Thermodynamic characterization of the adsorption of selected chiral compounds on immobilized amyloglucosidase in liquid chromatography

Immobilized amyloglucosidase was used as a chiral stationary phase (CSP). First, the retention and enantioselectivity of several model chiral amines and acids were investigated. We found that this CSP was unable to separate the enantiomers of acids, though all selected amines could be resolved. The adsorption of (R)- and (S)-propranolol and its influence on column temperature and 2-propanol content in the eluent were then studied in detail, using a three-step methodology. The adsorption was first evaluated using Scatchard plots; thereafter, the adsorption was characterized in detail by calculating the adsorption energy distribution. With this model-independent information, a better judgment could be made of the possible adsorption models selected in the last step, the model fitting to the data. In the case examined, the bi-Langmuir model (containing nonselective and enantioselective sites) describes the system well. The retention of (R)- and (S)-propranolol at low temperatures increases with the content of 2-propanol in the eluent, due to the increased saturation capacity of the enantioselective sites. The retention is an enthalpy-driven process at both types of sites, whereas the enantioseparation is due to differences between the entropy changes of the two enantiomers at the enantioselective sites. The enthalpy of adsorption at the nonselective sites is almost identical at the two concentrations of 2-propanol in the eluent. Enantioselective adsorption, on the other hand, is more exothermic at higher modifier content (20%). Thus, at high temperatures the retention decreases with increasing modifier content, whereas the opposite (unusual) trend is the case at low temperatures.     

Calculation of chromatographic band profiles with an implicit isotherm

The mechanisms of structure selective and enantioselective retentions of amines and acids on two chiral stationary phases based on wild type cellobiohydrolase I (CBH I) and its mutant D214N have been investigated. All the amino alcohols tested had an enantioselective site that overlaps with the catalytically active site of CBH I, whereas the enantioselectivity of prilocaine was not affected by the mutation. The hydroxyl group of the amino alcohols did not seem to be an important contributor to the total binding strength whereas a bromo substituent in the aromatic ring promotes a high enantioselectivity (alpha=7.05). Interestingly, the chiral recognition site of the acid warfarin overlaps with the binding site of the amino alcohols. Di-p-toluoyltartaric acid and dibenzoyltartaric acid were strongly retained probably due to electrostatic attraction, but no enantioselectivity was observed. The difference in retention characteristics for the amino alcohols on the two stationary phases was strongly pH-dependent. A change in elution order of different amino alcohols occurred when changing the pH from 5.0 to 7.0. The difference between the two phases was lower at low pH. The retention times could also be affected by ionic strength and by use of cellobiose as a mobile phase additive but no indication of ion-pair retention of the amines was observed, when adding hexanesulphonate as counter ion to the mobile phase. The temperature dependence of the retention of the enantiomers of propranolol at pH 7.0 on the mutant D214N was similar to what was earlier observed on the wild type CBH I at lower pH.     

Comparison of the separation efficiencies of chirobiotic T and TAG columns in the separation of unusual amino acids

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.     

Comparison of vancomycin-based stationary phases with different chiral selector coverage for enantioselective separation of selected drugs in high-performance liquid chromatography

Two vancomycin-based chiral stationary phases (CSPs) with different coverage of the chiral selector vancomycin (Chirobiotic V and Chirobiotic V2) were compared. beta-Blockers and profens, as structurally diverse groups of drugs, were chosen as analytes. Retention and enantioseparation of beta-blockers were studied in reversed-phase (RP) and polar-organic (PO) separation modes. Higher retention and better enantioresolution were obtained on the CSP with higher coverage of vancomycin in the both separation modes. Baseline separation of four beta-blockers (eight enantiomers) in the PO mode was achieved on the Chirobiotic V2 column within 15 min. The enantioseparation of profens did not bring so excellent and easy to interpret results. Higher retention of profens on the Chirobiotic V2 column was not always accompanied by an improvement of their chiral separation in the RP mode. The polar-organic mode was not suitable for these derivatives at all. The most interesting result was obtained with flobufen; its chiral center is further away from the rigid part of the molecule, which mostly causes difficulties in enantioselective recognition. Nevertheless, the enantiomers of flobufen were shown to be much better (baseline) resolved on the CSP with lower coverage of the chiral selector (Chirobiotic V).     

联苯类对映体在纤维素类手性固定相上的高效液 相色谱法直接拆分

涂敷纤维素-三(3,5-二甲基苯基氨基甲酸酯)于自制的球形氨丙基硅胶上,制备了手性固定相。用该固定相直接拆分了一系列外消旋联苯类保肝药物。考察了柱温和流动相中乙醇含量对样品的保留和立体选择性的影响,升高柱温或增大流动相中乙醇含量都会使对映体的保留因子和分离因子降低。氨考察榈的分离因子的对数与温度的倒数成线性关系。确定了对映体从流动相传递到固定相的吉布斯自由能变之差值、焓变之差值及熵变之差值。     

Direct high-performance liquid chromatographic separation of unusual secondary amino acids and a comparison of the performances of Chirobiotic T and TAG columns

Two macrocyclic glycopeptide antibiotic-type chiral stationary phases (CSPs) based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and TAG, respectively, were evaluated with regard to the high-performance liquid chromatographic separation of the enantiomers of 10 secondary alpha-amino acids (imino acids). The chromatographic results are given as the retention, separation and resolution factors, together with the enantioselective free energy difference corresponding to the separation of the enantiomers. By application of these two CSPs, excellent resolutions were achieved for the investigated compounds by using reversed-phase mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these particular amino acids ranged between 0.70 and -1.83 kJ mol(-1). It was established that better enantioseparations of the secondary alpha-amino acids were attained in most cases on the aglycone CSP.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

非手性-手性色谱-预测多反应监测法分析中药毛前胡的化学成分

中药毛前胡为伞形科植物短片藁本Ligusticum brachylobum Franch.的干燥根,主要用于治疗风热咳嗽痰多、痰热喘满、咯痰黄稠等证,富含香豆素类化学成分,含有多组对映异构体和非对映异构体.为了深入研究毛前胡的化学成分组成,特别是对映异构体的组成,研究建立了非手性-手性色谱-预测多反应监测法(achir...     

β-环糊精键合手性固定相分离苯并恶嗪类对映体

研究了在反相高效液相色谱模式下,基于点击化学的β-环糊精手性固定相对苯并恶嗪类对映体的手性分离情况。考察了流动相中有机改性剂的类型和比例、缓冲盐的浓度和pH值对分离的影响。考察结果表明: 乙腈作为有机改性剂比甲醇更有利于苯并恶嗪对映体的分离;乙酸三乙胺缓冲盐体积分数从0.1%增大到1.0%时,苯并恶嗪对映体的保留时间和分离度都随之减小,在pH 4.1时苯并恶嗪对映体具有最大分离度。因此确定乙腈和体积分数为0.1%的乙酸三乙胺缓冲盐流动相(pH 4.1)为最佳分离条件。分离机理研究结果表明,固定相和样品之间的包容络合相互作用以及样品和固定相之间的氢键作用,是样品得以分离的基础。本研究为进一步深入研究β-环糊精固定相提供了实验基础,同时也证明了点击化学在手性环糊精固定相制备中具有极大潜力。     

Adsorption behavior of a teicoplanin aglycone bonded stationary phase under harsh overload conditions

Silica-bonded teicoplanin aglycone allows enantioseparation of amino acids by reversed-phase liquid chromatography with a low organic solvent content. However, a reversible change in the adsorption behavior leading to a retention time shift (RTS) was observed when a preparative scale column was treated with harsh preparative chromatography-like conditions between finite-injection HPLC runs conducted under exactly the same conditions. This behavior was observed for all five investigated aliphatic and aromatic amino acids. In all cases, the retention times were prolonged after the overload conditions and the RTS was more pronounced for the later eluting d-enantiomer. We defined a standardized method for measuring the RTS and performed a systematic investigation on the influence of experimental conditions (type and concentration of pH modifier and organic modifier, temperature, pH) on the RTS. In this way a solvent composition--90/10 50 mM NH4Ac pH 5.8/MeOH--was identified that yielded no observable shift in retention time after overload conditions for both enantiomers. In order to treat the observed phenomenon on a mechanistic level, we applied band profile analysis based on the stochastic theory of chromatography and identified two different enantioselective sites. When the band profile analysis was performed on elution profiles obtained from runs with prolonged retention time after harsh overload conditions, the retention time shift could be attributed to both differentiable types of adsorption sites. One site was found to make both, enantioselective and non-selective contributions.     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

Existence of a low isoenantioselective temperature in complexation gas chromatography. Profound change of enantioselectivity of a nickel(II) chiral selector either bonded to, or dissolved in, poly(dimethylsiloxane)

The gas chromatographic enantiorecognition of the two enantiomeric pairs of Z- and E-2-ethyl-dioxaspiro[4,4]nonane (chalcogran), respectively, critically depends on whether the chiral selector nickel(II) bis[3-(heptafluorobutanoyl)-(1R)-camphorate] is chemically linked via a methylene spacer to poly(dimethylsiloxane) (Chirasil-Nickel 1), or is only dissolved in poly(dimethylsiloxane) (Chira-Nickel 2a). On 2a, the enantiomers are separated at ambient temperature with a large apparent enantioseparation factor alpha(app) whereas on Chirasil-Nickel 1 only a low alpha(app) is observed whereby the elution order of the enantiomers is reversed. Concise temperature-dependent studies show that on Chirasil-Nickel 1 a low isoenantioselective temperature, T(isoenant), of 80 degrees C is experimentally observed as the result of enthalpy/entropy compensation. For thermodynamic measurements, the method of the retention-increment R' has been employed. This concept separates achiral contributions to retention due to the poly(dimethylsiloxane) matrix, which are identical for enantiomers, from enantioselective contributions to retention, which are due to complexation of enantiomers with the chiral selector. From Van't Hoff plots, concise thermodynamic data of enantioselectivity, -Delta(D),(L)(DeltaG), -Delta(D,L)(DeltaH), Delta(D,L)(DeltaS) and T(isoenant) were obtained. It was found that minute changes in the structure of the spiroketals as well as miniscule differences of the nature of the chiral selector present in the stationary phase (chemically bonded versus physically dissolved) led to profound differences in enantioselectivity. The observation of a low isoenantioselective temperature T(isoenant) represents are rare phenomenon in enantioselective gas chromatography which both complicates the study of chiral recognition mechanisms and the correlation of the absolute configuration and retention due to the temperature-dependent reversal of the elution order.     

Practice and theory of enantioselective complexation gas chromatography

The practice and theory of enantioselective complexation GC is comprehensively reviewed for the first time. A multitude of racemic oxygen-, nitrogen- and sulfur-containing selectands can be separated without prior derivatization into enantiomers by complexation GC on optically active metal(II) bis[3-(perfluoroacyl)-(1R)-camphorate] selectors. Peak inversion is obtained when the selectors with opposite configuration are employed. Applications pertain to chiral analysis in asymmetric synthesis, enzymatic reactions, pheromone and flavour chemistry. Although the use of enantioselective complexation GC has diminished recently with the advent of modified cyclodextrins in enantioselective GC, the inherent principles of enantiorecognition together with other enantioselective phenomena can be elucidated easily by complexation GC. Using the concept of the retention-increment R' which allows the distinction between non-enantioselective and enantioselective contributions to retention, concise thermodynamic parameters of enantioselectivity - deltaD,L(deltaG) are accessible. The enantiomerization of configurationally labile enantiomers can be investigated and quantified by complexation GC. Four distinct enantioselective processes and four different coalescence phenomena have been discerned in complexation GC.     

Temperature and Solute Molecular Size Effects on the Retention and Enantioselectivity of a Series of D, L Dansyl Amino Acids on a Vancomycin-Based Chiral Stationary Phase

Summary The influence of column temperature (0–28 °C) and solute molecular size on the retention and enantioselectivity of a series of D, L dansyl amino acids with a non-polar side chain (valine, leucine, phenylalanine and tryptophan) were investigated using a vancomycin-based chiral stationary phase (CSP). The enthalpic and entropic terms for the solute-CSP association were determined from the linear vant Hoff plots. Two solute groups were distinguished in relation to these thermodynamic quantities: the solute group I (dansyl valine, dansyl leucine, dansyl phenylalanine) for which large negative values of enthalpic terms were obtained and the solute group II (dansyl tryptophan) for which H value was much less negative. The enthalpy-entropy compensation study revealed that the interaction mechanism was identical for the group I solute enantiomers but changed for D, L dansyl tryptophan. This was further exemplified as the group I compound enantiomers were resolved over the temperature range while the enantiomers of dansyl tryptophan were not separated in the operating conditions. Relationships between both the solute retention factors and apparent enantioselectivity, and the accessible surface area of the amino acid side chain indicated that when the solute molecular size increased (i) the retention was enhanced by the hydrophobic effect and (ii) the chiral discrimination decreased dependent, at least in part, on a steric hindrance phenomenon at the vancomycin aglycone pocket.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.     

Quick development of an analytical enantioselective high performance liquid chromatography separation and preparative scale-up for the flavonoid Naringenin

The HPLC enantioselective separation of (R/S)-Naringenin, a chiral flavonoid found in several fruits juices and well-known for its beneficial health-related properties, including antioxidant, anti-inflammatory, cancer chemopreventive, immunomodulating and antimicrobial activities, has been performed on both analytical and (semi)-preparative scale using an amylose derived Chiralpak AD chiral stationary phase (CSP). A standard screening protocol for cellulose and amylose based CSPs was firstly applied to analytical Chiralcel OD-H and Chiralpak AD-H, as well as to Lux Cellulose-1, Lux Cellulose-2 and Lux Amylose-2 in order to identify the best experimental condition for the subsequent scaling-up. Using Chiralpak AD-H and eluting with pure methanol (without acidic or basic additives) relatively short retention times, high enantioselectivity and good resolution (α=1.49, R(s)=3.48) were observed. Therefore, these experimental conditions were properly scaled-up to (semi)-preparative scale using both a pre-packed Regispack column and a Chiralpak AD column packed in house with bulk CSP. The developed preparative method proved to be superior to previously published methods in terms of elution times, separation and resolution and is suitable for obtaining a quick access to the desired enantiomers with high enantiomeric excess and amounts sufficient for biological investigations. Future scale-up options (enantioselective supercritical fluid chromatography or HPLC in the Simulated Moving Bed mode) were also evaluated. It could be shown that both methodologies have a high potential for future production of Naringenin enantiomers by enantioselective chromatography.     

Addition of organic modifiers to control retention order of enantiomers of dihydropyridines on chiral-AGP

Summary The retention order of the enantiomers of clevedipine (solute no 1), a shortacting blood pressure reducer, was controlled by type of organic modifier. With 1-propanol as, mobile phase modifier the (R)-form eluted first and by using methanol, the (S)-form was first to elute. These effects could also be seen for the hydrolysed analogue, to clevedipine, an acid (solute no 2). The reversal of retention order was then obtained when 1-propanol was replaced by acetonitrile. An oxidized analogue to clevedipine, a pyridine (solute no 3), was also tested regarding reversal of retention order of its enantiomers. In this case the retention order could not be controlled by type of organic modifier. The influence of the mobile phase buffer pH on enantioselective retention was also studied for the three substances. For clevidipine and the oxidized analogue, a high mobile phase pH favored enantioselective resolution while the opposite result was obtained for the hydrolysed analogue. Temperature studies were also performed, and enthalpies and entropies at different mobile phase pH:s using different organic modifiers were calculated, in order to promote an understanding of the thermodynamic driving forces for retention in the systems. Optimized chromatographic systems were used to determine less than 0.1% of an enantiomeric impurity in (R)- and (S)-clevidipine.     

Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBH I (T.r. Cel 7A) in enantioseparations

Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.     

Determination of methylamphetamine and related compounds in human urine by high performance liquid chromatography/electrospray/mass spectrometry

The influence of tetrabutylammonium (TBA) as a hydrophobic charged additive of the mobile phase was investigated in a chromatographic system involving d,l-dansyl amino acids as the test solute enantiomers and immobilized human serum albumin as the chiral stationary phase. By varying the column temperature, van't Hoff plots for solute retention and enantioselectivity were performed and thermodynamic parameters were calculated. An enthalpy-entropy compensation study revealed that the type of interaction between the analyte and stationary phase was independent of TBA concentration in the eluent. The counterion dependence on retention indicated that the coulombic interactions between dansyl amino acid and the site II binding cavity were of crucial importance in this association process. Also, the increasing variations of chiral discrimination with a TBA addition were attributed, by an analysis of the thermodynamic parameter trends, to a great facilitation of enantioselective H-bonding between solute and polar residues at the cavity rim.