High performance liquid chromatography of structural isomers using a cyclodextrin-poly(allylamine) coated silica column

Summary High performance liquid chromatography (HPLC) separation of substituted phenols by isocratic elution was investigated using a β-cyclodextrin-bonded column. The HPLC support was prepared in-house. This support was based on silica beads coated with a β-cyclodextrin-containing poly(allylamine). The retention behavior of some substituted phenol isomers and aromatic compounds was studied. The contribution of the amino groups of the polyamine and of the β-cyclodextrin cavity to the separation process is discussed. Two retention mechanisms, inclusion complex formation and acidbase interaction, were found. The effect of the composition and pH of the mobile phase on the separation was also examined.     

The role of β-cyclodextrin and hydroxypropyl β-cyclodextrin in the secondary chemical equilibria associated to the separation of β-carbolines by HPLC

The use of cyclodextrins (CDs) in HPLC as mobile phase additives provides a flexible alternative for the separation of chemically related compounds because these separations can be performed on conventional columns and are economically advantageous over the use of chiral stationary phases. The present paper describes the influence of the presence of β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HPβ-CD) on the separation of the β-carboline alkaloids norharmane, harmane and harmine. The nature of the stationary phase (reverse phases C₁ and C₁₈) affects the chromatographic separations and also the stability of the inclusion complexes that are developed. The changes in the proportion of the organic solvents at constant concentration of CDs (3 mM for β-CD and 15 mM for HPβ-CD) modify the retention factors (k′) for all alkaloids studied. The nature of the organic solvent in the mobile phase also changes the chromatographic parameters. The logarithm of the capacity factor (k′) is linearly increased with the proportion of water in the hydro-organic mobile phase (ethanolic or methanolic) but the slopes obtained vary depending on the CD added to the mobile phase. The role of competitive equilibria, i.e., chromatographic distribution and inclusion complexes formation is discussed. This paper was presented at XIIIth International Cyclodextrin Symposium. Torino, Italy, May, 14–17, 2006     

Chiral Separation of Duloxetine and Its R-Enantiomer by LC

The direct enantioseparation of duloxetine and its R-enantiomer was achieved by HPLC using hydroxypropyl-β-cyclodextrin (HP-β-CD) as a chiral selector and a vancomycin chiral stationary phase (Chirobiotic V). Operational parameters, such as the concentration of HP-β-CD, buffer pH, organic modifiers, temperature and flow rate, were varied in order to achieve the desired retention time and resolution. These two enantioseparation methods developed gave a baseline resolution of the enantiomers. Finally, the HPLC-CSP method was selected to determine the enantiomeric purity of duloxetine drug substance due to its much shorter analysis time and better resolution. The limit of detection of this method was 0.06 μg mL⁻¹.     

Optimization of the Synthesis of 2,6–Dinitro-4-trifluoromethylphenyl Ether Substituted Cyclodextrin Bonded Chiral Stationary Phases

The comparisons of five different chiral stationary phases (CSPs) based on 2,6-dinitro-4-trifluoromethylphenyl (DNP-TFM) ether substituted β-cyclodextrin are presented. The five CSPs differ from each other in the linkage/spacer chemistry, or on the position of the substituents on β-cyclodextrin, or in the sequence of the synthetic procedure. The results show that there are two optimum combinations: (1) DNP-TFM randomly substituted on the β-cyclodextrin as the chiral selector along with a carbamate linkage chain bonding it to the silica support; and (2) β-cyclodextrin derivatized by DNP-TFM substituents only on the C-2 and C-3 positions of the cyclodextrin with an ether linkage chain anchoring it to the silica gel. These two combinations show complementary separations for some enantiomers. The spacer chain effect is much more pronounced for the CSP based on the β-cyclodextrin derivatives with DNP-TFM substituents only on C-2 and C-3 positions than its randomly substituted counterpart. The sequence of derivatizing the cyclodextrin and attaching it to silica gel also affects its selectivity and efficiency. The β-cyclodextrin should be derivatized before it is linked to the silica gel.     

Enantioresolution of dihydropyridine substituted acid by supercritical fluid chromatography on hypercarb® with Z-(L)-arginine as chiral counter ion

Summary A chromatographic method is described for separation of substituted dihydropyridines on Hypercarb, porous-graphitic carbon as support and methanol-modified carbon dioxide containing Tween 60^? deactivation additive, as mobile phase. Enantiomers of a dihydropyridine substituted acid were resolved after addition of Z-(L)-arginine as ion-pairing counter ion. A 0.2 mM concentration was sufficient to achieve a resolution factor>2 or baseline resolution. The system was used to estimate the minor enantiomer below 0.1% level. Parts presented at HPLC 96 in San Francisco, CA, USA, June 16–21, 1996.     

Correlation between the chromatographic poperties of silica phenyl packing materials and the retention behaviour of methylated β-cyclodextrins

Summary The chromatographic properties of five columns packed with phenyl-bonded phases were characterized by an approach based on Tanaka's method for column to column comparisons in order to develop an LC analysis of partially methylated β-cyclodextrins. The retention behaviour of β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin was examined in order to evaluate the different interactions between phenyl stationary phase and this family of compound. Chromatograms show that the residual silanol activity of the packing materials plays a beneficial role in the partially methylated β-cyclodextrins separation process. It is concluded that knowledge of the interactions involved allows one to make a reasoned choice of the stationary phase in order to obtain the best possible analysis of three different commercial samples of partially methylated β-cyclodextrins.     

Interaction of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions

The main focus of this study was to explore the capability of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17α-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a β-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that β-cyclodextrin as well as γ-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.     

Chromatographic separation and enantiomeric resolution of flurbiprofen and its major metabolites

Summary The chromatographic separation and resolution of the enantiomers of flurbiprofen and its two major metabolites, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen was investigated using four different approaches: reversed-phase HPLC after pre-column derivatization with (R)-1-(naphthen-1-yl)ethylamine; reversed-phase HPLC using hydroxypropyl-β-cyclodextrin as a chiral mobile phase additive; chiral-phase HPLC using either an α₁-acid glycoprotein CSP (Chiral-AGP) or an amylose tris(3,5-dimethylphenylcarbamate) CSP (Chiralpak AD). Of all the approaches, only the direct method using the Chiralpak AD CSP demonstrated separation and enantiomeric resolution of all three analytes within an acceptable run time of 45 minutes. Enantiomeric resolution values of 1.67,3.67 and 3.44 were obtained for flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. Semi-preparative isolation of the individual enantiomers of both metabolites, followed by CD analysis, revealed that the elution order on the AD CSP wasR-beforeS-enantiomer for both metabolites and the same as that observed for flurbiprofen. The metabolite elution order was subsequently confirmed on the analysis of urine samples obtained from a healthy volunteer following oral administration of the individual drug enantiomers.     

A novel silica-based column packing for reversed-phase HPLC of basic compounds

Summary A novel bonded phase for reversed-phase HPLC was synthesized in two steps. Octylamine was first reacted with β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (β-ECTS) and then the intermediate product was coupled onto porous silica. The prepared packing was characterized by elemental analysis, solid-state¹³C NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes and methanol-water as binary mobile phase. The results showed that the stationary phase has excellent chromatographic properties and is resistant to hydrolysis between pH=2≈8. It can be used efficiently for the separation of basic compounds.     

Ion-pair high-performance liquid chromatography separation of β-endorphin-(6–17) from fourteen fragments

Summary In order to support metabolism studies of the proposed antipsychotic compound β-endorphin-(6–17), (Org 5878), the HPLC separation of this parent compound from fourteen peptide fragments was studied. The addition to the mobile phase of hydrophobic ion-pairing agents proved to be necessary to obtain adequate separation. The influence of the chromatographic parameters pH, type and concentration of the pairing agent, buffer concentration and temperature were investigated systematically. As a result the complete separation of Org 5878 and its fourteen fragments is reported.     

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.     

Separation and Determination of the Structural Isomers of Madecassoside by HPLC Using β-Cyclodextrin as Mobile Phase Additive

A reversed phase HPLC method has been investigated for separation and determination of the structural isomers of madecassoside (madecassoside and asiaticoside-B). The isomeric compounds can be isolated with high resolution by adding β-CD in mobile phase on a C₁₈ column. The effect of β-CD concentration on resolution is discussed. The functional group in the separation process is investigated. The correlation coefficient (R ²) of the calibration was 0.9995 over the range of 0.1–5.0 mg mL⁻¹. The method was successfully applied to characterize and determine the madecassoside in Centella extract.     

Determination of selected β-receptor antagonists in biological samples by solid-phase extraction with cholesterolic phase and LC/MS

A new method is presented for the determination of five selected β-receptor antagonists by HPLC, which emphasizes sample preparation via retention on a new type of silica gel sorbent used for solid-phase extraction (SPE). Sorbents of this type were obtained by the chemical modification of silica gels of various porosities by cholesterol ligands. The cholesterol-based packing material was investigated by spectroscopic methods and elemental analysis. The recoveries obtained with the extraction procedure were optimum over a relatively broad sample pH range (3.08–7.50). Analytical factors such as the sample loading, the washing step and elution conditions, the concentration of β-receptor antagonists to be extracted, and the type of sorbent were found to play significant roles in the sample preparation procedure and would therefore need to be controlled to achieve optimum recoveries of the analytes. Under optimum conditions, the recoveries of nadolol, acebutolol, esmolol, oxprenolol and propranolol from spiked buffers, blood and urine were reproducible and dependent on the polarity or hydrophilicity of the compounds. The above analytes were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV and ESI-ion trap mass spectrometry (MS) detection. The described method was found to be suitable for the routine measurement of compounds that are both polar and basic, and can be applied for the analysis of biological samples such as urine and blood in clinical, toxicological or forensic laboratories. The recovery measurements were performed on spiked human urine and serum, and on real samples of mouse blood serum.     

Preparation and Evaluation of 3,5-Dinitrobenzoyl-Bonded Silica Gel Stationary Phase for HPLC

Column packing materials are always a key factor influencing the development of high-performance liquid chromatography (HPLC). In this paper, a new preparation method of 3,5-dinitrobenzoyl-bonded silica gel stationary phase (DNB) for HPLC was developed by using N-(β-aminoethyl)-γ-aminopropyl-methyldimethoxy silane as coupling reagent. Its structure was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy, and thermal analysis. The surface concentration of 3,5-dinitrobenzoyl ligand is 2.082 μmol m⁻², according to the carbon content of elemental analysis. The chromatographic performance of new packing was evaluated by using different solute probes, such as alkylbenzenes, polycyclic hydrocarbons (PAHs), phenols, naphthalene derivatives, nitrophenol positional isomers, and sulfonamides. The results show that DNB was of the reversed-phase packing kind with weak hydrophobicity and versatile chromatographic property compared with octadecyl silane. The charge transfer between the dinitrobenzoyl ligand and the analytes plays a significant role in the separation of phenols and naphthalene derivatives. In addition, electrostatic, hydrogen-bonding, and dipole-dipole interactions are responsible for the above separations, which improve the selectivity of DNB for solutes. An advantage of DNB is that it is suitable for the separation of the basic compounds containing nitrogen atoms without a capped process because the spacer containing nitrogen atoms can shield the residual silanols from DNB. Translated from Chinese Journal of Chromatography, 2005, 23(3) (in Chinese)     

Retention and enantioselectivity properties of β-cyclodextrin polymers and derivatives on porous silica for reverse-phase liquid chromatographic separation of enantiomers

Summary Chiral stationary phases (CSP) based on porous silica coated with β-cyclodextrin polymer and methylated or acetylated derivatives have been prepared. Their enantiomer separation capability was tested and compared under reversed-phase mode elution conditions. Noticeable changes in retention properties werenoted and in many cases a reverse enantioselectivity was evidenced, as a consequence of a strong modification of the energy, geometry and number of points of contact between solutes and stationary phase sites of molecular recognition. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

基于巯基-烯点击化学法的β-环糊精固定相多模式色谱保留行为研究

基于巯基硅胶与单取代-6A-烯丙氨基-β-环糊精的巯基-烯点击化学反应,制备了β-环糊精(Click TE-CD)共价键合固定相。元素分析结果表明β-环糊精被成功键合到硅胶表面。以黄酮苷类化合物为模型,考察了Click TE-CD固定相在亲水、反相和超临界流体色谱等分离模式下的色谱保留行为。黄酮苷类化合物保留时间随流动相中乙腈含量的变化呈现典型的U型曲线,表明Click TE-CD固定相具有亲水/反相的双重保留特性。应用几何学方法测得Click TE-CD固定相在反相/亲水、亲水/超临界、反相/超临界混合模式下的正交性分别为69.8%、50.8%、50.8%。对比复杂中药样品降香提取物在反相、亲水、超临界等模式下的分离情况,结果表明Click TE-CD固定相在分离中药复杂样品方面具有极大潜力,可以在一根色谱柱上通过分离模式的改变,实现二维液相色谱的分离。Click TE-CD固定相不同分离模式的分离性能和较好的正交性表明该固定相具有在液相色谱方法发展和二维液相色谱分离方面应用的潜力。     

Improved Method of Molecular Imprinting of Cyclodextrin on Silica-gel Surface for the Preparation of Stable Stationary HPLC Phase

By using N-(3-triethoxysilyl)propylacrylamide (TPAAm), vinyl groups were introduced onto the surface of silica-gel. On the surface of this silica-gel, β-CyD was molecularly imprinted by using a redox initiator, and the composite was used as stationary phase of high performance liquid chromatography (HPLC). The pump pressure was sufficiently low and did not increase even after continuous elution for 24 h. In order to prepare still more stable columns, a new polymerization process was developed. There, the redox initiator was first mixed with the surface-modified silica-gel and then vinylated β-CyD, crosslinker, and the template were added. This modification promoted the immobilization of β-CyD copolymer to the silica-gel, resulting in still lower pump pressure. Concurrently, the imprinting efficiency was increased in comparison with previous method where the redox initiator was directly added to the mixture of the β-CyD–template complex, crosslinker, and surface-modified silica-gel. The molecularly imprinted β-CyD column, prepared by this new method, efficiently discriminated the enantiomers of N-benzyloxycarbonyltyrosine.     

High performance displacement chromatography of proteins: Separation of β-Lactoglobulins A and B

Summary β-Lactoglobulins A and B were separated by high performance displacement chromatography on an anion-exchanger column with chondroitin sulfate as the displacer. A sample of 100 mg containing a mixture of the two β-lactoglublins was separated on a column of 75×7.5 mm in a single chromatographic run. The separation process followed the rules of the classical displacement development: the two proteins formed contiguous rectangular bands and their concentrations were dependent on the displacer concentration. The results demonstrate that high performance displacement chromatography is a useful technique for the separation of proteins in preparative amounts with columns and instrumentation typically used in analytical HPLC. Furthermore, it has the potential to become the method of choice in large scale separation of proteins.     

Low temperature liquid chromatographic separation of oxygenated terpenes from terpene hydrocarbons

Summary The separation of β-pinene and menthone by HPLC on a silica gel column can be significantly improved with decreasing the column temperature. On a perparative scale complete separation of oxygenated terpenes from terpene hydrocarbons could be carried out under such conditions.     

Analytical and preparative methods for β-exotoxin fromBacillus Thuringensis

Summary Theβ-exotoxin fromBacillus thuringensis is a well known insecticide. The bioassay usually employed for its analysis is time-consuming and subject to many disturbances. The previously reported HPLC analysis has been shown to give erroneous results in some cases. A new analysis system has how been designed. The system is based on separation ofβ-exotoxin from the culture broth, and from various preparations containingβ-exotoxin by ion-pairing. For sample preparation in more complex matrices an method employing solid phase extraction was developed. In order to obtain a standard for quantitative analysis a preparative method for the isolation ofβ-exotoxin from concentrated culture broth after successive precipitations was also developed.