Characterization of nickel oxide deposited silica stationary phase
Firstly, the morphological structures of NiO@SiO2 and SiO2 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 SiO2 andNiO@SiO2. It can be seen that SiO2 contained only Si and O elements, while Ni element was detected on the surface of NiO@SiO2 microspheres after LPD. The image comparison of two materials demonstrated that NiO@SiO2 stationary phase was successfully prepared.
N2 adsorption analysis was performed to reveal the changes in the surface area and pore structure of NiO@SiO2. As can be seen from Table 1, the specific surface area of NiO@SiO2 was 354 m2·g− 1, which was higher significantly than that of SiO2 (180 m2·g− 1). However, the average pore volume and pore size of NiO@SiO2 were lower than those of the SiO2. In addition, NiO@SiO2 had a uniform pore structure after LPD (Fig. S2).
Table 1
Surface area, pore volume, and pore size of SiO2 and NiO@SiO2.
|
Specific surface area
(m2·g− 1)
|
Pore volume
(cm3·g− 1)
|
Pore size
(nm)
|
SiO2
|
180
|
1.43
|
24
|
NiO@SiO2
|
354
|
0.61
|
4
|
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@SiO2 and SiO2 stationary phases were investigated. The results were shown in Fig. 2a and 2b. 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 SiO2 was much weaker than that on NiO@SiO2 under high ACN content conditions. Especially, the presence of 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@SiO2 stationary phase was significantly enhanced in HILIC mode. These results demonstrated that the BZDs were retained on the NiO@SiO2 stationary phase through coordination interaction besides the hydrophilic interaction. Similarly, pyrimidines also showed coordination interaction with NiO@SiO2. 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@SiO2 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].
logk = logk w - φ (1)
logk = logk B - nlogXB (2)
Where k is the retention factor of the analytes; kw and kB are the assumed retention factors when pure ACN and pure water are used as the mobile phase, respectively; φ and XB 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 R2 (coefficient of determination) of analytes fitted for Eq. (1) and Eq. (2) was shown in Table S2. The R22 of most analytes was larger than R12 on SiO2 stationary phase. However, the result of theobromine was different from most analytes (R12 > R22), which indicated the partitioning and adsorption participated simultaneously in retaining the analytes on SiO2. The conclusion corresponded to previous studies on the retention mechanism of SiO2 in HILIC[19]. Likewise, the R22 of analytes on NiO@SiO2did not always exceed R12. Analytes on NiO@SiO2 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 was shown in Fig. 2c and 2d. At the selected pH (6, 7, and 8), analytes were protonated and positively charged. The zeta potential of NiO@SiO2 was measured and shown in Fig. S3. The isoelectric point of NiO@SiO2 stationary phase was around 6, while that of SiO2 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 mobile phase on retention if this was the effect of the aqueous part of mobile phase or the whole mobile phase. On the whole, pH had little effect on the retention of analytes on SiO2 and NiO@SiO2.
Influence Of Salt Concentration
Five concentrations (5, 10, 15, 20, 25 mmol·L− 1) of NH4Ac were added to mobile phase in order to explore the influence of ionic strength change on analyte retention. NH4Ac 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. (4)[32]:
logk = -slog[C] + d (4)
Where k is the retention factor of the analytes; [C] is the concentration of NH4Ac; s and d are constant.
The retention of analytes and the concentration of NH4Ac were fitted using Eq. (4) to investigate the interaction between analytes and stationary phase. The results were shown in Table 2. A good linear relationship (R2 > 0.9703) was observed between logk and log[C] for the two stationary phase. 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 dominated retention. On the contrary, ion-exchange played a major role while the slope was close to 1. Ion-exchange was not significant on SiO2 and NiO@SiO2 as the low slope was 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.
Table 2
Correlation coefficients and slopes for logk vs. log[C] on SiO2 and NiO@SiO2 stationary phases.
Analytes
|
SiO2
|
NiO@SiO2
|
Slope
|
y-Intercept
|
R2
|
Slope
|
y-Intercept
|
R2
|
4-nitroimidazole
|
-0.014
|
-0.884
|
0.9927
|
-0.045
|
-0.210
|
0.9928
|
Albendazole sulfone
|
-0.115
|
-0.841
|
0.9945
|
-0.088
|
-0.129
|
0.9989
|
Fenbendazole sulfone
|
0.027
|
-1.166
|
0.9995
|
-0.012
|
-0.779
|
0.9991
|
Albendazole sulfoxide
|
-0.018
|
-0.344
|
0.9948
|
-0.016
|
0.018
|
0.9989
|
Fenbendazole sulfoxide
|
0.035
|
-0.552
|
0.9994
|
-0.017
|
-0.192
|
0.9911
|
Caffeine
|
-0.025
|
-0.368
|
0.9902
|
-0.052
|
0.176
|
0.9989
|
Theobromine
|
-0.009
|
-0.270
|
0.9994
|
-0.049
|
0.305
|
0.9960
|
Theophylline
|
0.028
|
-0.386
|
0.9986
|
-0.115
|
0.214
|
0.9998
|
2'-deoxythymidine
|
0.004
|
-0.509
|
0.9703
|
-0.055
|
-0.080
|
0.9920
|
Adenosine
|
0.019
|
-0.215
|
0.9996
|
-0.035
|
0.196
|
0.9998
|
Uridine
|
0.056
|
-0.455
|
0.9992
|
-0.045
|
0.257
|
0.9926
|
Thymine
|
0.034
|
-0.523
|
0.9835
|
-0.047
|
0.077
|
0.9913
|
Uracil
|
0.045
|
-0.452
|
0.9997
|
-0.057
|
0.181
|
0.9968
|
As shown in Fig. 2e and 2f, the retention changes curve of analytes on SiO2 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 NH4Ac concentration on NiO@SiO2. 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. (5)), as follows:
$$lnk=-\frac{\varDelta {H}^{\varTheta }}{RT}+\frac{\varDelta {S}^{\varTheta }}{R}+ln\phi$$
5
Where k is the retention factor of the analytes; ∆HΘ and ∆SΘ 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 (8.314 J·(mol·K)−1); φ is the volume phase ratio of the system.
The result of fitting Eq. (5) was shown in Table 3. A linear relationship between lnk and 1/T was observed on the two stationary phases (R2 > 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 were also listed in Table 3. As seen from the result, all the analytes showed negative ∆HΘ on the SiO2, 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∆HΘresults were observed for albendazole sulfoxide and fenbendazole sulfoxide on the NiO@SiO2, which showed endothermic progress. Although positive ∆HΘcould not promote analytes to be retained on stationary phase, all the ∆GΘ 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 ∆HΘ was compensated by∆SΘ leading to enhanced retention on the NiO@SiO2[34].
Table 3
Parameters of Van’t Hoff equation on SiO2 and NiO@SiO2 stationary phases.
Analytes
|
SiO2
|
NiO@SiO2
|
∆HΘ
|
∆SΘ
|
∆GΘ
|
R2
|
∆HΘ
|
∆SΘ
|
∆GΘ
|
R2
|
Albendazole sulfone
|
-3.82
|
-21.72
|
2.11
|
0.9683
|
-6.07
|
-16.61
|
-1.53
|
0.9663
|
Fenbendazole sulfone
|
-1.78
|
-17.65
|
3.04
|
0.9439
|
-7.37
|
-21.35
|
-1.54
|
0.9874
|
Albendazole sulfoxide
|
-2.02
|
-3.60
|
-1.04
|
0.9547
|
8.30
|
36.43
|
-1.65
|
0.9635
|
Fenbendazole sulfoxide
|
-3.91
|
-13.74
|
-0.16
|
0.9791
|
8.59
|
32.02
|
-0.16
|
0.9860
|
Caffeine
|
-6.37
|
-18.48
|
-1.33
|
0.9968
|
-9.95
|
-22.18
|
-3.89
|
0.9961
|
Theobromine
|
-10.01
|
-41.51
|
1.33
|
0.9744
|
-8.98
|
-16.28
|
-4.54
|
0.9989
|
Theophylline
|
-4.18
|
-11.58
|
-1.01
|
0.9850
|
-7.96
|
-14.62
|
-3.97
|
0.9958
|
2'-deoxythymidine
|
-6.63
|
-23.11
|
-0.31
|
0.9831
|
-4.67
|
-11.27
|
-1.59
|
0.9811
|
2'-deoxyadenosine
|
-2.99
|
-4.57
|
-1.74
|
0.9668
|
-4.15
|
-4.04
|
-3.05
|
0.9977
|
Adenosine
|
-3.31
|
-6.23
|
-1.61
|
0.9624
|
-3.24
|
-0.38
|
-3.13
|
0.9808
|
Uridine
|
-6.68
|
-21.78
|
-0.73
|
0.9838
|
-4.60
|
-6.20
|
-2.90
|
0.9979
|
Thymine
|
-3.57
|
-12.00
|
-0.29
|
0.9868
|
-2.19
|
2.01
|
-2.74
|
0.9957
|
Uracil
|
-3.34
|
-9.92
|
-0.63
|
0.9812
|
-3.29
|
0.60
|
-3.45
|
0.9842
|
△H Θ ,△SΘ, and△GΘ are enthalpy, entropy and Gibbs free energy, respectively. △HΘ (kJ·mol− 1) and △SΘ (J·mol− 1·K) were calculated by the slope and intercept of the fit, respectively. △GΘ (kJ·mol− 1) = △HΘ-T△SΘ. The column phase ratio φ= (VW-VM)/VM, where VW was the geometric volume of the column, VM was the dead volume of the column.
Kinetics
Four analytes (theophylline, fenbendazole sulfoxide, 2'-deoxyadenosine, and thymine) were selected as probes to investigate their chromatographic kinetics on NiO@SiO2 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 about 0.07 mm·s− 1, the fenbendazole sulfoxide had the lowest H (54 µm) with a theoretical plate number of 2777 m− 1. 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@SiO2 column and the same chromatographic conditions were used for the tests. The retention time of four analytes on NiO@SiO2 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@SiO2 stationary phase was within the acceptable range.
The reproducibility of three batches of NiO@SiO2 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@SiO2 stationary phase to separate hydrophilic compounds and electron-donating compounds, comparative analyses of two typical HILIC columns including silica and commercial Zorbax NH2 columns were performed under the same chromatographic conditions with five representative mixtures used as probes.
Separation Of Imidazole Compounds
As mentioned above, NiO@SiO2had strong retention for imidazole compounds at a high level of ACN. Thus, four imidazole compounds were used to evaluate the separation performance of NiO@SiO2. As shown in Fig. 4a, four imidazole compounds were well separated within 5 min. Meanwhile, compared with silica column and Zorbax NH2 column, the retention of four imidazole compounds on NiO@SiO2 column was enhanced which might be attributed to the coordination interaction between Ni in NiO@SiO2and imidazole group.
Separation Of Hydrophobic And Hydrophilic Compounds
The performance of NiO@SiO2 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@SiO2 column. The elution order was not completely determined by the hydrophilicity of analytes, which might be due to the multimodal retention mechanisms of NiO@SiO2 column. Compared with silica column and commercial Zorbax NH2 column, the NiO@SiO2 column exhibited excellent selectivity for the five analytes under similar mobile phase conditions. Therefore, NiO@SiO2 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@SiO2 column for lipophilic clinical drugs was investigated. Figure 4c showed that nicorandil (analyte 1) and glimepiride (analyte 3) co-eluted on the Zorbax NH2 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@SiO2 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@SiO2 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@SiO2 column. As shown in Fig. 4d, baseline separation of 5 acid compounds was completed within 10 min on NiO@SiO2 column. In contrast, on 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 NH2 column and coordination effect on NiO@SiO2 column.
Separation Of Alkaloid Compounds
Three alkaloid compounds were selected to further elucidate the coordination interaction between analytes and NiO@SiO2 column (Fig. 4e). The alkaloid compounds were not separated on silica column, bu tthey were partially separated on Zorbax NH2 column under the same chromatographic conditions, which might be due to hydrogen bond interaction and ion-exchange interaction between the three alkaloids and Zorbax NH2 column. As shown in Fig. 4e, three alkaloid compounds were better separated within 6 min on NiO@SiO2 column than on above-mentioned two columns. The elution order of NiO@SiO2 column was different from that of silica column, which might be attributed to the coordination interaction between different nitrogen-containing groups and NiO@SiO2 stationary phase.