Cross‐linked polysiloxane‐coated stable bond O‐9‐(2,6‐diisopropylphenylcarbamoyl)quinine and quinidine chiral stationary phases as well as application in enantioselective cryo‐HPLC

In this work, brush‐type chiral stationary phases (CSPs) with O‐9‐(2,6‐diisopropylphenylcarbamoyl)‐modified quinidine (DIPPCQD‐brush/‐SH) and O‐9‐(2,6‐diisopropylphenylcarbamoyl)‐modified quinine (DIPPCQN‐brush/‐SH) were prepared as benchmarks for comparison with new corresponding polymeric CSPs with more stable bonding chemistry. These polymeric CSPs were prepared by coating a thin poly(3‐mercaptopropyl)‐methylsiloxane film together with the chiral selector onto vinyl‐modified silica. In a second step, immobilization of the quinine/quinidine derivatives as well as cross‐linking of the polysiloxane film to the vinyl‐silica is achieved by a double thiol‐ene click reaction. The polymeric CSPs exhibited similar enantioselectivity as the corresponding brush phases, but showed lower chromatographic efficiencies. Chiral acidic substances were separated into enantiomers (e.g., N‐protected amino acids, herbicides like dichlorprop) in accordance with an enantioselective anion‐exchange process. Oxidation of residual thiol groups of the polymer DIPPCQN‐CSP introduced sulfonic acid co‐ligands on the silica surface, which resulted in greatly reduced retention times. Acting as immobilized counterions, they allowed to reduce the concentration of counterions in the mobile phase, which is favorable for liquid chromatography (LC)–electrospray ionization–mass spectrometry application. Ibuprofen showed a single peak under ambient column temperature. However, application of cryogenic cooling of the column enabled to achieve baseline separation at –20°C column temperature. It can be explained by an enthalpically dominated separation, which leads to an increase in separation factors when the temperature is reduced. While it is quite uncommon to work at subzero degree column temperature, this work illustrates the potential to exploit such temperature regime for optimization of LC enantiomer separations.

quite uncommon to work at subzero degree column temperature, this work illustrates the potential to exploit such temperature regime for optimization of LC enantiomer separations.

K E Y W O R D S
click chemistry, cryogenic HPLC, enantiomer separation, polysiloxane-coated silica, thiol-ene click reaction

INTRODUCTION
Chiral separations, that is, separations of enantiomers, of organic molecules play an important role in various fields of research and industry, comprising pharmaceutical analysis, food and agricultural analysis, bioanalysis and metabolomics, forensics and toxicology, environmental analysis, and many more [1].Nowadays, numerous chiral stationary phases (CSPs) and commercially available enantioselective high-performance liquid chromatography (HPLC) columns are at our choice for this purpose [2][3][4][5][6][7].
The most popular chiral columns are based on cellulose and amylose derivatives [6,7], macrocyclic glycopeptide antibiotics (vancomycin, teicoplanin, and its aglycon) [8], cyclodextrins [8], cyclofructans [9], chiral crown ethers [10], synthetic donor-acceptor (Pirkle) type CSPs (e.g., Whelk O1) [11], and quinine (QN)/quinidine (QD) carbamate type chiral ion-exchangers [12][13][14].In recent years, also UHPLC versions based on sub-2 µm fully porous particles or sub-3 µm superficially porous particles of many of above-mentioned CSPs have been introduced into the market [15,16].On the other hand, there is still research going on focusing on the development of advanced selectors and new column technologies [17][18][19].Above-mentioned polysaccharide CSPs are obtained by coating of the polymeric selector [20]; when indicated, they are immobilized by cross-linking of the polysaccharide selectors to make them solvent resistant and more universally applicable [21,22].Other types of selectors are usually anchored to the supporting silica particle surface by silane linkers via a siloxane bond (Si-O-Si-R).Although relatively stable, this siloxane bond can be hydrolytically cleaved by strong acids and bases leading to a slight bleeding of the chiral selectors.Various strategies have been employed to improve the chemical bonding stability.Besides above-mentioned cross-linking strategies of polymeric selectors, bi-and trifunctional siloxane bondings [23], bipodal immobilization strategies [24], and immobilization via a polymer layer [25] that is immobilized by multi-point attachment are a few examples that have been considered to improve the longevity of CSPs and improve their electrospray ionization-mass spectrometry (ESI-MS) compatibility due to reduced background signal.More sta-ble chiral columns can lead to an increase in flexibility of conditions that can be employed and expand generally the applicability range.Most importantly, applicable conditions during method development are widened such as wider pH range and higher temperatures.
While temperature is widely accepted as an important factor in HPLC enantiomer separations, it is usually studied in a narrow range only (typically 5−40 • C).The main reason for it is that at the lower end column thermostats of HPLC equipment do not allow accurate cooling at lower temperatures.On the higher end, column stability is often limited and vendors recommend to use chiral columns not above 40 • C. Usually, LC enantiomer separations are enthalpy controlled [26][27][28][29].A reduction of the column temperature might be a valuable tool to increase separation factors and consequently resolutions.Temperatures at cryogenic conditions (i.e., sub-zero degree Celsius) are rarely employed in LC, because retention may get very strong leading to long analysis times.Furthermore, peak dispersion due to slow diffusion processes may get excessive, counteracting separations.Consequently, only a few studies reported the use of cryogenic temperatures for LC separations.For instance, cryogenic cooling was explored for its potential to trap peptides and proteins in reversed-phase liquid chromatography for refocusing/enrichment in biological applications [30].Ultralow-temperature HPLC (down to −176 • C) using low-molecular-weight hydrocarbons as mobile phases was employed by Motono et al. for the analysis of octane structural isomers [31].Cryogenic HPLC revealed that the retention of branched octanes was significantly reduced compared to the retention of n-octane.Several works suggested dynamic HPLC under cryogenic conditions to study stereolabile isomeric compounds and their interconversion kinetics [32][33][34][35].Also, supercritical fluid chromatography (SFC) enantioseparations at cryogenic temperatures have been reported [36,37].Enantiomers of compounds that showed low enantioselectivity could be baseline separated at cryogenic temperatures as low as −47 • C. SFC conditions and brush-type CSPs both were found favorable to retain good chromatographic efficiencies at low temperatures due to accelerated diffusivities and mass transfer under SFC conditions as well as fast adsorption-desorption kinetics of brush (Pirkle)-type phases such as Whelk O1.Sub-zero LC (e.g., at −30 • C) was also found favorable for enantiomer separations of 1,2-diacylglycerols as their 3,5-dinitrophenylurethane derivatives on a chiral column (Sumichiral OA-4100) [38].While there is a number of papers describing LC enantiomer separations under cryogenic conditions, such a concept was not overly popular probably due to the anticipation that at such extremely low temperatures the slow mass transfer and excessive peak dispersion contributions will dominate the separations and negatively impact enantiomer resolutions.
In this study, we present new QN and QD carbamate CSPs with orthogonal enantioselectivities to commercially available Chiralpak QN-AX and QD-AX (with tertbutylcarbamate residue at the C9-hydroxyl group of QN and QD) owing to their complementary sterically demanding and aromatic 2,6-diisopropylphenylcarbamoyl residue [39].
O-9-(2,6-Diisopropylphenylcarbamoyl)-modified quinine (DIPPCQN) and O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinidine (DIPPCQD) were employed as chiral selectors to prepare brush-type CSPs for comparison by thiol-ene click immobilization on 3-mercaptopropyl silica [40].With the aim to generate more stable columns for potential uses under more extreme experimental conditions, polymeric type analogs of these brush-CSPs were prepared.Thus, the title chiral selectors were immobilized onto silica via a polysiloxane film, which was coated and cross-linked to the silica surface by a double click immobilization reaction.The chromatographic performance of brush and polymeric CSPs as well as with their commercial tert-butylcarbamate counterparts was compared.The potential of enantiomer separations under sub-zero degree was illustrated by the enantiomer separation on a polymeric CSP with sulfonic acid co-ligand that, due to repulsive electrostatic interactions with the analyte, had low retentivity which was found favorable for LC under cryogenic conditions.

2.2.1
Immobilization of DIPPCQD to 3-mercaptopropyl silica (DIPPCQD-brush/-SH) Five grams of 3-mercaptopropyl-modified silica (3.7 µmol SH-groups, 0.74 µmol SH per gram modified silica) was dried overnight in vacuum at 60 • C. Chiral selector (1.58 g DIPPCQD; for synthesis, see Supporting Information) and 150 mg AIBN were also stored under vacuum at room temperature.Methanol for synthesis was sonicated for 30 min to remove dissolved air.At first, silica was suspended in 10 mL solvent and mixed with the chiral selector that has previously been dissolved in 5 mL methanol.AIBN was added directly to this mixture.Then methanol was added until having a total of 25 mL solvent for the reaction.The system was flushed with nitrogen for 10 min and then thiol click reaction was carried out at reflux for 7 h.The product was washed five times with hot methanol in a 75-mL Büchner filter (porosity 4) and then dried at 60 • C. Reaction success was indicated by an increase in weight of the modified silica (Δm = +0.97g) and confirmed by elemental (CHN) analysis (Table 1).

TA B L E 1 Elemental (CHN) analysis results and selector coverages of chiral stationary phases (CSP).
Stat

2.3.1
Immobilization of DIPPCQD to vinyl silica via polythiol polymer (DIPPCQD-poly/-SH) A homogeneous suspension of 2.5 g vinyl-modified silica (3.43 mmol, 0.93 mmol/g), 0.42 mL PMPMS (2.5 mmol thiol groups), 10.6 g DIPPCQD (2.0 mmol), and AIBN (0.6 mmol) was prepared in 25 mL methanol.Methanol was evaporated completely in a rotary evaporator and then thermally initiated thiol-click reaction was carried out at 60 • C under nitrogen atmosphere overnight.The product was washed several times with hot toluene (a good solvent for PMPMS) and hot methanol (a good solvent for the DIPPCQN selector).There was observed a weight increase of the silica after drying at 60 • C indicating success of the immobilization reaction (Δm = +0.73g; for CHN results, see Table 1).

Immobilization of DIPPCQN to vinyl silica via polythiol polymer (DIPPCQN-poly/-SH)
The corresponding DIPPCQN-carrying polymer-derived CSP was prepared as described above, however, in twice the amount.Weight increase was in similar range (Δm = +1.38 g, for CHN results, see Table 1).

2.3.3
Oxidation of polymer-derived CSP (synthesis of DIPPCQN-poly/-SO 3 H) Oxidation of residual thiols and thioether bonds of the DIPPCQN-poly/-SH CSP was performed by adding 8.1 mL of a mixture of formic acid and hydrogen peroxide (30% v/v) (9.5:0.5, v/v) dropwise into a suspension of 2.5 g DIPPCQN-poly/-SH in 25 mL methanol and 2.2 mL formic acid.The reaction was carried out under continuous stirring and ice cooling for 4 h.The silica gel assumed a slightly off-white appearance.It was washed with hot methanol/water (1:1, v/v) until the pH was neutral and afterward three times with hot methanol.Reduced adsorption tendency of the silica gel to the glass surface of the Büchner frit indicated success of the reaction (for CHN results after oxidation, see Table 1).

Column packing
Columns were packed at 800 bar with methanol as pressurizing solvent employing a pneumatic HPLC pump by Knauer.CSPs were suspended in 22 mL of isopropanol/acetic acid (10:1, v/v), sonicated for 3 min, and agitated for 30 min on an orbital shaker prior to packing.Stainless steel column hardware (150 × 4 mm ID) was sourced from Bischoff (Leonberg, Germany).
A mobile phase composed of methanol, acetic acid, and ammonium acetate (98/2/0.5, v/v/w) was used for chiral separation tests in polar organic elution mode.Columns were equilibrated and tested at a flow rate of 1 mL/min and temperature of 25 • C. Chiral test compounds (Ac-RS-Phe, Z-RS-Phe, and dichlorprop) were dissolved in methanol/acetone (100/0.05,v/v) and 5 µL of each solution were injected in triplicates.Compounds were detected at 254 nm.

Enantioselective cryo-HPLC
Enantioselective cryo-HPLC experiments were carried out on an Agilent 1100 HPLC system consisting of degasser, binary pump, autosampler, and column compartment.The HPLC was coupled to an Agilent MSD ion-trap mass spectrometer.A general setup of the system and cryogenic cooling unit is shown in Figure S10.For cryo-cooling of the column, a 1-L cryogenic storage dewar was filled with technical-grade methanol.Subsequently, the methanol in the dewar was cooled by liquid nitrogen to adjust the targeted temperature.The chromatographic column was submersed in the cooled methanol during analysis and the dewar was sealed with styrofoam and aluminum foil.
Temperature was checked and monitored with a TSIC306 digital temperature sensor and found to have <±1 • C temperature drift per hour under operating conditions.For void volume measurements, acetone was used, and t 0 was determined with and without column (with zero dead volume connector) at three different flow rates.The mobile phase volume (V m ) in the column was obtained by correcting the void volume by the extra-column volume.The volume of the stationary phase (V s ) in the column was obtained from the geometric column volume by subtracting the volume of the mobile phase, yielding the phase ratio ϕ as V s /V m .

Synthesis of stable bond DIPPCQN/DIPPCQD
Brush-type CSPs with 2,6-diisopropylphenyl quinine/ quinidine carbamate selectors are complementary in their retention profiles compared to their tert-butylcarbamate analogs, as described previously [39,41].Here, we describe the synthesis of more stable polymer-bonded CSP analogs.The chiral DIPPCQN and DIPPCQD selectors were synthesized by published protocols as described in the Supporting Information [40].The corresponding brush-type CSPs were obtained by radical addition reaction of the DIPPCQN and DIPPCQD as illustrated in Figure 1A.The surface coverage with chiral selector amounted to 0.34 mmol/g CSP and about 1.2 µmol/m 2 , respectively (see Table 1).
For the polymer bonding chemistry, a slurry of vinylmodified silica, poly(3-mercaptopropyl)methylsiloxane, and DIPCQN or DIPPCQD selector in methanol was prepared with a molar ratio of vinyl groups of silica to chiral selector of 1.7, thiols to total vinyl groups of 2.2, and selector to thiols of 0.8.In this reaction mixture, the selector was charged at 0.8 mmol/g modified silica, which is a factor of about 2.4 higher than the resulting surface coverage of the above brush-type CSPs.The slurry was carefully mixed to obtain a homogeneous dispersion by rotation on a rotary evaporator and then the methanol completely evaporated slowly.This ensured coverage of the silica surface with a thin polysiloxane film.Immobilization of the chiral selector by radical addition reaction took place by a solvent-free thiol-ene double click reaction (Figure 1B).At 60 • C, the initiator AIBN decays and forms radicals which start the reaction.The vinyl groups of both the selector and vinyl silica compete for reaction with the thiols of the polysiloxane film.In this way, the polysiloxane film gets immobilized on the vinyl silica and the chiral selector covalently anchored on the polythiol film, thus also immobilized.Elemental analysis indicates that 0.26 and 0.25 mmol DIPPCQD and DIPPCQN per g modified silica, respectively, were bonded (Table 1).This corresponds to a surface coverage of 0.9 and 0.8 µmol/m 2 , respectively, which is only little less than for the corresponding brush-type CSPs (Table 1).Immobilization yields of 33% and 31% for DIPPCQD-poly/-SH and DIPPCQN-poly/-SH, respectively, are lower than for brush-type CSPs (around 55%).It can be at least partially explained by the competition of the silica-vinyls for thiolene reaction with selector, which are present in 1.7-fold molar excess.The accomplished selector concentration on the surface, however, should be sufficient for effective chiral separations.
An aliquot of DIPPCQN-poly/-SH was subjected to performic acid oxidation.Residual thiols of the polysiloxane remaining unmodified after double-click reaction get oxidized to sulfonic acid residues.Thioethers of the polymer are oxidized to sulfonyl groups.Consequently, the surface becomes more polar and, due to sulfonic acid groups, the net surface charge significantly changed.Amino groups such as the quinuclidine nitrogen are not oxidized because they are protected from oxidation by protonation.The sulfonates on the DIPPCQN-poly/-SO 3 H CSP can interact with acidic analytes by repulsive ionic interactions, thereby modulating the net retention.It can be seen from Table 1 that selector and thiol contents are not significantly altered by the performic acid treatment.It indicates that the polysiloxane is indeed covalently linked to the vinyl-silica and not extracted by the hot toluene washing procedure (the polysiloxane is soluble in toluene).Furthermore, it can be concluded that the polysiloxane is not just cross-linked by disulfide bridges because they can be cleaved by performic acid oxidation to two sulfonic acids, which would lead to loss of polysiloxane film in the subsequent washing step.The improved chemical stability and lower background noise in liquid chromatography (LC)-ESI-MS of such polymer-bonded stationary phases compared to classical brush-type silica bonding was already investigated in detail by Zimmermann et al. [25].

Chromatographic evaluation and comparison with brush-type analogs
The synthesized CSPs were then evaluated for their LC performance using three chiral test substances, that is, Z-Phe, Ac-Phe, and dichlorprop.The chromatographic results are summarized in Table 2, and representative test chromatograms are depicted in Figure 2.
Baseline separation of enantiomers was accomplished for all test substances on all CSPs, except dichlorprop on DIPPCQN-poly/-SO 3 H.First, it is worthwhile noting that separation factors are larger on the DIPPCQN/QD brushtype CSPs compared to corresponding tert-butylcarbamate analogs (which are the chiral selector of commercial Chiralpak QN-AX/QD-AX) for Z-Phe and dichlorprop, while they are slightly lower for Ac-Phe (see Table 2).However, it turns out that with the bulky 2,6-diisopropylphenyl residue, a loss in plate numbers (around 20%) is observed for DIPPCQN/QD brush-type CSPs compared to corresponding tert-butylcarbamate analogs.It is hypothesized that the sterically demanding carbamate residue impacts negatively the association/dissociation kinetics at the WAX site of the selectors in the former CSPs.
When the chromatographic parameters of the polymerbonded CSPs (DIPPCQN/QD-poly/-SH) are compared with brush-type congeners (DIPPCQN/QD-brush/-SH), it is striking that retention factors are increased on the  polymer-bonded CSPs (cf. Figure 2A vs. D and Figure 2B vs. C, Table 2) probably due to additional nonspecific (hydrophobic) interaction of the analytes with the polysiloxane layer.The diffusion into the polymer network seems also unfavorable from kinetic viewpoint, which leads to a further loss in efficiencies (% loss in N around 50%).Separation factors are only slightly lower on the polymer-bonded CSPs (Table 2).It can also be seen from Figure 2 and Table 2 that enantiomer elution orders are reversed on corresponding QN-and QD-derived CSPs (Figure 2A vs. B and Figure 2C vs. D).Oxidation of the residual thiol groups of the DIPPCQNpoly/-SH CSP affords significantly reduced retention factors due to electrostatic repulsion of the negatively charged analytes (see Figure 2E vs. C and Table 2, cf.DIPPCQNpoly/-SH and DIPPCQN-poly/-SO 3 H CSPs).This may be an advantage for MS-hyphenation as this allows milder elution conditions comprising lower flow rates and reduced ion concentrations in the mobile phase [42,43].Separation factors are maintained and not negatively influenced by the polymer backbone oxidation (Table 2).The chromatographic efficiency on the other hand can be slightly improved by the oxidation.
In conclusion, the enantioselectivity of the DIP-PCQN/QD selectors can be conserved when instead of brush-type immobilization, polymer bonding via a reactive poly(3-mercaptopropyl)methylsiloxane film is pursued.The loss in chromatographic efficiency is quite commonly observed for polymer-bonded CSPs.A more stable bonding for extreme chromatographic conditions may in some applications outweigh this limitation of the new immobilization strategy, for example, in high-temperature LC.

Cryo-HPLC enantiomer separation of ibuprofen
Temperature is known and utilized as one of the instrumental factors for optimizing chromatographic separations.While it is more common, especially in the context of RP-type UHPLC, to work at elevated temperatures, LC enantiomer separations may benefit from lower temperatures.The majority of LC enantiomer separations take place under enthalpic control meaning that lowering the temperature leads to an increase in separation factors [27].While it is quite convincing from thermodynamic viewpoint to perform enantioseparations at low temperatures, poor kinetic performance at lower temperatures may counteract this straightforward optimization strategy.In this work, it was investigated whether the electrostatic repulsion principle of the DIPPCQN-poly/-SO 3 H CSP can, due to its very low retention factors, support enantiomer separations at cryogenic temperatures.A temperature study in the range of 25 to −20 • C was carried out with ibuprofen, which did not show enough enantioresolution at 25 • C (see Figure 3A).At 0 • C, a marginal separation was observed which improved when the temperature dropped to −10 • C, while at −20 • C a full baseline resolution was observed (see Figure 3B).The chromatographic parameters are summarized in Table 3.A van't Hoff analysis was then carried out to have a closer look into the factors, which drive the enantiomer separation [27].In general, the van't Hoff equation describes the relationship between natural logarithm of the retention factor ln k and the absolute temperature T (in K) in accordance with Equation (1): wherein ΔH 0 (J mol −1 ) and ΔS 0 (J K −1 ⋅mol −1 ) are the enthalpy and entropy change upon adsorption of the analyte to the stationary phase, respectively, R is the universal gas constant, and ϕ is the phase ratio (herein estimated from void volume measurements).Van't Hoff plots for the enantiomer separation of ibuprofen on DIPPCQNpoly/-SO 3 H CSP are shown in Figure 4. Estimates of enthalpic and entropic contributions to retention on the DIPPCQN-poly/-SO 3 H CSP, in accordance to Equation (1), are given in Table 4.Likewise, corresponding enthalpic and entropic contributions to the separation ΔΔH 0 and ΔΔS 0 , respectively, can be derived from Equation (2): The results are given in Table 4. From such derived ΔΔH 0 and ΔΔS 0 values, the isoeluotropic temperature (T iso ), at which the entropy and enthalpy changes compensate each other, can be calculated.It is 302.4K corresponding to 29.2 • C. At this temperature, the enantiomer zones completely coelute.Theoretically, above this temperature, the separation changes into an entropically dominated mechanism.However, under given mobile phase conditions, the practically relevant temperature range, in which the analyte is retained and separated, remains below this T iso .Overall, this study shows that temperature under cryogenic conditions may be a useful factor to optimize enantiomer separations in specific cases.

CONCLUDING REMARKS
New polymer-bonded CSPs based on DIPPCQN and DIP-PCQD as chiral selectors were prepared by coating of a poly(3-mercaptopropyl)methylsiloxane-polymer film onto the surface of vinyl silica along with admixed chiral   ble bonding can be obtained this way.While separation factors could be conserved as compared to corresponding brush phases, plate numbers were significantly lower, as often observed with polymer-bonded stationary phases.The specific stable-bonded CSPs could be of interest for separations that require harsh conditions.Herein, an oxidized CSP with sulfonate groups resulting from residual thiols was employed for enantiomer separations under cryogenic conditions.Enantiomers of ibuprofen could be baseline separated at −20 • C, while the zones of the two enantiomers almost completely merged at 25 • C. It shows that cryogenic temperatures can be employed as a tool for optimization of enantiomer separations.

A C K N O W L E D G M E N T S
Open access funding enabled and organized by Projekt DEAL.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T I Y S TAT E M E N T
The data that support the findings of this study are available in the Supporting Information Materials of this article.

F I G U R E 1
Overview of stationary phases and synthesis pathways: (A) Brush-type chiral stationary phases (CSPs) and (B) cross-linked polymer-type CSPs.
TA B L E 2