Separation of Cyclodextrins Using Cyclodextrin Bonded Phases

Abstract

Two different cyclodextrin bonded phases (α and β) were used for the separation of α-, β-and γ-cyclodextrins. The β-cyclodextrin phase was found to be, in general, more effective at resolving the cyclodextrins than the α-cyclodextrin bonded phase. Acetonitrile/water mixtures were used as mobile phases. The effect of mobile phase composition on retention and resolution is examined. The elution order was found to be size dependent. The results are discussed in terms of the overall retention mechanism.     

Enantiomer Separations by Normal HPLC Systems with Permethylated β-Cyclodextrin Dynamically Coated on Silica Solid Supports

Abstract

The paper describes the technique of solvent-generated liquid-solid chromatography with a chiral stationary phase consisting of permethylated β-cyclodextrin coated on a bare silica surface. The chiral adsorption layer was generated dynamically by pumping diluted solutions of permethylated β-cyclodextrin in binary (hexane/different alcohols) eluents through a column packed with microparticulate silica. This technique leads to column with excellent time stability and good reproducibility of the enantioselectivity. It was found that retention characteristics e. g. enantioselectivity and capacity depend very strongly on the type and concentration of the alcohol used as mobile phase component.     

High Performance Liquid Chromatographic Separation of Estradiol-17 α and -17/β in Biological Fluids; Application to Plasma,Milk and Urine of Cows

Abstract

A rapid HPLC technique was developed to separate estradiol epimers. In order to improve the sensitivity of the detection, a radioitmmunoassay was used.

Estrone, estradiol-17α and estradiol-17β were separated within 20 min using 10 ml of chloroform: acetone (90:10), as the mobile phase. The efficiency of the technique was assessed with ₃ steroids and the assay of collected fractions with antlsera specific to each estrogen. Using a non-specific radioimmunoassay, profiles of endogenous estrogens in different biological fluids (blood plasma, milk, urine) were obtained.

The efficiency of HPLC as a separation method and the high sensitivity of radioimmunoassay as a detector allows us to obtain profiles of estrogens from biological samples where steroid concentration is below lOOpg/ml.     

Chromatographic study of four sesquiterpenoids in volatile oil of Curcumae Rhizoma on reverse phase stationary phase with methyl-β-cyclodextrin as mobile additive

Abstract

The elution behavior of four sesquiterpenoids in volatile oil of Curcumae Rhizoma on reverse-phase high-performance liquid chromatography with methyl-β-cyclodextrin as mobile phase additive was studied, including germacrone, curzerene, furanodiene, and β-elemene. Stoichiometric ratio and apparent formation constants of inclusion complex formed by methyl-β-cyclodextrin and each analyte were calculated by varying the concentration of the additive in the mobile phase composed of methanol and water (90:10, v/v), in which the association constant for inclusion complex formed by the organic modifier methanol and methyl-β-cyclodextrin was also determined. Results showed that the stoichiometric ratio of all the inclusion complex was 1:1 when 0–9?mmol L^?1 of methyl-β-cyclodextrin was added in the mobile phase. Unusual retention behavior of the analyte germacrone was found, which was further investigated by the calculation of thermodynamic parameters. Meanwhile, enthalpy and entropy of the inclusion complexes and solute-stationary phase interactions were determined by linear van’t Hoff plots.     

High Performance Liquid Chromato-Graphy Separations of Nitrosamines. II. Acyclic Nitrosamines

Abstract

The separation of acyclic nitrosamines by HPLC using a β-cyclodextrin bonded silien gel column and a C₁₈ reversed phase column was evaluated. Four groups of nitrosamines were used to evaluate the utility of both columns: (1.) methylmethyl-, methylethyl-, methylpropyl and methylbutyl-; (2.) ethylmethyl-, ethylethyl-, and ethylpropyl-; (3.) propylmethyl-, propylpropyl-, and propylbutyl-; and (4.) butylmethyl-, butylethyl-, butylpropyl-, and butylbutyl nitrosamines.

The results show that both columns performed well in separating the nitrosamines in each group, with the C₁₈ column requiring a higher percentage of organic modifier (methanol) than the β-cyclodextrin column. The β-cyclodextrin column was superior in separating all the E to Z isomers of short chain alkyl nitrosamines, while the C₁₈ column was superior in separating long alkyl chain nitrosamines and their E and Z isomers.

It, was also found that, the longer the alkyl chain the longer the retention time, while nitrosamines having the same number of carbons, for example ethylbutyl-and propylpropyleluted at virtually the same time, using both columns.     

Analytical Method for Quantification β-Cyclodextrin in Milk, Cream and Butter by LC

A simple, rapid and sensitive liquid chromatographic method using a refractive index detector has been developed and validated for identification and quantification of β-cyclodextrin in milk, cream and butter. The chromatographic system consists of a YMC ODS-AQ column packed with C18 reversed phase silica packing material as stationary phase using a mixture of methanol and water 7:93% (v/v) as the mobile phase. Linearity was established for the β-cyclodextrin concentration in the range 0.01–4 mg mL^?1, with a coefficient (r) of 0.9998. Repeatability of the method was assessed; the coefficient of variation for β-cyclodextrin range were 1.24, 3.01, and 5.36% for milk, cream and butter. Recoveries ranged between 99.91 and 94.53%. This method was highly reproducible and reliable for quantification of treated milk with β-cyclodextrin for removal cholesterol from the milk fat.     

Effect of β-Cyclodextrin and Organic Modifier on Resolution of Nitro-Polycyclic Aromatic Hydrocarbons in Micellar Electrokinetic Chromatography

Abstract

Micellar electrokinetic chromatography with a photodiode array detector has been employed for the separation of nitro-polycyclic aromatic hydrocarbons, including 2-nitro-fluorene, 9-nitro-anthracene, 3-nitro-fluoranthene and 1-nitro-pyrene. Sodium deoxycholate was used as a micelle-forming reagent. The effects of β-cyclodextrin and the organic modifiers, methanol, acetonitrile and dimethylformamide, on the resolution of the nitro-polycyclic aromatic hydrocarbons were studied. Acetonitrile and dimethylformamide were useful modifiers to separate highly hydrophobic nitro-polycyclic aromatic hydrocarbons. Structure isomers, 3-nitro-fluoranthene and 1-nitro-pyrene, were not separated using β-cyclodextrin as modifier. The influence of temperature on the resolution of the nitro-polycyclic aromatic hydrocarbons were also investigated.     

Chiral Separations: A Comparison of Hplc and TLC

Abstract

Comparisons are made for separations attained in normalphase HPLC and TLC involving N-carbobenzyloxy-glycyl-L-proline and (+/?)?10-camphorsulfonic acid as chiral mobile phase additives/chiral counter ions (CMA). Possible reasons for differences in stereoselectivity of β-cyclodextrin CMA and chiral stationary phases (CSP) are discussed. In addition, differences in solubility and proposed separation mechanisms for native cyclodextrins (CD) versus derivatized CD are discussed. Inherent differences in high performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) methods are outlined. The pros and cons of using HPLC and TLC are considered.     

The Use of an α-Cyclodextrin Mobile Phase in the Thin-Layer Chromatographic Separation of Ortho,Meta, and Para Substituted Phenols

Abstract

An aqueous solution of α-cyclodextrin (cyclohexaamylose) is demonstrated to be a very effective mobile phase in thin-layer chromatographic separations. The chromatographic behaviors of twenty-six substituted phenolic and naphtholic compounds using polyamide thin-layer stationary sheets are described. The R_f values were found to be dependent upon both the structural features of the phenolic compounds and the concentration of α-cyclodextrin in the mobile phase. A possible mechanism that accounts for the observed chromatographic behavior is presented. The advantages and disadvantages of the aqueous α-cyclodextrin mobile phase over the traditional pure or mixed organic solvent systems typically employed are discussed.     

Thin Layer Chromatographic Separation of Ortho,Meta, and Para Substituted Benzoic Acids and Phenols with Aqueous Solutions of α-Cyclodextrin

Abstract

The use of aqueous solutions of α-cyclodextrin as the mobile phase in thin layer chromatography (TLC) is described. A series of eighteen substituted benzoic acid compounds were chromatographed on polyamide thin layer sheets. The R_f values were dependent on the concentration of α-cyclodextrin in the mobile phase as well as the structure and size of the individual molecules. Possible advantages of this technique over those which use pure or mixed solvent systems as the mobile phase are discussed.     

Simultaneous Determination of Dopamine,Norepinephrine, Tyramine and Octopamine by Reverse-Phase High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A method is described for the simultaneous separation and estimation of the catecholamines, norepinephrine and dopamine and their monohydroxy-equivalents, octopamine and tyramine. The method employs high-performance liquid chromatographic separation of the compounds on a C18 reverse-phase column with a mobile phase containing methanol as the organic modifier, octane sulphonate as an ion-pair reagent and acetic acid/ammonium hydroxide buffer. The influences of electrode potential and solvent pH on detector response were studied, and the optimal conditions identified as detector potential of 0.95 volts and pH 6.0. The technique of post-column mixing was introduced to provide optimal pH conditions for detector response without the constraint of on-column oxidation of catecholamines. The effects of buffer ionic strength on retention factors and detector response were also investigated and, on the basis of the results obtained, the optimal buffer strength was identified as 0.08–0.09 molar. The described procedure can be used for simultaneous estimation of catecholamines and monohydroxyphenolamines at concentrations between 200–5000 pg.     

Separation of Selected Dipeptides by High Performance Liquid Chromatography

Abstract

The use of β-cyclodextoin bonded column for the separation of a selected group of dipeptides was explored. The results show that the separation of the dipeptides using a raethanol/water (16:84) as the mobile phase and the above column is possible.     

Dynamically Generated Chiral Stationary Phase Systems with β-Cyclodextrin Derivatives

Abstract

It is demonstrated that chromatographic phase systems with a dynamically generated stationary phase with β-cyclodextrin derivatives are a powerful and versatile tools for enantioseparations. In this technique a small amount of β-cyclodextrin derivatives (di-, tri-methylated and peracetylated) is added to the eluent. These compounds are strongly adsorbed on the ODS surface where they form new adsorption sites with strong stereoselective interactions with enantiomers leading to a change in retention properties and selectivity of the phase system. This technique leads to columns with excellent time stability and good reproducibility of the enantioselectivity. The choice of a suitable β-cyclodextrin derivative offers a wide range possibilities to optimize enantioselectivity. It is shown that the kinetics of the sorption-desorption process of the solute on the chiral stationary phase largely contribute to the efficiency of the columns making an appropriate choice of the flow rate an important parameter for the optimization of the separations.     

Synthesis,chromatographic evaluation and hydrophilic interaction/reversed-phase mixed-mode behavior of a “Click β-cyclodextrin” stationary phase

A native β-cyclodextrin (β-CD) stationary phase was prepared by covalently bonding β-CD on silica particles via Huisgen [3 + 2] dipolar cycloaddition between the organic azide and terminal alkyne, the so-called Click chemistry. The resulting β-CD bonded silica (Click β-CD) was characterized by FT-IR, solid state ¹³C cross polarization/magic-angle spinning (CP/MAS) NMR and elemental analyses, which proved the successful immobilization of β-CD on the silica support with Click chemistry. The retentive properties of Click β-CD were investigated under hydrophilic interaction liquid chromatography (HILIC) mode in different mobile phase conditions with a set of polar compounds including nucleosides, organic acids and alkaloids. The effects of water content, concentration of the salt and pH of the buffer solution on the retention time were studied and the results demonstrated the typical retention behavior of HILIC on Click β-CD. Separation of very polar components, such as nucleosides and oligosaccharides, and chiral separation under HILIC mode were successfully achieved. In addition, Click β-CD was chromatographically evaluated with a set of flavone glycosides. The retention curves depending on the mobile phase of acetonitrile content were “U” curves, which is an indication of HILIC/RPLC mixed-mode retention behavior. The difference of the separation selectivity between HILIC and RPLC was described as orthogonality by using geometric approach and the orthogonality reached 69.4%. The mixed-mode HPLC properties and excellent orthogonality demonstrated the flexibility in HPLC methods development and great potential in two-dimensional liquid chromatography separation.     

β-环糊精流动相添加剂在手性分离中的应用综述

介绍了β-环糊精的基本性质,综述了β-环糊精及其衍生物作为流动相添加剂在高效液相色谱和高效毛细管电泳手性分离中的应用,并探讨了其作为手性流动相添加剂的特点.指出β-环糊精是良好的手性识别体,不仅可作为色谱手性固定相,还可作为流动相添加剂,用于手性对映体的拆分.     

Novel β-cyclodextrin derivative functionalized polymethacrylate-based monolithic columns for enantioselective separation of ibuprofen and naproxen enantiomers in capillary electrochromatography

A novel enantioselective polymethacrylate-based monolithic column for capillary electrochromatography was prepared by ring-opening reaction of epoxy groups from poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith with a novel β-cyclodextrin derivative bearing 4-dimethylamino-1,8-naphthalimide functionalities. Conditions for the ring-opening reaction with respect to different reaction parameters were thoroughly optimized to obtain high electroosmotic flow, separation efficiency and enantioselectivity for the analytes. The nonaqueous mobile phase composition regarding acetonitrile–methanol ratio and the concentration of electrolyte were examined to manipulate the hydrophobic inclusion and anion-exchange interaction between the analytes and chiral stationary phase. It was observed that in addition to β-cyclodextrin cavity, the electrostatic interaction exhibited pronounced influence on the enantioseparation of acidic analytes. Acidic enantiomers (ibuprofen and naproxen) could be separated with separation factor (α) values up to 1.08 and a maximum separation efficiency of 86 000 plates/m could be achieved.     

The Determination of Aspartame in Diet Soft Drinks by High Performance Liquid Chromatography

Abstract

A simple procedure for the qualitative and quantitative determination of aspartame (Nutrasweet) in diet soft drinks is described. A high performance liquid chromatography method is used which requires a 250 × 4.6 mm β-cyclodextrin bonded silica gel column and a mobile phase of methanol/1% triethyl ammonium acetate (pH 4.5). The effluent was monitored at 214 nm. The method was applied for the analysis of aspartame in diet Coke, diet Pepsi, diet 7-Up and the sweetner Equal. Determination of sodium benzoate caramel coloring and caffeine is also possible by this procedure.     

Chromatographic and Spectroscopic Studies on β-Cyclodextrin Functionalized Ionic Liquid as Chiral Stationary Phase: Enantioseparation of Flavonoids

In this study, β-cyclodextrin functionalized ionic liquid was prepared by adding 1-benzylimidazole onto 6-monotosyl-6-deoxy-β-cyclodextrin (β-CDOTs) to obtain β-CD-BIMOTs. β-CD-BIMOTs were then bonded onto the modified silica to produce chiral stationary phases (β-CD-BIMOTs-CSP). The performance of β-CD-BIMOTs-CSP was evaluated by observing the enantioseparation of flavonoids. The performance of β-CD-BIMOTs stationary phase was also compared with native β-CD stationary phase. For the selected flavonoids, flavanone and hesperetin obtained a high resolution factor in reverse phase mode. Meanwhile, naringenin and eriodictyol attained partial enantioseparation in polar organic mode. In order to understand the mechanism of separation, the interaction of selected flavonoids and β-CD-BIMOTs was studied using spectroscopic methods (¹H NMR, NOESY and UV–Vis spectrophotometry). The enantioseparated flavanone and hesperetin were found to form an inclusion complex with β-CD-BIMOTs. However, naringenin and eriodictyol were not enantioseparated due to the formation of hydrogen bonding at exterior torus of β-CD-BIMOTs.

     

Separation of Mycotoxins,Polycyclic Aromatic Hydrocarbons,Quinones, and Heterocyclic Compounds on Cyclodextrin Bonded Phases: An Alternative LC Packing

Abstract

β-Cyclodextrin and γ-cyclodextrin chiral bonded phases were previously shown to be useful in the separation of enantiomers, diastereomers and structural isomers. In this work it is demonstrated that these stationary phases are also useful in more routine separations. As such, they provide an alternative to the popular reverse phase packings. Because the selectivity of cyclodextrin packings is often unique they can be used to compliment conventional columns, particularly when separating complex mixtures where peak overlap is a problem. The separation of several important classes of compounds is used to demonstrate the general utility of this packing.     

高效液相色谱手性流动相添加剂分离乳酸对映体

分别将β-环糊精、2,6－二甲基－β－环瑚精和2,3,6－三甲基－β－环糊精作为手性流动相添加剂,系统地研究了D,L-乳酸在反相HPLC系统中的拆分,考察了流动相种类,pH值和手性流动相添加剂的浓度对手性分离的影响,建立了甲基化β-环糊精动态手性固定相分离乳酸对映体的方法。