This work focuses on the effects of the mobile phase pH and the counterion concentration in buffer solution on retention in hydrophilic interaction liquid chromatography (HILIC) mode. Analytes with various acid-base properties and a silica-based Diol stationary phase were used. Retention and separation selectivity changes with ionization of the analytes and the adsorbent’s groups were discussed. It was demonstrated that the Diol phase behaved as a cation exchanger at pH 5.76 because of its residual dissociated silanols, while the phase provided almost no charge at lower pH (2.85). Separation efficiency and asymmetry factors for ionized compounds were also affected by the changes of mobile phase pH and counterion concentration. Separation conditions for the mixture of analytes of various acid-base properties were established.
Hydrophilic interaction liquid chromatography (HILIC) is a convenient method for analyzing multicomponent mixtures containing polar substances of different structure and charge. It can be successfully applied to various fields, such as proteomics, metabolomics, and the food and pharmaceutical industries (1). As the scale of HILIC application increases, it is important to comprehend the factors that influence retention of an analyte and separation selectivity. They include the nature of the stationary phase and mobile phase composition. A typical HILIC stationary phase is usually a silica-based material containing unmodified silanol groups or various bonded polar groups. Modified phases can also contain residual silanol groups (2,3). The mobile phase is a water-acetonitrile mixture with an acetonitrile content of more than 60 vol.%. The pH of the eluent in HILIC is usually controlled by using acetate or formate buffer solutions.
The influence of the factors (stationary and mobile phase) on retention and selectivity is described in terms of the retention mechanism involved. The retention mechanisms in HILIC are complex, as it is a combination of partitioning, electrostatic, and other adsorption interactions. The thickness of the water layer adsorbed on the stationary phase plays a key role in partitioning, depending on the adsorbent’s ability to retain water, mobile phase composition, and temperature (4). For compounds with a high contribution of partitioning, elution order is more in accordance with log P or log D parameters (5).
Electrostatic interactions may be dominant for ionizable compounds on charged stationary phases. The main parameters influencing these interactions are pH and buffer concentration of the mobile phase (6–9). The effect of pH changes on solute retention depends on its acid-base nature and the ionization state of adsorbent groups. Changes in anion and cation retention with pH variation could be explained by dissociation or protonation of the adsorbent’s functional groups. Weak acids and bases under certain pH could be ionized; as a result, they could be more hydrophilic than in molecular form. Therefore, a change of solute hydrophilicity affects its ability to interact with the adsorbed water layer, and thus its retention (6–8,10).
The influence of buffer concentration on retention is a superposition of multiple effects, with the predominant effect on the strength of the electrostatic interactions. On the one hand, increasing the concentration of buffer enhances the shielding of ionized stationary phase groups, and, as a result, decreases electrostatic interactions (both attraction and repulsion) (8,9). However, increasing the buffer concentration increases the thickness of the “water-rich layer” on the stationary phase surface, and therefore, it increases the partitioning of the analyte (4).
These two parameters of mobile phase (pH and buffer concentration) can also significantly affect the asymmetry of chromatographic peaks and column efficiency (2). Thus, the aim of this work was to study the effects on chromatographic parameters produced by changing salt concentration and pH of mobile phase on a diol column.
A Dionex UltiMate 3000 Liquid Chromatography system (Dionex/Thermo Scientific) equipped with a gradient pump, an autosampler, and a diode array detector, and a Vanquish Flex liquid chromatograph equipped with a gradient pump, an autosampler, and a column thermostat with fluorescence and diode array detectors, were used for HILIC with acetate or formate buffer as an eluent. Data acquisition and processing were controlled by Chromeleon 7.0 (Dionex/Thermo Scientific).
All chemicals were of reagent or analytical reagent grade and have been purchased from Merck and TCI Chemicals. The DIOL 300 column (6 μm, pore diameter 300 Å, 4.6 × 250 mm) has been provided by BioChemMack ST. This stationary phase is a non end-capped silica phase with hydroxyl groups in the functional layer (Scheme 1).
SCHEME 1: Diol stationary phase structure.
Mobile phases contained 90 vol.% of acetonitrile, and formate or acetate buffer solutions (Table I). The choice of pH and buffer concentration for the experiments are discussed in the “Results and Discussion” section. In situ preparation of each buffer system was provided by titration of the particular amount of the acid (formic or acetic) with sodium hydroxide solution using a pH-meter calibrated by three standard water solutions with pH 4.01, 6.86, and 9.18.
TABLE I: Buffer compositions used in this study
Eleven compounds were used for the experiments (Table II). Among them, there were quaternary ammonium salts, sulfonic acids, weak acids, and bases. Test compounds from each group differed by hydrophilicity evaluated by Hansh distribution parameters (log P, log D). They were chosen to cover the variety of analytes usually tested in HILIC. Additionally, several compounds with a fixed charge were selected for testing the ionic behavior of the stationary phase.
TABLE II: Structures of test compounds, their log P, log D, and pKa values and concentrations in a mixture
All the standard solutions were prepared with concentrations of individual analytes in the range of 10–500 ppm in water-acetonitrile 10:90 v/v to avoid their precipitation during analysis with high acetonitrile content. Toluene was used as a dead time marker. Other chromatographic conditions were fixed. The flow rate was 1 mL/min. The UV detector working wavelength was 254 nm. Column temperature was 25 °C. The injection volume was 20 μL.
In a medium with a high organic solvent content, dissociation constants (pKa) of analytes, buffer components, and adsorbent functional groups may change significantly (11). For evaluating pKa and pH in water-acetonitrile mixtures, we define the measurement of the pH in the mixed aqueous:organic mixture using a pH meter calibrated using aqueous standards as pHs. Designation for conventional scale is pH. In this work, the pH values were measured both in aqueous solutions (pHw) and water-acetonitrile mixtures (pHs).
We used mobile phases containing 90 vol.% of acetonitrile, and formate or acetate buffer solutions. They allow adjusting pH in a wider range, providing different charge status for silica substrate, which is not possible using only one buffer system. Certainly, the different abilities of formate and acetate, to influence the thickness of the “water-rich layer” on the stationary phase surface and to form ion pairs, should be taken into account (12,13).
The pH values (2.85 and 5.76) were chosen as boundary values, which are possible to make using formate or acetate buffer systems with reasonable buffer capacity. Formic acid, pKa 3.75 was used for the preparation of buffer solutions with pHw 2.85 (pHs 5.6), which value is (pKa [HCOOH] – 0.9) and about two units less than the average pKa of silanol groups pKa of 4.7). Acetic acid (pKa 4.76) was used for the buffer solutions with pHw 5.76 (pHs 9.2), which value is (pKa [AcOH]+ 1).
Three concentration levels of each buffer solution were used (Table I). Such buffer concentrations were chosen in order to provide similar acetate and formate anion (salt) concentrations in the mobile phase. Salt concentrations were chosen at such a level to provide reasonable retention factors for test analytes. Buffer concentrations were calculated from salt concentrations using a proportion shown in Table I:
The range of salt concentration differed for acetate and formate; top and bottom concentration limit was higher for acetate. It was due to less buffer capacity provided by the acetate buffer solution (Table I). The pHs value of buffer solution with the smallest buffer capacity as well as the others was checked during the experiment, and it appeared to be stable.
Sodium was selected as a cation in the buffer systems, and it acted as the eluting ion in the ion-exchange process. Traditionally used ammonium cation was not suitable, due to its ability to deprotonate around a pHs of 9 (10). We used sodium cation, which concentration was equal to that of formate or acetate anion concentration after the buffers preparation (Table I).
We paid close attention to the ion-exchange process when varying pH and concentration of buffer solution of the mobile phase. The process is based on the ion-exchange equilibrium:
which describes the exchange of mobile phase counterions M+ with protonated base BH+. In our system M+ was Na+. An expression for the distribution coefficient (Dix) between the stationary phase and mobile phase through the ion exchange constant and acid-base constant leads to equation 3:
From this equation, it follows that the base retention factor, which is directly proportional to the distribution coefficient, varies with the inverse of the counterion (eluting ion) concentration in the mobile phase. A response of the retention factor on 1/[Na+] (k’=f(1/[Na+]) should be a straight line passing through the origin (assuming that no other retention mechanism exists). Alternatively, the presence of other retention mechanisms (for example, partitioning and adsorption) would be indicated by an intercept on the k’-axis, which corresponds to an infinite eluting ion concentration. Because in this case the ion-exchange effects are eliminated, this plot must arise from the other mechanisms that exist, as it was proposed in (14). This was applied in (15) and the following works to compare ion-exchange and HILIC impacts for catecholamines in bare silica and zwitterionic columns. In the equation:
the free term b indicates the retention via nonionic interactions, including partitioning and adsorption. The percent contribution of hydrophilic interactions to the overall retention can be calculated by dividing free term b on k’ value obtained in the presence of a particular eluting ion concentration. The percent contribution of ion exchange mechanism is calculated as follows 1–b/k’.
The compounds chosen for the experiments (Table II) represented five groups according to their structure and acid-base properties. There were neutral (uracil, uridine, cytosine), positively charged (TMPAC, VBTMAC, B1), negatively charged (SPTS, SPSS), weak acids (xanthine, benzoic acid), and a weak base (benzylamine). The series for increasing hydrophilicity for compounds with the fixed charge are as follows: uracil < uridine < cytosine, VBTMAC < TMPAC < B1, SPSS < SPTS. According to pKa for benzoic acid, it can be expected that it was protonated at pHw 2.85 and deprotonated at pHw 5.76. Benzylamine and xanthine can be expected as protonated under both aqueous pH values. To interpret the retention behavior of the weak acids and bases, changes of dissociation constants in the medium with high amount of acetonitrile were considered.
The effect of sodium concentration in mobile phase on retention of the selected compounds (Table II) was evaluated at two pHw levels: 2.85 and 5.76. If we expect a negative charge from a silica-based diol column, then increasing sodium concentration should result in decreasing cation retention via ion exchange mechanism and increasing anions retention because of shielding surface charge. The secondary effect could be increasing surface water layer thickness with increasing ion concentration in the mobile phase. This lowers the energy required to remove the water from the mobile phase system because of enhancing its polarity and thus decreasing solubility in acetonitrile creating the potential for more adsorbed water on the surface. The increased water layer then results in increased partition, which could override and charge repulsion and result in increased retention for both cations and anions. It was observed that retention factors for cations, benzylamine, and anions fluctuated near the mean value with increasing eluting ion and buffer concentration (Figure 1, Table III). This showed the absence of any electrostatic interactions at low pH. Thus, the Diol column behaved as a neutral one. Increasing ion strength in this case led to the small increase of the retention for neutrals, thiamine, and xanthine, indicating the slightly enhanced thickness of the stationary phase adsorbed water layer (4). Similar observations were made for Diol Luna HILIC column at pHw 3 in the presence of percent acetonitrile (9). The retention factors obtained for compounds from the same group (neutrals, positively, and negatively charged) were in accordance with their hydrophilicity (see section “Mobile Phase and Test Analytes”). Retention factors increased from uracil to cytosine, from VBTMPAC to B1, and from SPSS to SPTS in all the studied conditions (Tables III and IV).
FIGURE 1: Retention factor values for test compounds using 112–447 mM formate buffer solutions pH 2.85. Conditions: stationary phase—DIOL 300 (4.6 × 250 mm); mobile phase—sodium formate buffer pH 2.85/acetonitrile 10:90 v/v; flow rate: 1 mL/min; UV detection at 254 nm.
TABLE III: Chromatographic parameters for eleven compounds at pHw 2.85 at three buffer concentration levels. Elution conditions are presented in Figure 1.
TABLE IV: Chromatographic parameters for eleven compounds at pHw 5.76 at three buffer concentration levels. Elution conditions are presented in Figure 3.
In addition to retention changes with buffer concentration, changes in efficiency varied for some of the compounds (Table III). A 4x increase of buffer concentration from 112 mM to 447 mM in aqueous portion of the eluent led to 1.5-6x increase in efficiency for charged compounds (SPSS, SPTS, VBTMAC, TMPAC, B1, and benzylamine). This might result from better partitioning kinetics for charged analytes reflecting enhanced surface water layer or surface silanol shielding with higher ion concentration. The decrease in efficiency was obtained for benzoic acid only. For other compounds, there were no significant changes. Good asymmetry values (As) from 0.7 to 1.2 were obtained for all of the compounds except B1, because of its strong retention.
From the initial set of 11 analytes, we used eight compounds in the model mixture including representatives from each class for providing structural and charge variety. The given chromatograms demonstrate the separation ability in tested conditions. Slight changes in the retention of test compounds with the increasing salt concentration led to insignificant changes in selectivity and resolution of the mixture (Figure 2). Poor peak shapes for the bases were obtained with a low buffer concentration. The best peak separation was obtained with the use of mobile phase containing 224 mM sodium formate buffer pHw 2.85/acetonitrile 10:90 v/v, while the best efficiency for basic analytes was achieved with the highest buffer concentration.
FIGURE 2: Separation of test mixture (Table II) at three buffer concentrations at pH 2.85. Conditions: stationary phase—DIOL 300 (4.6 × 250 mm); mobile phase—sodium formate buffer pH 2.85/acetonitrile 10:90 v/v; concentration of buffer in aqueous portion: 112 mM (orange line); 224 mM (yellow line); 447 mM (green line); flow rate: 1 mL/min; UV detection at 254 nm.
At pHw 5.76, we expected a negative charge from dissociated silanols of the substrate, and thus increased surface hydrophilicity and adsorbed aqueous layer thickness. While adjusting sodium acetate buffer concentrations at three levels, we observed significant changes of retention time for the charged analytes, and slight changes for neutral ones (Figure 3, Table IV). For neutral compounds (uracil, uridine, cytosine) the retention factors slightly increased with increasing buffer concentration. The effect was similar to that observed for pHw 2.85.
FIGURE 3: Retention factor values for test compounds using 27.5–82.5 mM acetate buffer solutions pH 5.76. Conditions: stationary phase —DIOL 300 (4.6 × 250 mm); mobile phase— sodium acetate buffer pH 5.76/acetonitrile 10:90 v/v; flow rate: 1 mL/min; UV detection at 254 nm.
For positively charged analytes (TMPAC, VBTMAC, B1, benzylamine), the retention time increased with the decrease of eluting sodium ion concentration. This indicated the existence of ion exchange mechanisms. As compared to the absence of electrostatic interactions at pHw 2.85, in the case of pHw 5.76, the changes of cation retention with concentration showed that the diol stationary phase had a negative charge from the dissociated residual silanol groups. The same effect was demonstrated for a column with similar functionality (Inertsil diol column) (3), where keeping the aqueous mobile phase at pHw 5.0 in the presence of 90 vol.% of acetonitrile, an increase in buffer concentration from 5 to 20 mM led to the substantial decrease of thiamine retention factor.
Plots k’=f(1/[Na+]) were built (Figure 4) in order to evaluate the contribution of ion-exchange mechanism in cation retention (equation 3). A non-zero intercept on the k’-axis indicated the presence of nonionic retention mechanisms. From the experimentally measured values of k’ for a particular solute and counterion concentration, the contribution of ion-exchange in percent could be determined (see Figure 4). The procedure is described in section “Mobile Phase and Test Analytes.” Significant contribution of 80–85% was obtained for the quaternary ammonium cations at 2.5 mM sodium concentration in mobile phase. For benzylamine, the contribution of ion exchange mechanism was only 26% to the overall retention. The reason is discussed in section “Effect of Mobile Phase pH.”
FIGURE 4: k’ – 1/[Na+] plots obtained for the counterion concentration range 2.5–7.5 mM in mobile phase. Percents marked are the contribution of ion exchange mechanism into cations’ retention in mobile phase containing 2.5 mM [Na+].
For negatively charged analytes (SPSS, SPTS), benzoic acid, and xanthine, the increase of sodium ion concentration led to the significant increase of the retention time (Figure 3). Shielding of dissociated silanol groups with the increased eluted ion concentration led in this case to reducing the repulsion between the analyte and the stationary phase. Changes in retention factor for benzoic acid with increasing buffer concentration at pHw 5.76 were expected according to its dissociation contact (pKa 4.2).
The variation of xanthine retention factor was more significant than it was for neutrals (uracil, uridine, cytosine) because of the changes of retention were more likely governed by electrostatic repulsion. It could not be explained in terms of xanthine dissociation using pHw and pKa for water solutions, where xanthine pKa 7.6 is about two units higher than the fixed pHw 5.76. We should mention that xanthine has two tautomeric forms, which equilibrium shifts toward the presence of anionic form in 90 vol.% of acetonitrile under these working conditions. Retention mechanisms of xanthine should be additionally evaluated. According to its behavior at both lower and higher pH, xanthine belonged to the group of weak acids in this study.
Additionally, efficiency also varied with buffer concentration (Table IV). Efficiency increased with buffer concentration for ionic compounds (SPSS, SPTS, VBTMAC, TMPAC) as it was observed at pHw 2.85. The effect could be explained by decreasing electrostatic interactions impact between the analytes and the stationary phase produced by higher eluting ion concentration. The results are in agreement with Kawachi and associates’ observation (2) that reducing ion-exchange interactions is important to obtain better column efficiency in HILIC. Moreover, the increase in concentration enhanced symmetry for VBTMAC and TMPAC. Efficiency for benzoic acid also increased opposite to its behavior at pHw 2.85. Since almost all the retention factors increased at higher pH despite the lower total buffer concentration at pHw 5.76, it is likely that an increased water layer thickness due to the more hydrophilic silica substrate, provided greater partition interactions for benzoic acid.
Selectivity at pHw 5.76 varied greatly as shown by the significant changes in retention (Figure 3) and differed from that at pHw 2.85 (Figure 5). For example, elution order of TMPAC 5, cytosine 7, and xanthine 4 changed from 5-7-4 to 7-5-4 and to 7-4-5 with the decrease of buffer concentration. Moreover, selectivity of benzylamine 6, uridine 3, and benzoic acid 1 varied with increasing concentration. Uridine and benzylamine were not separated using the mobile phase with 2.5 mM sodium concentration (or 27.5 mM of buffer in aqueous portion of mobile phase), while at higher concentrations of 5 and 7.5 mM, the peaks were well separated with benzoic acid eluted after them. Thiamine was excluded from the mixture because of its extremely high retention time (more than 1 h). The best peak resolution was obtained with mobile phase containing 5 mM of sodium at pHw 5.76.
FIGURE 5: Separation of test mixture (Table II) at three buffer concentrations at pH 5.76. Conditions: stationary phase—DIOL 300 (4.6 × 250 mm); mobile phase—sodium acetate buffer pH 5.76/acetonitrile 10:90 v/v; concentration of buffer in aqueous portion: 27.5 mM (orange line); 55 mM (yellow line); 82.5 mM (green line); flow rate: 1 mL/min; UV detection at 254 nm.
The experiment on the variation of pH was conducted while the eluting sodium ion concentration was kept constant (at 2.5 mM level in the mobile phase). Although the column behaved as a neutral phase at pH 2.85, from the IEX point of view, it didn’t matter which concentration of sodium was chosen, while at any other pH between boundaries (pH 2.85 and 5.76) the salt concentration might influence the retention of ionizable compounds significantly. So, we fixed the concentration of sodium suggesting the expansion of pH studied under which a part of residual silanols might be dissociated. Also, providing constant ion strength allows for equalizing adsorbed water layer caused by ion concentration in the mobile phase and observing its further changes due to other reasons, such as stationary phase dissociation. Thus, in these experiments, the eluting sodium ion concentration in mobile phase was kept constant. Providing those boundary pH values we had to use different buffering systems where formate and acetate anions both belonged to weak kosmotropes were prepared in the same concentration. Total buffer and acetic acid concentration for pHw 5.76 had to be lower than those for formate at pHw 2.85.
In this study, we compared the retention factors of the test analytes on the Diol column with pHw of the mobile phase being 2.85 and 5.76 (Tables III and IV). The retention changes were significantly affected by stationary phase charge and acid-base properties of the analytes (Figures 6 and 7). Retention factors increased for neutrals, weak acids, and cations, and decreased for strong acids and a weak base with increasing pH. Enhanced retention for neutrals at higher pH reflects the stationary phase silanols dissociation followed by increased polarity and adsorbed water layer providing better partition. Thus, stationary phase charge status is a dominant factor influencing retention in this case.
FIGURE 6: Retention factor values for test compounds using mobile phases with 2.5 mM sodium concentration at pH 2.85 and 5.76. Conditions: stationary phase—DIOL 300 (4.6 × 250 mm); mobile phase—224 mM sodium formate buffer pH 2.85, or 27.5 mM sodium acetate buffer pH 5.76/acetonitrile 10/:90 v/v; flow rate 1 mL/ min; UV detection at 254 nm.
FIGURE 7: The chromatograms of the test mixture (Table II) at fixed sodium concentration in mobile phase of 2.5 mM. Conditions: stationary phase—DIOL 300 (4.6. × 250 mm); mobile phase—sodium formate buffer pH 2.85 or sodium acetate buffer pH 5.76/acetonitrile 10:90 v/v; flow rate 1 mL/ min; UV detection at 254 nm.
Cytosine belonged to neutrals while it has pKa equal to 4.4. If cytosine was protonated at pHw 2.85 and deprotonated at pHw 5.76 its retention time would decrease significantly due to the decrease of its hydrophilicity as for weak bases (12). However, we observed a slight increase in cytosine retention factor with increasing pH as it was noted for uracil and uridine. For permanently charged cations and anions, significant changes in retention can be caused mostly by changes in the charge of the stationary phase (14), although partitioning, adsorption interactions and hydrogen bond formation should be considered in addition to electrostatic interactions. As it was demonstrated by the effect of counterion concentration (see sections “Effect of Buffer Concentration at pHw 2.85” and “Effect of Buffer Concentration at pHw 5.76”), at pHw 2.85, no electrostatic interactions were observed, while at pHw 5.76, the diol column behaved as cation exchanger. For the strong sulfonic acids SPTS and SPSS, ion-exchange is dominant, therefore they are more repelled from the negative surface charge.
Weak acids (benzoic acid and xanthine) should retain lower at 5.76 because of repulsion if ion-exchange is dominant or retain more if partitioning is dominant. Their resulting retention was affected by these two opposite effects with partitioning prevailing. Thus, it was demonstrated that an increase in solute ionization must have a more significant effect on acid retention than the effect of any changes in the ionization of the stationary phase. The same observation was made, for example, by Karatapanis and associates (3) for L-ascorbic acid on Inertsil diol column, and by McCalley (6) for 3,4,5-trihydroxybenzoic acid on the silica column using a mobile phase containing 85-90 vol.% of acetonitrile in the pH range 3-6.
With the strong bases VBTMAC, TMPAC, and B1 both ion-exchange and partition resulted in four times increased retention factors. The effect of increasing pH by three units on charged analytes retention was more significant than that provided by sodium concentration increase from 2.5 mM to 7.5 mM in the mobile phase at 5.76. Thiamine behaved like a compound with a fixed charge in the studied pH range. Similar results were reported in reference (12).
The weak base benzylamine was expected to be fully ionized according to its aqueous pKa 9.3 and eluent pH 5.76 values. The high content of acetonitrile gave an effective pHs of 9.2, rendering benzylamine mostly neutral and thus reducing its hydrophilicity. According to (17), basic compounds pKa values can be about 1 unit lower in 90% acetonitrile than the aqueous-based pKa. This, coupled with the effective pHs of nine results in an overall less charged molecule that will be less likely to partition into the aqueous phase or interact with negatively charged silanol groups on the surface.
As it was displayed earlier (Figure 4), the contribution of ion-exchange to benzylamine retention was only 26% at 5.76 and 2.5 mM sodium concentration in the mobile phase. The rest was attributed to other interactions. VBTMAC and TMPAC have log D parameters close to that for benzylamine (Table II), but the contribution for them was 50 % higher. Similar simultaneous effects of both decreasing solute ionization and increasing adsorbent ionization was observed in (14).
Such changes in retention with increasing pH led to differences in selectivity (Figure 7). With 2.5 mM sodium concentration level in mobile phase, all the peaks were fully separated at pHw 2.85. At pHw 5.76, low resolution was obtained for benzoic acid 1, uridine 3, and benzylamine 6. Retention time for B1 at pHw 5.76 was 1 h.
This work focused on the effects of the mobile phase pH and the ion concentration of the buffer solution on the retention of analytes with various acid-base properties when using the diol silica-based stationary phase.
Changing buffer concentration allowed us to evaluate the charge of diol stationary phase. At lower pHw 2.85, there was almost no charge, while at higher pHw 5.76, adsorbent behaved as cation exchanger because of dissociated residual silanols. This influenced the retention time of charged compounds. Increasing silanol dissociation led to enhancing the attraction of cations (VBTMAC, TMPAC, B1) and the repulsion of anions (SPTS, SPSS). For weak acids and a weak base, both ionization of the analytes and adsorbent affected the retention. The ionization process increased hydrophilicity of benzoic acid and xanthine, and changed their partitioning, which overpowered electrostatic repulsion. Increasing retention of neutrals with rising pH and buffer concentration was linked to the enhanced thickness of stationary phase adsorbed water layer. Explanations given were made by taking into account the shift of pKa values of the analytes and the buffer components in water acetonitrile mixtures in HILIC. The necessity of understanding the potenital impact of pHs scale while working in HILIC was emphasized.
Separation efficiency and asymmetry parameters were also affected by changes of mobile phase pH and counterion concentration. Strong effects were observed for compounds with fixed and variable charge. The efficiency increased 1.5–6x up to 18,000 plates per column for charged solutes when the electrostatic interactions were suppressed with high concentration of counterions in mobile phase or pH provided less charge on the stationary phase. It was presumably related to the change of molecule and stationary phase’s ionization state.
As a result, we were able to find the conditions suitable for the separation of the mixture containing analytes with various acid-base properties by varying these two parameters of the mobile phase. The best separation of eight compounds was achieved when using mobile phase containing 224 mM sodium formate buffer pH 2.85/acetonitrile 10/90 v/v at 30 min with good efficiency up to 18,000 plates per column. These results may be useful for practitioners when solving complex analytical problems.
This work was supported by the Russian Science Foundation through the grant No. 20-13-00140 using the equipment of the Central Collective Use Center of Moscow State University, “Technologies for Obtaining New Nanostructured Materials and Their Comprehensive Study,” acquired by Moscow State University under the program for updating the instrumentation base within the framework of the national project “Science” and the Development Program of Moscow State University. The authors acknowledge S.M. Staroverov and BioChemMack ST (Moscow, Russia) for providing the DIOL 300 column.
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Anna Shemiakina is a PhD student with the Analytical Chemistry Division, in the Chemistry Department of Lomonosov Moscow State University, in Moscow, Russia.
Aigu Xie is a third year student, at Northeast Normal University, in Changchun, Jilin, China. She was formerly associated with the Chemistry Department of Lomonosov Moscow State University, in Moscow, Russia.
Grigoriy Maksimov is with the Analytical Chemistry Division, in the Chemistry Department at Lomonosov Moscow State University, in Moscow, Russia.
Alla Chernobrovkina is an Associate Professor with the Analytical Chemistry Division of the Chemistry Department at Lomonosov Moscow State University, in Moscow, Russia. Direct correspondence to: chernobrovkina@analyt.chem.msu.ru
Recent Developments in HPLC & UHPLC
June 5th 2024In the world of liquid chromatography, innovative strides in column technology continue to take place. We are also reminded that there is always more to learn about “well-known” methodologies, and our craft is continuously influenced by important social concerns.