, v/v), flow rate: 0.6 mL min−1 , column temperature: 25 ◦ C.
are negative for C18, because there are fewer hydrogen-bond acting sites present. Three hydrophilic stationary phases gave positive a and b of varying size, while two IL bonded stationary phases are more HBD basic (bigger a and smaller b) than APS-2 Hypersil. This may relate to the associated anion or absorbed water layer, because the mobile phase is less HBD basic than methylimidazolium Ntf2 -. 3.4.3. Thermodynamic analysis The Van’t Hoff plot is one of the most employed thermodynamic methods to investigate physicochemical process mechanisms [35,36], and changes in enthalpy and entropy can be calculated through the slope and intercept of the plot by the Van’t Hoff equation, expressed as: lnk = −
H S + + ln∅ RT R
R is the gas constant, ∅ represents the phase ratio (vs / vm ), H and S measure the change of enthalpy and entropy, respectively, of solutes transferring from the mobile phase to the stationary phase. As shown in Fig. 7, in accordance with previous works, increasing the temperature of the column from 10 to 50 ◦ C, the retention of solutes such as pyridine, melamine and benzoic acid showed continuous declination. The Van’t Hoff plots associated with additional solutes are presented in Fig. S2. Interestingly, p-nitrobenzoic acid showed an opposite trend, indicating that the retention mechanism may differ from other solutes. Apart from methylparaben, all solutes exhibit a Van’t Hoff plot with high correlation coefficients (R2 >0.97), therefore, no obvious
variations in the retention mechanism occurred in the temperature range studied. Furthermore, the H and S values of seven solutes were calculated, and are listed in Table 3, with different mechanisms observed for the solutes. Negative H and S values were observed for four solutes (cytidine, adenosine, pyridine and melamine), indicating an enthalpy-driven mechanism for their exothermic retention processes, the retention was assumed to be principally based on hydrophilic partition. Conversely, positive H and S values were observed for p-nitrobenzoic acid, indicating an entropy-driven process. A positive H value indicates a more energetically favorable system for p-nitrobenzoic acid to remain in the mobile phase. A large positive S suggest an increasing disorder of the system when p-nitrobenzoic acid moved into the stationary phase, appearing as an adsorption process. A positive S may result from two aspects when p-nitrobenzoic acid is adsorbed onto the stationary phase. First, the degrees of freedom decreased for p-nitrobenzoic acid, this is a process that increased the order of the system. Second, a portion of water molecules adsorbed on the surface of stationary phases are replaced by p-nitrobenzoic acid and moved into the mobile phase, which greatly increases the disorder of the system. Furthermore, according to the retention variation tendency for p-nitrobenzoic acid when the chromatographic conditions change, the retention of p-nitrobenzoic acid is considered to be principally based on ion-exchange interactions. A negative H and positive S were observed for the retention process of benzoic acid, indicating an enthalpy entropy dual driven mechanism, hence, a mixed hydrophilic partitioning and adsorption mechanism is considered. Lastly, the values of H and S for methylparaben are meaningless because of the poor correlation coefficients derived
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Table 2 LSER coefficients of C18, APS-2 Hypersil, Sil-C4Im-C9Co and Sil-C9Im-C4Co. Stationary phases
c
e
s
a
b
v
d−
d+
n
r2
C18 APS-2 Hypersil Sil-C4Im-C9Co Sil-C9Im-C4Co
−0.683 −0.609 −0.852 −1.077
0.226 0.043 0.374 0.601
−0.484 0.350 0.432 0.589
−0.444 0.327 0.648 0.940
−0.377 0.950 0.382 0.239
0.766 −1.445 −1.212 −1.542
−0.619 1.552 1.558 1.724
0.035 −0.242 −0.154 −0.135
35 35 35 35
0.892 0.980 0.973 0.952
Fig. 7. Chromatogram of solutes under different temperature. Mobile phase: ACN /100 mM NH4 FA (90/10, v/v), flow rate: 0.6 mL min−1 , UV 254 nm. (1) pyridine, (2) melamine, (3) benzoic acid, (4) p-nitrobenzoic acid. Table 3 H and S for the retention process of analytes on Sil-C4Im-C9C and Sil-C9Im-C4Co. Sil-C4Im-C9Co Analytes Methylparaben Cytidine Adenosine Pyridine Melamine Benzoic acid p-Nitrobenzoic acid
H (kJ/mol) −0.24 −9.27 −10.11 −9.79 −9.37 −4.82 4.72
from the Van’t Hoff plot, the relative contribution of each of the partition and adsorption mechanisms may change when the temperature increased. 3.5. Application 3.5.1. Application under HILIC mode First, the prepared Sil-C9Im-C4Co and Sil-C4Im-C9Co were employed to separate carboxylic acids, nucleoside and bases under HILIC mode, and a commercial Acclaim Mixed-Mode WAX was used for comparison. As shown in Fig. 8, separations of seven carboxylic acids were achieved on prepared stationary phases in a shorter time when compared with Acclaim Mixed-Mode WAX. And the selectivity are different for Sil-C9Im-C4Co (˛4,5 = 1.59, ˛5,6 = 1.55, ˛6,7 = 1.19), Sil-C4Im-C9Co (˛4,5 = 1.42, ˛5,6 = 1.55, ˛6,7 = 1.30) and Acclaim Mixed-Mode WAX (˛4,5 = 3.60, ˛5,6 = 1.03, ˛6,7 = 1.41). Six nucleosides and bases were approximately baseline separated on three stationary phases, but peak orders are different. Adenine is
Sil-C9Im-C4Co S (J/mol K) −2.44 −18.42 −16.85 −35.94 −19.24 6.22 39.09
H (kJ/mol) −8.38 −9.53 −10.92 −10.87 −9.67 −5.95 3.66
S(J/mol K) 29.26 −19.39 −20.01 −40.39 −21.07 2.04 36.50
more strongly retained on the prepared columns, and an approximate calculation of the interactions between adenine and the stationary phases was performed through the LSER coefficients obtained above. The results showed that dipole–dipole and − interactions were significantly stronger between adenine and the prepared stationary phases (data not shown). Hence, Sil-C9ImC4Co and Sil-C4Im-C9Co can provide comparable performance to Acclaim Mixed-Mode WAX under HILIC mode, however, with different selectivity. 3.5.2. Application under RPLC mode Alkylbenzenes and PAHs are the most common solutes used when researching the performances of stationary phases under RPLC mode. As shown in Fig. 9, Sil-C9Im-C4Co provide satisfactory performance in separating five alkylbenzenes and four PAHs. Sil-C4Im-C9Co showed weak hydrophobicity and fail to separate alkylbenzenes, however, four PAHs were separated. The difference between Sil-C9Im-C4Co and Sil-C4Im-C9Co under RPLC mode may
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Fig. 8. Separation of carboxylic acids, nucleoside and bases under HILIC mode. (a) ACN /66.6 mM NH4 FA buffer (80/20, v/v), pH 3.5, 0.6 mL min−1 , UV 240 nm; (b) ACN /66.6 mM NH4 FA buffer (87/13, v/v), pH 3.5, 0.6 mL min−1 , UV 240 nm; (1) ibuprofen, (2) p-aminobenzoic acid, (3) trans-cinnamic acid, (4) Benzoic acid, (5) p-trifluoromethyl benzoic acid, (6) p-nitrobenzoic acid, (7) o-nitrobenzoic acid, (8) Uracil, (9) uridine, (10) cytosine, (11) adenine, (12) cytosine, (13) guanosine.
Fig. 9. Separation of alkylbenzenes and PAHs under RPLC mode. Mobile phase: ACN/H2 O (40/60, v/v) for Sil-C9Im-C4Co and Sil-C4Im-C9Co, ACN/H2 O (50/50, v/v) for Acclaim Mixed-Mode WAX, flow rate: 0.6 mL min−1 , column temperature:25 ◦ C (1) benzene, (2) toluene, (3) ethylbenzene, (4) propyl benzene, (5) butyl benzene, (6) naphthalene, (7) phenanthrene, (8) pyrene.
result from the different hydrophobicity of the prepared silane reagents and discriminatory bonded densities. Acclaim MixedMode WAX performed better than prepared stationary phases under RPLC mode.
3.5.3. Application under IEC mode The evaluation of Sil-C9Im-C4Co and Sil-C4Im-C9Co indicated that they can retain solutes through strong ion-exchange interactions, therefore, in this section, these stationary phases were
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indicate that the stationary phases developed in this work could be used to detect melamine in dairy products. 4. Conclusions
Fig. 10. Separation of inorganic anions under IEC. Mobile phase: ACN/5 mM Na2 SO4 solution (10/90, v/v) for Sil-C9Im-C4Co and SilC4Im-C9Co, ACN/50 mM pH 6.0 Na3 PO4 solution (50/50, v/v) for Acclaim MixedMode WAX, 0.5 mL min−1 , UV 210 nm; (1) BrO3 − , (2) NO2 − , (3) NO3 − , (4) IO3 − , (5) SCN− .
In this work, two IL silane reagents and two carboxylic reagents possessing alkyl chains of varying length were prepared via photoinitiated thio-ene chemistry and bonded to silica to construct two novel stationary phases, Sil-C9Im-C4Co and Sil-C4Im-C9Co. Both two columns can retain solutes through hydrophobic, hydrophilic, − stacking and ion-exchanged interactions, which could be influenced by chromatographic conditions. Sil-C9Im-C4Co and Sil-C4Im-C9Co provide different selectivity when compared to commercial RPLC and HILIC stationary phases. The alkyl chain length of silane reagents can influence the selectivity of stationary phases, as anion-exchange interaction is stronger on Sil-C9ImC4Co than Sil-C4Im-C9Co. Thermodynamic analysis indicated that hydrophilic solutes retain on new stationary phases through partition mechanism while carboxyl acids through mixed mechanisms of partition and adsorption. Furthermore, the prepared stationary phases successfully separated hydrophobic solutes, hydrophilic solutes and inorganic anions under mixed mode of RPLC, HILIC and IEC. Declaration of competing interest None. Acknowledgements This work was financially supported by theNational Natural Science Foundation of China (no. 21277110). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 460372. References
Fig. 11. Chromatograms of milk powder extract on Sil-C9Im-C4Co and Sil-C4ImC9Co. Mobile phase, ACN/66.6 mM NH4 FA buffer (90/10, v/v); pH 4.1, flow-rate, 0.6 mL min−1 ; detection wavelength, 215 nm.
employed to separate inorganic anions under IEC mode. As shown in Fig. 10, five inorganic anions were well separated on the prepared columns, while the anions were weakly retained on Acclaim Mixed-Mode WAX and poorly separated. Hence, the prepared stationary phases can provide excellent performance when employed in ion-exchange chromatography. 3.5.4. Detection of melamine in milk powder extract Melamine is a heterocyclic compound with a high nitrogen content. As a result of the deficiencies of traditional methods to quantify protein content in dairy products, melamine may be illegally added to increase the detected protein content. However, accumulated melamine in the human body may cause severe kidney failure. Therefore, new stationary phases have been developed to detect melamine in milk powder extract. As shown in Fig. 11, melamine is adequately retained on Sil-C9Im-C4Co and Sil-C4ImC9Co and is somewhat unaffected by other compounds. The results
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