Ion-pair LC of sugars at alkaline pH and pulsed electrochemical detection

Summary Sugars, sugar acids, amino sugars and oligomers are separated as ion-pairs with hydrophobic counter ions at alkaline pH using PLRP-S and Hypercarb as solid phases. Important parameters for regulation of retention and selectivity are nature and concentration of the counter ion, pH (hydroxide concentration) and temperature. Reversals in the elution order wer obtained in some cases. Oligosaccharides are highly retained in these systems. The addition of organic modifiers to the mobile phase for elution of the solutes were found to interfere with the pulsed electrochemical detection (PED). Anions added to the mobile phase compete with the solutes for ion-pair retention, hence, decreasing the capacity factors, and phosphate could be used for this purpose in the separation of maltooligomers (M2-M10) from corn syrup.     

Retention behaviour of imidazolium ionic liquid cations on 1.7 μm ethylene bridged hybrid silica column using acetonitrile-rich and water-rich mobile phases

The potential of 1.7 μm ethylene bridged hybrid silica phase was investigated for the separation of twelve imidazolium-based ionic liquid cations. U-shaped retention profile was observed for all solutes with an increase in retention at both low and high acetonitrile content. Chromatographic behaviour of imidazolium cations in both hydrophilic interaction chromatography (HILIC) and per aqueous liquid chromatography (PALC) modes was studied by varying key parameters such as buffer concentration and pH, acid additive, organic modifier and column temperature. Experimental data provided some evidences that under PALC conditions cationic solutes are retained predominantly by mixed hydrophobic/ion-exchange interactions. In the HILIC mode, both partitioning and ion-exchange interactions are responsible for the retention of solutes. Compared to PALC, HILIC provided significantly higher efficiencies with less or even no peak tailing, better separation selectivity and greater resistance to overload. In PALC mode gradient elution was required to achieve adequate retentivity of all solutes but selectivity was not sufficient to distinguish between solutes with very similar hydrophobicity. In contrast, under HILIC conditions twelve solutes were almost completely resolved in less than 4 min by using isocratic elution. Summarizing, it could be concluded that ethylene bridged hybrid silica column providing a dual retention mechanism offers the possibility of selecting between the two retention modes with opposite separation selectivity, just by changing the composition of the mobile phase.     

Toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an interface chip

This paper reports a thermal lens microscope (TLM) detection coupled with capillary electrophoresis (CE) by using an interface chip (IFChip) to achieve highly sensitive detection with high reproducibility. Fused silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel on the IFChip. In comparison with an on-capillary detection method in CE-TLM, ca. 10-fold improvements in the reproducibility for peak height were obtained by using IFChips. The detection limit of an azo dye was estimated to be 3.6 x 10(-7)M (100 ppb), which was above 100-times lower than that of conventional absorbance detection. Toward further improvement of the detectability for nonfluorescent compounds, on-line sample preconcentration by sweeping was applied to the CE-TLM using the IFChip. Due to the sweeping effect, 3900000-fold increase in the sensitivity was successfully achieved.     

Retention behavior of basic compounds on alkylphosphonate-modified magnesia-zirconia composite stationary phase in RPHPLC

Summary The chromatographic properties of an alkylphosphonate-modified magnesia-zirconia composite stationary phase have been investigated by reversed-phase high-performance liquid chromatography with basic compounds as probes. The influence of organic modifier composition and mobile phase pH was studied. The new stationary phase, similar to a silica-based reversed-phase stationary phase, has hydrophobic properties, but greater pH stability. Use of the phase results in more symmetric peaks for basic compounds. A possible mechanism of retention of basic solutes on the new stationary phase is discussed. The chromatographic behavior of the basic solutes depends mainly on hydrophobic interactions between the solutes and the hydrophobic moiety of the stationary phase. Br?nsted acidic and basic sites on the surface of the new stationary phase play an important role in the retention of ionized solutes by ion-exchange interaction. Promising separations of some basic compounds have been achieved by use of methanolic TRIS buffer, pH 10.0, as the mobile phase.     

Sweeping of neutral analytes via complexation with borate in capillary zone electrophoresis

Summary The sweeping concept is extended to capillary zone electrophoresis (CZE) separation of neutral solutes involving complexation with borate. Analogous to the pseudostationary phase in electrokinetic chromatography (EKC), the complexing agent (borate) serves as carrier for sweeping and separation in CZE. Therefore, similar to the retention factor in EKC, the focusing effect in the present system is directly related to the association constant between the analyte and complexing agent. Theoretical and some preliminary experimental studies gerenally suggest that the electrophoretic mobility of the complex and the concentration of the complexing agent affect the resulting length of narrowed zones. Moreover, sweeping using borate is affected by pH since borate complexation is pH dependent. From around 10 to 40-fold improvement in peak heights has been observed experimentally for some neutral test analytes (monosaccharides, catechols, and nucleosides)     

On-line sample concentration in micellar electrokinetic chromatography using cationic surfactants

Two on-line sample concentration techniques, sample stacking and sweeping, were evaluated using cationic surfactants as pseudostationary phases in micellar electrokinetic chromatography. As cationic surfactant micelles, tetradecyltrimethylammonium bromide and cetyltrimethylammonium chloride were employed. About 10-fold and 1000-fold increases in detection sensitivity in terms of peak heights were observed by sample stacking and sweeping, respectively, without suppression of the electroosmotic flow. In particular, the concentration limits of detection (S/N=3) for test naphthalenesulfonic acids obtained with sweeping were from 0.96 to 0.47 ppb with UV detection without any preconcentration procedure.     

High-sensitivity analyses of metabolites in biological samples by capillary electrophoresis using dynamic pH junction-sweeping

Emerging fields of biochemical research, such as metabolomics, present challenges to current separation technologies because of the large number of metabolites present in a cell and their often low (submicromolar) concentration. Although capillary electrophoresis (CE) holds great promise as the method of choice for high-resolution separations of biological samples, it suffers from poor concentration sensitivity, especially with the use of UV detection. In CE, sweeping and dynamic pH junction represent two complementary on-line focusing techniques that have been used for sensitivity enhancement of hydrophobic and weakly acidic analytes, respectively. However, the application of either the sweeping or dynamic pH junction technique alone might, in some cases, be less effective for the analysis of certain sample mixtures. Recent work in the development of a hyphenated dynamic pH junction-sweeping technique is presented as an effective on-line method of preconcentration suitable for both hydrophilic (anionic) and hydrophobic (neutral) analytes. Sensitive analyses of flavin metabolites by CE with laser-induced fluorescence (LIF) detection is demonstrated in various biological matrixes, including cell extracts of Bacillus subtilis, pooled human plasma, as well as heat-deproteinized flavoenzymes. Enhanced analyte band narrowing and improved sensitivity is achieved for flavins using dynamic pH junction-sweeping compared to either sweeping or dynamic pH junction alone. This results in over a 1200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (LOD, defined as S/N = 3) of about 4.0 x 10(-12) M. Strategies for sensitive and more comprehensive analyses of other cell metabolites, including nucleotides, coenzymes, and steroids, are also discussed when using on-line focusing techniques in conjunction with multiplexed CE and UV detection.     

Dynamic pH junction-sweeping capillary electrophoresis for online preconcentration of toxic pyrrolizidine alkaloids in Chinese herbal medicine

There is a need to develop simple yet effective preconcentration methods to enhance concentration sensitivity for CE analysis of trace level analytes in real samples, particularly when commonly available but less sensitive detection methods, e.g., UV detection, are used. In this report, a hyphenated online preconcentration strategy combining dynamic pH junction with sweeping (i.e., dynamic pH junction-sweeping) was employed for the analysis of four toxic pyrrolizidine alkaloids (PAs) of senkirkine, senecionine, retrorsine, and seneciphylline in Chinese herbal medicine (Kuan donghua). Direct electrokinetically focusing of a large sample volume injection (up to 20% of capillary length) on the capillary was performed using the dynamic pH junction-sweeping method. A sample matrix consisting of 10 mM phosphate with 20% methanol at pH 4.0 and a BGE containing 20 mM borate, 30 mM SDS, and 20% methanol at pH 9.1 were utilized to realize dynamic pH junction-sweeping for PAs. This online preconcentration strategy resulted in sensitivity enhancement factors ranging from 23.8- to 90.0-fold for the four toxic PAs, giving an LOD as low as 30 ppb for the PAs. Critical factors such as sample matrix type, pH, and salt concentration were also examined to achieve higher sensitivity enhancement, shorter analysis time, and better resolution. The results indicate that the proposed dynamic pH junction-sweeping technique is a powerful alternative approach for identification and determination of trace levels of these toxic PAs and other hydrophobic, protonatable compounds in real samples.     

Is hydrophilic interaction chromatography with silica columns a viable alternative to reversed-phase liquid chromatography for the analysis of ionisable compounds?

The separation of acidic, neutral and particularly basic solutes was investigated using a bare silica column, mostly under hydrophilic interaction chromatography (HILIC) conditions with water concentrations >2.5% and with >70% acetonitrile (ACN). Profound changes in selectivity could be obtained by judicious selection of the buffer and its pH. Acidic solutes had low retention or showed exclusion in ammonium formate buffers, but were strongly retained when using trifluoroacetic acid (TFA) buffers, possibly due to suppression of repulsion of the solute anions from ionised silanol groups at the low (s)(s)pH of TFA solutions of aqueous ACN. At high buffer pH, the ionisation of weak bases was suppressed, reducing ionic (and possibly hydrophilic retention) leading to further opportunities for manipulation of selectivity. Peak shapes of basic solutes were excellent in ammonium formate buffers, and overloading effects, which are a major problem for charged bases in RPLC, were relatively insignificant in analytical separations using this buffer. HILIC separations were ideal for fast analysis of ionised bases, due to the low viscosity of mobile phases with high ACN content, and the favourable Van Deemter curves which resulted from higher solute diffusivities.     

Insights into the retention mechanism on an octadecylsiloxane-bonded silica stationary phase (HyPURITY C18) in reversed-phase liquid chromatography

Plots of the retention factor against mobile phase composition were used to organize a varied group of solutes into three categories according to their retention mechanism on an octadecylsiloxane-bonded silica stationary phase HyPURITY C18 with methanol-water and acetonitrile-water mobile phase compositions containing 10-70% (v/v) organic solvent. The solutes in category 1 could be fit to a general retention model, Eq. (2), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent composition with a discontinuity at low organic solvent compositions. The solutes in category 3 exhibited a pronounced step or plateau in the middle region of the retention plots with a retention mechanism similar to category 1 solutes at mobile phase compositions after the discontinuity and a different retention mechanism before the discontinuity. Selecting solutes and appropriate composition ranges from the three categories where a single retention mechanism was operative allowed modeling of the experimental retention factors using the solvation parameter model. These models were then used to predict retention factors for solutes not included in the models. The overwhelming number of residual values [log k (experimental) - log k (model predicted)] were negative and could be explained by contributions from steric repulsion, defined as the inability of the solute to insert itself fully into the stationary phase because of its bulkiness (i.e., volume and/or shape). Steric repulsion is shown to strongly depend on the mobile phase composition and was more significant for mobile phases with a low volume fraction of organic solvent in general and for mobile phases containing methanol rather than acetonitrile. For mobile phases containing less than about 20 % (v/v) organic solvent the mobile phase was unable to completely wet the stationary phase resulting in a significant change in the phase ratio and for acetonitrile (but less so methanol) changes in the solvation environment indicated by a discontinuity in the system maps.     

Ion-exchange and hydrophobic interactions affecting selectivity for neutral and charged solutes on three structurally similar agglomerated ion-exchange and mixed-mode stationary phases

The nature and extent of mixed-mode retention mechanisms evident for three structurally related, agglomerated, particle-based stationary phases were evaluated. These three agglomerated phases were Thermo Fisher ScientificIon PacAS11-HC – strong anion exchange, Thermo Fisher Scientific IonPac CS10 – strong cation-exchange PS-DVB, and the Thermo Fisher Scientific Acclaim Trinity P1silica-based substrate, which is commercially marketed as a mixed-mode stationary phase. All studied phases can exhibit zwitterionic and hydrophobic properties, which contribute to the retention of charged organic analytes. A systematic approach was devised to investigate the relative ion-exchange capacities and hydrophobicities for each of the three phases, together with the effect of eluent pH upon selectivity, using a specifically selected range of anionic, cationic and neutral aromatic compounds. Investigation of the strong anion-exchange column and the Trinity P1 mixed-mode substrate, in relation to ion-exchange capacity and pH effects, demonstrated similar retention behaviour for both the anionic and ampholytic solutes, as expected from the structurally related phases. Further evaluation revealed that the ion-exchange selectivity of the mixed-mode phase exhibited properties similar to that of the strong anion-exchange column, with secondary cation-exchange selectivity, albeit with medium to high anion-exchange and cation-exchange capacities, allowing selective retention for each of the anionic, cationic and ampholytic solutes. Observed mixed-mode retention upon the examined phases was found to be a sum of anion- and cation-exchange interactions, secondary ion-exchange and hydrophobic interactions, with possible additional hydrogen bonding. Hydrophobic evaluation of the three phases revealed log P values of 0.38–0.48, suggesting low to medium hydrophobicity. These stationary phases were also benchmarked against traditional reversed-phase substrates namely, octadecylsilica YMC-Pac Pro C18 and neutral μPS-DVB resin IonPac NS1-5u, yielding log P values of 0.57 and 0.52, respectively.     

Development of a solid-phase microextraction/reflection-absorption infrared spectroscopic method for the detection of chlorinated aromatic amines in aqueous solutions.

In this paper, the reflection-absorption infrared (IR) spectroscopic method combined with the principle of solid-phase micro-extraction (SPME) is proposed to detect chlorinated aromatic amines in aqueous solutions. This proposed method provides simplicity in both the optical system and equipment setup. Compared to the SPME/attenuated total reflection-IR method, this method reduces the cost for internal-reflection elements and optical systems. Meanwhile, it has no SPME/transmission IR method problems, which require high polymer film preparation techniques to obtain a standing film that has no physical/chemical property changes when immersed in an aqueous solution. The typical linear coefficients obtained using this method for chloroanilines in aqueous solutions are around 0.995 and the detection can be lower than 100 ppb. The thickness of the hydrophobic film is relatively important in the SPME/ATR-IR method, but the uncertainty caused by the film thickness can be partially eliminated in the proposed method. This is because the IR signals are proportional to the film thickness and can be corrected using hydrophobic film signals. The low detection limits have also indicated that this proposed method can compete with the currently existing IR methods, but allowing much simpler detection.     

Effects of pH in reversed-phase liquid chromatography

The effects of concomitant variations in pH and organic modifier concentration on retention, efficiency and peak symmetry are considered for reversed-phase liquid chromatography (RPLC) on octadecyl-modified silica (ODS) columns. A number of factors are discussed, which make the systematic exploitation of pH effects in RPLC more complicated than the optimization of solvent composition. If the pH is varied, a second factor (usually the concentration of organic modifier) will need to be varied simultaneously to maintain retention (capacity factors) in the optimum range. When pH is considered as a parameter in RPLC, not only its effects on retention, but also the variations in efficiency (plate count) and peak shape (asymmetry) need to be considered. These parameters turn out to vary drastically between individual solutes and between different experimental conditions. The results of a study involving a number of acidic, basic and neutral solutes, two different ODS columns and mixtures of either methanol or acetonitrile with aqueous buffers are reported. In the earlier part of the study, using methanol as the organic modifier, reproducible data for retention, peak width and peak symmetry were obtained and these data are reported. In the later part of the study, using acetonitrile, a gradual change in retention as a function of time was observed, this effect coinciding with a decrease in column efficiency. It is concluded that ODS columns are subject to considerable degradation during studies in which the pH is varied. Although this effect can be described mathematically, the preferred solution is thought to be the use of pH-stable columns.     

Modelling retention in liquid chromatography as a function of solvent composition and pH of the mobile phase

The aim of this work was to develop a model that accurately describes retention in liquid chromatography (LC) as a function of pH and solvent composition throughout a large parameter space. The variation of retention as a function of the solvent composition, keeping other factors constants, has been extensively studied. The linear relationship established between retention factors of solutes and the polarity parameter of the mobile phase, E(N)T, has proved to predict accurately retention in LC as a function of the organic solvent content. Moreover, correlation between retention and the mobile phase pH, measured in the hydroorganic mixture, can be established allowing prediction of the chromatographic behavior as a function of the eluent pH. The combination of these relationships could be useful for modelling retention in LC as a function of solvent composition and pH. For that purpose, the retention behavior on an octadecyl silica column of a group of diuretic compounds covering a wide range of physico-chemical properties were studied using acetonitrile as organic modifier. The suggested model accurately describes retention of ionizable solutes as concomitant effects of variables included and is applicable to all solutes studied. We also aimed to establish an experimental design that allows to reproduce to a good approximation the real retention surface from a limited number of experiments, that is from a limited number of chromatograms. Ultimately, our intention is to use the model and experimental design for the simultaneous interpretive optimization of pH and proportion of organic solvent of the mobile phase to be used in the proposed separation.     

Enantioseparations by capillary electrophoresis using chiral glycosidic surfactants. I. Evaluation of cyclohexyl-pentyl-beta-D-maltoside surfactant.

Chiral cyclohexyl-pentyl-beta-D-maltoside (CYMAL-5) surfactant was evaluated in the enantioseparation of charged racemic species by capillary electrophoresis. CYMAL-5 is a glycosidic surfactant (GS) with a chiral maltose polar head group and a cyclohexyl-pentyl hydrophobic tail. At concentrations above its critical micellar concentration (CMC), CYMAL-5 produces neutral micelles in aqueous media. The neutral micelles migrate at the velocity of the electroosmotic flow (EOF). As expected, the CYMAL-5 system was only useful for the enantioseparation of charged chiral solutes. The enantioresolution of the CYMAL-5 can be manipulated over a wide range of electrolyte composition, e.g., pH, ionic strength and surfactant concentration. In the presence of EOF, and in all cases, there is an optimum surfactant concentration for maximum enantioresolution, which is located at low surfactant concentration for strongly hydrophobic solutes and at high surfactant concentration for relatively hydrophilic solutes. The presence of an optimum surfactant concentration for maximum enantioresolution is attributed to the EOF. At low pH values where the EOF is negligible, enantioresolution increased with increasing surfactant concentration in the useful concentration range in a way similar to chromatography.     

Effect of Temperature on the Chromatographic Behavior of Epirubicin and its Analogues on High Purity Silica Using Reversed-Phase Solvents

The influence of temperature on the retention behavior of epirubicin and its analogues on high purity silica with reversed-phase solvents has been systematically investigated. It was found that temperature effects on retention are highly dependent on the type and concentration of organic modifier, as well as the pH of the mobile phase. In organic-rich mobile phases, the type of organic modifier plays an important role. With an aprotic solvent as modifier, retention times show anomalous increases with elevated temperature. At the same time, both efficiency and resolution are significantly improved but this is not the situation with a protic solvent as modifier. In addition, temperature shows different effects on retention time and selectivity when the pH is changed, and temperature-dependent selectivity reversal is found at higher pHs. In aqueous-rich mobile phases, regardless of the nature of the organic solvent and pH, retention of solutes drops as temperature is raised. It seems that the effect of temperature on chromatographic behavior of the solutes on bare silica using mobile phases containing various organic modifiers or pHs, results from a number of different retention mechanisms.     

Off-Line and On-Line Preconcentration Techniques for the Determination of Phenylureas in Freshwaters

Abstract

Phenylurea herbicides are analysed by reversed-phase liquid chromatography using UV detection at 244 nm after a concentration step in order to determine ppb or sub-ppb levels in drinking and river waters. With an average UV detection limit of 5 ng, a 500 ml sample volume is necessary to reach the 10 ppt level for spiked LC grade water samples and enables easy determination of concentrations below the ppb level for river water samples. Off-line and on-line methods are compared for the concentration step. Off-line concentration consists in a liquid sorption on n-octadecyl silica (C18) and elution by a suitable organic solvent. Polar phenylureas have low retention volumes on C18 silica and consequently the length of the concentration column has to be 10 cm to concentrate them at the ppb level from 100 ml of water and longer for lower levels of detection. Nevertheless, we show that increasing the size of the concentration column does not improve the limits of detection because of the numerous interferences also concentrated when percolating high volumes of water. On-line technology can be used only with short precolumns and requires a sorbent with a great retention for phenylureas. The copolymer-based PRP-1 is found to be an excellent sorbent and it is then possible to apply on-line precolumn technology with preconcentration through two precolumns (10 × 21 mm ID) in series, the first one being packed with C18 silica and the other with the PRP-1 polymer. Interfering compounds are then trapped onto the first precolumn acting as a filter and common phenylurea-breakthrough volumes on the PRP-1 precolumn are higher than 500 ml. Knowing the amounts preconcentrated on both precolumns and using UV and electrochemical detection help the identification of phenylureas in river water.     

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Br?nsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.     

Retention behavior and selectivity of a latex nanoparticle pseudostationary phase for electrokinetic chromatography

The retention characteristics and separation selectivity of a novel latex nanoparticle (NP) pseudostationary phase (PSP) for electrokinetic chromatography have been characterized. The anionic NPs have very low or no affinity for cationic solutes, but show significant interactions and retention based on hydrophobic interactions. Retention factors of alkyl-phenyl ketones increase linearly with the concentration of the NPs and have zero or near zero y-intercepts as expected for electrokinetic chromatography with non-micellar PSPs. The retention factors of these solutes and representative pharmaceuticals decrease logarithmically with increases in the concentration of ACN in the background electrolyte, as expected for reversed-phase retention. Linear solvation energy relationship analysis indicates that the NPs are less cohesive than would be expected for polymeric PSPs with similar structure but that the overall separation selectivity can be expected to be similar to polymer PSPs with similar backbone chemistry. The results indicate that the hydrophobic core of the NPs is non-cohesive and is highly accessible to solutes, whereas the ionic head groups are not as accessible and do not contribute substantially to retention or selectivity.     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。