A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silica-based C₁₈ material proved superior to the polymer materials tested.     

A study of the behaviour of some new column materials in the chromatographic analysis ofCatharanthus alkaloids

Summary New column-packing materials specially designed for the HPLC analysis of basic compounds have been tested for the analysis ofCatharanthus alkaloids. Mobile phase optimization was performed for each column tested. The influence of mobile phase pH, nature and content of the organic modifier and salt concentration on retention, selectivity and resolution was studied. An important factor in the separation proved to be the pH of the eluent, because of the widely different pK _a values of the analytes. Complete separation was easily achieved on ODS columns, but polymeric materials also gave acceptable results. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Effect of ionic liquid additives to mobile phase on separation and system efficiency for HPLC of selected alkaloids on different stationary phases

The silica-based stationary phases with favorable physical characteristics are the most popular in liquid chromatography. However, there are several problems with silica-based materials: severe peak tailing in the chromatography of basic compounds, non-reproducibility for the same chemistry columns, and limited pH stability. Ionic liquids (ILs) as mobile phase components can reduce peak tailing by masking residual free silanol groups. The chromatographic behavior of some alkaloids from different classes was studied on C18, phenyl, and pentafluorophenyl columns with different kinds and concentrations of ionic liquids as additives to aqueous mobile phases. Ionic liquids with different alkyl substituents on different cations or with different counterions as eluent additives were investigated. The addition of ionic liquids has great effects on the separation of alkaloids: decrease in band tailing, increase in system efficiency, and improved resolution. The retention, separation selectivity, and sequence of alkaloid elution were different when using eluents containing various ILs. The increase of IL concentration caused an increase in silanol blocking, thus conducted to decrease the interaction between alkaloid cations and free silanol groups, and caused a decrease of alkaloids retention, improvement of peak symmetry, and increase of theoretical plate number in most cases. The effect of ILs on stationary phases with different properties was also examined. The different properties of stationary phases resulted in differences in analyte retention, separation selectivity, peak shape, and system efficiency. The best shape of peaks and the highest theoretical plate number for most investigated alkaloids in mobile phases containing IL was obtained on pentafluorophenyl (PFP) phase.     

Analysis of basic compounds at high pH values by reversed-phase liquid chromatography

Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C18, was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Preparation and evaluation of n-octadecylphosphonic acid-modified magnesia-zirconia stationary phases for reversed-phase liquid chromatography

Three n-octadecylphosphonic acid-modified magnesia-zirconia reversed stationary phases (C₁₈PZM) are prepared via the strong Lewis base interactions between organophosphonate and magnesia-zirconia composite. And two of them are end-capped by using trimethylchlorosilane as end-capping agent in different procedures. Stability studies at extreme high pH conditions (pH 9-12) show that both the non-endcapped and endcapped columns are quite stable at pH 12 mobile phase. The reversed-phase liquid chromatographic behavior of three C₁₈PZM stationary phases are comparatively investigated in detail using a variety of basic compounds as probes. The retention of basic compounds on the three phases is studied over a wide range of pHs. And the possible retention mechanisms of basic compounds on the three stationary phases are discussed. The results show that the basic solutes retain by a hydrophobic and cation-exchange interaction mixed mechanism on three stationary phases when they are operated in eluents at pH values near to the pK_a of the Brönsted conjugate acid form of the analyte, suggesting that inherent zirconol groups on ZM are not expected to interact with bases via cation-exchange interaction at lower pH. Nonetheless, the non-endcapped phase differs markedly from the edncapped ones in retention and selectivity of basic solutes using eluents at pH 4.1, implying a complex retention mechanism at this pH. The cation-exchange sites under such conditions are more likely due to the adsorbed Lewis base anionic buffer constituents (acetate) on accessible ZM surface sites than the chemisorbed phosphonate. Although the three phases exhibit very similar chromatographic behavior with eluents at pH 10.1, and show in general satisfactory separation of basic compounds and alkaloids studied, the performance for a specific analyte, however, differs largely from column to column.     

Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

手性固定相AD、AS和OD的拆分性能

手性固定相-高效液相色谱法在手性药物、手性农药等的分离分析中应用广泛。本文采用3种多糖衍生物的手性固定相(即EnantioPak AD、AS和OD)对20种手性化合物开展手性分离研究,进而探讨样品分子结构、多糖骨架和衍生基团对手性分离的影响。结果表明,除化合物13外,其余化合物在EnantioPak AD上均实现基线分离,分离度多在2.0以上,在正己烷-醇流动相中加入酸碱添加剂可改善和优化酸性或碱性化合物的分离效果;芳香醇(化合物13~16)随着侧链碳数增加在色谱柱上的保留减弱,其分离度呈现增加的趋势;对比8种化合物在3种手性固定相上的分离结果可知,EnantioPak AD表现出更优的分离性能。这为深入研究和了解多糖手性固定相、拓展其手性分离应用提供了参考。     

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。     

Tuning retention and selectivity in reversed-phase liquid chromatography by using functionalized multi-walled carbon nanotubes

Aim of this work was to explore the possibility of retention and selectivity tuning in reversed-phase liquid chromatography by means of chemically modified multi-walled carbon nanotubes (MWCNTs). These were synthesized by derivatizing pristine MWCNTs with amino-terminated alkyl chains containing polar embedded groups. A novel hybrid material based on functionalized MWCNTs (MWCNTs-R-NH₂) was prepared, characterized and tested. The idea was to design a mixed-mode separation medium basing its sorption properties on the peculiar characteristics of MWCNTs combined with the chemical interactions provided by the functional chains introduced on the nanotube skeleton. MWCNTs-R-NH₂ were easily grafted to silica microspheres by gamma radiation (using a ⁶⁰Co source) in the presence of polybutadiene as the linking agent. The composite was characterized by scanning electron microscopy (SEM) and Brunauer, Emmett and Teller (BET) analysis in terms of structural morphology, surface area and porosity. The MWCNTs-R-NH₂ sorbent was tested as stationary phase. The reversed-phase behaviour was first proved by analysis of alkylbenzenes, while the key role of CNT derivatization in addressing the selectivity/affinity towards the solutes was evidenced by testing three classes of analytes, viz. barbiturates, steroid hormones and alkaloids. These compounds, with different molecular structure and polarity, were here analysed for the first time on CNT-based LC stationary phases. The behaviour of the novel sorbent was compared in terms of retention capability and resolution with that observed using unmodified MWCNTs, pointing out the mixed-mode characteristics of the MWCNTs-R-NH₂ material. The same test mixtures were analysed also on a conventional mono-modal separation sorbent (C18) to highlight the particular behaviour of the (derivatized)MWCNTs-based stationary phases. The novel material showed better performance in separation of polar compounds, i.e. barbiturates and alkaloids, than the unmodified MWCNTs and than the C18 column. Results showed that MWCNT functionalization is powerful to modulate retention/selectivity in reversed-phase liquid chromatography.     

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     

Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides

The application of enhanced fluidity liquid (EFL) mobile phases to improving isocratic chromatographic separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC) mode is described. The EFL mobile phase was created by adding carbon dioxide to a methanol/buffer solution. Previous work has shown that EFL mobile phases typically increase the efficiency and the speed of the separation. Herein, an increase in resolution with the addition of carbon dioxide is also observed. This increase in resolution was achieved through increased selectivity and retention with minimal change in separation efficiency. The addition of CO₂ to the mobile phase effectively decreases its polarity, thereby promoting retention in HILIC. Conventional organic solvents of similar nonpolar nature cannot be used to achieve similar results because they are not miscible with methanol and water. The separation of nucleosides with methanol/aqueous buffer/CO₂ mobile phases was also compared to that using acetonitrile/buffer mobile phases. A marked decrease in the necessary separation time was noted for methanol/aqueous buffer/CO₂ mobile phases compared to acetonitrile/buffer mobile phases. There was also an unusual reversal in the elution order of uridine and adenosine when CO₂ was included in the mobile phase.     

Zirconia—A stationary phase capable of the separation of polar markers of myocardial metabolism in hydrophilic interaction chromatography

Creatine, phosphocreatine, and adenine nucleotides are highly polar markers of myocardial metabolism that are poorly retained on RP silica sorbents. Zirconia represents an alternative material to silica with high promise to be used in hydrophilic interaction chromatography (HILIC). This study describes a first systematic investigation of the ability of ZrO₂ to separate creatine, phosphocreatine, adenosine 5′‐monophosphate, adenosine 5′‐diphosphate, and adenosine 5′‐triphosphate and compares the results with those obtained on TiO₂. All analytes showed a HILIC‐like retention pattern when mobile phases of different strengths were tested. Stronger retention and better column performance were achieved in organic‐rich mobile phases as compared to aqueous conditions, where poor retention and insufficient column performance were observed. The effect of mobile phase pH and ionic strength was evaluated as well. The analysis of myocardial tissue demonstrated that all compounds were separated in a relevant biological material and thus proved ZrO₂ as a promising phase for HILIC of biological samples that deserves further investigation.     

Enantioseparations of basic and bifunctional pharmaceuticals by capillary electrochromatography using polysaccharide stationary phases

A fast screening strategy was developed in capillary electrochromatography (CEC) for the chiral separation of basic and bifunctional compounds. The screening conditions were determined on polysaccharide chiral stationary phases using 15 pharmaceutical compounds. The content and type of organic modifier, as well as the pH of the mobile phase appeared to have the largest influence on the chiral resolution. It was seen that for acidic compounds, our approach was not suitable. A generic mobile phase for basic and bifunctional compounds was determined. The testing on 20 additional compounds showed that the proposed mobile phase performed well since enantioselectivity was observed for 86% of the investigated compounds. A comparison of CEC and reversed-phase liquid chromatography (RPLC) results was attempted to demonstrate the potential of the used technique for chiral method development.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.     

Characterization of chromatographic supports for the analysis of basic compounds

Reversed‐phase liquid chromatography (RP‐HPLC) has become a powerful and widely employed technique for the analysis of a great variety of substances and, in particular, of basic compounds. These compounds are present in various areas. In pharmacy, 80% of drugs are estimated to possess a basic function. Basic compounds can strongly interact with free silanol groups on the surface of the silica particles. These ion exchange interactions produce peak tailing which affects resolution, sensitivity, and reproducibility. For these reasons, many new stationary phases have been designed to reduce access to silanol groups. The main problem facing the analyst is to effectively select the best column for a particular type of separation. In order to characterize and evaluate the properties of these packings, several tests are proposed in the literature, which can be divided into two main categories: general tests and particular tests. In this work, a particular test was developed for the characterization of base deactivated RP‐HPLC stationary phases. For this purpose, a set of 14 basic test substances was selected and five different chromatographic supports were tested with three isocratic mobile phases. Furthermore, in order to undertake a complete characterization of these supports, batch and column reproducibility were also studied. Principal Component Analysis was applied to evaluate both the performance of the test compounds and of the stationary phases.     

Separation of the enantiomers of 4-aryl-7,7-dimethyl- and 1,7,7-trimethyl-1,2,3,4,5,6,7,8-octahydroquinazoline-2,5-diones by chiral HPLC

Summary Analytical HPLC methods using derivatized cellulose chiral stationary phases have been developed for separation of the enantiomers of 25 racemic 4-aryl-7,7-dimethyl- or 1,77-trimethyl-1,2,3,4,5,6,7,8-octahydroquinazoline-2,5-diones, condensed derivatives of dihydropyrimidines. The enantiomers of the compounds were resolved by normal-phase chromatography on silica-based cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD) and amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) columns with mobile phases consisting of mixtures ofn-hexane and an alcohol (2-propanol, ethanol, or methanol) in different proportions. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. The effect of the concentration of alcohol in the mobile phase was studied. The resolution obtained on the two columns was complementary.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography: III. enantiomeric separation of basic compounds with the polar organic mobile phase

The separation of basic compounds into their enantiomers was achieved using capillary electrochromatography in 50 or 75 microm inner diameter (ID) fused-silica capillaries packed with silica a stationary phase derivatized with vancomycin and mobile phases composed of mixtures of polar organic solvents containing 13 mM ammonium acetate. Enantiomer resolution, electroosmotic flow, and the number of theoretical plates were strongly influenced by the type and concentration of the organic solvent. Mobile phases composed of 13 mM ammonium acetate dissolved in mixtures of acetonitrile/methanol, ethanol, n-propanol, or isopropanol were tested and the highest enantioresolutions were achieved using the first mobile phase, allowing the separation of almost all investigated enantiomers (9 from 11 basic compounds). The use of capillaries with different ID (50 and 75 microm ID) packed with the same chiral stationary phase revealed that a higher number of theoretical plates and higher enantioresolution was achieved with the tube with lowest ID.     

氮氧化硅反相色谱固定相材料的制备及色谱分离性能评价

硅胶是目前高效液相色谱(HPLC)固定相中应用最为广泛的基质材料,其流动相的最佳使用范围为pH 2～8。在高pH(pH＞8)的流动相条件下,流动相中的氢氧根会进攻硅胶基质表面残余的硅醇基,导致硅胶基质固定相骨架溶解。在前期的研究中,我们将高温氨气处理多孔硅胶微球得到的氮氧化硅材料用作HPLC固定相,氮氧化硅在高pH流动相条件下表现出了较硅胶更高的稳定性。本文利用元素分析对氮氧化硅材料的氮化程度及含氮量进行系统的表征,并考察了氮氧化硅材料在不同pH条件下的静态稳定性。利用十八烷基二甲基氯硅烷试剂对氮氧化硅材料表面进行疏水性修饰,并以元素分析和核磁共振波谱对表面键合氮氧化硅材料进行了表征。考察了不同碳链的烷基苯、酸性化合物、碱性化合物在疏水改性的C₁₈氮氧化硅反相色谱固定相上的色谱分离性能。进一步分别以酸、碱和中性化合物为分析对象,比较了C₁₈-SiON1050(10)与C₁₈-SiO₂色谱保留的差异。     

Facile separation of enantiomers,geometrical isomers,and routine compounds on stable cyclodextrin LC bonded phases

The effectiveness of employing stationary phases composed of chemically bonded cyclodextrin molecules in the high performance liquid chromatographic separation of a variety of different types of compounds is summarized. Over one hundred compounds, including optical, geometrical, and structural isomers, diastereomers, and epimers were successfully separated from each other via use of beta- or gamma-cyclodextrin bonded phases and aqueous methanolic mobile phases. The mechanism of separation is based upon inclusion complex formation between the compounds being separated and the cyclodextrin molecules bonded to the stationary phase. The effects of temperature, mobile phase composition and flow rate upon the chromatographic selectivity and resolution are described. The results indicate that the cyclodextrin columns may be more versatile, flexible, and effective compared to the conventional normal or reversed phase columns.