Micelles for signal enhancement and novel selectivity of dansyl amino acids

Fluorescence intensity of various chemical species is enhanced in the microenvironment provided by micelles. Parameters which affect fluorescence intensities are examined by using dansyl (Dns) amino acids as the probe. The retention behavior of Dns-amino acids in micellar LC is examined by using ion-exchange-induced stationary phases. The type and concentration of micellar agent and modifier ion as well as concentration of acetonitrile in the mobile phase affect the retention and signal intensity of Dns-amino acids. The order of elution of Dns-amino acids obtained with the micellar mobile phase is very different from that observed in conventional reversed-phase LC. Fluorescence intensities of Dns-amino acids are enhanced by the micellar mobile phase.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

Simultaneous isocratic separation of phenolic acids and flavonoids using micellar liquid chromatography

The simultaneous isocratic separation of a mixture of five phenolic acids and four flavonoids (two important groups of natural polyphenolic compounds with very different polarities) was investigated in three different RPLC modes using a hydro‐organic mobile phase, and mobile phases containing SDS at concentrations below and above the critical micellar concentration (submicellar LC and micellar LC (MLC), respectively). In the hydro‐organic mode, methanol and acetonitrile; in the submicellar mode methanol; and in the micellar mode, methanol and 1‐propanol were examined individually as organic modifiers. Regarding the other modes, MLC provided more appropriate resolutions and analysis time and was preferred for the separation of the selected compounds. Optimization of separation in MLC was performed using an interpretative approach for each alcohol. In this way, the retention of phenolic acids and flavonoids were modeled using the retention factors obtained from five different mobile phases, then the Pareto optimality method was applied to find the best compatibility between analysis time and quality of separation. The results of this study showed some promising advantages of MLC for the simultaneous separation of phenolic acids and flavonoids, including low consumption of organic solvent, good resolution, short analysis time, and no requirement of gradient elution.     

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。     

Modelling and prediction of retention in high-performance liquid chromatography by using neural networks

Summary Multi-layer feed-forward neural networks trained with an error back-propagation algorithm have been used to model retention behaviour of liquid chromatography as a function of the composition of the mobile phases. Conventional hydro-organic and micellar mobile phases were considered. Accurate retention modelling and prediction have been achieved using mobile phases defined by two, three and four parameters. With micellar mobile phases, the parameters involved included the concentrations of surfactant and organic modifier, pH and temperature. It is shown that neural networks provide a competitive tool to model varied inherent nonlinear relationships of retention behaviour with respect to the mobile phase parameters. The soft models defined by the weights of the networks are capable of accommodating all types of linear and nonlinear relationships, neural networks being specially useful when the relationships between retention behaviour and the mobile phase parameters are unknown. However, to train neural networks more experimental points than with hard-modelling methods are required, hence the use of the networks is recommended only for those cases where adequate theoretical or empirical models do not exist.     

Fluorescence enhancement of dansylamino acids by bovine serum albumin as mobile phase additive in microcolumn liquid chromatography

Summary The effects of acetonitrile and bovine serum albumin (BSA) concentrations on the signal intensity and retention behavior of dansylamino acids have been examined by using -cyclodextrin-bonded silica gel as the stationary phase in microcolumn liquid chromatography. Fluorescence intensities of dansylamino acids were enhanced by BSA as a mobile phase additive, e.g., detection limits of dansyl derivatives of L-Ala and L-Phe were improved by a factor of 12–18.     

甘氨酸衍生化β-环糊精键合硅胶液相色谱固定相的合成与评价

 β-环糊精键合硅胶经对甲苯磺酰化后,与甘氨酸反应得到甘氨酸衍生化β-环糊精键合硅胶固定相(GCDS)。考察了GCDS对位置异构体、丹磺酰化氨基酸异构体、苯丙酸类药物等的分离性能,研究了流动相中甲醇浓度、pH以及柱温对色谱保留行为的影响。     

Hexadecyltrimethylammonium bromide/ethanol/water systems as mobie phase in reverse phase high performance liquid chromatography. Study of metal-diethyldithiocarbamate complexes retention

Summary This paper presents the, chromatographic retention of Co(II), Ni(II) and Cu(II) as diethyldithiocarbamate complexes in the presence of hexadecyltrimethylammonium bromide/ethanol/water systems, as mobile phase, by reversed phase high performance liquid chromatography. The presence of an organic modifier reduces the retention times and improves the efficiency. In order to evaluate the interaction between the metal complexes and the mixed micellar system the values of solute binding constants are calculated in, two ways: a) Arunyanart and Cline-Love's treatment and b) multiple regression analysis taking account of the ethanol percentages.     

Micellar Liquid Chromatography Study of Quantitative Retention–Activity Relationships for Antihypertensive Drugs

Use of micellar mobile phases in reversed-phase liquid chromatography (RPLC) results in hydrophobic and electrostatic sites for interaction. Modified stationary phases in micellar liquid chromatography (MLC) are structurally similar to biomembranes. To confirm this we focused on the effects of the type and concentration of surfactant (Brij 35, SDS, and CTAB) and mobile phase pH on the retention of antihypertensive drugs on modified C₁₈ stationary phases. Quantitative retention-activity relationships are proposed for the drugs and the different surfactants and compared with those obtained using aqueous–organic mobile phases. Finally, a correlation was obtained between the logarithm of retention factors (log k) and the toxicity (LD₅₀) of antihypertensive drugs. Revised: 14 September 2005 and 4 April 2006     

Separation optimization of quercetin,hesperetin and chrysin in honey by micellar liquid chromatography and experimental design

The chemometrics approach was applied for the separation optimization of flavonoid markers (quercetin, hesperetin and chrysin) in honey using micellar liquid chromatography (MLC). The investigated method combines SPE of flavonoids from honey using C₁₈ cartridge and their separation and quantification by micellar liquid chromatography. A two level full factorial design was carried out to evaluate the effect of four experimental factors including concentration of SDS, alkyl chain length of the alcohol used as the organic modifier (N), volume percentage of the organic modifier (V_m) and volume percentage of acetic acid (AcOH) in mobile phase on analytes retention times. Experiments for analytes retention times modeling and optimization of separation were performed according to central composite design. Multiple linear regression method was used for the construction of the best model based on experimental retention times. Pareto optimal method was used to find suitable compatibility between resolution and analysis time of analytes in honey. The optimum mobile phase composition for separation and determination of analytes in honey were [SDS]=0.124 mol/L; 7.8% v/v ethanol and 5.0% v/v AcOH. Limits of detection and linear range of flavonoid markers were 0.0079–0.0126, 0.05–50.0 mg/L, respectively.     

A chromatographic approach to analyze dansyl amino acid-HP-beta-CD association using macrocyclic antibiotic as the stationary phase

The retention mechanism for a series of D,L-dansyl amino acids in high-performance liquid chromatography is investigated using a teicoplanin stationary phase and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as the mobile phase additive (0-16mM). A theoretical treatment is developed to determine the HP-beta-CD influence on the equilibrium between the teicoplanin phase and the aqueous medium, respectively. From the experimental data, the association constants of the D,L-dansyl amino acids-HP-beta-CD inclusion complexes are determined and discussed in relation to the enantiomer structure. A thermodynamic study confirms that both the retention and complexation mechanisms are independent of the dansyl amino acid molecular structure and its absolute carbon configuration.     

Signal enhancement by on-column fluorimetric detection in gradient elution of dansyl amino acids in liquid chromatography

Summary Fluorimetric detection in the presence of a stationary phase has been applied to gradient elution of dansyl amino acids in liquid chromatography. A 1.5 mm ID quartz tube packed with the same materials as the separation column was employed for the flow cell. Conventional-size columns were employed. The peak height of analytes increased with increasing retention owing to focusing and environmental effects of the stationary phase, leading to improvements in sensitivity, which was pronounced for analytes eluting late. The lower the gradient, the larger the improvement in sensitivity achieved. Detection limits were improved by a factor of up to 5.1 by fluorimetric detection using the packed flow cell, compared with those achieved using a common empty flow cell.     

Signal enhancement by on-column fluorimetric detection in gradient elution of dansyl amino acids in liquid chromatography

Summary Fluorimetric detection in the presence of a stationary phase has been applied to gradient elution of dansyl amino acids in liquid chromatography. A 1.5 mm ID quartz tube packed with the same materials as the separation column was employed for the flow cell. Conventional-size columns were employed. The peak height of analytes increased with increasing retention owing to focusing and environmental effects of the stationary phase, leading to improvements in sensitivity, which was pronounced for analytes eluting late. The lower the gradient, the larger the improvement in sensitivity achieved. Detection limits were improved by a factor of up to 5.1 by fluorimetric detection using the packed flow cell, compared with those achieved using a common empty flow cell.     

Enantioseparation of dansyl amino acids by ultra-high pressure liquid chromatography using cationic β-cyclodextrins as chiral additives

This work reports the application of ultra-high pressure liquid chromatography (UHPLC) for reasonably fast enantiorecognition of some dansyl amino acids by employing three cationic β-cyclodextrins (β-CDs) as chiral additives. Good resolutions were obtained on an Agilent C18 column (2.1 mm i.d.; 1.8 μm; 50 mm length) with 1% (v/v) triethylammonium acetate buffered at pH 4.7 and acetonitrile as the mobile phase. Most of the analytes could be baseline resolved within 10 min. Increased cationic CD concentration or acetonitrile proportion in the mobile phase results in a decreased retention factor but accentuated selectivity. Furthermore, molecular mechanics calculation was performed and found to be consistent with the experimental results.     

Enantiomeric separation by capillary electrochromatography. I. Chiral separation of dansyl amino acids and organochlorine pesticides on a diol-silica dynamically coated with hydroxypropyl-beta-cyclodextrin

In this work, a commercially available diol-silica stationary phase was converted in situ to a chiral stationary phase by dynamically coating it with hydroxypropyl-beta-cyclodextrin (HP-beta-CD). This stationary phase was shown useful for the capillary electrochromatography (CEC) separation of neutral and anionic enantiomers such as some organochlorine pesticides and dansyl amino acids, respectively. The inclusion of HP-beta-CD in the mobile phase to produce the in situ chiral stationary phase allowed the rapid separation of the anionic dansyl amino acid enantiomers at relatively low electroosmotic flow (EOF). The formation of host-guest complexes between the dansyl amino acids and the neutral HP-beta-CD in the mobile phase lowered the actual charge-to-mass ratios of the anionic solutes, thus speeding up their transport by the EOF across the packed capillary column. Several parameters affecting enantioseparation were investigated, including the concentration of HP-beta-CD, ionic strength, pH, and organic modifier content of the mobile phase.     

Determination of Steroids in Human Urine by Micellar Liquid Chromatography

Abstract

A simple and sensitive method for the determination of steroids using micellar liquid chromatography is described. The steroids, including hydroxycorticosterone. corticosterone, northisterone, testosterone, mexdroprogesterone acetate and progesterone, were separated by reversed-phase using a micelles mobile phase following UV detection at 245 nm. The parameters affecting retention of the test solutes such as the concentration of sodium dodecyl sulfate (SDS) and n-butanol-1 in the mobile phase were investigated. It was found that the retention of the solutes was dependent on the composition of mobile phase. The linear calibration plots range from 0.1 to 10 μg ml^?1 in mobile phase containing 5.0 × 10^?2 mol l^?1 SDS/9 % n-butanol-1 at pH 6.0, and the detection limit in order of 0.1 μg ml^?1 was obtained. The proposed method was used for the determination of steroids in urine using direct injection of samples without previous treatment.     

大环糖肽抗生素键合相高效液相色谱法拆分7种氨基带保护基的氨基酸对映体

采用高效液相色谱法在装有大环糖肽抗生素键合相的手性柱上拆分了7种氨基带有芴甲氧羰基(fluorenylmethoxycarbonyl,Fmoc)保护的氨基酸对映体。比较了Fmoc-缬氨酸和相应的不带保护基的缬氨酸对映体在不同流动相体系中的色谱保留行为;考察了甲醇-醋酸-三乙胺流动相体系中醋酸和三乙胺的浓度以及它们二者的浓度之比对N-Fmoc氨基酸对映体拆分效果的影响。实验结果表明分离温度及流动相流速的变化也会对分离结果产生影响。该法简便快速,已成功地用于这类氨基酸的光学纯度测定。     

Separation and mechanism elucidation for six structure-like matrine-type alkaloids by micellar liquid chromatography

A mixed micellar liquid chromatography (MLC) method, the mobile phase consisting of anionic surfactant SDS and nonionic surfactant Brij35, was firstly developed for the separation and determination of six structure-like matrine-type alkaloids, including matrine, oxymatrine, sophocarpine, oxysophocarpine, sophoridine, and oxysophoridine. The factors influencing the resolution of the six alkaloids were systematically investigated and optimized, including the micellar composition and concentration, column temperature, the type and amount of organic solvent, and the pH values in the mobile phases. Under the optimized separation conditions, the six matrine-type alkaloids could be easily isocratically eluted with a baseline separation within 22 min. Under the designated conditions (SDS concentration from 10 to 50 mM, Brij35 from 5 to 30 mM, pH 3 and 5% 1-propanol), the hydrophobic selectivity was negatively correlated with the concentration of Brij35 but not with SDS. The functional group selectivity of the carbonyl group, double bond, and diastereomers, all decreased with the increase in percentage of SDS in the mixed micellar phase, because the strong electrostatic force masks other molecular forces which can discriminate the retention of the analytes. Therefore, such a combination in surfactants of MLC is a powerful strategy to increase the selectivity by adjusting the balance among the various molecular interaction forces influencing analytes' retention. Finally, the developed method was successfully used to separate and determine the contents of main alkaloids in Sophora medicinal plants, S. flavescens Ait. In summary, the mixed MLC is a valuable approach to separate and determine the structure-like multi-component natural samples.     

RP-LC with On-Line Recycled Mobile Phase for Analysis of Amino Acids Pre-Derivatized with o-Phthalaldehyde

Reversed-phase high-performance liquid chromatography (RPLC) of 17 amino acids and ammonium ion, pre-derivatized with o-phthalaldehyde and ethanethiol, has been investigated using on-line recycled mobile phase. The mobile phase was sodium phosphate buffer, pH 7.2, containing acetonitrile and 1 mM potassium peroxodisulfate. Fifty consecutive separations on an octadecyl silica gel column, kept at 35 °C, could be achieved by use of 50 mL mobile phase, at a flow rate of 2.0 mL min⁻¹, without significant changes of peak height and retention times of valine (Val), methionine (Met), and isoleucine (Ileu). RSD of peak heights and retention times were, respectively, 7.6 and 1.1% for Val, 9.6 and 0.9% for Met, and 4.9 and 0.8% for Ileu. The volume of mobile phase used was only one sixtieth that used for conventional RPLC. Analysis of Val, Met, and Ileu in beverages was achieved by use of this method. The results were in agreement with those obtained by conventional RPLC. This method could be also used for analysis of the other amino acids.     

Multi-criteria decision making in micellar liquid chromatographic separation of chlorophenols

Simultaneous optimization of separation quality and analysis time of the micellar liquid chromatography of nine chlorophenol isomers was investigated. The effect on retention of three experimental parameters was studied using multivariate analysis. The factors studied were the concentration of sodium dodecyl sulfate, propanol content, and pH of the mobile phase. The experiments were performed according to the face-centered cube central composite design and the inverse form of the experimental retention times of analytes was fitted to polynomial models. The results of the analysis of variance showed that the models obtained explain over 99% of the variance observed in the chromatograms. The good predictive ability of the models was verified by high correlation coefficient (R2 > 0.99) and F ratio values for the plots of predicted cross-validated versus experimental retention times. The study showed that the use of the Pareto-Optimality method, an approach from multi-criteria decision making, allows selection of the best possible combinations of separation quality and analysis time in micellar liquid chromatography of chlorophenols.