Enantioselective separation of thalidomide on an immobilized α1-acid glycoprotein chiral stationary phaseglycoprotein chiral stationary phase

Summary An easy and rapid enantioselective separation for assay of racemic thalidomide on an immobilized α₁-acid glycoprotein chiral stationary phase (GPA CSP) is described. The effects of tetrahydrofuran (THF) as organic modifier, buffer concentration to control the ionic strenth, and mobile phase pH were studied. These variations have consequences in terms of chromatographic retention (k), resolution (R _s), selectivity (α), and peak asymmetry (USP tailing factor). The main condition affecting chromatographic retention was mobile phase pH. At pH 4.5, no separation of thalidomide enantiomers was achieved whereas at pH 7.9 chiral separation was optimum. Peak tailing was directly related to changes in pH and to addition of THF as mobile phase modifier. Results also indicated that the resolution factor is THF concentration-dependent, and that the separation factor (α) is the best parameter for evaluating enantioselectivity. The best mobile phase was pH 7.0, 30 mM ammonium acetate containing 0.3% THF. Under these conditions validation including linearity, recovery, and precision was performed. The suitability of this method has been successfully proved in a limited in-vivo study after intravenous administration of thalidomide to a New Zealand male rabbit.     

高效液相色谱手性流动相添加法拆分阿卓乳酸对映体

采用C₁₈反相色谱柱,以磺丁基醚-β-环糊精（SBE-β-CD）作为手性流动相添加剂,建立了阿卓乳酸对映体的高效液相色谱拆分方法。考察了环糊精衍生物类型、手性添加剂浓度、流动相pH、流速和柱温对手性分离的影响,同时探讨了高效液相色谱采用磺丁基醚-β-环糊精分离阿卓乳酸对映体的分离机制及包结常数,确定了色谱条件为YMC-Pack ODS-A C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相为含25 mmol/LSBE-β-CD的0.1 mol/L磷酸盐缓冲液（pH 2.68）-乙腈（90：10,v/v）,流速为0.6 mL/min,柱温为30 ℃,紫外检测波长为220 nm。对映体的保留时间分别为26.65和28.28 min,分离度为1.68。本方法分离度好,简便易行,且比使用手性固定相分离更加经济。     

Investigation of the chromatographic behaviour of some selenium species—Comparison with their octanol-water partitioning

The retention behaviour of selenites (Se(IV)), selenates (Se(VI)), seleno-dl-methionine (Se-Met), selenocystine (Se-Cyst), selenocystamine (Se-CM) and selenourea (Se-U) was investigated using a Discovery end-capped reversed-phase column as stationary phase and different mobile phase conditions. Extrapolated to 100% aqueous mobile phase retention factors (log k_w) of the investigated Se species, determined using different methanol fractions (φ) as organic modifier, were compared with the corresponding actual values. The proper operation of this column even at 100% aqueous phase proved to be valuable for the accurate determination of log k_w values of Se-CM and Se-Cyst, presenting a convex curvature log k = f(φ) at low MeOH fractions, often neglected in the extrapolation procedure. The effect of the presence of n-decylamine as well as saturation of the mobile phase with n-octanol was also studied. For ampholytic Se-Met and Se-Cyst the effect of n-decylamine in retention reflected the predominance of zwitterionic nature in the case of Se-Met in contrary to the non-zwitterionic species found in the case of Se-Cyst, in accordance with our previous findings concerning partitioning experiments in the n-octanol/water system. Finally, an attempt was made to correlate log k_w values with the logarithm of n-octanol/water distribution coefficient, log D, of the investigated Se species and an indicative log D value of Se-U was derived.     

Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicity relationships studies of selected β-blockers

The addition of the homologous series of perfluorinated acids-trifluoroacetic acid (TFAA), pentafluoropropionic acid (PFPA), heptafluorobutyric acid (HFBA) to mobile phases for reversed-phase high-performance liquid chromatography (RP-HPLC) of β-blockers was tested. Acidic modifiers were responsible for acidification of mobile phase (pH 3) ensuring the protonation of the β-blockers and further ion pairs creation. The effect of the type and concentration of mobile phase additives on retention parameters, the efficiency of the peaks, their symmetry and separation selectivity of the β-blockers mixture were all studied. It appeared that at increasing acid concentration, the retention factor, for all compounds investigated, increased to varying degrees. It should be stressed that the presence of acids more significantly affected the retention of the most hydrophobic β-blockers. Differences in hydrophobicity of drugs can be maximized through variation of the hydrophobicity of additives. Thus, the relative increase in the retention depends on either concentration and hydrophobicity of the anionic mobile phase additive or hydrophobicity of analytes. According to QSRR (quantitative structure retention relationship) methodology, chromatographic lipophilicity parameters: isocratic log k and log k_w values (extrapolated retention to pure water) were correlated with the molecular (log P_o/w) and apparent (log P_app) octanol–water partition coefficients obtained experimentally by countercurrent chromatography (CCC) or predicted by Pallas software. The obtained, satisfactory retention-hydrophobicity correlations indicate that, in the case of the basic drugs examined in RP-HPLC systems modified with perfluorinated acids, the retention is mainly governed by their hydrophobicity.     

The stereoselective synthesis of (E)-octafluoro-1,3,5-hexatriene and (3E,5E,7E)-dodecafluoro-1,3,5,7,9-decapentaene

(E)-(1,2-Difluoro-1,2-ethenediyl)bis[tributylstannane], 3, readily undergoes a Pd(PPh₃)₄/CuI-catalyzed cross-coupling reaction with iodotrifluoroethene to yield (E)-octafluoro-1,3,5-hexatriene, 4, in high isomeric purity. (1Z,3E,5Z)-(1,2,3,4,5,6-Hexafluoro-1,3,5-hexenetriyl)bis[tributylstannane], 7, was sequentially prepared from (1Z,3E,5Z)-(1,2,3,4,5,6-hexafluoro-1,3,5-hexenetriyl)bis[triethylsilane], 5, which was prepared via a Pd(PPh₃)₄/CuI-catalyzed cross-coupling reaction of 3 with (E)-1,2-difluoro-1-iodo-2-triethylsilylethene, 6. Pd(PPh₃)₄/CuI cross-coupling of 7 with iodotrifluoroethene gave (3E,5E,7E)-dodecafluoro-1,3,5,7,9-decapentaene, 8.     

Prediction of the chromatographic behaviour for a series of diuretic compounds

Summary The proportion of organic modifier and the pH of the acetonitrile-water mixtures used as mobile phases were optimized in order to separate a group of diuretic compounds covering a wide range of physyco-chemical properties. The Linear Solvation Energy Relationship (LSER) formalism based either on the multiparameter π^*, β and α scales or the single solvent polarity parameterE _T ^N , have been used to predict their chromatographic behaviour as a function of the percentage of acetonitrile in the eluent. Moreover, correlation established between retention and pH of the aqueous-organic mobile phases have been used to predict the chromatographic behaviour of the diuretic compounds studied as a function of the eluent pH. Linear correlation between a function of the eluent pH. Linear correlation between the chromatographic retention and theE _T ^N polarity parameter of mobile phases containing different percentages of organic modifier has been obtained Based on the knowledge of the acid-base dissociation constant the relation between retention and mobile phase pH has also been linearized. These relationship allowed an important reduction of the experimental retention data needed for developing a given separation and a great improvement in chromatographic optimization schemes.     

Use of beta-cyclodextrin bonded phase with s-triazine moiety in the spacer for separation of aromatic carboxylic acid isomers by high-performance liquid chromatography

The separation and retention behavior of five aromatic carboxylic acid isomers was investigated by means of high-performance liquid chromatography (HPLC) using a β-cyclodextrin bonded phase with s-triazine ring in the spacer. The influence of mobile phase pH on the retention was examined. The presence of s-triazine moiety in the spacer enhances greatly the selectivity of the isomers of aromatic carboxylic acids. Baseline separations of the five aromatic carboxylic acid isomers were achieved. In particular, the isomers of toluic, aminobenzoic, nitrobenzoic and hydroxybenzoic acid were successfully and effectively separated. The chromatographic results indicate that, in addition to inclusion complexation, π-π interaction and hydrogen bonding interaction between the bonded phase and analytes play significant roles in the retention of these acid isomers. Different elution orders were observed for these acidic solutes with different substituents. Possible retention mechanisms are discussed.     

Stabilization of the trivalent oxidation state of copper by tridentate imine–oxime–amine ligands

Two tridentate imine–oxime–amine ligands have been synthesized and their corresponding copper(II) complexes have been isolated. These copper(II) complexes are readily oxidized both chemically and electrochemically to give relatively stable copper(III) complexes. In the pH range 1.5–3.0 the electron transfer process is electrochemically reversible with ΔE_p = 60 mV and i_pa/i_pc ∼ 1. Plots of E_1/2 versus pH are linear with a slope = −60 indicating the involvement of one proton in the electron transfer process. Aqueous solutions of copper(III) complexes have high molar absorption at λ_max with ε > 10⁴ M⁻¹ cm⁻¹. Solid samples of the complexes are diamagnetic consistent with a d⁸ square planar geometry. It seems that only imine–oxime nitrogens are coordinated to copper(II) with the NH₂ group being free as indicated by i.r. spectra. Substitution of a –CH₃ group on the carbon atom adjacent to the oxime group by the more electron donating group –CH(CH₃)₂ lowers electrode potential by more than 90 mV. This is consistent with an earlier observation that electron-donating substituents on the carbon atom adjacent to the oxime group lower the potential of Cu^III/Cu^II couples and stabilize the higher oxidation state.     

Separation of C1-C5 aliphatic carboxylic acids on a highly sulfonated styrene-divinylbenzene copolymer resin column with a C6 aliphatic carboxylic acid solution as the mobile phase

Simultaneous separation of C₁-C₅ aliphatic carboxylic acids (formic, acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acids) on a highly sulfonated styrene-divinylbenzene copolymer resin column (TSKgel SCX,  mm i.d.) was performed with C₆ aliphatic carboxylic acids (3-methyl-n-valeric, iso-caproic and caproic acids) solutions as the mobile phases. Using 0.05 mM sulfuric acid at pH 4.0 as the mobile phase, although good separation of these C₁-C₅ acids was achieved, peaks of the C₅ acids (iso-valeric and valeric acids) with large hydrophobicity tailed strongly. In contrast, using 1 mM C₆ acids at pH ca. 4.0 as the mobile phases, although vacant peaks corresponding to the C₆ acids in the mobile phase appeared, the peak shapes of the C₅ acids were improved greatly. Excellent simultaneous separation, symmetrical peaks and relatively high-sensitivity conductimetric detection for these C₁-C₅ acids were achieved on the TSKgel SCX column in 15 min with 1 mM iso-caproic acid at pH 4.0 as the mobile phase.     

Silanol group effect in C18 bonded phases in HPLC

Summary If any residual (free) silanol groups remain at the surface of silica gel after bonding treatment, they may affect the retention of solutes since the dissociated groups (SiO^–) will attract cations. The silanol group effect on the retention of cationic solutes will increase with increasing pH of the mobile phase but the effect will decrease with increasing hydrophobic-ion concentration at the C₁₈ surface because such ions can mask the residual silanol groups. A method for the separation of metal complexes with 2-(5-bromo-2-pyridylazo)-diethylaminephenol (5-Br-PADAP) has been developed. The hydrophobic ion in the MeOH/H₂O mobile phase was tetrabutylammonium (TBA).     

Simultaneous analysis and retention behavior of major isoflavonoids in Radix Puerariae lobatae and Radix Puerariae thomsonii by high performance liquid chromatography with cyclodextrins as a mobile phase modifier

In order to differentiate two species of Radix Puerariae (Radix Puerariae lobatae and Radix Puerariae thomsonii) and to determine major isoflavonoids (puerarin, daidzin, daidzein and genistein) in the samples, a simple high performance liquid chromatography (HPLC) method with isocratic elution employing cyclodextrins (CDs) as mobile phase additives was developed. Various factors affecting the retention of isoflavonoids in the C₁₈ reversed-phase column, such as the nature of CDs, the concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD) and the methanol percentage in the mobile phase, were studied. Experimental results confirmed that HP-β-CD, as a very effective mobile phase additive, could markedly reduce the retention of isoflavonoids, especially daidzein and genistein. The elution of four isoflavonoids could be achieved on a Kromasil^® C₁₈ column within 56 min by using the methanol–water contained 5 mM HP-β-CD (25/75, v/v) mixture as the mobile phase. The formation of the inclusion complexes between isoflavonoids and HP-β-CD explained the modification of the retention of analytes. The apparent formation constants determined by HPLC confirmed that the stoichiometry of HP-β-CD-isoflavonoid complexes was 1:1, and the stability of the complexes depended on the size and property of isoflavonoids. The optimized method was successfully applied for the simultaneous determination of major isoflavonoids in P. lobatae and P. thomsonii samples. This work provides a useful method for the analysis of traditional Chinese herbs.     

Determining scaling in known phase diagrams of nonionic microemulsions to aid constructing unknown

Microemulsions based on nonionic surfactants of the ethylene oxide alkyl ether type C_mE_n, have been studied thoroughly for around 30 years. Thanks to the considerable amount of published data available on these systems, it is possible to observe trends to make predictions of phase diagrams not yet determined. Strey and Kahlweit, and subsequently Sottmann and Strey, with coworkers have studied and published phase diagrams for systems with a fixed ratio of oil to water, varying the surfactant, the so-called Kahlweit fish-cut diagrams. Some properties of the phase diagrams can be scaled to become general and not system dependent. Here are shown two examples of scaling data from phase diagrams and the use of trends to determine phase diagrams, both inside and outside a dataset. The trends of microemulsions with fixed ratio of surfactant to oil, the so-called Lund-cut diagrams, are also investigated. The trends are used to determine a new phase diagram and this is compared with previously unpublished experimental data on C₁₂E₅-Octadecane-Water system. The scalings and trends make it possible to get good estimations of many of the important properties of the phase diagrams, both temperatures and surfactant concentrations of interest, by investigating one sample in the 3-phase region of the balanced fish-cut diagram.     

Nano size crystals of goethite, α-FeOOH: Synthesis and thermal transformation

An aqueous suspension of amorphous iron(III) hydroxide was kept at room temperature (298 K) for 23 years. During this period of time the pH of the liquid phase changed from 4.3 to 2.85, and nano size crystals of goethite, α-FeOOH crystallised from the amorphous iron(III) hydroxide. Transmission electron microscopy (TEM) investigations, Mössbauer spectra, and powder X-ray diffraction using Co Kα radiation showed that the only iron containing crystalline phase present in the recovered product was α-FeOOH. The size of these nano particles range from 10 to 100 nm measured by TEM. The thermal decomposition of α-FeOOH was investigated by time-resolved in situ synchrotron radiation powder X-ray diffraction and the data showed that the sample of α-FeOOH transformed to α-Fe₂O₃ in the temperature range 444-584 K. A quantitative phase analysis shows the increase in scattered X-ray intensity from α-Fe₂O₃ to follow the decrease of intensity from α-FeOOH in agreement with the topotactic phase transition.     

Study on the chromatographic behavior of water-soluble vitamins on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

In this paper, the chromatographic behavior of some water-soluble vitamins was studied on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS, 5 μm particle size, the bonded amount 0.071 mmol g⁻¹) by using vitamin standards as probes for HPLC. The comparative study of the separation of these compounds was done by using CABS and ODS as stationary phases under the same chromatographic conditions. The better separation of six vitamins including: B₁, B₂, B₆, B₁₂, C, and nicotinic acid (B₅), on CABS can be achieved by using isocratic mode with methanol-phosphate buffer (25:75, (v/v)) as mobile phase within 20 min. The results show that the calix[8]arene-bonded phase exhibits high selectivity for water-soluble vitamins. We found that the elution order of B₂ (12.08 min) and B₁₂ (16.42 min) on CABS was very different from that of B₁₂ (7.76 min) and B₂ (18.47 min) on ODS, which indicate that different retention mechanisms exist in the chromatographic processes of the two stationary phases. According to the chromatographic data, it can be concluded that various chromatographic retention mechanisms are responsible for the separation of above compounds on CABS, such as hydrophobic interaction, hydrogen bonding interaction, and π-π interaction. The new packing has two advantages over ODS. On one hand, the polar and ionized analytes, such as C and B₅, exhibited stronger affinities to CABS because of hydrogen bonding interaction. On the other hand, the retention of B₂ and B₁₂ became shorter on CABS with weaker hydrophobicity in comparison with ODS. The new material exhibits the promising application in the separation of water-soluble vitamins.     

Modeling of the three-phase equilibrium in systems of the type water + nonionic surfactant + alkane

Liquid–liquid equilibria of systems water (A) + C_iE_j surfactant (B) + n-alkane (C) have been modeled by a mass-action law model previously developed and so far successfully applied to a series of binary water + C_iE_j systems and to the ternary system water + C₄E₁ + n-dodecane. These calculations provide the basis for the presented modeling. The aqueous systems give information about the association constants and the χ_AB-parameter of the Flory–Huggins theory and the ternary C₄E₁-system provides universal temperature functions for the χ_AC- and the χ_BC-parameter. The three-phase equilibrium for seven ternary C_iE_j systems (i = 6–12, j = 3–6) has been calculated by fitting one additional parameter for each of both temperature functions to the characteristic “fish-tail” point. The agreement with the experimental data is reasonably well. For systems with very small three-phase areas the results can considerably be improved by individual temperature functions that incorporate the experimental temperature maximum of the “fish” into the parameter fit. Based on the parameters of the system water + C₈E₄ + n-C₈H₁₈ the “fish-shaped” phase diagram of the system water + C₈E₄ + n-C₁₄H₃₀ was predicted reasonably well.     

Synthesis, optical properties and electronic structures of polyoxometalates K3P(Mo1−xWx)12O40 (0?x?1)

Various compositions of solid solutions K₃P(Mo_1−xW_x)₁₂O₄₀ (0?x?1) were prepared using two solid state synthetic routes. The crystallite size was determined by linewidth refinements of X-ray diffraction patterns using the Warren-Averbach method, and the grain size distribution by laser scattering experiments. Optical properties were determined by diffuse reflectance measurements in the UV-visible range. The optical gap E_g was found to increase exponentially from ∼2.5 to ∼3.30 eV with increasing x, and is systematically shifted to a higher energy when the grain size decreases. The relation between E_g and x was analyzed by calculating the HOMO-LUMO gaps of the [P(Mo_1−xW_x)₁₂O₄₀]³⁻ anions on the basis of tight-binding electronic structure calculations.     

NMR study of phase separation in D2O/ethanol solutions of poly(N-isopropylmethacrylamide) induced by solvent composition and temperature

¹H and ¹³C NMR spectroscopies were applied to investigate phase separation in solutions of poly(N-isopropylmethacrylamide) (PIPMAm) in D₂O/ethanol (EtOH) mixtures induced by solvent composition (cononsolvency) and temperature. Effects of EtOH content in D₂O/EtOH mixtures and temperature on the appearance and extent of the phase separation were characterized. Differences in mesoglobules formed during the phase separation induced by cononsolvency and temperature were found. For temperature-induced phase separation, ¹³C spin-spin relaxation times showed that besides the free EtOH expelled from the PIPMAm mesoglobules, there are also EtOH molecules bound in these mesoglobules. On the other hand, virtually no bound EtOH molecules were detected for mesoglobules formed as a consequence of the cononsolvency. For PIPMAm random copolymers containing negatively charged methacrylate units the phase separation induced by solvent composition was not observed.     

Selective separation of hydroxy polychlorinated biphenyls (HO-PCBs) by the structural recognition on the molecularly imprinted polymers: direct separation of the thyroid hormone active analogues from mixtures

We developed novel separation media for hydroxy polychlorinated biphenyls (HO-PCBs) using the molecular imprinting techniques. The results of evaluation for the molecularly imprinted polymers (MIPs) by the liquid chromatography (LC) suggested that MIPs had selective separation ability for certain HO-PCB analogues. The results of the LC evaluations and molecular modeling indicated that the molecular volumes and pK_a values of template molecules were related with the retention factor of HO-PCBs. Additionally, according to the detail evaluation toward the selective separation behaviors of MIPs, these HO-PCB analogues have low pK_a values dependent on their chemical structures. In other words, the prepared MIPs had selective recognition ability against the analogues, which have an OH group on a phenyl carbon and two chlorine atoms on the both neighboring carbons of the carbon attached with the OH group. Moreover, these analogues may have a potential for thyroid hormone activities so that we attempted to separate these analogues directly from mixtures of HO-PCBs using a prepared MIP.     

Chromatographic fingerprints of industrial toluic acids established for their quality control

The chromatographic fingerprints of industrial o-toluic acid, m-toluic acid and p-toluic acid have been established by HPLC-UV detection according to their impurity groups. HPLC separation of all relative substances involved in the groups was developed on a Kromasil C₁₈ column by using methanol-water-NH₄Ac-HAc buffer (100 mM, pH 4.70) 15/65/20 (v/v/v) as the mobile phase at a flow rate of 1.5 mL/min, and detection was operated by UV adsorption at a wavelength of 254 nm. The ultraviolet spectra corresponding to each chromatographic peak were also recorded for further identification of all components. Whether the limits of relative impurities residues in a toluic acid product are qualified or not can be intuitively estimated by analyzing its chromatogram with comparison to the fingerprint. This protocol has successfully provided some Chinese manufacturers with a simple and feasible method for quality control of toluic acids for industrial use.     

Studies on the chromatographic behavior of nucleosides and bases on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

The chromatographic behavior of some nucleosides, pyrimidines and purines on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS) were studied by high performance liquid chromatography. Their retention behavior on CABS were compared with those on ODS. The influence of mobile phase variables, such as methanol content, pH and ionic strength on the retention behavior were studied. Some nucleosides, pyrimidines and purines on CABS were successfully separated. The results show that the calix[8]arene-bonded phase exhibits high selectivities for the above analytes in high aqueous mobile phases. According to the chromatographic data, it is indicated that hydrophobic interaction, hydrogen-bonding interaction, and dipole-dipole interaction are mainly responsible for the retention behavior. In addition, in some extent, the vertical stacking action of the analytes on CABS can also change the retention behavior. CABS was superior to ODS in the routine fast separation of nucleosides and bases.