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Article

Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles

by
Thomas H. Walter
*,
Cheryl Boissel
,
Jessica A. Field
and
Nicole L. Lawrence
Waters Corporation, Milford, MA 01757, USA
*
Author to whom correspondence should be addressed.
Separations 2023, 10(3), 175; https://doi.org/10.3390/separations10030175
Submission received: 8 February 2023 / Revised: 27 February 2023 / Accepted: 2 March 2023 / Published: 6 March 2023
(This article belongs to the Section Chromatographic Separations)
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Abstract

:
One of the fundamental attributes of a liquid chromatography column is its stability when exposed to acidic and basic mobile phases. However, there have been relatively few reports to date on the stability of hydrophilic interaction chromatography (HILIC) columns. Here, we report the results of stability evaluations carried out for HILIC columns packed with ethylene-bridged hybrid or silica particles using accelerated conditions, employing a 100% aqueous pH 11.3 ammonium bicarbonate mobile phase at 70 °C. Under these conditions, the primary mode of column failure was a loss of efficiency due to the formation of voids resulting from the hydrolysis of the particles. We investigated the dependence of stability on the surface area of both unbonded and sulfobetaine-bonded ethylene-bridged hybrid stationary phases. The results show a clear trend of stability increasing as the surface area decreases. Several commercially available HILIC columns that are recommended for use with high-pH mobile phases were also evaluated. The results show times to 50% loss of the initial efficiency ranging from 0.3 to 9.9 h. Columns containing unbonded, sulfobetaine-bonded or diol-bonded ethylene-bridged hybrid stationary phases had longer lifetimes than amino-bonded silica or sulfobetaine-bonded, hybrid-coated, superficially porous silica columns.

1. Introduction

Hydrophilic interaction chromatography (HILIC) is a mode of liquid chromatography that is widely employed for separating compounds that are too polar to be retained using reversed-phase chromatography [1,2]. In HILIC, a polar stationary phase is used with a less polar organic/aqueous mobile phase. Commercially available HILIC stationary phases include materials based on silica, organic polymers and hybrid organic/inorganic materials [3,4,5,6]. Carbon [7,8,9], zirconia [10,11], magnesia-zirconia [11], titania [12] and aluminosilicates [13] have also been investigated for HILIC. Silica-based stationary phases have been the most widely reported in the HILIC literature [14]. Silica particles generally have an excellent pressure tolerance, allowing them to be packed to form efficient and mechanically stable columns. However, they are not recommended for use with alkaline mobile phases because silica is hydrolyzed in basic solutions [15]. The hydrolysis reaction produces silicic acid (Si(OH)4) and related oligomers, which may dissolve in the mobile phase and elute from the column. Over time, this weakens the packed bed structure, leading to the formation of voids and resulting in a large decrease in efficiency [16]. The hydrolysis products have been observed as column bleed using evaporative light scattering or charged aerosol detectors [17,18,19,20]. Changes in retention times have also been observed [13,21,22].
Due to these issues, silica-based columns are not often used with high-pH mobile phases. However, such mobile phases offer substantial benefits, including avoiding the degradation of acid-labile compounds, modifying separation selectivity and increasing sensitivity when using electrospray ionization mass spectrometry detection [23]. High-pH mobile phases have also been shown to provide narrower peaks and improved peak symmetry for organic acids and phosphorylated analytes in metabolomics studies [24,25,26,27]. This may be due to a decrease in ionic interactions with stainless steel surfaces in the LC system and column at pH values above the isoelectric point of the surface oxide layer (ca 7) [28].
We recently reported a study of the effects of temperature, mobile phase pH and water content on the stability of an unbonded silica HILIC column [29]. The results showed that the silica was hydrolyzed when exposed to basic mobile phases with water concentrations of 20–60%. This caused large decreases (>50%) in the column efficiency as a result of the formation of voids. Smaller changes in retention factors (<25%) were also observed. Using a mobile phase containing 60% acetonitrile and 40% aqueous ammonium bicarbonate buffer (10 mM,   p w w H   11.30) at 70 °C, six different silica-based HILIC columns showed efficiency losses ranging from 40 to 70% after 3 h of exposure. In contrast, columns packed with unbonded or amide-bonded ethylene-bridged hybrid (BEH) particles [30,31] showed efficiency losses of less than 4%.
Here, we describe the development of a harsher accelerated base stability test for HILIC columns. The key difference vs. the previously described test is the use of a higher water content mobile phase. Our goal was to identify conditions that cause >50% efficiency loss for an ACQUITY™ UPLC™ BEH™ HILIC Column after approximately 5 h of exposure to the basic solution. Using these conditions, we then compared the stability of unbonded and sulfobetaine-modified BEH stationary phases prepared from particles with different surface areas that ranged from 92 to 399 m2/g. In addition, we evaluated several commercially available HILIC columns recommended for use at an alkaline pH with upper limits ranging from pH 9 to 12. The results provide insight into the relative base stability of these columns as well as the stationary phase characteristics that impact stability.

2. Materials and Methods

2.1. Chemicals

LC-MS grade acetonitrile (ACN) was obtained from Fisher Scientific (Hampton, NH, USA). Ammonium formate (AF), ammonium bicarbonate (AmBic), ammonium hydroxide, formic acid and all test compounds were sourced from Millipore-Sigma (Burlington, MA, USA). Deionized water was produced using a Millipore Milli-Q™ System (Burlington, MA, USA).

2.2. Instrumentation and Columns

All chromatographic evaluations were performed using an ACQUITY UPLC H-Class System equipped with an ACQUITY UPLC Column Manager and an ACQUITY UPLC Photodiode Array Detector (Waters Corporation, Milford, MA, USA). ACQUITY UPLC BEH HILIC Columns (1.7 µm, 2.1 × 50 mm), Atlantis™ Premier BEH Z-HILIC Columns (1.7 µm, 2.1 × 50 mm) and Torus™ Diol (1.7 µm, 2.1 × 50 mm) Columns were obtained from Waters Corporation (Milford, MA, USA). Columns packed with unbonded and sulfobetaine-bonded BEH particles with different surface areas were prepared by Waters Chemistry R&D group, and were packed in conventional stainless steel UPLC column hardware. Poroshell™ 120 HILIC-Z Columns (1.9 μm, 2.1 × 50 mm) were obtained from Avantor (Radnor, PA, USA). Luna™ NH2 Columns (3 μm, 2.0 × 50 mm) were from Phenomenex (Torrance, CA, USA).
Multipoint N2 sorption measurements were carried out using an ASAP 2420 analyzer (Micromeritics Instrument Corporation, Norcross, GA, USA). The results were used to determine the surface areas and average pore diameters for the stationary phases. The surface areas were calculated using the Brunauer–Emmett–Teller method [32], and the average pore diameters were calculated from the desorption leg of the isotherm using the Barrett–Joyner–Halenda method [33].

2.3. Sample and Mobile Phase Preparation

The samples contained acenaphthene (25 μg/mL), adenine (25 μg/mL), cytosine (50 μg/mL), trimethylphenylammonium chloride (TMPA) (1000 μg/mL) and sodium p-toluene sulfonate (TS) (400 μg/mL) dissolved in 95/5 v/v ACN/200 mM AF, pH 3.00 (aq). The analytes were split between two separate samples to avoid coelutions during the stability evaluations.
The pH values of the buffers were determined as aqueous solutions with the pH meter calibrated using aqueous reference buffers, designated as p w w H values. The p w w H 3.00 AF buffer was prepared by dissolving AF in water to provide a concentration of 200 mM. Formic acid was then added to adjust the pH. The p w w H 11.30 AmBic buffer was prepared by dissolving AmBic in water to provide a concentration of 100 mM. Then, 30% ammonium hydroxide was added to adjust the pH. This solution was diluted with water to prepare the 10 mM p w w H 11.30 AmBic buffer. The 20/80 and 10/90 v/v ACN/ p w w H 11.30 AmBic (aq) buffer mobile phases were prepared by combining weighed amounts of ACN, 100 mM AmBic buffer and water to produce the desired compositions. Both mobile phases contained 10 mM AmBic in the final mixture. As previously shown, the AmBic solutions containing ACN have p w s H values that are slightly lower than the p w w H   value [29].

2.4. Method Details

The accelerated base stability tests were carried out using the generalized gradient program shown in Table 1, which is similar to the program used in our previous study [29]. The duration of the initial isocratic segment (x) was adjusted as necessary based on the retention time of the most-retained analyte for a particular column. Samples containing acenaphthene (the hold-up time marker), cytosine, adenine, TMPA and TS were separated using a 95/5 v/v ACN/200 mM AF, p w w H 3.00, (aq) mobile phase at a flow rate of 0.4 mL/min with UV absorbance detection (254 nm). The injection volume was 1 μL. The peaks were identified based on their UV spectra, which were obtained using a photodiode array detector. A challenge solution of varying composition was then passed through the column at 0.4 mL/min for 20.57 min (75 column volumes), followed by washing with 50/50 and 90/10 v/v ACN/water, then equilibration with the 95/5 v/v ACN/200 mM AF, p w w H 3.00 (aq) test mobile phase (all at 0.4 mL/min). This cycle was repeated until the column showed an average efficiency loss greater than 50%. The column temperature was maintained at 70 °C throughout. The USP efficiencies, USP tailing factors and retention factors of the compounds were determined vs. the time exposed to the challenge solution. The trends in the relative efficiency changes were generally found to be similar for all test compounds; therefore, the average values were used to determine the time at which the efficiency dropped below 50% of its initial value (t½). The only exception was the Torus Diol column, which demonstrated an initial significant increase in the TMPA efficiency before it dropped; therefore, results for this analyte were not included in the average efficiency. The retention factor trends were not similar for all test compounds; consequently, these data were treated individually.

3. Results

3.1. Effect of Mobile Phase Aqueous Content on the Stability of Unbonded 130 Å BEH Columns

We previously demonstrated the strong effect of mobile phase aqueous content on the stability of an unbonded silica column [29]. The time to loss of 50% of the initial efficiency (t½) varied from an average of 13.3 h for a mobile phase containing 80% ACN/20% aqueous AmBic (10 mM p w w H   11.00) to 1.0 h for 40% ACN/60% of the same aqueous buffer (both at 70 °C). Using a challenge mobile phase containing 60% ACN/40% aqueous AmBic buffer (10 mM p w w H   11.30), ACQUITY UPLC BEH HILIC Columns were found to exhibit only an average 3.8% efficiency loss after 3.1 h. To increase the rate of hydrolysis, we investigated the effect of varying the aqueous content of the challenge mobile phase from 80 to 100%. The results are shown in Figure 1 as plots of efficiency as a % of the initial value vs. the time exposed to the challenge solution. With an 80% aqueous content, the average efficiency decreased by only 13% after 30.5 h of exposure. When the aqueous content was increased to 90%, t½ was found to be 18.2 h. Further increasing the aqueous content to 100% resulted in a t½ of 6.5 h. Due to the relatively fast efficiency loss for the 100% aqueous challenge mobile phase, we chose this composition for comparing the base stability of columns that are recommended for use with high-pH mobile phases.
In addition to the drop in efficiency, the tailing factors of all test compounds increased by approximately 50–200% at the time of the steepest change in efficiency. An example is shown in the Supporting Information, Figure S1. As the tailing factor changes occurred at the same time as the steepest decline in efficiency, they did not provide additional information on the rate of hydrolysis. Consequently, we focused on using efficiency as an indicator of the rate of hydrolysis of the stationary phases.
Changes in retention were also observed, but they were generally smaller and more gradual than the efficiency changes (see Supporting Information, Figure S2). The retention factors for the neutral compounds adenine and cytosine decreased <20% at t½, while the anionic compound TS showed a <10% increase in retention factor over the first several hours before gradually returning to nearly the initial value. The retention factor of the cationic probe TMPA decreased 40–50% during the duration of the tests.
Representative chromatograms are shown in Figure 2, illustrating the changes in peak shape, peak width and retention resulting from exposure to the 100% and 80% aqueous challenge mobile phases. The chromatogram in Figure 2B shows that exposure to the 100% aqueous pH 11.30 mobile phase at 70 °C for 6.5 h caused pronounced peak broadening and splitting. In contrast, little change was observed after exposure to the 80% aqueous mobile phase for 30.5 h.

3.2. Dependence of Base Stability and Retention of Unbonded BEH Columns on Surface Area

In our previous study, we observed that the rate of base-catalyzed efficiency loss appeared to increase with increasing surface area for silica-based stationary phases [29]. To investigate the dependence of base stability on the surface area of unbonded BEH particles, we evaluated columns packed with five different materials with surface areas ranging from 92 to 399 m2/g. The key characteristics of these stationary phases (designated by their approximate average pore size in Å as BEH65, BEH80, BEH95, BEH130 and BEH300) are shown in Table 2. The retention factors for the test analytes exhibited linear correlations with the surface area, as shown in Figure 3. The correlation coefficients ranged from 0.970 to 0.996.
For each of the unbonded BEH materials, two columns were evaluated for base stability using a 100% aqueous pH 11.30 AmBic challenge mobile phase at 70 °C. The t½ values are shown as a function of the surface area in Figure 4. A trend is evident, with t½ values increasing with decreasing surface area. For each material, the two columns provided identical t½ values, indicating excellent reproducibility for the method.
Changes in retention were also observed, and the trends were similar for all five materials (see Supporting Information, Figure S3). The retention factors for the neutral compounds adenine and cytosine decreased <20%, while that of the anionic compound TS generally showed smaller changes. The retention factor of the cationic probe TMPA decreased 25–40% during the duration of the tests.

3.3. Dependence of Base Stability and Retention of Sulfobetaine-Modified BEH Columns on Surface Area

We also investigated the dependence of retention and t½ on surface area for three sulfobetaine-modified stationary phases prepared from BEH particles with different surface areas. The key characteristics of these materials are summarized in Table 2, designated as BEH95 S, BEH130 S and BEH300 S based on the average pore size, in Å, of the precursor particles. The dependence of the retention factors on surface area (determined for the bonded materials) is shown in Figure 5. The correlation coefficients ranged from 0.944 to 0.999. The BEH95 S stationary phase is available commercially in Atlantis Premier BEH Z-HILIC Columns. The base stability of this material was previously reported using a challenge mobile phase of 60/40 v/v ACN/10 mM AmBic pH 11.00 at 70 °C [34]. Under those conditions, no significant loss of efficiency was observed after 34 h of contact with the challenge mobile phase. With the 100% aqueous challenge mobile phase, the BEH95 S column was found to lose half of its initial efficiency after 5.7 h. Columns packed with BEH130 S and BEH300 S stationary phases exhibited t½ values of 11.5 h and 31.9 h, respectively (see Figure 4). As observed for the unbonded BEH materials, the BEH sulfobetaine stationary phases showed a trend of increasing t½ values with decreasing surface area.
Changes in retention were also observed during the base stability tests, with similar trends observed for the three materials (see Supporting Information, Figure S4). The retention factors for the neutral compounds adenine and cytosine and the anionic compound TS varied <10% during the duration of the tests. The retention factor of the cationic probe TMPA increased 50–70%, showing a trend opposite to that of the unbonded BEH columns.

3.4. Comparison of the Stability of Commercially Available HILIC Columns Recommended for Use at High-pH

We used the 100% aqueous p w w H 11.30 AmBic buffer challenge mobile phase at 70 °C to evaluate the stability of several HILIC columns that are recommended by their manufacturers for use at pH values ranging up to 9–12. The properties of the stationary phases are summarized in Table 2. The Luna NH2 stationary phase is a crosslinked, amine-bonded phase on fully porous 100 Å/400 m2/g silica particles [4]. Poroshell 120 HILIC-Z is a sulfobetaine-bonded phase on hybrid-coated 100 Å/95 m2/g superficially porous silica [35]. Torus Diol is a diol-bonded phase on 130 Å/180 m2/g BEH particles which was designed for use in supercritical fluid chromatography. It has been shown to perform well under HILIC conditions [36,37]. Atlantis Premier BEH Z-HILIC Columns contain the BEH95 S stationary phase, while ACQUITY UPLC BEH HILIC Columns are packed with BEH130 particles. Of these columns, the highest recommended upper pH limit is 12 for the Poroshell 120 HILIC-Z column, followed by 11.0 for the Luna NH2 column, 10 for the Atlantis Premier BEH Z-HILIC Column and 9 for the BEH HILIC Column.
The base stability results are shown in Figure 6. The Luna NH2 column was found to lose 83% of its initial efficiency after the first cycle of exposure to the 100% aqueous pH 11.30 challenge solution (0.3 h). The Poroshell 120 HILIC-Z column lasted longer, failing after 2.1 h. The Atlantis Premier BEH Z-HILIC Column had a t½ value of 5.5 h, while the ACQUITY BEH HILIC Column lasted for 6.5 h. The Torus Diol column showed the greatest stability with a t½ value of 9.9 h.
Changes in retention were also observed during these tests (see Supporting Information Figure S5). The trends for the unbonded BEH and the BEH Z-HILIC Columns were described above. For the Luna NH2 column, the retention factors for adenine, cytosine and TS decreased 8% at t½ while that of the cationic probe TMPA increased by 180%. The Poroshell 120 HILIC-Z column showed a 43% decrease in the retention factor of TS at t½ while k increased 6% for cytosine, 24% for adenine and 225% for TMPA. The Torus Diol Column showed the following retention factor changes at t½: −50% for TS, −5% for adenine, +20% for cytosine and +385% for TMPA.

4. Discussion

As in our previous study, the primary failure mode that we observed resulting from exposure of the HILIC columns to the basic mobile phase was a loss of efficiency. This is in agreement with studies of silica- and hybrid-based reversed-phase columns and is caused by the hydrolysis of the silica or hybrid particles, involving the formation of silicic acid and the removal of the hydrolysis product by the mobile phase [30,38,39,40]. Eventually, this results in collapse of the packed bed, forming voids that cause the loss of efficiency. We observed visible voids at the inlet ends of the packed beds when we removed the end fittings from the columns after completing the stability tests. Decreases in retention for the neutral compounds adenine and cytosine likely occur because of the loss of particle mass. In several cases, we observed large increases in the retention factor of the cation TMPA, which may be explained by the formation of additional silanol groups due to the hydrolysis of siloxane bonds in the particles [21]. The trend of decreasing retention of TMPA observed for the unbonded BEH Columns is unexpected, and the cause requires further elucidation. In most cases, we observed that the retention factor of the anion TS moved in the opposite direction from TMPA, suggesting that a change in surface charge occurred. For the surface-modified stationary phases, the loss of the attached groups may also cause retention changes.
As shown in our earlier study [29], the water concentration has a large effect on the rate of efficiency loss, with higher concentrations resulting in a shorter time to column failure, as measured by t½. This is likely due to differences in the solubility of the hydrolysis products, with silicic acid and related oligomers being more soluble in water than in acetonitrile/water mixtures. With the higher solubility of the hydrolysis products, particle degradation would be expected to occur at a faster rate. While typical HILIC mobile phases have water concentrations of less than approximately 50%, higher concentrations are used in gradient methods employed to separate compounds that cover a wide range of polarities. Important examples of such a method are polar metabolomics studies, which have reported gradients increasing to 80–100% water in combination with alkaline buffers [24,25,26,27]. Our results suggest that when using alkaline buffers with silica- or hybrid-based HILIC columns, the water content of the mobile phase should be kept as low as possible to maximize the column lifetime.
The results presented here show a clear trend of t½ values increasing as the surface area of the stationary phase decreases. In previous studies of reversed-phase columns, a similar trend was reported in a comparison of six different stationary phases; however, it was ascribed to a difference in the manufacturing processes used for the particles [40]. It had been concluded that silicas produced by aggregating sols were more stable at a high pH than those made by other processes. In that study, the three sol-based silicas had surface areas that were approximately half the surface area of the non-sol-based silicas (170–180 vs. 320–340 m2/g). In the work reported here, all the BEH particles were synthesized in the same non-sol based process, differing only in the pore enlargement conditions. Our observations suggest that it is the surface area of the stationary phase that affects its base stability, not the process by which it was made (i.e., sol aggregation vs. other approaches). The dependence of base stability on surface area is in agreement with prior studies on the solubility of silica gel in water [41]. While base stability may be increased by using low-surface-area stationary phases, the concomitant decreases in retention must also be considered. To achieve adequate retention for a range of compounds, it is necessary to choose a surface area that balances these opposing trends.
Comparing the results for the sulfobetaine-modifed BEH stationary phases to those for the unmodified BEH materials, similar stability was observed when considering materials of approximately the same surface areas except for the BEH300/BEH300 S pair. Note that here we used the surface areas of the materials after surface modification, while it is more common to report the surface areas of the precursor particles [34]. The increase in weight from the surface modification results in a proportional decrease in the surface area [42]. The observation that columns packed with modified and unmodified HILIC materials of comparable surface area have similar base stability is in contrast to the results reported for reversed-phase columns. In the latter case, surface modification with hydrophobic groups was shown to increase the base stability of the materials, with the rate of silica hydrolysis decreasing with increasing surface concentration of the bonded groups [39,40]. The different behavior seen here for the sulfobetaine-modified stationary phases is likely due to the hydrophilicity of these groups. Due to their hydrophilic character, they do not repel water from the particle surface. However, the longer t½ value of the BEH300 S material vs. BEH300 suggests that the surface modification may provide some degree of steric protection of the particle surface from the mobile phase. Such steric protection was previously reported for silica particles derivatized with cyclofructan 6 [21].
Considering the base stability results for the different commercially available columns, several factors likely explain the results. The silica-based Luna NH2 column showed a very short lifetime under our test conditions. The short lifetime relative to the BEH-based columns is consistent with our previous results for other silica-based HILIC columns [29], as well as a report by other researchers [43]. The high surface area of this material (400 m2/g) is likely another factor contributing to the short lifetime. The Poroshell 120 HILIC-Z stationary phase is based on hybrid-coated superficially porous silica particles and showed modestly improved stability compared to the Luna NH2 column. This material is reported to have a relatively low surface area (95 m2/g), which likely contributed to the greater stability. However, compared to the BEH95 S stationary phase, which has a surface area of 172 m2/g, the Poroshell 120 HILIC-Z material had a lower stability (t½ = 2.1 h vs. 5.5 h). This suggests that the hybrid-coated, superficially porous silica particles are less base-stable than the homogeneously hybrid BEH particles. The comparable stability of the unmodified BEH130 and the BEH95 S columns is attributed to their common BEH substrate and similar surface areas (181 and 172 m2/g, respectively). Finally, the observation that the Torus Diol column had the longest t½ value is consistent with it being based on BEH particles and having a relatively low surface area (129 m2/g).

5. Conclusions

These results demonstrate that the aqueous content of the mobile phase has a major impact on the base stability of silica- and BEH-based HILIC columns. Using mobile phases containing an aqueous pH 11.30 ammonium bicarbonate buffer and a temperature of 70 °C, the time to loss of 50% of the initial efficiency (t½) for a BEH130 column decreased from >30 h with 20/80 acetonitrile/buffer to 6.5 h with 100% buffer. Using a 100% aqueous pH 11.30 ammonium bicarbonate mobile phase at 70 °C, we observed that the t½ values for both unmodified and sulfobetaine-modified BEH columns show a dependence on surface area, with t½ increasing as the surface area decreases. However, retention drops with a decreasing surface area, so the surface area must be high enough to provide adequate retention for the intended use of the column.
Using a 100% aqueous pH 11.30 ammonium bicarbonate mobile phase and a temperature of 70 °C, we found t½ values ranging from 0.3 to 9.9 h for five commercially available HILIC columns recommended for use with basic mobile phases. Silica-based Luna NH2 columns were found to have the shortest lifetime (0.3 h), followed by Poroshell 120 HILIC-Z sulfobetaine-bonded hybrid-coated superficially porous silica columns (2.1 h). Columns based on fully porous Atlantis BEH Z-HILIC and BEH HILIC (unbonded) stationary phases had similar t½ values of 5.5 and 6.5 h, respectively. The longest t½ value (9.9 h) was observed for a Torus Diol (130 Å) column. These results demonstrate the greater base stability of HILIC columns packed with BEH-based stationary phases relative to those packed with silica- and hybrid-coated, silica-based materials. They also suggest directions for further improvements in base stability for HILIC columns.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/separations10030175/s1, Figure S1: Comparison of relative changes in average tailing factor and efficiency for a BEH130 HILIC Column exposed to 100% aqueous pH 11.30 10 mM AmBic at 70 °C; Figure S2: Relative changes in retention factors for BEH130 HILIC Columns exposed at 70 °C to basic challenge solutions with different water concentrations; Figure S3: Relative changes in retention factors for columns packed with BEH materials of different surface area exposed at 70 °C to 100% aqueous pH 11.30 10 mM AmBic; Figure S4: Relative changes in retention factors for columns packed with BEH Sulfobetaine materials of different surface area exposed at 70 °C to 100% aqueous pH 11.30 10 mM AmBic; Figure S5: Relative changes in retention factors for three commercially available HILIC columns exposed at 70 °C to 100% aqueous pH 11.30 10 mM AmBic.

Author Contributions

Conceptualization, T.H.W.; formal analysis, T.H.W.; methodology, T.H.W., C.B., J.A.F. and N.L.L.; investigation, C.B. and J.A.F.; visualization, T.H.W.; writing—original draft preparation, T.H.W.; writing—review and editing, C.B., J.A.F., N.L.L. and T.H.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon reasonable request from the corresponding author.

Acknowledgments

The authors acknowledge Erik Goulet and Steve McBride for carrying out the multipoint N2 sorption analyses and Mike Dion for packing the experimental columns.

Conflicts of Interest

The authors are employed by Waters Corp., the manufacturer of several of the columns that were evaluated. ACQUITY: Atlantis, BEH, Torus and UPLC are trademarks of Waters Technologies Corporation. Poroshell 120 is a trademark of Agilent. Luna is a trademark of Phenomenex.

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Separations 10 00175 g001 550
Figure 1. Effect of the water concentration of the challenge solution on the stability of 2.1 × 50 mm 1.7 μm ACQUITY UPLC BEH HILIC Columns, shown as % of initial efficiency vs. time of exposure to the basic solution. The columns were exposed at 70 °C to solutions containing an aqueous pH 11.30 ammonium bicarbonate (AmBic) buffer (green circles—20/80 v/v ACN/buffer; orange triangles—10/90 v/v ACN/buffer; purple squares—100% buffer).
Figure 1. Effect of the water concentration of the challenge solution on the stability of 2.1 × 50 mm 1.7 μm ACQUITY UPLC BEH HILIC Columns, shown as % of initial efficiency vs. time of exposure to the basic solution. The columns were exposed at 70 °C to solutions containing an aqueous pH 11.30 ammonium bicarbonate (AmBic) buffer (green circles—20/80 v/v ACN/buffer; orange triangles—10/90 v/v ACN/buffer; purple squares—100% buffer).
Separations 10 00175 g001
Separations 10 00175 g002 550
Figure 2. Chromatograms showing the changes caused by the stability test for 2.1 × 50 mm 1.7 μm ACQUITY UPLC BEH HILIC Columns. (A) Initial chromatogram; (B) chromatogram after 6.5 h of exposure to 100% aqueous pH 11.30 AmBic buffer at 70 °C; (C) initial chromatogram; and (D) chromatogram after 30.5 h of exposure to 80% aqueous pH 11.30 AmBic buffer at 70 °C. Peak identification, left to right: acenaphthene, p-toluene sulfonate, adenine, cytosine.
Figure 2. Chromatograms showing the changes caused by the stability test for 2.1 × 50 mm 1.7 μm ACQUITY UPLC BEH HILIC Columns. (A) Initial chromatogram; (B) chromatogram after 6.5 h of exposure to 100% aqueous pH 11.30 AmBic buffer at 70 °C; (C) initial chromatogram; and (D) chromatogram after 30.5 h of exposure to 80% aqueous pH 11.30 AmBic buffer at 70 °C. Peak identification, left to right: acenaphthene, p-toluene sulfonate, adenine, cytosine.
Separations 10 00175 g002
Separations 10 00175 g003 550
Figure 3. Dependence of retention factors on surface area for unbonded BEH Columns at 70 °C with a 95/5 v/v ACN/200 mM ammonium formate pH 3.00 (aq) mobile phase (blue squares—TMPA; black triangles, cytosine; gray diamonds—adenine; red circles—TS). The dotted lines show the best linear fits for the data.
Figure 3. Dependence of retention factors on surface area for unbonded BEH Columns at 70 °C with a 95/5 v/v ACN/200 mM ammonium formate pH 3.00 (aq) mobile phase (blue squares—TMPA; black triangles, cytosine; gray diamonds—adenine; red circles—TS). The dotted lines show the best linear fits for the data.
Separations 10 00175 g003
Separations 10 00175 g004 550
Figure 4. Dependence of time to 50% efficiency loss (t½) on surface area for unbonded BEH and BEH Sulfobetaine Columns at 70 °C with a 10 mM AmBic pH 11.30 (aq) mobile phase (purple circles—unbonded BEH; blue triangles—BEH Sulfobetaine).
Figure 4. Dependence of time to 50% efficiency loss (t½) on surface area for unbonded BEH and BEH Sulfobetaine Columns at 70 °C with a 10 mM AmBic pH 11.30 (aq) mobile phase (purple circles—unbonded BEH; blue triangles—BEH Sulfobetaine).
Separations 10 00175 g004
Separations 10 00175 g005 550
Figure 5. Dependence of retention factors on surface area for sulfobetaine-bonded BEH Columns at 70 °C with a 95/5 v/v ACN/200 mM ammonium formate pH 3.00 (aq) mobile phase (blue squares—TMPA; gray diamonds—adenine; black triangles—cytosine; red circles—TS). The dotted lines show the best linear fits for the data.
Figure 5. Dependence of retention factors on surface area for sulfobetaine-bonded BEH Columns at 70 °C with a 95/5 v/v ACN/200 mM ammonium formate pH 3.00 (aq) mobile phase (blue squares—TMPA; gray diamonds—adenine; black triangles—cytosine; red circles—TS). The dotted lines show the best linear fits for the data.
Separations 10 00175 g005
Separations 10 00175 g006 550
Figure 6. Column efficiency (% of initial value) vs. time exposed to 100% aqueous pH 11.30 AmBic buffer at 70 °C for five commercially available HILIC columns (gray squares—Luna NH2 Column; orange triangles—Poroshell 120 HILIC-Z Column; blue X—Atlantis Premier BEH Z-HILIC Column; purple circles—ACQUITY BEH HILIC Column; green diamonds—Torus Diol Column).
Figure 6. Column efficiency (% of initial value) vs. time exposed to 100% aqueous pH 11.30 AmBic buffer at 70 °C for five commercially available HILIC columns (gray squares—Luna NH2 Column; orange triangles—Poroshell 120 HILIC-Z Column; blue X—Atlantis Premier BEH Z-HILIC Column; purple circles—ACQUITY BEH HILIC Column; green diamonds—Torus Diol Column).
Separations 10 00175 g006
Table
Table 1. Generalized gradient program for the base stability test.
Table 1. Generalized gradient program for the base stability test.
Time (min)Flow Rate (mL/min)%A—Water%B—Acetonitrile%C—200 mM
Ammonium
Formate pH 3 (aq)		%D–Challenge SolutionCurve 1
Initial0.4000.095.05.00.0Initial
x0.4000.00.00.0100.011
20.57 + x0.4000.00.00.0100.011
22.24 + x0.40050.050.00.00.06
25.54 + x0.40050.050.00.00.06
27.20 + x0.40010.090.00.00.06
30.50 + x0.40010.090.00.00.06
42.90 + x0.4000.095.05.00.06
1 Curve indicates the shape of the change of the solvent composition for each gradient segment. Curve 11 is a step change at the end of the segment, and Curve 6 is a linear change during the segment.
Table
Table 2. Chemical and physical properties of the stationary phases evaluated.
Table 2. Chemical and physical properties of the stationary phases evaluated.
Stationary PhaseParticle
Chemistry		MorphologySurface
Chemistry		Surface
Concentration (μmol/m2)		Average Particle
Diameter (μm) 1		Average Pore
Diameter (Å)		Surface Area (m2/g)Recommended pH Range
BEH65BEHFully porousUnbonded 1.766399
BEH80BEHFully porousUnbonded 1.779362
BEH95BEHFully porousUnbonded 1.799273
BEH130BEHFully porousUnbonded 1.71381811–9
BEH300BEHFully porousUnbonded 1.731192
BEH95 SBEHFully porousSulfobetaine2.961.771 2172 22–10
BEH130 SBEHFully porousSulfobetaine4.161.7108 2133 2
BEH300 SBEHFully porousSulfobetaine3.401.7239 285 2
Torus DiolBEHFully porousDiol4.861.7123 2129 2
Luna NH2silicaFully porousAmino5.80 33 3100 3400 31.5–11.0 3
Poroshell 120 HILIC-Zhybrid-coated
silica		superficially porousSulfobetaineNA1.9 3100 395 32–12 3
1 nominal value; 2 measured for bonded material; 3 value reported by manufacturer.
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Walter, T.H.; Boissel, C.; Field, J.A.; Lawrence, N.L. Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles. Separations 2023, 10, 175. https://doi.org/10.3390/separations10030175

AMA Style

Walter TH, Boissel C, Field JA, Lawrence NL. Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles. Separations. 2023; 10(3):175. https://doi.org/10.3390/separations10030175

Chicago/Turabian Style

Walter, Thomas H., Cheryl Boissel, Jessica A. Field, and Nicole L. Lawrence. 2023. "Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles" Separations 10, no. 3: 175. https://doi.org/10.3390/separations10030175

APA Style

Walter, T. H., Boissel, C., Field, J. A., & Lawrence, N. L. (2023). Further Evaluation of the Base Stability of Hydrophilic Interaction Chromatography Columns Packed with Silica or Ethylene-Bridged Hybrid Particles. Separations, 10(3), 175. https://doi.org/10.3390/separations10030175

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