Preparation, Characterization, and Chromatographic Evaluation of Nickel Oxide Deposited Silica Stationary Phase in Hydrophilic Interaction Liquid Chromatography

Nickel oxide deposited silica stationary phase was prepared by liquid phase deposition and characterized. Its chromatographic performance was evaluated using several compounds such as typical polar compounds and benzimidazoles as probes. The effects of mobile phase variables such as acetonitrile content, salt concentration, and pH on the chromatographic behavior, thermodynamics, and kinetics of these compounds were investigated to reveal the retention mechanism. The results showed that the prepared stationary phase exhibited a retention behavior of hydrophilic interaction liquid chromatography, and that multiple retention mechanisms including partitioning, adsorption, ion-exchange, electrostatic attraction, and coordination interactions contributed to solute retention. The coordination of nickel oxide electron-accepting sites and the electron-donating solutes resulted in the mixed-mode retention on stationary phase, which could be very useful for enhancing the chromatographic selectivity for the analytes. The batch-to-batch reproducibility was acceptable with the relative standard deviations of probe retention of less than 9.89%. The prepared nickel oxide deposited silica stationary phase was successfully employed for the separation of several compounds, and it showed better separation effect and different selectivity from silica column and commercial Zorbax NH2 column.


Introduction
High-performance liquid chromatography (HPLC) is widely used in many fields such as food [1], pharmaceutics [2], biology [3], and environment [4]. Since the stationary phase is the key component of HPLC, it is essential to develop a novel stationary phase.
Typical HPLC stationary phases are often prepared from silica due to the properties of porous silica spheres, such as large specific surface area, large pore volume, uniform particle size, narrow pore size distribution, and favorable pore connectivity [5][6][7]. Metal oxides exhibit good chemical and thermal stability with some special adsorption sites on their surfaces, including Bronsted acid site, Bronsted base site, and Lewis acid site [8][9][10]. Therefore, some metal oxidebased stationary phases have been developed and used for normal-phase, reversed-phase, ion exchange, and affinity chromatography [11]. However, their undesirable surface and pore properties limit their wide application in chromatography. Some metal oxide-coated silica stationary phases such as Al 2 O 3 /SiO 2 [12], TiO 2 /SiO 2 [13], and ZrO 2 /SiO 2 [10] have been developed, and they exhibit the chromatographic performances different from traditional SiO 2 . They incorporate the advantages of silica with unique chemical properties of metal oxides, and show substantial potential to separate polar compounds and hydrophilic compounds [14].
The polar and hydrophilic compounds are poorly retained, and they cannot be well separated in commonly used reverse-phase liquid chromatography (RPLC) [15]. In recent years, hydrophilic interaction liquid chromatography (HILIC) has been widely employed, and it shows excellent separation ability for polar compounds with good solubility in mobile phase and high compatibility with MS [16,17]. Several metal oxides such as TiO 2 and ZrO 2 have been used for separating a variety of polar compounds, and their retention mechanism has been investigated in HILIC mode [18,19]. Zhou et al. investigated the effect of mobile phase composition on analyte retention and separation on TiO 2 column and found the existence of ligand-exchange and HILIC interactions, additionally, fifteen nucleotides and their intermediates were well separated on the TiO 2 column within 26 min [20]. Kucera et al. studied the retention behavior of some organic acids on the zirconia column in HILIC mode and found that the analytes were primarily retained on the zirconia column by surface adsorption and on the silica column by partitioning [21]. Later, three polar metabolites were separated using zirconia columns in HILIC mode [22]. Therefore, the retention in the metal oxides stationary phase has been confirmed to be a multimodal process in HILIC mode. The mixed-mode HILIC stationary phase can provide better separation selectivity for a wide range of compounds on a single column. However, little attention has been paid to the application of metal oxide-coated silica in HILIC mode.
Recently, nickel oxide (NiO) has been used for sample preparation [23,24]. NiO-related materials have been confirmed to have a strong affinity to polyhistidine, and have been applied to the purification of histidine-labeled proteins [25,26]. In our previous research, NiO-deposited silica is prepared by liquid phase deposition (LPD) and used as a solid-phase extraction adsorbent for the extraction of some compounds with imidazole groups such as benzimidazoles (BZDs) [27,28], which is mainly based on the coordination interaction between the empty orbit of Ni (II) in NiO and the electron-donating group of imidazole. Therefore, nickel oxide-deposited silica (NiO@ SiO 2 ) is hydrophilic, ionizable, and electron-acceptable, and thus NiO@SiO 2 is expected to be used as HILIC stationary phase for the separation of diverse compounds, especially hydrophilic molecules. However, few reports on chromatographic separation using NiO-related materials are available.
In this study, NiO@SiO 2 stationary phase was prepared by depositing NiO on the surface of silica using LPD and characterized. Then, the chromatographic separation performance of NiO@SiO 2 was evaluated and compared with that of bare silica and commercial Zorbax NH 2 HILIC columns.

Apparatus
High-performance liquid chromatography (Shimadzu) was equipped with LC-20AD binary pumps, SPD-20A variable wavelength UV-visible detector, SIL-20AC auto-sampler, DGU-20A5 degasser, and CTO-20A column oven. The data processing was performed by software LabSolutions. The specific surface and pore size of the stationary phase was measured with a specific surface area analyzer (Beijing JWGB Sci. & Tech. Co., Ltd., Beijing, China) using the Brunauer-Emmett-Teller and Barrett-Joyner-Halenda equations. The surface morphology and energy spectra of the materials were analyzed using Field Emission Scanning Electron Microscope (FE-SEM, Zeiss SIGMA) and its attached energy dispersive spectrum (EDS, Oxford Ultim-Max 40). Zeta potential was measured by Malvern ZS Zeta-Sizer Nano. The mobile phase was filtered and sonicated before chromatographic analysis.

Preparation of Nickel Oxide Deposited Silica Stationary Phase
NiO@SiO 2 stationary phase was prepared by LPD [27] and the preparation scheme is shown in Fig. S1. Prior to use, spherical SiO 2 were activated by HCl /H 2 O (1:1, v/v), and then washed with deionized water to neutral and dried. The saturated NiF 2 solution and the 0.6 mol·L −1 H 3 BO 3 solution were mixed thoroughly at a ratio of 3/5 (v/v) at room temperature to prepare the precursor solution for LPD. Subsequently, activated silica (2.0 g) was added to the precursor solution (80 mL) and the mixture was continuously shaken (200 rpm) at 40 ℃ for 24 h. The composite was filtered, washed with deionized water 3 times, and dried at 120 °C. The above-mentioned LPD steps were repeated 3 times to obtain NiO@SiO 2 . NiO@SiO 2 and SiO 2 were simultaneously annealed at 200 °C for 1 h. The temperature was raised from room temperature (25 °C) to 200 °C at a rate of 1 °C·min −1 and maintained at 200 °C for 1 h.

Column Packing
The NiO@SiO 2 stationary phase and SiO 2 stationary phase were packed by slurry method, respectively. Two grams (2.0 g) of stationary phase (NiO@SiO 2 or SiO 2 ) was dispersed in 10 mL of isopropanol by sonicating for 1.0 min. The slurry was then packed into a stainless-steel column (150 mm × 4.6 mm i.d.) under a constant pressure of 40 MPa for 15 min with methanol as the pushing solvent. The two chromatographic columns were flushed with methanol at a flow rate of 0.5 mL·min −1 for 24 h before subsequent experiments.

Characterization of Nickel Oxide Deposited Silica Stationary Phase
Firstly, the morphological structures of NiO@SiO 2 and SiO 2 were compared by FE-SEM. As shown in Fig. 1a, the nickel oxide nanoparticles were uniformly deposited on the silica surface. Figure 1b were EDS mappings of SiO 2 and NiO@ SiO 2 . It can be seen that SiO 2 contained only Si and O elements, while the Ni element was detected on the surface of NiO@SiO 2 microspheres after LPD. The image comparison of two materials demonstrated that NiO@SiO 2 stationary phase was successfully prepared. N 2 adsorption analysis was performed to reveal the changes in the surface area and pore structure of NiO@SiO 2 . As can be seen from Table 1, the specific surface area of NiO@SiO 2 was 354 m 2 ·g −1 , which was significantly higher than that of SiO 2 (180 m 2 ·g −1 ). However, the average pore volume and pore size of NiO@SiO 2 were lower than those of the SiO 2 . In addition, NiO@SiO 2 had a uniform pore structure after LPD (Fig. S2).

Investigation of Retention Behavior
In this section, the retention behavior was evaluated by investigating the influence of some chromatographic parameters on retention, including ACN content, pH, salt concentration in the mobile phase and column temperature and flow rates using 15 compounds as probes.

Influence of ACN Content
In the mobile phases with different ACN contents (70%, 80%, 90%, 92%, and 95%), the retentions of 15 compounds on NiO@SiO 2 and SiO 2 stationary phases were investigated. The results are shown in Fig. 2a and b. The retentions of analytes on the two stationary phases became stronger with the increase of the ACN content in the mobile phase, and when the ACN content in the mobile phase was more than 90%, the retentions of analytes on the two stationary phases were significantly enhanced, indicating a typical HILIC mode. Moreover, the retention of analytes on SiO 2 was much weaker than that on NiO@SiO 2 under high ACN content conditions. The presence of a benzene ring in the BZDs structure resulted in its weaker hydrophilic properties than nucleosides, bases, and other polar analytes (Table S1 LogP). However, the retention of BZDs on NiO@SiO 2 stationary phase was significantly enhanced in HILIC mode. These results demonstrated that the BZDs were retained on the NiO@SiO 2 stationary phase through coordination interaction besides the hydrophilic interaction. Similarly, pyrimidines also showed coordination interaction with NiO@ SiO 2 . For example, the retention of cytosine was the strongest among the three pyrimidine analytes since the cytosine structure had more nitrogen-containing groups. Therefore, their retentions on the NiO@SiO 2 were not merely based on the hydrophilic interaction in HILIC mode.
At present, both partitioning and adsorption models are often used to explore the retention mechanism of HILIC, which was described by Eqs. (1) and (2), respectively [29,30].
where k is the retention factor of the analytes; k w and k B are the assumed retention factors when pure ACN and pure water are used as the mobile phase, respectively; φ and X B are the volume fraction and mole fraction of water in the mobile phase, respectively; n is the cross-sectional area occupied by the solute molecule on the surface of the stationary phase water layer.
Based on the above two empirical equations, linear regression was performed to reveal the relationship between the retention of analytes and the water content in the mobile phase. The R 2 (coefficient of determination) of analytes fitted for Eqs. (1) and (2) is shown in Table S2. The R 2 2 of most analytes was larger than R 1 2 on SiO 2 stationary phase. However, the result of theobromine was different from most analytes (R 1 2 > R 2 2 ), which indicated the partitioning and adsorption participated simultaneously in retaining the analytes on SiO 2 . The conclusion corresponded to previous studies on the retention mechanism of SiO 2 in HILIC [19]. Likewise, the R 2 2 of analytes on NiO@SiO 2 did not always exceed R 1 2 . Analytes on NiO@SiO 2 were also affected by both partitioning and adsorption interactions.

Influence of pH
The pH in the mobile phase affects the charge state of analytes and stationary phase, which in turn affects the retention of analytes on stationary phase. The change of k (retention factor) for analytes is shown in Fig. 2c and d. At the selected pH (6, 7, and 8), analytes were protonated and positively charged. The zeta potential of NiO@ SiO 2 is measured and shown in Fig. S3. The isoelectric point of NiO@SiO 2 stationary phase was around 6, while that of SiO 2 was generally around 3 [31]. The surfaces of two stationary phases were negatively charged under the experiment pH conditions, thus these two stationary phases could generate electrostatic attraction with positively charged analytes. When pH increased, the retention of analytes on the two stationary phases changed little. This was because negative charge on the surface of two stationary phases increased, while positive charge of analytes reduced. Therefore, the number of total charges in the system hardly fluctuated leading to electrostatic attraction interaction almost unchanged. It is not clear in the testing of the effect of pH in the mobile phase on retention if this was the effect of the aqueous part of the mobile phase or the whole mobile phase. On the whole, pH had little effect on the retention of analytes on SiO 2 and NiO@SiO 2 .

Influence of Salt Concentration
Five concentrations (5, 10, 15, 20, 25 mmol·L −1 ) of NH 4 Ac were added to mobile phase to explore the influence of ionic strength change on analyte retention. NH 4 Ac was chosen because it is a volatile salt, which is usually combined with ESI-MS. The theoretical model of the ion-exchange and hydrophilic mechanism can be expressed by Eq. (3) [32]: where k is the retention factor of the analytes; [C] is the concentration of NH 4 Ac; s and d are constant. The retention of analytes and the concentration of NH 4 Ac were fitted using Eq. (3) to investigate the interaction between analytes and stationary phase. The results are shown in Table 2. A good linear relationship (R 2 > 0.9703) was observed between logk and log[C] for the two stationary phases. The type of interaction between analytes and stationary phase could be judged by the slope of the fitting line. A slope close to 0 indicated that hydrophilic interaction On the contrary, ion-exchange played a major role while the slope was close to 1. Ion-exchange was not significant on SiO 2 and NiO@SiO 2 as the low slope is observed in Table 2. Rather the effect was more likely a subtle salt effect on the water layer thickness or surface adsorption. The hypothesis of subtle effect on the water layer was supported by uracil, which would not be charged under these conditions. As shown in Fig. 2e and f, the retention changes curve of analytes on SiO 2 remained flat. The result was consistent with previous work that addition of low concentrations of buffer solution caused only small increase in the retention of the water layer [33]. Differently, k of analytes declined with the increasing NH 4 Ac concentration on NiO@SiO 2 . As previously mentioned, shielded electrostatic attraction interaction, occupied coordination site or weakened hydrophilic interaction would be the reason for retention reduction.

Thermodynamics
For exploring the retention behavior of the analytes on two stationary phases from the perspective of thermodynamics, four column temperatures including 35 °C, 45 °C, 55 °C, and 65 °C were selected. Commonly, the relationship between retention factor for analytes and column temperature can be depicted by van't Hoff equation (Eq. (4)), as follows: where k is the retention factor of the analytes; ∆H Θ and ∆S Θ are the standard enthalpy change and entropy change in the process of solute transfer from the mobile phase to the stationary phase, respectively; R is the molar gas constant  Table 3. A linear relationship between lnk and 1/T was observed on the two stationary phases (R 2 > 0.9439). This suggested that standard enthalpy change and entropy change were not varied over the investigated temperature range studied. The standard enthalpy change and entropy change could be calculated from the slope and intercept of line, respectively. The parameters are also listed in Table 3. As seen from the result, all the analytes showed negative ∆H Θ on the SiO 2 , which indicated that the progress of analytes transferring from mobile phase to stationary phase was exothermic and lower temperature benefitted for increased retention. Two positive ∆H Θ results were observed for albendazole sulfoxide and fenbendazole sulfoxide on the NiO@SiO 2 , which showed endothermic progress. Although positive ∆H Θ could not promote analytes to be retained on stationary phase, all the ∆G Θ of analytes were negative (which meant that the progress of analytes from mobile phase to stationary phase was spontaneous). This was because the loss of ∆H Θ was compensated by ∆S Θ leading to enhanced retention on the NiO@SiO 2 [34].

Kinetics
Four analytes (theophylline, fenbendazole sulfoxide, 2′-deoxyadenosine, and thymine) were selected as probes to investigate their chromatographic kinetics on NiO@ SiO 2 stationary phase. As shown in Fig. 3, theophylline, 2′-deoxyadenosine, and thymine had the lowest plate height (H) when the linear velocity was about 0.14 mm·s −1 . Their lowest H were 43 μm, 54 μm, and 55 μm with the corresponding theoretical plate numbers of 3488 m −1 , 2777 m −1 , and 2727 m −1 , respectively. When the linear velocity was The H of four analytes increased with the increase of flow rate when the flow rate was bigger than the optimum flow rate. Obviously, fenbendazole sulfoxide was more sensitive to flow rate than the other three analytes. When the actual flow rate was 1 mL·min −1 , the mass transfer resistance was the main factor that caused the chromatographic peak to broaden.

Stability and Reproducibility
Four analytes (fenbendazole sulfoxide, theophylline, thymine, and 2'-deoxyadenosine) were selected to investigate the stability and reproducibility of the NiO@SiO 2 column and the same chromatographic conditions were used for the tests. The retention time of four analytes on NiO@SiO 2 column was continuously monitored within one month (at least 6000 column volumes). The relative standard deviations (RSDs) of retention factor for four analytes were 1.37%, 0.72%, 0.51%, and 0.30%. The stability of NiO@SiO 2 stationary phase was within the acceptable range. The reproducibility of three batches of NiO@SiO 2 columns prepared by the same method was investigated under the same chromatographic conditions with the above four analytes as probes. As shown in Table S3, the RSDs of the retention factor of different batches were less than 9.89%, indicating the proposed material was reproducible.

Evaluation of Separation Performance
To investigate the potential of NiO@SiO 2 stationary phase to separate hydrophilic compounds and electron-donating compounds, comparative analyses of two typical HILIC columns including silica and commercial Zorbax NH 2 columns were performed under the same chromatographic conditions with five representative mixtures used as probes.

Separation of Imidazole Compounds
As mentioned above, NiO@SiO 2 had strong retention for imidazole compounds at a high level of ACN. Thus, four imidazole compounds were used to evaluate the separation performance of NiO@SiO 2 . As shown in Fig. 4a, four imidazole compounds were well separated within 5 min. Meanwhile, compared with silica column and Zorbax NH 2 column, the retention of four imidazole compounds on NiO@ SiO 2 column was enhanced which might be attributed to the coordination interaction between Ni in NiO@SiO 2 and imidazole group.

Separation of Hydrophobic and Hydrophilic Compounds
The performance of NiO@SiO 2 column was further evaluated with the mixture including both hydrophobic (fenbendazole sulfoxide and gliclazide) and hydrophilic compounds (uracil, 2′-deoxythymidine, and 2′-deoxyadenosine) (Fig. 4b). The retention of hydrophilic compounds (peak 1 and 2) was stronger than that of hydrophobic compounds (peak 3, 4, and 5) on NiO@SiO 2 column. The elution order was not completely determined by the hydrophilicity of analytes, which might be due to the multimodal retention mechanisms of NiO@SiO 2 column. Compared with silica column and commercial Zorbax NH 2 column, the NiO@SiO 2 column exhibited excellent selectivity for the five analytes under similar mobile phase conditions. Therefore, NiO@SiO 2 column had the potential to be used for simultaneous separation of both hydrophilic and hydrophobic compounds.

Separation of Clinical Drugs
The separation selectivity of the developed NiO@SiO 2 column for lipophilic clinical drugs was investigated. Figure 4c showed that nicorandil (analyte 1) and glimepiride (analyte 3) co-eluted on the Zorbax NH 2 column, and that the other five analytes were barely retained on the silica column except ibuprofen (analyte 6). However, these lipophilic drugs were retained and were basically separated on the NiO@SiO 2 column, which might be ascribed to the formation of coordination interaction between electron-deficient Ni (II) and electron-rich compounds, thus enhancing the retention. These results suggested that NiO@SiO 2 was a promising stationary phase for the separation of clinical drugs.

Separation of Acid Compounds
Five acid compounds were used to further evaluate the separation performance of NiO@SiO 2 column. As shown in Fig. 4d, baseline separation of 5 acid compounds was completed within 10 min on NiO@SiO 2 column. In contrast, on the silica column, the five compounds failed to be separated, and they were eluted almost at a dead time. Different elution orders on three columns indicated that specific interactions between acids and different polar stationary phases could be responsible for the selectivity difference. For example, the hydrogen bond effect on Zorbax NH 2 column and coordination effect on NiO@SiO 2 column.

Separation of Alkaloid Compounds
Three alkaloid compounds were selected to further elucidate the coordination interaction between analytes and NiO@SiO 2 column (Fig. 4e). The alkaloid compounds were not separated on silica column, but they were partially separated on Zorbax NH 2 column under the same chromatographic conditions, which might be due to hydrogen bond interaction and ion-exchange interaction between the three alkaloids and Zorbax NH 2 column. As shown in Fig. 4e, three alkaloid compounds were better separated within 6 min on NiO@SiO 2 column than on abovementioned two columns. The elution order of NiO@SiO 2 column was different from that of silica column, which might be attributed to the coordination interaction between different nitrogen-containing groups and NiO@SiO 2 stationary phase.

Conclusion
In this study, a nickel oxide deposited silica (NiO@SiO 2 ) stationary phase was successfully prepared by liquid phase deposition method and applied for HILIC chromatography. The evaluation of its chromatographic performance in HILIC mode indicated that the prepared NiO@SiO 2 stationary phase possessed a typical hydrophilic retention mechanism and some multimodal interactions including partitioning, adsorption, ion-exchange, electrostatic attraction, and coordination interactions. Compared with silica and commercial Zorbax NH 2 columns, NiO@SiO 2 column exhibited the unique multimodal retention capabilities, a wider range of retention behaviors, and better selectivity for some hydrophobic and hydrophilic compounds. Therefore, NiO@SiO 2 had great potential to separate multiple compounds.