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Jan 21, 2015 - Certain chromatographic procedures require aggressive liquid ... achieve these objectives, reversed-phase (RP) stationary phases.
Journal of Chromatographic Science 2015;53:1107– 1122 doi:10.1093/chromsci/bmu173 Advance Access publication January 21, 2015

Article

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for Liquid Chromatography. Part II: Chemical and Thermal Stability Endler M. Borges1,2* and Dietrich A. Volmer3 1

Nu´cleo Biotecnoloo´gico, Universidade do Oeste de Santa Catarina, Rua Paese, 198, Bairro Universita´rio—Bloco K, Videira, SC CEP 89560-000, Brazil, 2Laborato´rio de Toxicologia e Essencialidade de Metais, Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Avenida do Cafe´ s/n, Monte Alegre, 14040-903 Ribeira˜o Preto, SP, Brazil, and 3Institute of Bioanalytical Chemistry, Saarland University, Campus B2.2, 66123 Saarbru¨cken, Germany *Author to whom correspondence should be addressed. Email: [email protected] Received 27 August 2014; revised 27 October 2014

In the first part of this review, stationary phases (silica, hybrid silica, hydride silica and non-silica stationary phases) were characterized and compared with respect to selectivity, efficiency, resolution, solvent consumption and analysis time. The present review focuses on the thermal and chemical stability of stationary phases. Stationary phases of high chemical and thermal stability are required for separations that are carried over a wide pH and/or temperature range.

Introduction Certain chromatographic procedures require aggressive liquid chromatography (LC) eluents and/or elevated temperature conditions, to improve selectivity and efficiency, to reduce the amount of organic modifier or to decrease analysis times. To achieve these objectives, reversed-phase (RP) stationary phases of high chemical and thermal stability are necessary (interested readers are referred to Classens and van Straten’s extensive review (1) of the subject). In LC, column temperature can be increased by isothermal or gradient programing, to improve kinetic performance, reduce mobile phase viscosity or to change selectivity. The benefits of temperature programing have been reviewed in a series of recent articles (2– 12). Nowadays, due to the development of highly stable stationary phases and improved instrumentation, separations can be conducted from sub-ambient temperatures to up to .2008C. In the first part of this review, stationary phases were reviewed (13). This second part focuses on thermal and chemical stabilities of the previously described stationary phases. An overview of the commercially available stationary phases, which tolerate high temperatures and a large pH range, is shown in Table I (11).

Stationary phase chemical stability The limited stability of silica-based stationary phases in neutral to high alkaline mobile phases is caused by dissolution effects, i.e., increased solubility at high pH values. Numerous attempts have been made to increase the stability of stationary phases for alkaline mobile phases. Initially, increased ligand surface coverage (bonding density) on the silica surface was conducted to improve thermal and chemical stability (14). Due to steric hindrance, however, it was impossible to react all of the available silanol groups on the surface of the silica with silane, even after applying multiple end-capping reactions; .50% of the

initial silanol groups remained intact in these experiments (14). Furthermore, even stationary phases having a low number of surface silanol groups, which are usually considered to be chemically reactive, are still unstable in alkaline media. For example, the Luna 5 m C18 (2) stationary phase was shown to degrade after passage of 4,700 column volumes of the alkaline mobile phase acetonitrile/phosphate ( pH 10; 20 mM; 45 : 55 v/v) at 308C (15). Claessens and van Straten (1) have shown that the firstgeneration hybrid stationary phase (XTerra MS C18), which exhibited fewer residual silanol groups due to substitution of –SiOH by –SiCH3 groups, were more stable in an alkaline mobile phase (methanol/carbonate buffer; pH 10, 100 mM, 50:50, v/v) than other stationary phases prepared from non-hybrid silica (e.g., Nucleosil C18, C18 Hypersil, Nova-Pak C18 Lichrospher C18, Zorbax ODS and Zorbax Rx-C18). An exception was Zorbax Extend-C18 stationary phase, which is prepared by functionalization of silica with bidentate silanes. This column exhibited stability similar to XTerra MS C18. Second-generation hybrid stationary phases, which exhibit even fewer residual silanol groups due to substitution of –SiOH by – CH2 – CH2 – groups, are more stable than their firstgeneration counterparts. They are also more stable than silica stationary phases. Figure 1 illustrates the performance of the new generation silica hybrid XBridge C18 stationary phase, which was substantially more stable (no loss of acenaphthene efficiency after 200 h of testing) than the first-generation silica hybrid phases such as XTerra MS C18 and Gemini C18, and the traditional silica-based phases (e.g., Luna 5 m C18 (2), YMC Pro C18), which typically exhibited 70% loss of efficiency for acenaphthene after 20 h of testing (16). The examples shown in Figure 2 use aqueous sodium hydroxide as mobile phase and the stability decreased in the order XBridge C18 . XSelect C18 . Gemini NX C18 . XTerra C18 (17). The examples also demonstrate that C18 phases are more stable than phenyl and phenyl-hexyl stationary phases. It should be pointed out that assessment of phase stability using 100% aqueous conditions is not advised as certain phases undergo different degrees of phase collapse/de-wetting, in particular when low pressures are employed (18–20). The use of 100% aqueous mobile phases can result in reduced exposure of the phase to the alkaline mobile phase. Interestingly, AkzoNobel claimed that their Kromasilw Eternity phase is more stable than Waters’ second-generation hybrid

# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Table I Commercially Available LC Columns for High Temperature Operation (Most of the Data Were Collected from Reference (11)) Base material

Column (manufacturer)

Available stationary phases

Tmax (8C)

Silica, sterically protected

Zorbax StableBond (Agilent Technologies)

C18 C8, C3, CN, phenyl XB-C18 XB-C18 XB-C18 and XB-C8 C18, C8, Phenyl-hexyl, RP-Amide, pentafluorophenylpropy, CN, PEPTIDE ES-C18, protein C18 C18, cholesterol C8, C18 C18 C18 C18, C6Phenyl C18 CARB, OS PBD, PS Carb, Diamondbond C18 PBD, PS Carbon, Carbon C18 PS/DVB PS/DVB PS/DVB DVB, C18-DVB Graphitized carbon Diammond

90 80 90 90 90 90 200 100 150 150 150 150 150 100 150 200 150 200 150 150 150 150 200 200

Silica, polydentate Silica, hybrid particle technology

Silica, encapsulated Titanium oxide Zirconium oxide

Poroshell (Agilent Technologies) Kinetex (Phenomenex) AerisTM WIDEPORE (Phenomenex) Halo (Advanced Materials Technology) Blaze 200 (Selerity Technologies) Cogent (MicroSolv Technologies) YMC Triart (YMC) Kromasil Eternity (AkzoNobel) Gemini NX (Phenomenex) Xbridge (Waters Corporation) Pathfinder (Shant Laboratories) Zirchrom (Zirchrom Separations) Zirchrom (Zirchrom Separations) Discovery Zirconia (Supelco)

Polystyrene/divinylbenzene

Graphitized carbon Diammond

PLRP-S (Polymer Laboratories) PRP-1 (Hamilton) Shodex ET-RP1 (Showa Denko) Jordi RP (Jordi Associates) Hypercarb (Thermo Electron) Flare (Diamond Analytics)

Figure 1. Stationary phase stability: efficiency loss as a function of time of purge with an aggressive mobile phase (analyte: acenaphthene; mobile phase: triethylamine, pH 10; 50 mM at 508C). Figure reprinted with permission from reference (16).

phase as determined by gradient elution chromatography using acetonitrile/ammonium carbonate ( pH 10, 10 mM) at 408C for 8,000 min (Figure 3) (21). Phenomenex evaluated its hybrid stationary phase, Gemini NX, using the same conditions and test compounds as described for Kromasilw (Figure 4) for a significantly longer time (40,000 min) and observed that Gemini NX lost 20% of its efficiency after 30,000 min of testing. One possible explanation for apparent differences of stabilities are differences of test conditions as well as choice of analytes (22). In the described case, Kromasilw Eternity stationary phases were hybrid-encapsulated stationary phases, which have a hybrid surface similar to XBridge and a silica core (13).

Buffer type and pH effects It has been demonstrated that parameters such as pH of mobile phase, temperature, buffer concentration and type of buffer have 1108 Borges and Volmer

Figure 2. Results of accelerated base stability testing for six stationary phases, showing percent change of retention factor (k) for decanophenone versus exposure time (h) to aqueous sodium hydroxide (20 mM; pH 12.3) at 508C. The stationary phases were purged at 0.85 mL/min for 1.8 h, washed for 10 min at 0.43 mL/min. Mobile phase: acetonitrile/water, 50:50 v/v. Columns: XTerra MS C18 (50  3 mm), Gemini C18 and Xbridge C18 (50  4.6); all other columns 30  3 mm. Figure reprinted with permission from reference (17).

significant influence on the dissolution rate of the silica (1, 15). The type of buffer is a very important factor that strongly affects the stability of the stationary phase; it was shown to be as important as the mobile phase pH. Buffers that are strong Lewis bases, such as phosphate and carbonate, are usually more aggressive than weak Lewis buffers (e.g., amino buffers). For example, Borges and Collins (23) evaluated the stability of stationary phases, which were prepared by thermal immobilization of poly(methyloctylsiloxane) on a silica surface. They observed that these stationary phases failed after 10 h of continuous purging of mobile phase with 20 mM phosphate buffer ( pH 11), while the same stationary phases were almost not affected after 100 h of continuous purging with 20 mM phosphate ( pH 7) and 20 mM triethylamine buffer ( pH 11.5).

Claessens et al. (24) have shown that a mobile phase consisting of a methanol/phosphate buffer resulted in a higher degradation rate of the stationary phase (Zorbax SB-CN, 150  4.6 mm, 5 mm) as compared with a mobile phase of the same composition, but buffered with weak Lewis base (Figure 5). Until 2004, zirconium-based stationary phases such as ZirChrom PDB, ZirChrom DiamondBond C18 and ZirChrom Carb were the only inorganic-based oxide materials compatible with the use of strong Lewis buffers such as phosphate and carbonate. For example, Figure 6 illustrates that at pH 12, XTerra RP18 was quickly degraded, while Zirchrom PBD remained stable (25).

Figure 3. Efficiency loss for (A) predinisole and (B) amitriptyline as a function of purging time. Columns: Kromasil Eternity-5-C18 and Waters XBridge C18 (250  4.6 mm, 5 mm). Gradient elution: 0 min 100% A, 15 min 100% B, 16 min 100% A, 20 min 100% A, where A ¼ 90:10 acetonitrile/ammonium bicarbonate (10 mM; pH 10.5) and B ¼ 10:90 acetonitrile/ammonium bicarbonate (10 mM; pH 10.5). Flow rate: 1 mL/min. Temperature: 458C (Courtesy of Kromasil. Reprinted with permission).

Figure 4. Stability test at alkaline conditions using Gemini and Gemini NX columns. The test evaluated efficiency loss of amitriptyline and predinisole as a function of purging time, where both test compounds exhibited the same pattern (Figure provided by Phenomenex. Reprinted with permission).

Figure 5. Effect of buffer type on stationary phase stability. (A) Phosphate buffer, (a) initial, (b) after 1,060 mL and (c) after 3,540 mL. (B) Tris, (a) initial, (b) after 1,420 mL and (c) 3,600 mL. (C) Citrate, (a) initial, (b) after 2,740 mL and (c) after 4,120 mL. (D) HEPES, (a) initial, (b) after 1,360 mL and (c) 5,210 mL. Column: Zorbax SB-CN 150  4.6 mm. The columns were purged with acetonitrile/phosphate buffer ( pH 7; 250 mM; 20:80 v/v) at 608C using a flow rate of 1 mL/min. Before testing, the column was first flushed with at least 20 column volumes of methanol/water (60:40), before equilibration with 20 column volumes of phosphate buffer ( pH 7; 250 mM; 20:80 v/v). Evaluation performed for tricyclic antidepressants: 1 ¼ uracil, 2 ¼ doxepin, 3 ¼ trimipramine, 4 ¼ amitriptyline, 5 ¼ nortriptyline. Figure reprinted with permission from reference (24).

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for LC 1109

Figure 6. High pH stability of PBD–zirconia versus Waters XTerra RP 18 column. LC conditions: ZirChromw-PBD; mobile phase, 28/72 ACN/20 mM potassium phosphate at pH 12; Zorbax EXTEND; 35/65 ACN/20 mM potassium phosphate at pH 12; flow rate, 1.0 mL/min; temperature, 408C; detection at 254 nm. Solutes: (1) labetolol; (2) atenolol; (3) acebutolol; (4) metoprolol; (5) oxprenolol; (6) quinidine; (7) lidocaine; (8) alprenolol and (9) propranolol. Figure reprinted with permission from reference (25).

The pH value of a mobile phase consisting of an organic modifier and an aqueous buffer is referred to as swpH, whereas the buffer in a fully aqueous mixture is designated as w wpH (26, 27). s The w pH of a mobile phase varies depending on the type of buffer ( phosphate, carbonate, borate, Tris, citrate, etc.) and the percentage of organic modifier. Mobile phases prepared with inorganic buffers ( phosphate, borate, etc.) exhibit swpH values w pH of aqueous phases before addition of the orhigher than the w ganic modifier. In contrast, mobile phases prepared with amino buffers (ammonium, Tris, tricine, triethylamine, piperazine, etc.) s have w pH values that are lower than w wpH of the aqueous phase s before addition of the organic modifier, while the w pH values of a mixture of methanol with an aqueous buffer are different from swpH of a mobile phase prepared with acetonitrile (Table II). Thus, the higher stability of the stationary phases observed when amino and organic buffer are employed can be readily explained by the lower swpH values of the mobile phases as compared with those prepared from inorganic buffers. Figure 7 illustrates the dramatic shifts of both retention and selectivity, which are possible by varying the mobile phase pH. In these experiments, the selectivity of a mixture of acidic, neutral and basic compounds was significantly changed for different mobile phase pH (28). At low pH, basic compounds eluted first from the column, whereas at high pH, the acidic compounds came out first. Clearly, these selectivity shifts can be conveniently used during method 1110 Borges and Volmer

development. For the particular compound mixture shown in Figure 7, the optimum separation was obtained at pH 8.0. In the same context, it is interesting to note that in reversed phase liquid chromatography (RPLC) basic compounds are commonly analyzed at a mobile phase pH value, which is two units higher than the compound’s w wpH value. For example, for analysis of nortriptyline (w wpH 10), it was expected that at pH 12, the analyte was in its neutral form, thus increasing retention through s increased hydrophobic interactions. However, the w pH value of s the basic analyte decreased in the mobile phase (wpKa) and nortriptyline was already in neutral at swpH 10. As a result, nortriptyline exhibited constant retention behavior for swpH 10 because in aqueous acetonitrile, swpKa and swpH were lower and higher, w respectively, than w wpKa and wpH values. Table III highlights effects of type and composition of organic modifier on pKa of various basic solutes. In all cases, the addition of organic modifier resulted in a reduction of the bases’ pKa (26, 27). Thus, basic solutes can be readily analyzed in their neutral form in mobile phases with w wpH slightly lower than the compound’s w wpKa values as previously shown in Figure 7. In RPLC mode, basic solutes are more strongly retained using alkaline mobile phases, because they are in their neutral forms and hydrophobic interactions thus dominate. In electrospray ionization (ESI) LC-MS, elution of analytes from the column using higher amounts of organic modifier in the mobile phase is

Table II s wpH Variation as Function of Organic Modifier Concentration in the Mobile Phase at 208C (Taken from References (26, 27)) pH 0% % Methanol H3PO4 – H2PO2 4 22 H2PO2 4 –HPO4 2 H3Cit –H2Cit H2Cit2 – Cit22 HCit2—Cit32 HAc –Ac2 NHþ 4 –NH3 % Acetonitrile H3PO4 – H2PO4 2 H2PO42 –HPO22 4 H3Cit –H2Cit2 2 2 H2Cit – Cit 2 HCit2—Cit32 HAc –Ac2 NHþ 4 –NH3 H2CO2 –HCO2 2 Piperazina (pKa1) Piperazina (pKa2) Tris Borate 22 HCO2 3 –CO3

20%

40%

2.11 7.20 3.13 4.76 6.40 4.76 9.24

2.63 7.55 3.44 5.12 6.83 5.05 9.11

3.09 8.04 3.84 5.53 7.39 5.43 8.97

2.11 7.2 3.13 4.76 6.4 4.76 9.24 3.72 5.37 9.76 8.08 9.23 10.35

2.28 7.48 3.15 4.32 6.55 5.39 9.79 3.96 5.26 9.62 7.94 9.85 10.82

2.55 7.66 3.43 4.65 6.84 5.8 9.39 4.4 5.19 9.53 7.85 10.43 11.31

50% 3.35 8.36 4.07 5.8 7.66 5.66 8.89

60% 3.68 8.75 4.30 6.10 7.96 5.92 8.82 2.54 8.01 3.64 5.01 7.28 6.47 9.25 4.87 5.06 9.42 7.72 11.00 11.62

80% 4.3 9.58 4.77 6.64 8.60 6.46 8.63

advantageous, because of the improved desolvation process in ESI under these conditions. Analysis of basic compounds on RP columns by ESI-MS has traditionally been accomplished by means of acidic mobile phases (e.g., by addition of 0.1% formic acid to the solvents). Basic compounds are often ionized in the bulk solution prior to ESI, which enhances ion formation (29). On the other hand, some studies have reported higher ESI response at high pH (29 –36). Peng and Farkas (35) illustrated how separations conducted at alkaline conditions could afford increased response for positive ion ESI-MS (Figure 8). They applied a Gemini C18 stationary phase (150  3 mm, 5 mm) to analyze a series of basic pharmaceutical compounds in gradient elution mode using a buffered acetonitrile mobile phase. Higher resolution and larger peak areas were obtained for 10 mM ammonium hydrogencarbonate buffer as compared with 0.1% formic acid in the mobile phase. This increase was attributed to increased retention and better peak shape for alkaline buffer as compared with acid mobile phase. Figure 8A shows that the analytes were more retained and had higher peak height for the alkaline mobile phase as compared with the acidic mobile phase, while Figure 8B demonstrates the better peak shape for the alkaline mobile phase as compared with acid mobile phase.

Figure 7. Separation of a mixture of acidic, basic and neutral compounds using different mobile phase pH values. Column: XTerra RP18 (150  3.9 mm, 5 mm). Mobile phase: 20 mM buffer/acetonitrile 65:35 v/v; potassium phosphate buffers were used at pH 2.5, 7.0 and 8.0, ammonium acetate was used for the pH 5.0 mobile phase, potassium borate for pH 9.5 mobile phase and pyrrolidine hydrochloride for pH 10.6. Detection wavelengths: 210 nm for pH 2.5, 5.0 and 8.0, 230 nm for pH 10.6. Flow rate: 1.0 mL/min. Peaks: 1 ¼ acetaminophen, 2 ¼ lidocaine, 3 ¼ doxepin, 4 ¼ imipramine, 5 ¼ nortriptyline, 6 ¼ ibuprofen. Figure reprinted with permission from reference (28).

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for LC 1111

Table III s wpKa Values for Several Basic Solutes as Function of Organic Modifier Concentration and Type of Organic Modifier at 258C (Taken from References (26, 27))

Methanol Nortriptyline Diphenhydramine Quinine Codeine Procainamide Benzylamine Protriptyline Amitriptyline Acetonitrile Nortriptyline Diphenhydramine Quinine Codeine Procainamide Benzylamine

70%

DpKa

60%

DpK

40

DpK

30%

DpK

20%

DpK

Literature

9.25 8.24 7.94

20.94 20.92 20.54

8.45 8.86 9.73 8.27

20.87 20.59 20.98 21.05

9.43 8.48 8.11 7.56 8.68 9.01 9.88 8.53 60% 9.72 8.69 8.34 7.54 8.99 8.78

20.76 20.68 20.37 20.64 20.64 20.44 20.83 20.79 DpK 20.61 20.56 20.22 20.71 20.43 20.77

9.72 8.78 8.33 7.8 8.93 9.15 10.21 8.92 40% 9.87 8.92 8.47 7.84 9.16 9.11

20.47 20.38 20.15 20.4 20.39 20.3 20.5 20.4 DpK 20.46 20.33 20.09 20.41 20.26 20.44

9.84 8.89 8.38 7.9 9.05 9.23 10.31 9.09

20.35 20.27 20.1 20.3 20.27 20.22 20.4 20.23

9.96 9 8.44 8.06 9.15 9.33 10.43 9.27 20% 10.11 9.16 8.59 8.09 9.34 9.38

20.23 20.16 20.04 20.14 20.17 20.12 20.28 20.05 DpK 20.22 20.09 20.03 20.16 20.08 20.17

10.0 –10.11 9.00 –9.40 8.39 –8.52 7.83 –8.21 9.20 –9.40 9.33 –9.73 10.7 9.40 –9.45 Literature 10 –10.11 9 –9.4 8.39 –8.52 7.83 –8.21 9.2 –9.42 9.33 –9.73

Similar results were obtained by Rainville et al. (32), who analyzed 24 basic, acidic and neutral pharmaceuticals with an Acquity BEH C18 (50  2.1 mm, 1.7 mm) stationary phase using gradient elution with acidic or alkaline mobile phases (formic acid, 0.1% v/v, or ammonium hydroxide, 0.1% v/v). Of the 24 compounds, 21 showed larger average peak areas for the basic mobile phase. The increase of peak area ranged from 1.2- to 9.6-fold. The observed increase was not necessarily related to the late elution of the analyte and the resulting higher organic composition at basic conditions. For example, Figure 9 demonstrates a 7- and 10-fold increase of signal-to-noise ratio for propranolol and lidocaine, respectively, for basic versus acidic conditions, clearly showing that low limits of quantification (LOQ) can be obtained from basic eluents. Temperature effects Wiese et al. (6) employed the linear elution strength (LES) model, which was adapted from temperature programing in gas chromatography (GC), for systematic method development in high-temperature liquid chromatography (HT-LC). For example, Figure 10 illustrates that LES can be used to substitute organic modifier gradients or, in combination with organic modifier gradients, to improve selectivity (37). In HT-LC, the mobile phase must be pre-heated. Without preheating, the center of the column would be at lower temperature caused by cold mobile phase entering the column, thus creating a temperature gradient (Tcenter , Twall ), resulting in viscosity and retention time differences. This thermal divergence is avoided by preheating the mobile phase to the column temperature (38) as shown in Figure 11 (39). Nowadays, ovens are able to preheat the mobile phases adding only minimal dead volume to the chromatographic system (i.e., the CTO-20AC oven from Shimadzu, which only adds 10 mL to the dead volume). There are also systems especially designated for preheating the mobile phase, such as Polartherm 9000 from Selerity Technologies and Metalox 200C from ZirChrom Separations (40). HT-LC also results in flatter van Deemter curves. Figure 12A shows the dramatic effect of temperature on the mass transfer within the stationary phase (38). This improvement in efficiency 1112 Borges and Volmer

is especially pronounced at higher linear velocities, where the C term is the dominant contribution to reduced plate height (h ¼ plate height/particle size). Elevated temperatures increase interphase mass transfer, resulting in higher column efficiency at higher flow rates. At the same time, higher mobile phase linear velocities also give a dramatic reduction of eluent viscosity as the temperature is increased. In addition, the curve minimum reduced plate heights (hopt) shift to faster flow rates as the temperature increases as shown in Figure 12A, while the absolute values of hopt are virtually unaffected by temperature rise as shown in Figure 12A (40). In the first part of this review (13), polymeric stationary phases were neglected because they are usually associated with poor efficiency as compared with silica. However, increasing the analysis temperature on polymeric phases leads to an increased optimum mobile phase velocity and significantly decreased in hopt as shown in Figure 12B (38). In this figure, hopt dropped from 3.4 at 258C (optimal flow 0.4 mL/min) to 2.3 at 1508C (optimal flow 2 mL/min). The performance of the polymeric phase improved significantly as a function of temperature in the range of 100 – 1508C, while values for hopt for the silica-based stationary phase remained unchanged with an increase in temperature (38). Analyte degradation was not common in HT-LC. However, some functional sites of chromatographic material catalyzed analyte degradations. Figure 13 illustrates this situation for analysis of thalidomide carried out using a carbon-clad zirconium dioxide stationary phase as well as a polystyrene divinylbenzene stationary phase (41). On the carbon-clad zirconium dioxide stationary phase, the thalidomide peak areas decreased with increasing temperature (41) (Figure 13A). On the other hand, on the polystyrene divinylbenzene stationary phase, the thalidomide peak area remained constant with an increase of temperature (Figure 13B) (41). This clearly illustrates that the stationary phase has a pivotal role on analyte stability on silica- and metalbased stationary phases, where sites might be able to catalyze degradation, while polymeric stationary phases do not possess such sites. If the column is heated to temperatures up to 2008C, hydrolysis of the bonded phase, dissolution of the support material or column hardware degradation may occur, which is summarized

Figure 8. Effect of acidic and alkaline conditions on positive ion ESI-MS. (A) Overlaid XICs for each of the 14 basic compounds eluted in mobile phases containing either 0.1% formic acid (gray line) or 10 mM ammonium carbonate pH 10 (solid line). (B) Peak asymmetry for 14 basic compounds eluted in mobile phase of pH 2.7 and 10, respectively. Figure reproduced with permission from reference (35).

as column bleed (42). Such column bleed can significantly influence LC-MS detection. Teutenberg et al. (42) have shown that a polymeric stationary phase (PLRP-S from Polymer Laboratories) exhibited less bleeding than zirconium dioxide and graphitized carbon stationary phases, as shown in Figure 14, where detection was carried out using a charged aerosol detector (CAD). CAD response is believed to be directly proportional to the analyte concentration (42). The prototype titanium dioxide stationary

phase used in the study is now commercially available as Sachtoporew-RP. It is very important to mention that some of the practical problems with HT-LC are caused by the tendency of column manufacturers to employ polyether ether ketone (PEEK) mounted frits or internal PEEK ferrules, which are not readily accessible by the user. However, under the temperatures and pressures used in HT-LC, the PEEK material can start leaking Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for LC 1113

Figure 9. Signal-to-noise ratio for propranolol (A) and lidocaine (B) spiked into plasma, prepared by protein-precipitation on positive ion ESI-MS. Conventional acidic compared with basic mobile phase. Column: Acquity BEH C18 (2.1 mm  50 mm, 1.7 mm). Mobile phase A consisted of water with formic acid (0.1%, v/v) or water with ammonium hydroxide (0.1%, v/v). Mobile phase B consisted of methanol. The column was maintained at 458C and eluted under linear gradient conditions from 5 to 95% B over 2.0 min at a flow rate of 0.6 mL/min. Figure reprinted with permission from reference (32).

after a single run, or as worst case scenario, coats the frit and blocks mobile phase flow (43). For example, Teutenberg et al. (44) have shown that Hypercarb columns lost their efficiency very fast after heating cycles. This was caused by the limited thermal stability of the PEEK frits; these Hypercarb columns were very stable for several heating cycles during 1 week, however, when metal frits were used instead (45). Temperature is another important variable that affects the rate of silica dissolution. Higher temperatures increase solubility of silica in the mobile phase, even at neutral pH (1, 7, 10, 23, 46 – 49). When a Zorbax Rx-C18 stationary phase was exposed to an acetonitrile/phosphate buffer ( pH 7; 250 mM; 20:80 v/v), 1114 Borges and Volmer

Figure 10. Chromatograms illustrating utility of temperature and flow-rate gradients compared with conventional solvent gradient approach. (A) Solvent gradient separation; (B) isocratic-temperature gradient separation and (C) combination of temperature and flow-rate gradient. Chromatographic conditions, column: Dionex Acclaim C18 (2.1  100 mm, 3 mm), mobile phase: A, 10 mM NH4OAc ( pH 5.5); B, acetonitrile; flow rate: 0.5 mL/min for A and B; sample concentration: 0.1 mg/mL of each analyte; injection volume: 25 mL; elution order: (1) aufamerazine, (2) sulfamethazine, (3) sulfamethizole, (4) sulfamethoxazole, (5) sulfamethoxine, (6) furosemide, (7) prednisolone and (8) indapamide. Figure reprinted with permission from reference (37).

virtually no silica was dissolved at 408C. However, at 608C, rapid dissolution of silica into the mobile phase was seen (15). Borges and Collins (5) showed that a stationary phase, which was virtually unaffected after 100 h of purging with a methanol/ triethylamine buffer ( pH 11; 20 mM; 65:35 v/v) at 238C, was

Figure 11. Chromatograms showing the effect of efficient (A) and inefficient (B) preheating on peak shape. Solutes: benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene; column Zirchrom-DB-C18 50  4.6 mm; flow rate, 4 mL/min; temperature 1508C; preheating tube 127 mm i.d.; length of preheating tube 2 m (a) and 1 m (b). Figure reprinted with permission from reference (39).

entirely destroyed after only 50 h of purging with the same mobile phase at 608C. They also showed that a methanol/phosphate buffer (pH 7; 20 mM; 65:35 v/v) at 608C was as aggressive as the same mobile phase at pH 12 at only 238C. Borges and Collins (5) observed that a methanol/carbonate buffer (pH 10; 5 mM) (65:35, v/v) at 238C was more aggressive than the same mobile phase using borate instead of carbonate, at 238C, as previously described by Claessens et al. (24). Interestingly, Borges and Collins (23) observed that both mobile phases were equally aggressive at 608C. Hybrid stationary phases have been frequently used at high temperatures, efficiency and/or retention losses as a function of time have not been reported. For example, Al-Khateeb and Smith (50) applied a XTerra stationary phase to analyze hydrophobic steroids using methanol/water mobile phases at 50–1108C, as well as a XBridge phenyl stationary phase at 50–2008C. In both cases, no degradation of the stationary phase was observed. Another interesting example was shown by Wiese et al. (6), who examined Acquity Phenyl (100  2.1 mm, 1.7 mm) and XBridge C18 (75  4.6 mm, 2.5 mm) columns using water/acetonitrile at 50 – 1808C. They reported no stationary degradation during the course of the experiments, even for long exposure times at high temperatures (Figure 15) (51). In addition, it was shown that isothermal variation of temperature was a powerful tool to modify selectivity and reduce analysis time.

Figure 12. Reduced plate height versus flow rate for acetophenone: (A) Blaze 200 (3 mm particles) and (B) ET-RP1 (5 mm particles) column. Mobile phase: water/ acetonitrile; injection volume: 2 mL; detection: diode array detector (DAD) 210 nm. The mobile phase composition was modified to keep the k-value nearly constant at 1.8. Figure reprinted with permission from reference (38).

Hybrid and non-silica stationary phases are very stable at elevated temperatures and can be readily used with non-buffered mobile phases at high temperatures. Zhang et al. (52) utilized XBridge C18 (up to 1808C), Acquity C18 (2008C), Triart C18 (1508C) and Zirchrom PBD under isothermal and temperature gradient conditions, by isotope ratio mass spectrometry coupled with HT-LC, without observing column deterioration or column bleed. The study nicely demonstrated that new hybrid stationary phases (XBridge C18 and Acquity C18), special chemical bonding and end-capping (Triart C18) and zircoinia (Zirchrom PBD) can be implemented at temperature .1008C with a non-buffered mobile phase. Polar and ionized compounds are especially sensitive to temperature effects in RPLC. Small changes (20–608C) of temperature can result in large variations of selectivity, improved peak shapes, reduced peak widths and analysis times (9, 53, 54). For example, for a separation of several basic pharmaceutical drugs at 208C, a total analysis time of 45 min was observed, while the same mixture was separated in ,7 min, when the temperature was raised to 608C, with peaks heights 8-fold higher than at 208C (12). Large molecules such as peptides and proteins also Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for LC 1115

Figure 14. Dependence of detector response on temperature programed measurements for four different HPLC columns (C18 column not included). Detector: CAD; flow rate of mobile phase ( pure water): 0.5 mL/min; temperature program: 5 min at 308C, from 5 to 10 min from 30 to 2008C, 10 min constant at 2008C, then cooling down to 308C. Figure reprinted with permission from reference (42).

An example of high thermal stability of zirconium-based phases is given in Figure 16 (59). Figure 16A illustrates that at 508C, steroids analyzed on a Discovery Zr–Carbon C18 column took a long time to elute and gave broad peaks. On the other hand, at 1508C, fast separation with sharp peaks was obtained (Figure 16B). In addition, no stationary phase degradation was reported during the course of this study. Buffer concentration effect

Figure 13. Monitoring of on-column degradation of thalidomide on a (A) ZirChrom Carb column (150  4.6 mm, 3 mm) from (a) 608C to (g) 1808C in 208C increments. Chromatographic conditions: flow rate, 1 mL/min; mobile phase, 75% deionized water/25% acetonitrile (þ0.1% formic acid each); detector, DAD at 300 nm. (B) PLRP-S column (150  4.6 mm, 3 mm) from (a) 408C to (h) 1808C in 208C increments. Chromatographic conditions: flow rate, 0.2 mL/min; mobile phase, 80% deionized water/20% acetonitrile (þ0.1% formic acid each); detector, DAD at 300 nm. Figure reprinted with permission from reference (41).

exhibited large variations of retention factors after small changes of temperature (55, 56). While HT-LC using latest generation stationary phases offers many advantages and stabilities at higher temperatures are not an issue for non-buffered mobile phases (vide supra), the same, unfortunately, does not hold true for neutral and alkaline mobile phases, where temperature increases can dramatically accelerate degradation of the phase. For example, the ZirChrom PDB phase was virtually unchanged in an alkaline mobile phase at pH 10 –12 ( phosphate, carbonate buffer) and 408C (25); it was also virtually unaffected in non-buffered mobile phases at 1008C (57). However, ZirChrom PDB quickly degraded at pH 7 ( phosphate buffer) and 1208C (58). 1116 Borges and Volmer

Kirkland et al. (15) demonstrated that solubility of silica increases with buffer concentration, even though these effects were not particularly strong. For example, a 250 mM buffer solution dissolved ca. 1/3 more silica than a 50 mM solution at 608C and pH 7. Much larger amounts of silica were solubilized with sodium phosphate buffer in comparison to Tris. Only moderate differences were seen between 250 and 50 mM phosphate buffer concentrations, but larger silica solubility differences were observed for 250 and 50 mM Tris buffers. Borges and Collins (23) observed that effects of methanol/triethylamine buffer ( pH 11.5; 20 mM; 65:35 v/v) at 238C were not much stronger as compared with the same mobile phase at a buffer concentration of 250 mM (9). These observations also support the conclusion that organic buffers may significantly prolong the lifetime of silica-based columns. However, mobile phase buffer concentrations should always be adequate to ensure that pH does not vary during separation. Stationary phase stability in acidic mobile phases Bare silica is very stable in acidic solutions, with the exception of exposure to hydrofluoric acid (23). In contrast, siloxane bonds of derivatized silica materials are susceptible to hydrolysis in acidic mobile phases (19). Typically, shorter chain bonded ligands such as the groups used in end-capping reactions (trimethylsilyl, TMS), C4, phenylpropyl, cyanopropyl and pentafluorophenylpropyl are more susceptible to hydrolysis than ligands with longer chains such as C8 and C18 (60 – 62). Substitution of the typical methyl side groups (R) in silanes (XSi(R) 2R1) with

Figure 16. Effects of isothermal increase of temperature on separation of levonorgestrel and related compounds. Column: Discovery Zr – Carbon C18 (150  4.6 mm, 3.5 mm). (A) 508C; eluent 60% methanol in water; flow rate 0.5 mL/min. (B) 1508C; eluent 50% methanol in water; flow rate 1.5 mL/min. Figure reprinted with permission from reference (59).

Figure 15. Isothermal separations of five sulfonamides and uracil. Stationary phase: Waters XBridge C18 (75  4.6 mm, 2.5 mm); mobile phase: deionized water with 0.1% formic acid; flow rate: 1.0 mL/min; injection volume: 2 mL (60 and 808C) and 1 mL (from 100 to 1808C); detection: UV at 270 nm. Analytes: (1) uracil, (2) sulfadiazine, (3) sulfathiazole, (4) sulfamerazine, (5) sulfamethoxazole and (6) sulfamethazine. Figure reprinted with permission from reference (51).

bulky groups (e.g., isopropyl, iPr) increases stationary phase stability in acidic media. At this point, it must also be emphasized that zirconia, titania and porous graphitic carbon (PGC) are very stable in acid solutions, because they do not have bonded functionalities that would undergo hydrolysis at low pH. Zorbax StableBond-C18 is prepared using XSi(iPr)2C18 and is consequently a very stable phase under acidic conditions, because the Si –C bond on the silica surface is sterically protected by the bulky iPr group. This was demonstrated by Vanhoenacker and Sandra (11), who examined Zorbax StableBond-C18 (50  2.1 mm, 1.8 mm) and found the phase to be stable using nonbuffered mobile phases at 80 and 50 – 908C in gradient elution as well as gradient temperature mode (40 – 908C). Guillarme et al. (63) also used the same sterically protected stationary phase (150  4.6 mm, 5 mm) with an acetonitrile/water (35:65 v/v) mobile phase at 2 mL/min and 908C or, alternatively, acetonitrile/water (28:72 v/v) at 2.5 mL/min and 1208C. At 908C, the stationary phase remained unchanged after 7,000 column volumes. At 1208C, the stationary phase lost 75% of its efficiency over the same time period. The authors concluded that Zorbax SB can be routinely used at 90 –1008C without any significant deterioration of chromatographic performance in nonbuffered mobile phases, but stationary phase lifetime would probably be extended under acidic conditions [0.1% formic acid or trifluoroacetic acid (TFA)]. In this specific case, it was claimed that the acid mobile phase preserves the stationary phases rather than degrades it.

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for LC 1117

In general, hydrolysis of siloxane bonds is believed to solely catalyzed by protons. Ma and Carr (61) offered a similar line of thought. The authors showed that metal cations, released from acidic corrosion of the stainless steel inlet frit, greatly accelerated hydrolysis of siloxane bonds. These metal cations (and not the high acidity per se!) were the main cause for phase instability. Ma and Carr were able to support their view by removing the stainless steel inlet frit (and using a titanium frit), which greatly reduced or totally eliminated corrosion and improved stability of both stationary phase and column hardware. For analysis of macromolecular, it is widely reported that separations at high temperatures provide much better performance than those at or near ambient conditions (64). It is also a common assumption that the use of isopropanol reduces detrimental

interactions of peptides and proteins with the stationary phase. For example, an Aeris Widepore material was used to achieve excellent separation of proteins at high temperatures with a mobile phase containing isopropanol (64). Interestingly, Ma and Carr (61) observed that the use of isopropanol in the mobile phase prolonged the stationary phase life. Thus, it can be concluded that macromolecular analyses are readily possible using sterically protected stationary phases and isopropanol addition to the mobile phase further increases stability and efficiency at high temperatures. Nowadays, sterically protected bonding is widely used in core –shell stationary phases (65). The sterically protected bonding used in core –shell stationary phases give high stability at high temperature and acidic conditions. Indeed, these stationary phases can be used safely under acidic conditions at 75–908C. Figure 17 illustrates that a HALO Peptide ES-C18 stationary phase remained unchanged after 775 injections; Figure 18 demonstrates that Halo Protein C4 (Figure 18A) and HALO Peptide ES-C18 (Figure 18B) remained unchanged after purging 15,000 column volumes.

Type C stationary phase thermal stability at acidic conditions

Figure 17. Stability of HALO Peptide ES-C18 column, 100  2.1 mm, 2.7 mm particle size; flow rate: 0.5 mL/min; temperature: 608C; mobile phase A: water/0.1% TFA; B: 70/30 water/0.1% TFA; gradient: 9 – 55% B in 10 min; injection volume: 5 mL. Analytes: 1 ¼ Gly-Tyr, 2 ¼ Val-Tyr-Val, 3 ¼ Met-enk, 4 ¼ angiotensin II, 5 ¼ Leu-enk ribonuclease, 6 ¼ porcine, 7 ¼ insulin (Courtesy of Stephanie Schuster from Advanced Materials Technology).

Silica produced until the 1990s contained high concentrations of metals, which caused silanol groups to be highly acidic and, thus, afforded poor peak shapes for basic compounds. These silica materials were termed ‘Type A’ silica. Subsequently, new production technologies enabled synthesis of materials with much lower metal concentration, giving ‘Type B’ silica stationary phases. Type C silica replaced surface OH groups with SiH groups, making it significantly more stable than ordinary silica, as silica dissolution takes place at free silanol groups. Type C silica is commercialized under Cogent trade name by MicroSolv Technologies. Soukup and Jandera (66, 67) illustrated the high thermal and chemical stability of Type C stationary phases by studying retention of several flavonoids and phenolic acids. Experiments were carried out at 35 – 1008C with an acidic mobile phase

Figure 18. Halo Protein stationary phase stability under acidic and HT-LC conditions: (A) HALO Protein C4 column: 100  2.1 mm; mobile phase gradient, 25–40% acetonitrile/0.1% aqueous FA in 10 min; (B) HALO Protein ES-C18 column: 2.1  100 mm; mobile phase gradient: 25–45% acetonitrile/0.1% aqueous TFA in 5 min; temperature: 908C; flow rate: 1.0 mL/min; detector: 215 nm (Courtesy of Stephanie Schuster from Advanced Materials Technology).

1118 Borges and Volmer

(10 mM acetic acid). The authors did not observe any degradation of the Type C stationary phases throughout the study, as shown in Figure 19, where the increased temperature readily

yielded reduced analysis time and increased efficiency and selectivity.

Thermal and chemical stability of silica, hybrid silica and non-silica stationary phases

Figure 19. Separation of flavonoid compounds in RP mode. Conditions: mobile phase, 10 mmol/L ammonium acetate in 65:35% water/acetonitrile, pH of aqueous part adjusted to 3.26 using formic acid; flow rate: 0.5 mL/min; UV detection at 275 nm; injection volume: 10 mL. VR ¼ retention volume; VC ¼ volume of empty column. Peaks: 1 ¼ 7-Hydroxyflavone, 2 ¼ flavone, 3 ¼ apigenin, 4 ¼ biochanin A, 5 ¼ vanillin, 6 ¼ 4-hydroxycoumarin, 7 ¼ hesperidin, 8 ¼ esculin, 9 ¼ naringin. 10 ¼ hesperetin, 11 ¼ naringenin and 12 ¼ Scopoletin. Figure reprinted with permission from reference (67).

Teutenberg et al. (46) described the use of heating cycles to assess thermal and chemical stability of new generation stationary phases, which were claimed by their manufacturers to be extremely stable to extreme chemical and thermal conditions. The protocol assessed the stability of a range of stationary phases in buffered and non-buffered acid and alkaline conditions. The columns were subjected to heating cycles at 1508C for 5 h. The column was then cooled to 258C and its chromatographic performance evaluated using Neue’s test procedure (68). Stationary phases were initially exposed to five heating cycles using a non-buffered mobile phase (methanol/water, 10:90 v/v), then five heating cycles using an acidic mobile phase (methanol/phosphate, 10:90 v/v; 20 mM; pH 2.2) and finally five heating cycles at alkaline conditions (methanol/phosphate, 10:90 v/v; 20 mM; pH 12). Two studies carried out by Teutenberg’s group (21, 46) indicated that hybrid stationary phases (XBridge C18, Gemini NX C18 and YMC Triart) were stable at high temperatures in nonbuffered media and exhibited only minor deterioration under acidic conditions. These results for XBridge C18, Gemini NX C18 and YMC Triart are illustrated in Figure 20A –C, respectively. However, both phases suffered major degradation under alkaline

Figure 20. Stability of hybrid and a polymeric stationary phases: (A) column performance of XBridge C18: (a) test chromatogram obtained before column was heated to 1508C; (b) test chromatogram after 5th heating cycle, (c) after 10th heating cycle, (d) after 15th heating cycle. (B) Column performance of Gemini NX C18: (a) test chromatogram obtained before column was heated to 1508C; (b) test chromatogram obtained after 5th heating cycle, (c) after 10th heating cycle and (d) after 15th heating cycle. (C) Column performance of YMC Triart C18 column: (a) brand-new, (b) after neutral heating phase, (c) after acidic heating phase and (d) after first cycle of basic heating phase. (D) Column performance of Showa Denko Shodex ET-RP1 4D polymer-based column: (a) brand-new, (b) after neutral heating phase, (c) after acidic heating phase and (d) after basic heating phase. Analytes: uracil (1), methyl benzoate (2), n-butyl benzoate (3) and n-hexyl benzoate (4). Analytes for (A), (B) and (C) 1 ¼ dihydroxyacetone, 2 ¼ propyl paraben, 3 ¼ propranolol, 4 ¼ dipropyl phthalate, 5 ¼ naphthalene, 6 ¼ acenaphthene, 7 ¼ amitriptyline, for (D) uracil (1), methyl benzoate (2), n-butyl benzoate (3) and n-hexyl benzoate (4). For (A), (B) and (C) mobile phase: 65/35 (v/v) methanol–phosphate buffer ( pH 7; 10 mM), flow rate: 1 mL min21, for (D) mobile phase: 65/35 (v/v) acetonitrile/water, flow rate: 0.6 mL min21. UV detection was carried out at 254 nm for all experiments. (A) and (B) were adapted from reference (21), (C) and (D) were adapted from reference (46) (reprinted with permission).

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for LC 1119

conditions at high temperatures. Stationary phases based on traditional silica materials and first-generation hybrid stationary phases (Zorbax Stable Bond C18 and Gemini C18) exhibited deterioration even under non-buffered conditions, while ZirChrom PDB was observed as the most stable column for alkaline conditions. Haun et al. (21) used the above methodology to evaluate the following stationary phases: XSelect CSH C18, XBridge Amide, XBridge C18, Eternity-2.5-C18, YMC Triart C18 and Shodex ET-RP1 4D. The authors found XSelect CSH C18 and XBridge Amide to be less stable than XBridge C18. They also observed that the hybrid phases Eternity-2.5-C18 had low stability due to column hardware problems associated with the heating cycles (i.e., the frits were damaged by heating and cooling), while the polymeric stationary phase Shodex ET-RP1 4D provided higher chemical and thermal stability than hybrid stationary phases (compare Figure 20D with Figure 20A –C). Polymer encapsulated silica, such as Pathfinder C18, is also known as polymer encapsulated silica, made from organic and inorganic building blocks, one forming the internal silica core and another forming the external polymer capsule. They appear to have seen little use (43) and Teutenberg et al. (46) observed that they were degraded during the non-buffered heating cycles. As shown in Figure 20, polymeric stationary phases have reached the ‘state of art’ of chemical and thermal stability. However, these phases have low mechanical stability under high pressure (69) as typically seen in ultra high performance liquid chromatography (UHPLC) analysis (1,200 – 1,500 bar is routinely employed today (70)). With respect to high mechanical stability, the diamond stationary phases commercialized by Diamond Analytics under the Flare trade name are very promising materials, because they have a hardness, 22-times harder than ordinary silica. It is also more stable than PGC stationary phases because diamond has tetrahedral bonded carbon atoms with an sp3 configuration, while PGC has sp2 configuration bonded carbon atoms (71). These stationary phases have high thermal and chemical stability and have been used over a wide pH and temperature range (72). Unfortunately, there are only few studies on the stability of these phases, but Flare stationary phases have been shown to be stable at 1208C using a mobile phase of 60:40:0.1 water/acetonitrile/triethanolamine (v/v/v) at pH 11.3 (73). Conclusion The development of new, second-generation hybrid silica, Type C silica, PGC and zirconia polymer-coated stationary phases has provided chromatographers with tools to exploit a much wider range of experimental design (i.e., temperature and pH). Useful mobile phases now include buffers and solvents that were previously impossible to use, for example, alkaline eluents. These columns therefore make it much easier to achieve desired chromatographic selectivity or resolution targets for a particular analytical problem. They also provide reproducible separations at elevated temperatures under acidic conditions without column deterioration. The ability to perform analyses at low and high pH values as well as different temperatures considerably increases the likelihood of success when generic gradient programs are employed, for example in discovery applications and impurity profiling in pharmaceutical analyses. 1120 Borges and Volmer

Acknowledgments E.M.B. acknowledges financial support and a fellowship (11/07466-0) from FAPESP (‘Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo’), ‘Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico’ (CNPq 150098/2014-6) and ‘Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´ vel Superior’ (CAPES). We thank Prof. Melvin R. Euerby (University of Strathclyde), Stephanie Schuster (Advanced Materials Technology) and Fernando Barbisa Jr (‘Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto’) for helpful discussions and support, friendship and encouragement. D.A.V. acknowledges research support by the ‘Alfried Krupp von Bohlen und Halbach-Stiftung’.

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