Band splitting in overloaded isocratic elution chromatography II. New competitive adsorption isotherms

The equations of two new binary competitive isotherms models are derived. The first of these models assumes that the isotherms of the two pure, single compounds have distinct monolayer capacities. Its derivation is based on kinetic arguments. The ideal adsorbed solution (IAS) framework was applied to derive the second model that is a thermodynamically consistent competitive isotherm. This second model predicts the competitive adsorption isotherm behavior of a mixture of two compounds that have single-component adsorption behavior following a BET and/or a Langmuir isotherms. Both models apply well to the binary adsorption of ethylbenzoate and 4-tert.-butylphenol on a Kromasil-C18 column (with methanol-water, 62:38, v/v, as the mobile phase). The best single-solute adsorption isotherms of these two compounds are the liquid-solid extended multilayer BET and the Langmuir isotherms, respectively. The kinetic and thermodynamic new competitive models were compared, regarding the accuracy of their prediction of the elution band profiles of mixtures of these two compounds. A better agreement between experimental and calculated profiles was observed with the kinetic model. The IAS model failed because the behavior of the ethylbenzoate/4-tert.-butylphenol adsorbed phase mixture is probably non-ideal. The most striking result is the qualitative prediction by these models of the peak splitting of 4-tert.-butylphenol during its elution in presence of ethylbenzoate.     

Determination of khellin and visnagin in Ammi visnaga fruits by capillary electrophoresis

A sensitive method for the simultaneous determination of phenolic xenoestrogens such as bisphenol A, 2,4-dichlorophenol, 4-tert,-butylphenol, 4-n2-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-octylphenol, 4-nonylphenol was developed using reversed-phase LC and coulometric-array detection. Stepwise gradient elution with phosphoric acid in water-acetonitrile was used. The calibration curves were linear in the range of 5.0 (or 10.0)-1000 ng ml(-1) with correlation coefficients of 0.9978-0.9999, the limits of detection were 0.01-0.02 ng ml(-1). Sample clean-up was performed by solid-phase extraction (SPE) using 3M Empore extraction disks. Three commercial sorbents, C18, SDB-XD (styrene-divinylbenzene polymer) and SDB-RPS (sulfonated styrene-divinylbenzene polymer) were compared. The highest recoveries were obtained with SDB-RPS. They were above 70% with a relative standard deviation of less than 15%. The proposed method was applied to the determination of phenolic xenoestrogens in various water samples.     

Microanalytical systems for separations of stratum corneum ceramides

The small amount of lipids from human skin obtained with noninvasive sampling method led us to investigate microanalytical separation techniques. The lipid class analysis was performed with a micro polyvinyl alcohol-silica (PVA-Sil) column. The gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 microL/min. In addition an evaporative light scattering detector (ELSD) was modified for micro-LC. All solvents contained 0.1% of triethylamine and formic acid in stoichiometric amount, which increased the ELSD response. In these conditions, the cholesterol eluted before free fatty acid, and squalene and triglycerides close to the dead volume. The various ceramide classes eluted following the order of the increased number of hydroxyl groups. The LOD for ceramides was 2.2 ng. The advantages of this method are the use of a normal stationary phase more reliable due to its chemical stability, its surface homogeneity and its development in microchromatography without chlorinated solvents which offers small LOD and the whole profile of lipids present in stratum corneum (SC). A method using a narrow-bore PVA-Sil column was used to collect ceramide fraction. Then the molecular species were analysed with a porous graphitic carbon column in capillary LC using a gradient from CH3OH/CHCl3 70:30 v/v to CHCl3 at 2%/min with a flow rate at 5 microL/min. The LOD obtained for ceramide was 1 ng. Both methods were assessed with SC samples obtained by rinsing a 5.7 cm2 area of the forearm with 25 mL of ethanol.     

Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.     

Optimization of a field flow pre-concentration system by experimental design for the determination of copper in sea water by flow-injection-atomic absorption spectrometry

A Plackett–Burman 2^7×3/32 design for seven factors (sample pH, sample flow rate, eluent volume, eluent concentration, eluent flow rate, ethanol percentage in the eluent and mini-column diameter) was carried out in order to find the significant variables affecting the field flow pre-concentration system (FFPS) and the flow injection elution manifold for copper determination in seawater samples by flame atomic absorption spectrometry. By using the optimized flow systems, seawater samples were collected and pre-concentrated in situ by passing them with a peristaltic pump through a mini-column packed with Amberlite XAD-4 impregnated with the complexing agent 4-(2-pyridylazo) resorcinol. Thus, copper is pre-concentrated without the interference of the saline matrix. Once in the laboratory, the mini-columns loaded with copper are incorporated into a flow injection system and eluted with a small volume of a 40% (v/v) ethanolic solution of 3 mol l⁻¹ hydrochloric acid into the nebulizer-burner system of a flame atomic absorption spectrometer. Analysis of certified reference materials (SLEW-3 and NASS-5) showed good agreement with the certified value.     

Determination of lead and mercury in sea water by preconcentration in a flow injection system followed by atomic absorption spectrometry detection

The capabilities of three solid chelating reagents were compared for the preconcentration of lead and mercury in high salinity aqueous samples (sea waters). The tested materials were 7-(4-ethyl-1-methyloctyl)-8-hydroxiquinoline (Kelex 100) adsorbed on Bondapack C18 (Kelex-100/C18), 8-hydroxiquinoline immobilized on vinyl co-polymer Toyopearl gel (TSK) and the commercial polystyrene/DVB ion exchange resin with paired iminodiacetate groups (Chelex-100). The two metals preconcentration and final determination were carried out in a flow injection system, coupled on-line to an atomic absorption spectrometric detector. Analytes were preconcentrated in the minicolumn, packed with the materials under investigation, while elution was achieved by injection of 500 μl of an adequate mineral acid solution. The different packing materials and minicolumn designs have been evaluated in terms of sensitivity for simultaneous preconcentration of both metals in sea water. Regarding the solid support, the best results were obtained for the TSK solid phase. Concerning the minicolumn design, the behavior was different for lead and mercury. Lead was quantitatively eluted with 0.5 M HCl and best performance was achieved when packing the solid material in a minicolumn with relatively small volume (1 cm length and 2.5 mm i.d.). In the case of mercury, bigger minicolumn volumes (5.5 cm length and 5.0 mm i.d.) and mixtures, 2 M HCl+1 M HNO₃, were required for its quantitative recovery and elution. The system has been evaluated for quantitative determination of the two metals under study in different Asturian coastal aqueous samples.     

Elution behavior of polyethylene in polar mobile phases on a non-polar sorbent

Linear polyethylene standards in the range of 1-500 kg/mol, dissolved in 1,2,4-trichlorobenzene, were injected into a column packed with oligo(dimethylsiloxane) modified silica gel. Fifteen polar solvents (cyclohexanone, cyclohexylacetate, cyclohexanol, nonylalcohol, dimethylformamide, dimethyl sulfoxide, ethylene- and diethylene glycol monobutyl ether, benzylalcohol, hexylacetate, bis(2-ethyl-hexyl)phthalate, N,N-dimethylacetamide, propylene carbonate, dipropylene glycol and N-methyl-pyrrolidone) were evaluated as mobile phases. Depending on the type of mobile phase evaluated, different elution behaviors are observed for polyethylene: (1) polyethylene was eluted in the size exclusion mode, (2) polyethylene was eluted together with the sample solvent peak at constant elution volume, (3) polyethylene was partially or fully retained on the column. The retained polymer was easily removed from the column by injecting a small volume of trichlorobenzene. The use of ethylene glycol monobutyl ether as the mobile phase enabled separation of the polyethylene from polypropylene. In this case polypropylene is eluted in the size exclusion mode, while polyethylene is eluted at a constant elution volume or remains in the column.     

On the minimization of the band-broadening contributions of a modern, very high pressure liquid chromatograph

The contributions of the volume of sample injected, the mobile phase flow rate, the inner diameter of the needle seat capillary and that of the connector capillary, the heat exchanger, and the detector cell volume to the widths of bands eluted from the 1290 Infinity HPLC instrument were investigated in depth. Four sample volumes (0.16, 0.80, 4.0, and 20 μL), three flow rates (0.04, 0.4, and 4.0 mL/min), two needle seat capillary I.D. (100 mm × 115 and 140 μm), three sets of connector capillary I.D. (350 mm × 80, 115, and 140 μm placed upstream the column, and 220 mm × 80, 115, and 140 μm downstream the column), two UV detector cell volumes (0.8 and 2.4 μL), and the presence/absence of the heat exchanger (1.6 μL) between the inlet connector capillary tube and the column were combined to generate up to 4 × 3 × 2 × 3 × 2 × 2=288 system configurations for this instrument. For each configuration, 5 consecutive injections were performed in order to assess the injection-to-injection repeatability, providing 1440 elution band profiles which are analyzed. The results demonstrate that the band broadening contribution of the instrument depends mostly on the detector cell volume and on the inner diameter of the needle seat capillary tube. The impact of these two contributions is particularly important at high flow rates (4 mL/min). Best efficiencies are obtained with a small sample volume, below 1 μL, which avoids volume overload of the instrument, or with large sample volumes, which maximize the radial concentration gradients of the sample across the instrument channels, in the vicinity of the anfractuosities of the channel walls. The injection of large sample volumes reveals the imperfection of current injection systems, the performance of which is remote from the one expected to provide an ideal rectangular injection (～+4 μL(2)). Although the present behavior of the instrument is satisfactory, serious improvements would become necessary to operate the next generation of more efficient columns that might be commercialized soon.     

Application of NaClO-treated multiwalled carbon nanotubes as solid phase extraction sorbents for preconcentration of trace 2,4-dichlorophenoxyacetic acid in aqueous samples

Multiwalled carbon nanotubes functionalized by oxidation of original multiwalled carbon nanotubes with NaClO were prepared and their application as solid phase extraction sorbent for 2,4-dichlorophenoxyacetic acid (2,4-D) was investigated systemically, and a new method was developed for the determination of trace 2,4-D in water samples based on extraction and preconcentration of 2,4-D with solid phase extraction columns packed with NaClO-treated multiwalled carbon nanotubes prior to its determination by HPLC. The optimum experimental parameters for preconcentration of 2,4-D, including the column activating conditions, the amount of the sorbent, pH of the sample, elution composition, and elution volume, were investigated. The results indicated 2,4-D could be quantitatively retained by 100 mg NaClO-treated multiwalled carbon nanotubes at pH 5, and then eluted completely with 10 mL 3:1 (v/v) methanol-ammonium acetate solution (0.3 mol/L). The detection limit of this method for 2,4-D was 0.15 μg/L, and the relative standard deviation was 2.3% for fortified tap water samples and 2.5% for fortified riverine water sample at the 10 μg/L level. The method was validated using fortified tap water and riverine water samples with known amount of 2,4-D at the 0.4, 10, and 30 μg/L levels, respectively.     

Preparative isolation of polyphenolic compounds from Vitis vinifera by centrifugal partition chromatography

This study deals with a centrifugal partition chromatography developed for the separation of phenolic compounds from Vitis vinifera. EtOAc grape seed extracts were separated using the solvent system hexane-ethyl acetate-ethanol-water (1:8:2:7; v/v) in two fractions: one containing about 75% of flavanol monomers (catechin and epicatechin) corresponding to 18% of crude extract and another fraction B-type dimers (22% of crude extract). From the stalk extracts, we could separate stilbenoid compounds (resveratrol and its oligomers; 12% of crude extract) which were eluted in less than 30 min from flavanols (which required a few hours of additional elution). Using the same solvent system but in different ratios (4:5:3:3; v/v), we isolated the trans-resveratrol (7@1000; 90% purity).     

免疫亲和萃取-超高效液相色谱-串联质谱法分离测定中成药及中药材中的5种黄曲霉毒素

利用免疫亲和萃取结合超高效液相色谱-串联四极杆质谱技术(UHPLC-ESI/QqQ-MS/MS)建立了中成药及中药材中5种黄曲霉毒素(B1、B2、G1、G2和M1)的提取、分离、确证与定量方法。样品经80%(体积分数)的甲醇水溶液提取和免疫亲和固相萃取后,采用UHPLC-ESI/QqQ-MS/MS的多反应监测模式实现分离、鉴定和外标法定量。5种目标毒素标准溶液的检出限(LOD)为0.05～0.3 μg/L。在0.5～100 μg/L的基质添加浓度范围内具有良好的线性关系(r2＞0.99);以甘草为例,当添加水平为1.0 μg/kg和5.0 μg/kg时,得到62.3%～82.4%的回收率(相对标准偏差(RSD)＜10%, n=6)。该方法灵敏度高、选择性和重复性好、回收率较高、检测速度快,适用于中成药及中药材等复杂基体中多种黄曲霉毒素的快速分析与筛查。     

凝胶色谱与多角激光光散射联用研究聚氧化乙烯的扩展效应和链构象

采用凝胶色谱与多角激光光散射联用的方法,测定了一系列不同分子量的聚乙二醇(PEG)和聚氧化乙烯(PEO)在色谱柱中的扩展效应.扩展因子随PEG/PEO分子量的增加而增大,经扩展效应改正后得到了样品的准确分子量和分子量分布.同时建立了PEO的Z均回转半径Rgz与重均分子量Mw之间的单分散标度关系:Rgz=0.0272 Mw0.56,结果表明,长链PEO在水溶液中由于排除体积效应采取溶胀的无规线团构象.     

Separation and on-line concentration of bisphenol A and alkylphenols by micellar electrokinetic chromatography with cationic surfactant

The separation and on-line concentration of bisphenol A and three alkylphenols were investigated by micellar electrokinetic chromatography with cationic surfactant. Tetradecyltrimethylammonium bromide was used as surfactant and the separation conditions were optimized by the addition of the organic solvents and cyclodextrins to the running solution. The separation of hydrophobic analytes and 4-nonylphenol isomers was improved by the addition of 20% acetonitrile and 20 mM beta-cyclodextrin to the running solution. When the sweeping with the running solution used as the on-line concentration procedure, 56-, 67- and 29-fold increase in detection sensitivity of bisphenol A, 4-tert.-butylphenol and 4-(1,1,3,3-tetramethylbutyl)phenol, respectively. The detection limits were 0.030, 0.098 and 0.159 mg/l, respectively.     

Use of expanded bed adsorption to purify flavonoids from Ginkgo biloba L.

Three techniques (liquid–liquid extraction, packed bed adsorption and expanded bed adsorption) have been compared for the purification of flavonoids from the leaves of Ginkgo biloba L. A crude Ginkgo extract was obtained by refluxing with ethanol for 3 h. The yield of flavonoids achieved by this crude extraction was about 19% (w/w) and the purity of flavonoids in the concentrated extract was between 1.9 and 2.3% (w/w). The crude extract was then dissolved in deionized water and centrifuged where necessary to prepare clarified feedstock for further purification. For the method using liquid–liquid extraction with ethyl acetate, the purity, concentration ratio and yield of flavonoids were 25.4–31.0%, 16–18 and >98%, respectively. For the method using packed bed adsorption, Amberlite XAD7HP was selected as the adsorbent and clarified extract was used as the feedstock. The dynamic adsorption breakthrough curves and elution profiles were measured. For a feedstock containing flavonoids at a concentration of 0.25 mg/mL, the appropriate loading volume to reach a 5% breakthrough point during the adsorption stage was estimated to be 550–600 mL for a packed bed of volume 53 mL and a flow rate of 183 cm/h. The results from the elution stage indicated that the majority of impurities were eluted by ethanol concentrations of 40% (v/v) or below and efficient separation of flavonoids from the impurities could be achieved by elution of the flavonoids with 50–80% ethanol reaching an average purity of ∼25%. The recovery yield of flavonoids using the packed bed purification method was about 60% of the flavonoids present in the clarified feedstock (corresponding to around 30% for the total flavonoids in the unclarified crude extract). For the method using expanded bed adsorption also conducted with Amberlite XAD7HP as the adsorbent, the optimal operation conditions scouted during the packed bed experiments were used but unclarified crude extract could be loaded directly into the column. For an expanded bed with a settled bed height of 30 cm, the loss of flavonoids in the column flow-through was about 30%. The two-step elution protocol again proved to be effective in separating the adsorbed impurities and flavonoids. More than 96% of the bound impurities were completely removed by 40% ethanol in the first elution stage and less than 4% remained in the final product eluted by 90% ethanol in the second elution stage. Also, ∼74% of the adsorbed flavonoids on column (corresponding to 51% of the total flavonoids in the unclarified feedstock) were recovered in the product. In addition to higher recovery yield, the average process time to obtain the same amount of product was decreased in the expanded bed adsorption (EBA) process. The results suggest that the adoption of EBA procedures can greatly simplify the process flow sheet and in addition reduce the cost and time to purify flavonoids from Ginkgo biloba. These results clearly demonstrate the potential for the use of EBA to purify pharmaceuticals from plant sources.     

Gel permeation chromatography: On the shape of the calibration plot and molecular size separability

Data obtained from the calibration of GPC columns of different permeabilities with standard polystyrenes are reported. For single columns the logarithm molecular weight–elution volume plot is linear for approximately one and one-half decades in molecular weight. GPC separations are such that the separability of two samples of similar molecular weight improves as their mean molecular weight decreases. Because of this the analysis of high molecular weight polymers can best be accomplished on a series of columns in which each column has a high permeability limit. The elution volume for columns in series is shown to be the sum of the elution volumes of the individual columns. As higher molecular weights are eluted a pronounced tailing effect is observed.     

Preconcentration of cobalt with 8-hydroxyquinoline and gas chromatographic stationary phase Chromosorb 105 and its determination by graphite furnace atomic absorption spectrometry

This paper presents a study of the adsorption characteristics of a commercially available GC stationary phase Chromosorb 105 for Co²⁺, which can be successfully applied to the preconcentration of Co²⁺ in water samples followed by GFAAS determination. After reacting with 8-hydroxyquinoline to form a complex at pH 8.0, Co²⁺ in water can be retained on a minicolumn packed with Chromosorb 105 and eluted with 2.5 ml of a mixture of ethanol and 2 mol l⁻¹ HNO₃ (2+1, v/v). The recoveries of Co²⁺ from 200 ml of tap water, river water and bottled natural mineral water samples are quantitative. Conditions for quantitative and reproducible preconcentration, elution and subsequent GFAAS determination were studied. A highly sensitive, simple method for preconcentration and GFAAS determination of trace amount of cobalt in natural water samples using a Chromosorb 105 packed minicolumn has been proposed. The high retention efficiency (95%) for Co²⁺ provides a sensitivity enhancement of 80 for a 200 ml sample volume with a detection limit of 13.4 ng l⁻¹ (3σ) and a quantification limit of 44.5 ng l⁻¹ (10σ).     

Separation of polythionates and the gold thiosulfate complex in gold thiosulfate leach solutions by ion-interaction chromatography

A method for the separation of the polythionates (SxO6(2-), x = 3-5) in gold thiosulfate leach solutions using ion-interaction chromatography with conductivity and ultraviolet (UV) detection is described. Polythionates were eluted within 18 min using an eluent comprising an acetonitrile step gradient at 0.0 min from 15% v/v to 28% v/v, 3 mM TBAOH, and 2.5 mM sodium carbonate, operated using a Dionex NS1-5 micron column with guard. The developed method was capable of separating the gold thiosulfate complex ion in standard solutions, but quantification of this species in realistic leach solutions proved impractical due to a self-elution effect that caused the gold peak to be eluted as a broad band. Detection limits for polythionates using a 10 microL injection volume ranged between 1-6 mg L(-1) (5-23 microM) for conductivity and 0.8-13 mg L(-1) (4-68 microM) for UV detection, based on a signal-to-noise ratio of 2. Calibration was linear over the ranges 5-2000, 10-2000 and 25-2500 mg L(-1) for trithionate, tetrathionate and pentathionate, respectively. The technique was applied successfully to leach liquors containing 0.5 M ammonium thiosulfate, 2 M ammonia, 0.05 M copper sulfate and 20 % m/v gold ore.     

Magnesium sulfate aerosols studied by FTIR spectroscopy: hygroscopic properties, supersaturated structures, and implications for seawater aerosols

Supersaturated MgSO4 aerosols and dilute MgSO4 solutions were studied by FTIR spectroscopic techniques (i.e., aerosol flow tube (AFT) and attenuated total reflection (ATR)). The hygroscopic properties of MgSO4 aerosols were investigated with results in good agreement with previous measurements by a scanning electrodynamic balance (SEDB). Well-defined spectral evolutions with changing relative humidity (RH) for the v3 band of SO4(2-) and the water O-H stretching envelope could be directly related to the observed hygroscopic properties of MgSO4 aerosols. When the RH decreased from approximately 55 to approximately 40%, the v1 band of SO4(2-) in supersaturated MgSO4 aerosols was observed to transform from a sharp peak at approximately 983 cm(-1) into a wide band at approximately 1005 cm(-1). The sharp peak at approximately 983 cm(-1) was mainly assigned to such associated complexes of Mg2+ and SO4(2-) as double solvent-separated ion pairs (2SIPs), solvent-shared ion pairs (SIPs), and simple contact ion pairs (CIPs) in supersaturated MgSO4 aerosols, while the wide band at approximately 1005 cm(-1) was due to polymeric CIPs chains, probably the main component of gels formed in MgSO4 aerosols at low RHs. Relating to this v1 band transformation, the peak position of the v(3) band was first shown to be a sensitive indicator of CIPs formation, spanning across approximately 40 cm(-1) on the formation of polymeric CIPs chains, which could also be supported by aerosol composition analysis in the form of water-to-solute molar ratios (WSR). In the water O-H stretching envelope, the absorbance intensities at 3371 and 3251 cm(-1) were selected to represent contributions from weak and strong hydrogen bonds, respectively. The absorbance intensity ratio changing with RH of 3371 to 3251 cm(-1) could be related to the previous observations with the v1 and v3 bands of SO4(2-). As a result, the formation of CIPs with various structures in large amounts was supposed to significantly weaken hydrogen bonds in supersaturated MgSO4 aerosols, while 2SIPs and SIPs were not expected to have similar effects even when occurring in abundance. In comparison with MgSO4 aerosols, the peak positions of the v3 band of SO4(2-) in artificial seawater aerosols implied that the MgSO4 component should be contained as gels or concentrated solutions in the fissures of microcrystals of sea salts for freshly formed seawater aerosols at low RHs.     

Use of Magnetic Nanoparticles and a Microplate Reader with Fluorescence Detection to Detect C‐reactive Protein

Two approaches based on magnetic nanoparticles (MNPs) have been compared to analyze C‐reactive protein (CRP). Both the non‐eluted and eluted MNP‐1°Ab‐CRP‐2°Ab/FITC bioconjugates were measured by a microplate reader with fluorescence detection. The linear ranges for the non‐elution and elution methods were 10‐200 and 0.1‐2.0 μg/mL, respectively. The concentration limits of detection for the nonelution and elution methods were 2.91 and 0.04 μg/mL, respectively. The non‐ elution method gave better precision and recovery than the elution method, and also showed comparable results to that of ELISA assay. The non‐elution method is simple and only needs minute volumes of sample and buffer. There is no need to dissociate the fluorescence probes from the bioconjugates, and the fluorescence signals can be directly measured on the MNP‐1°Ab‐CRP‐2°Ab/FITC bioconjugates. Meanwhile, samples with high CRP concentrations are not necessarily to be diluted before analysis.