超临界流体色谱法分析非对映异构体d4T-5’-N-磷酰化苯丙氨酸甲酯

采用超临界CO2流体色谱技术,分析d4T-5’-N-磷酰化苯丙氨酸甲酯手性磷的非对映异构体。色谱柱为Hpersil ODS2(250 mm×4.6 mm,5μm),流动相为夹带改性剂甲醇、乙醇和异丙醇的超临界CO2流体。以容量因子、选择性和分离度为指标,考察改性剂、背压和柱温对分离的影响。在甲醇、乙醇和异丙醇3种改性剂中,甲醇为最好的改性剂,其中在7%甲醇改性剂下,该化合物的分离度可达到3.35。在7%甲醇改性剂条件下,考察了压力(10～20 MPa)和温度(303.15～318.15 K)的影响。在优化的分离条件(改性剂为7%甲醇,流速为2 mL/min,柱温为308.15 K,背压为15 M Pa)下,d4T-5’-N-磷酰化苯丙氨酸甲酯的两种非对映异构体完全达到基线分离,分离时间约15 min。     

Liquid chromatographic separation and thermodynamic investigation of stereoisomers of darunavir on Chiralpak AD‐Hcolumn

Liquid chromatographic separation of stereoisomers of darunavir on Chiralpak AD‐H, a column containing the stationary phase coated with amylose tris(3,5‐dimethylphenylcarbamate) as a chiral selector, was studied under normal‐phase conditions at different temperatures between 20 and 50°C. The effect of quality and quantity of different polar organic modifiers viz: methanol, ethanol, 1‐propanol, and 2‐propanol in the mobile phase as well as column temperature on retention, separation, and resolution was investigated and optimized. The optimum separation was accomplished using a mobile phase composed of n‐hexane/ethanol/diethyl amine (80:20:0.1 v/v/v) at 40°C. Apparent thermodynamic parameters ΔH⁰ and ΔS* were derived from the Van't Hoff plots (lnk′ versus 1/T) and used to explain the strength of interactions between the stereoisomers and amylose tris(3,5‐dimethylphenylcarbamate) coated chiral stationary phase.     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

Enantiomeric separation of six chiral pesticides that contain chiral sulfur/phosphorus atoms by supercritical fluid chromatography

Six chiral pesticides containing chiral sulfur/phosphorus atoms were separated by supercritical fluid chromatography with supercritical CO₂ as the main mobile phase component. The effect of the chiral stationary phase, different type and concentration of modifiers, column temperature, and backpressure on the separation efficiency was investigated to obtain the appropriate separation condition. Five chiral pesticides (isofenphos‐methyl, isocarbophos, flufiprole, fipronil, and ethiprole) were baseline separated under experimental conditions, while isofenphos only obtained partial separation. The Chiralpak AD‐3 column showed a better chiral separation ability than others for chiral pesticides containing chiral sulfur/phosphorus atoms. When different modifiers at the same concentration were used, the retention factor of pesticides except flufiprole decreased in the order of isopropanol, ethanol, methanol; meanwhile, the retention factor of flufiprole increased in the order of isopropanol, ethanol, methanol. For a given modifier, the retention factor and resolution decreased on the whole with the increase of its concentration. The enantiomer separation of five chiral pesticides was an “enthalpy‐driven” process, and the separation factor decreased as the temperature increased. The backpressure of the mobile phase had little effect on the separation factor and resolution.     

Chiral separation of helical chromenes with chloromethyl phenylcarbamate polysaccharide‐based stationary phases

Two chloromethyl phenylcarbamate‐based chiral stationary phases, one containing an amylose‐type chiral selector (Lux Amylose 2, from Phenomenex) and the other a cellulose‐type one (Lux Cellulose‐4, from Phenomenex), were successfully used for the chiral resolution of three helical chromenes featuring a helicene‐like structure. The compound bearing a phenyl substituent on the helicene‐like structure was enantioresolved at 25°C with Lux Cellulose‐4 and a n‐hexane/1‐propanol 99:1 v/v eluent. With a n‐hexane/2‐propanol 99.8:0.2 v/v mobile phase, the same column (operated at 35°C) provided the separation of the four isomers of the compound having a hexyl residue on the helicene‐like motif and an additional asymmetric carbon. Lux Amylose‐2 was necessary for the enantioseparation of the compound having the sole hexyl residue on the helical scaffold. For the last compound a n‐hexane/2‐propanol 99.8:0.2 v/v eluent was used, and the column temperature was fixed at 5°C. The enantiomer elution order was appraised by using electronic circular dichroism and theoretical calculations. Notably, different thermodynamics of retention and enantioseparation were observed for molecules with pronounced structural similarity, that is, the enantiomer pairs of the compound containing the additional asymmetric carbon atom. Indeed, both entropically and enthalpically controlled adsorption and separation processes were observed.     

Competitional hydrophobicity driven separations under RP‐LC mechanism: Application to sulfonylurea congeners

Retention of a model set of sulfonylurea compounds has been studied under RP‐LC conditions, considering competitional effects brought by different alcohols (ethanol, 1‐propanol, 2‐propanol, 1‐butanol, 1‐pentanol, and 1‐octanol) used as additives in the organic component of the mobile phase (methanol). The capacity factors determined for the model compounds decreased with the increase of the hydrophobic character of the organic additive in the mobile phase. The amount of the additive within the organic component of the mobile phase was kept constant (1% as volumetric ratio). Retention was studied at different mobile phase compositions (aqueous to organic component ratios). Different functional fitting models were used to correlate retention to the content of the organic component in the mobile phase. Extrapolation of retention expressed as capacity factor to a mobile phase composition free of organic component is well correlated to the hydrophobic characteristics of the organic additives. The adsorption model was used for tuning the experimental find‐outs. The possibility of controlling retention through the competitive effects induced by hydrophobic additives in the mobile phase is highlighted.     

Glycolipid class profiling by packed-column subcritical fluid chromatography

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.     

Enantioseparation of racemic paroxol on an amylose‐based chiral stationary phase by supercritical fluid chromatography

The separation of racemic paroxol, a key precursor of trans‐(?)‐paroxetine, on Chiralpak AD‐H, an amylose‐based chiral stationary phase, by supercritical fluid chromatography was studied. Pulse experiments were investigated using supercritical carbon dioxide modified with methanol (MeOH), ethanol and 2‐propanol at 35°C and 15 MPa. Retention and separation factors were determined under analytical conditions for different mobile phase compositions. Among the modifiers used, MeOH was shown to be the best additive, and 5% v/v of MeOH was the preferable concentration at which selectivity of 1.14 and resolution of 3.0 was obtained. In order to evaluate the potential with respect to preparative separations, the adsorption isotherms of individual enantiomers of paroxol were estimated using the elution by characteristic point method. Isotherm parameters were determined from the overloaded elution profiles that were collected at pressure ranging from 15 to 24 MPa. The isotherms obtained were further validated by comparing experimentally recorded elution profiles with the predictions based on the equilibrium‐dispersive model. The results are important to the process design and optimization of preparative supercritical fluid chromatography application.     

Enantiomeric separation of proton pump inhibitors on new generation chiral columns using LC and supercritical fluid chromatography

The enantioselectivity of proton pump inhibitors, namely, omeprazole, lansoprazole, rabeprazole, pantoprazole, tenatoprazole, and ilaprazole were studied using new generation chiral packing materials: CHIRALPAK IA, CHIRALPAK IB, and CHIRALPAK IC. Two versatile techniques, HPLC and supercritical fluid chromatography (SFC) were used in this study. CHIRALPAK IC has shown superior selectivity under both LC and SFC conditions, whereas CHIRALPAK IA has shown good selectivity in SFC when compared to LC under primary screening conditions. The chiral recognition ability in LC and SFC modes were found to be in the order CHIRALPAK IC > CHIRALPAK IA > CHIRALPAK IB. In addition to diode array detection, chiral detection was carried out using a laser polarimeter and the elution orders were found to be the same in both LC and SFC elution modes. Mobile phase modifiers and column temperature effects were also studied. In SFC, modifiers (cosolvent) elution strength was found to be in the order ethanol > methanol > 2‐propanol > acetonitrile. In both LC and SFC, a decrease in retention and increase in resolution with an increase in temperature was noticed for all the proton pump inhibitors.     

超临界流体色谱对吴茱萸中吲哚类生物碱的快速分析

建立了超临界流体色谱快速分析吴茱萸中吲哚类生物碱的方法。以标准品混合物和复杂样品为对象比较4种色谱柱的分离效果,进行色谱柱的筛选;考察了进样体积、改性剂、添加剂、温度和背压对保留行为的影响。结果表明,进样体积对峰形影响显著;添加剂对保留时间和色谱峰形影响有限;改变改性剂能使保留时间显著改变;降低温度,升高背压,保留时间减小。经过优化,确定采用Waters ACQUITY UPC² BEH色谱柱,以甲醇为改性剂,在35 ℃柱温和2.07×10⁷ Pa背压条件下,15 min内完成复杂样品的分析。同时采用超高效液相色谱完成复杂样品的快速分析。结果表明,超临界流体色谱可用于天然产物的高效快速分析,同时该方法与超高效液相色谱在分离选择上的差异有助于天然产物分析方法的拓展。     

新型含苯并噻唑α-氨基膦酸酯类化合物在淀粉类手性固定相上的液相色谱分离

本文运用涂敷型(Chiralpak AD-H)和键合型(Chiralpak IA)两种淀粉类手性固定相高效液相色谱法,进行了新型含苯并噻唑α-氨基膦酸酯类化合物的手性分离。从色谱分离的保留因子(k)、分离系数(α)和分离度(Rs)三个方面考察了两种类型色谱柱的分离性能,上述化合物在Chiralpak IA柱上能够得到较好的基线分离。同时,讨论了温度、流动相极性和目标分析物的结构等因素对Chiralpak IA柱分离性能的影响。由于键合型固定相较稳定的性能,使某些非常规的溶剂(如THF)成功地应用于手性α-氨基膦酸酯类化合物的分离。     

Retention mechanisms in super/subcritical fluid chromatography on packed columns

Whereas the retention rules of achiral compounds are well defined in high-performance liquid chromatography, on the basis of the nature of the stationary phase, some difficulties appear in super/subcritical fluid chromatography on packed columns. This is mainly due to the supposed effect of volatility on retention behaviours in supercritical fluid chromatography (SFC) and to the nature of carbon dioxide, which is not polar, thus SFC is classified as a normal-phase separation technique. Moreover, additional effects are not well known and described. They are mainly related to density changes of the mobile phase or to adsorption of fluid on the stationary phase causing a modification of its surface. It is admitted that pressure or temperature modifications induce variation in the eluotropic strength of the mobile phase, but effects of flow rate or column length on retention factor changes are more surprising. Nevertheless, the retention behaviour in SFC first depends on the stationary phase nature. Working with polar stationary phases induces normal-phase retention behaviour, whereas using non-polar bonded phases induces reversed-phase retention behaviour. These rules are verified for most carbon dioxide-based mobile phases in common use (CO(2)/MeOH, CO(2)/acetonitrile or CO(2)/EtOH). Moreover, the absence of water in the mobile phase favours the interactions between the compounds and the stationary phase, compared to what occurs in hydro-organic liquids. Other stationary phases such as aromatic phases and polymers display intermediate behaviours. In this paper, all these behaviours are discussed, mainly by using log k-log k plots, which allow a simple comparison of stationary phase properties. Some examples are presented to illustrate these retention properties.     

Chiral separation of omeprazole and several related benzimidazoles using supercritical fluid chromatography

A study of the enantiomeric separation of omeprazole and several related benzimidazoles, using supercritical fluid chromatography (SFC), on the amylose based column Chiralpak AD is presented in this work. The effect of the organic modifier as well as temperature on the retention and enantioresolution was investigated. Alcohol-type modifiers provided the best results, allowing the enantiomeric separation of all the compounds studied with resolutions that were in most cases higher than 2, and analysis times lower than 10 minutes. An investigation of the temperature effect revealed that the isoelution temperature was below the working temperature range in only two cases, and hence it was better to work at the highest temperature permitted.     

手性固定相法拆分咔唑羧酸衍生物对映异构体

用直链淀粉－三（３,５－二甲基苯基氨基甲酸酯）手性固定相,在正相条件下首次拆分了６种咔唑羧酸衍生物对映异构体。考察了流动相中不同的酸性添加剂、不同的王丙醇浓度对样品保留和拆分的影响。     

Optimization and validation of a fast ultrahigh‐pressure liquid chromatographic method for simultaneous determination of selected sulphonamides in water samples using a fully porous sub‐2 μm column at elevated temperature

High temperature in HPLC is considered a valuable tool helping to overcome the increase in the column backpressure when using small packing particles such as sub‐2 μm, as it allows reduction in the mobile‐phase viscosity. In this study, a fast analytical method based on HPLC‐UV was developed using a sub‐2 μm column at elevated temperature for the simultaneous determination of nine sulphonamides. Owing to the lower viscosity of the mobile phase, the separation could be achieved in 3 min at 60°C for all analytes. The effect of temperature, the organic modifier percentage and the flow rate on the retention time was studied. The method developed was used for the determination of selected sulphonamides in surface and wastewater samples. Sample preparation was carried out by solid‐phase extraction on Oasis HLB cartridges. The method developed was validated based on the linearity, precision, accuracy, detection and quantification limits. The recovery ranged from 70.6 to 96 % with standard deviations not higher than 4.7%, except for sulphanilamide. Limits of detection ranged from 1 to 10 μg/L after optimization of all analytical steps. This method has the highest performance in terms of analytical speed compared with other published HPLC‐UV methods for the determination of sulphonamides in water.     

Retention indices in supercritical fluid chromatography on a polystyrene-divinylbenzene column

Summary The application of alkylarylketones and n-alkanes as retention index scale standards in supercritical fluid chromatography has been examined using a packed polystyrene-divinylbenzene column. The two scales gave comparable results but the alkylarylketone scale gave comparable results but the alkylarylketone scale was considered to have a wider application as it could also be used when modifiers were added to the mobile phase. The changes in the retention indices of a number of aromatic test compounds were used to compare the selectivity of the separation under different temperature and pressure conditions and in the presence of modifiers.     

Efficient separation of tanshinones by polyvinylpyrrolidone‐stabilized graphene‐modified micellar electrokinetic chromatography

In this work, a PVP‐stabilized graphene was used in MEKC for the separation of tanshinones. Seven structurally similar tanshinones were studied, that is, tanshinone IIB, dihydrotanshinone I, tanshinone I, cryptotanshinone, 1,2‐dihydrotanshinone I, miltirone, and tanshinone IIA. To achieve optimal conditions, graphene concentration, sample solvent composition, SDS concentration, 2‐propanolconcentration, and buffer pH were investigated. At a separation voltage of 30 kV and a 41.5 cm effective length fused‐silica capillary, good resolution within 12 min was performed using 10 mM borate buffer (pH 9.3) containing 30 mM SDS, 10% v/v 2‐propanol and 6 μg/mL graphene. The method was validated in terms of linearity (r² > 0.9970), intra‐ and inter‐day precision were less than 3.56 and 4.83%, respectively. The proposed method was then successfully applied to Danshentong capsule, an herbal preparation from Salvia miltiorrhiza. Our results indicated the high separation efficiency of PVP‐stabilized graphene provided new opportunities for the analysis of complex samples.     

Enantioseparation of paroxetine intermediate on an amylose-derived chiral stationary phase by supercritical fluid chromatography

The enantioseparation of trans-3-ethoxycarbonyl-4-(4′-fluorophenyl)-1-methyl piperidine-2,6-dione (3), which is one of the important racemic precursors of trans-(−)-paroxetine, has been investigated using supercritical fluid chromatography on a Daicel Chiralpak AD column. Supercritical CO₂ modified with methanol, ethanol and 2-propanol were used as mobile phase. The influence of type and concentration of alcohol modifier on retention factor, enantioselectivity and resolution were studied. Among methanol, ethanol and 2-propanol, 2-propanol was proved to be the most favorable modifier, and 9.5% (v/v) of 2-propanol was the preferred concentration at which racemate 3 could be separated with resolution of 15.86 and retention factor of 6.323. The effects of pressure and temperature were investigated at 9.5% (v/v) of 2-propanol in the pressure range of 12–24 MPa and temperature range of 303.15–318.15 K. It was found that the lower pressure and temperature were favorable to the enantioseparation. Using van’t Hoff plot, the isoenantioselective temperature was calculated to be 410 K. The enantioseparation process was “enthalpically driven” under experimental conditions. Finally, the retention factors were satisfactorily correlated by a simplified lattice–fluid model with average absolute relative deviation (AARD%) of both enantiomers smaller than 1.76%.     

Separation of stereoisomers of several furan derivatives by capillary gas chromatography-mass spectrometry,supercritical fluid chromatography,and liquid chromatography using chiral stationary phases

The direct separation of several stereoisomers (enantiomers and geometrical isomers) of furan derivatives, important intermediates for the synthesis of physiologically active natural products, was achieved using capillary gas chromatography/mass spectrometry with a per-O-methyl-beta-cyclodextrin, supercritical fluid chromatography and high-performance liquid chromatography with a tris(3,5-dimethylphenylcarbamate) of cellulose or amylose for the chiral stational phases, respectively. The temperature dependence of the peak resolution (Rs) and the retention factor (k) over the range of 110-130 degrees was studied using crotyl furfuryl ether in gas chromatography. Successive increases in the Rs value and of the difference between the k value of the E-isomer and the k value of the Z-isomer were observed when the gradient temperature was decreased. The per-O-methyl-beta-cyclodextrin column was suitable for use with volatile furan ethers whose molecular masses are between 150 and 180. In conclusion, the separation of thermally unstable furan derivatives was accomplished using supercritical fluid chromatography and high-performance liquid chromatography.