Comparison of High-Temperature Gradient Heart-Cutting and Comprehensive LC × LC Systems for the Separation of Phenolic Antioxidants

Natural phenolic antioxidants were separated using comprehensive 2D HPLC on a Purospher Star RP-18e column in the first dimension and on two parallel Zirconia Carbon columns working in alternating cycles in the second dimension. The combination of the two columns provides great differences in separation selectivity in each dimension and an almost orthogonal 2D system. Temperature and solvent gradients were compared for the separation of the first-dimension fraction in the stop-flow heart-cutting 2D setup. Temperature gradients provide shorter separation times in comparison with solvent gradients. However, the time required for post-run column equilibration is too long for comprehensive LC × LC. High-temperature isocratic separation was employed in the second dimension of the comprehensive setup, allowing improvement of the fraction transfer frequency between the two dimensions and shorter 2D separation time in comparison to the earlier published method. The approach was applied to the analysis of beer and wine.     

New potential humic acid stationary phase toward drug components: Development of a chemometric‐assisted RP‐HPLC method for the determination of paracetamol and caffeine in tablet formulations

A new humic acid based stationary phase has been used, for the first time, to achieve the separation and quantification of paracetamol and caffeine in pharmaceutical preparations under reversed‐phase high‐performance liquid chromatography conditions. Central composite design was applied as a powerful tool to optimize the most dominant parameters that influence the resolution of reversed‐phase high‐performance liquid chromatography, that is, mobile phase composition (acetonitrile percentage in water), flow rate, and column temperature. The optimum conditions were obtained as 21.69%, 1.5 mL/min, and 15°C, respectively, with the aid of a second‐order quadratic model and desirability function. Under the optimum conditions, the peaks could be baseline separated within 10 min. For the developed reversed‐phase high‐performance liquid chromatography method, the linearity was investigated in the concentration ranges of 2–160 mg/mL (R² = 0.999) for paracetamol and 2–9.9 mg/mL (R² = 0.991) for caffeine. Mean recoveries for paracetamol and caffeine were 95.90 and 95.68%, respectively. The limits of detection and quantification were 4.1 × 10^‐4 and 1.3 × 10^‐3 mg/mL for paracetamol and 1.6 × 10^‐4 and 5.0 × 10^‐4 mg/mL for caffeine. The results showed that the new humic acid based stationary phase is very suitable for the separation of paracetamol and caffeine in pharmaceutical preparations and, thus it can be used effectively in the pharmaceutical industry.     

应用高效离子交换色谱快速分离定量不同致病力的青枯菌

应用高效离子交换色谱和激光光散射仪在线检测,快速分离定量不同致病力的青枯菌。青枯菌经过高效离子交换色谱分离得到3个特征峰,通过2,3,5-三苯基氯化四氮唑(TTC)平板鉴定和采用剪叶法回接番茄组培苗感染试验,发现这3个色谱峰所对应的青枯菌在致病力方面存在差异;其中峰3组分的致病力最强,峰1组分的致病力最弱。通过对青枯菌进样量与激光光散射仪的响应信号(峰面积)之间的线性关系研究,发现当青枯菌进样菌数为9×106~9×108时,菌数与色谱峰面积之间呈现出良好的线性关系,相关系数r=0.99。该项应用研究为不同致病力青枯菌的快速定量提供了一种新的分析方法。     

Study on Separation/Enrichment and Determination of Mercury(II) using Microcrystalline Thymolphthalein Loaded with Ternary Association Complex

The paper presents a novel method for the separation/enrichment of Hg²⁺ using microcrystalline thymolphthalein loaded with ternary association complex prior to the determination by spectrophotometry. The effects of different parameters, such as the dosages of KI and dodecyl trimethyl ammonium bromide (DTAB) and thymolphthalein, various salts and acidity etc. on the enrichment yield of Hg²⁺ have been investigated to select the experimental conditions. The results showed that in the presence of 1.0 g NaCl, when the dosage of 0.1 M KI solution was 1.50 mL and 5.0 × 10⁻³ M dodecyl trimethyl ammonium bromide (DTAB) solution was 1.50 mL respectively, the water‐insoluble ternary association complex of (DTAB)₂(HgI₄) which produced by Hg²⁺ and I⁻, DTAB cation (DTAB⁺) was quantificationally absorbed on the surface of microcrystalline thymolphthalein Therefore, Hg²⁺ was separated completely from Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Fe³⁺, Al³⁺, Pb²⁺, Bi³⁺ and Cr³⁺ etc. by contolling acidity. The possible enrichment mechanism of Hg²⁺ was deduced. The proposed method has been successfully applied to the determination of Hg²⁺ in the sample of industrial waster water, and the results agreed well with the dithizone method. The recoveries were 94.5%∼106.5%, and the RSD was 2.0%∼2.8%.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

Determination of Doxepin in human plasma using cloud‐point extraction with high‐performance liquid chromatography and ultraviolet detection

A new straightforward method based on cloud‐point extraction has been developed, optimized, and validated for the determination of doxepin in human plasma by high‐performance liquid chromatography separation and UV detection. The nonionic surfactant Triton X‐114 was chosen as the extraction solvent. Chromatography separation was performed on a μBondapak^R C₁₈ column (4.6 mm id × 300 mm, 3 μm particle size), which was used for isocratic elution at a detection wavelength of 289 nm. Under the optimum conditions, the linear range of doxepin in human plasma was 0.1–0.9 μg/mL. Also, the detection limit, preconcentration factor, and enrichment factor were 0.08 μg/mL, 50, and 49.0, respectively.     

Determination of aliphatic amines in air by membrane enrichment directly coupled to a gas chromatograph

Summary A method is described for the determination of shortchain aliphatic amines in ambient air based on impinger sampling in dilute H₂SO₄, selective enrichment across a PTFE gas membrane and quantification by gas chromatography. The enrichment step is carried out in a flow system directly connected to the chromatograph. The separation is performed on a packed column with nitrogen selective detection. The enrichment per sample volume was in the range 7.3 to 8.2 mL^–1 for C₁–C₆ amines. Detection limits were ca 3–10 nM with enrichment of a 2.9 mL liquid sample. After impinger sampling of 5 m³ air in 10 mL absorption solution, this corresponds, to 0.4–0.8 ng/m³ (ca 0.2–0.5 ppt by volume) in air.     

Angiotensin I-Converting Enzyme Inhibitory Proteins and Peptides from the Rhizomes of Zingiberaceae Plants

Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC₅₀ of 7.30 × 10⁻⁷ mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (−20–60 °C), showed >80% activity across a broad pH range of 4–12 (optimal at pH 4–5) and appeared as a competitive inhibitor of ACE (K _i of 9.1 × 10⁻⁵ mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC₅₀ of 0.38 ± 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC₅₀ of 0.011 ± 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K _i of 1.25 × 10⁻⁶ mg protein/mL).     

Determination of airborne mercury-203

A method is described for the determination of airborne compounds of²⁰³Hg. The method is based on the sorption of Hg on hopcalite (activated copper oxide/manganese oxide) with subsequent monitoring for the gamma-radiation of²⁰³Hg. Aerosol activity in the air is separated by sucking the air through a filter prior to the absorption tube. The sensitivity of the method permits the assessment of less than one tenth of the maximum permissible concentration in air. It is shown experimentally that the separation procedure used is suitable for the determination of airborne mercuric acetate and its thermal decomposition products.     

Study of antagonism between some intestinal bacteria with high-speed micellar electrokinetic chromatography

In this work, high-speed micellar electrokinetic chromatography with LIF detection was applied to study the antagonism between three intestinal bacteria, Escherichia coli (E. coli), Bacillus licheniformis (B. licheniformis) and Bacillus subtilis (B. subtilis). The fluorescent derivatization for the bacteria was performed by labeling the bacteria with FITC. In a high-speed capillary electrophoresis (HSCE) device, the three bacteria could be completely separated within 4 min under the separation mode MEKC. The BGE was 1 × TBE containing 30 mM SDS and 1.5 × 10^–5 g/mL polyethylene oxide. The limits of detection for E. coli, B. licheniformis and B. subtilis were 2.80 × 10⁶ CFU/mL, 1.60 × 10⁶ CFU/mL and 1.90 × 10⁶ CFU/mL respectively. Lastly, the method was applied to investigate the antagonism between the three bacteria. The bacteria were mixed and cultured for 7 days. The samples were separated and determined every day to study the interaction between bacteria. The results showed that B. licheniformis and B. subtilis could not inhibit each other, but they could effectively inhibit the reproduction of E. coli. The method developed in this work was quick, sensitive and convenient, and it had great potential in the application of antagonism study for bacteria.     

Neutral Lipid Class Fractionation and Further Separation of Simple Neutral Glycolipids by OPTLC

Abstract

In lipid preparation from biological sources extraction and chromatographic methods are widely used. The use of the OPTLC method is introduced in the present paper. The method is suitable for the class separation of the neutral fraction of a total lipid extract with a single isocratic run. With a step or an exponential gradient the simple neutral glycolipids can be separated. Either elution can be performed on a 10 × 20 cm plate on 12 parallel samples. The chromatograms were evaluated by densitometric scanning after staining with orcinol-H²SO₄ reagent.     

Graphite furnace-atomic absorption spectrophotometric determination of palladium in soil

A new and sensitive procedure for the graphite furnace-atomic absorption spectrophotometric (GF-AAS) determination of Pd in soil at nanogram level is described. The method is based on prior separation and enrichment of the metal as Pd(II)-SnCl₃ ^–-N-butylacetamide (BAA) complex into 1-pentanol (PN) by solvent extraction method. The value of the molar absorptivity of the complex in three solvents, i.e. ethyl acetate, 1-pentanol, chloroform, lie in the range of (0.70–2.75) × 10⁴ L mol^–1 cm^–1 at λ_max 360–440 nm. The metal could be enriched into organic solvent, i.e. PN, up to 10-folds. The sensitivity (A = 0.0044) of the method in the term of the peak height was 0.5 ng Pd/mL of the aqueous solution at an enrichment factor (EF) of 5. Optimization of analytical variables during enrichment and GF-AAS determination of the metal are discussed. The method has been applied for the analysis of Pd to soil samples derived from roads and highways in Germany.     

A Novel Electrogenerated Chemiluminescence Reaction Scheme Using Core-Shell LuminoI-Based SiO2 Nanoparticles as a Regulator and Its Analytical Application

A novel core-shell luminol-based SiO2 nanoparticle While these nanoparticles were used as electrogenerated was synthesized by two step micro-emulsion method. chemiluminescence （ECL） reagent, the electrochemical （EC） reaction as well as the subsequent chemiluminescence （CL） reaction not only could be separated spatially, but also presented high efficiency for analytical purpose. In this case, the core-shell luminol-based SiO2 nanoparticles offered more potential to avoid the contradiction between the EC and the CL reaction conditions. A new ECL method based on the nanoparticle was developed, and isoniazid was selected as a model analyte to illustrate the characteristics of this new ECL method. Under the selected conditions, the proposed ECL response to isoniazid concentration was linear in the range of 1.0 ×10^-10 to 1.0 × 10^-6 g/mL with 2 × 10^-11g/mL detection limit.     

Separation of HIV‐1 gag virus‐like particles from vesicular particles impurities by hydroxyl‐functionalized monoliths

The downstream processing of enveloped virus‐like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl‐functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV‐1 gag virus‐like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10¹²/mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double‐stranded DNA (88–90%) and host cell protein impurities (39–61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV‐1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10¹¹ particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10⁹ particles.     

Immunomagnetic nanoparticle-based sandwich chemiluminescence-ELISA for the enrichment and quantification of E. coli

Rapid methods for the quantification of Escherichia coli are required for the monitoring of faecal contamination in water to secure public health. The immunomagnetic separation (IMS) offers rapid enrichment and purification of bacteria in complex matrices and is compatible with immunoassays. By means of this technique, non-target cells and matrix components which might interfere with subsequent analytical methods are removed. We present the synthesis of magnetic nanoparticles (MNPs) and covalent coupling to antibodies against the enterobacterial common antigen (ECA) for use with IMS. Quantification was carried out with a chemiluminescence-based sandwich enzyme-linked immunosorbent assay (ELISA). Our anti-ECA-MNPs allow for a group-specific enrichment of bacterial cells, which can be combined with a species-specific analytical method. The particles were used along with commercially available magnetic columns for the selective enrichment of E. coli from 10-mL water samples. The volumetric enrichment factor was 9. For enriched samples, the limit of detection was reduced from 5.0?×?10⁶ cells·mL^-1 to 2.6?×?10⁵ cells·mL^-1. Using 200 µL anti-ECA-MNPs, we determined a recovery of 97?±?6% for a sample containing 10⁶ cells·mL^-1 and 89?±?2% for a sample containing 10⁷ cells·mL^-1. The overall time for cell enrichment and detection was 3 h 45 min.     

Metabolic footprinting of tumorigenic and nontumorigenic uroepithelial cells using two-dimensional gas chromatography time-of-flight mass spectrometry

In this study, gas chromatography mass spectrometry (GC-MS) and two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) were employed for the metabolic footprinting of a pair of immortalized human uroepithelial cells namely HUC-1 (nontumorigenic) and HUC T-2 (tumorigenic). Both HUC-1 and HUC T-2 cell lines were cultivated in 1 mL of Ham’s F-12 media. Subsequent to 48 h of incubation, 200 μL of cell culture supernatant was protein-precipitated using 1.7 mL of methanol and an aliquot of 1.5 mL of the mixture was separated, dried, trimethylsilyl-derivatized, and analyzed using GC-MS and GC×GC-TOFMS. Metabolic profiles were analyzed using multivariate data analysis techniques to evaluate the changes of the metabolomes. Both GC-MS and GC×GC-TOFMS analyses showed distinct differences in metabolic phenotypes of the normal and tumorigenic human bladder cells (partial least squares-discriminant analysis (PLS-DA) of GC×GC-TOFMS data; two latent variables, R ² X = 0.418, R ² Y = 0.977 and Q ² (cumulative) = 0.852). Twenty metabolites were identified as being statistically different between the two cell types. These metabolites revealed that several key metabolic pathways were perturbed in tumorigenic urothelial cells as compared to the normal cells. Application of GC×GC-TOFMS offered several advantages compared to classical one-dimensional GC-MS which include enhanced chromatographic resolution (without increase in analytical run time), increase in sensitivity, improved identification of metabolites, and also separation of reagent artifacts from the metabolite peaks. Our results reinforced the advantages of GC×GC-TOFMS and the role of metabolomics in characterizing bladder cancer biology using in vitro cell culture models.     

Determination of active ingredients in hawthorn and hawthorn piece by capillary electrophoresis with electrochemical detection

Capillary-zone electrophoresis with electrochemical detection has been used for the separation and determination of (-)-epicatechin, rutin, hyperin, chlorogenic acid, and quercetin in hawthorn and hawthorn piece. The effects of several important factors, including the running buffer acidity, the separation voltage, and the working electrode potential, were evaluated to acquire the optimum analytical conditions. The working electrode was a 300-μm carbon-disk electrode at a working potential of +0.95 V (vs. SCE). Under the optimum conditions, the analytes can be well separated within 16 min in a 75-cm-long fused-silica capillary. The current response was linear over two orders of magnitude with detection limits (S/N = 3) ranging from 6.00 × 10⁻⁸ to 3.75 × 10⁻⁷ g/mL for all analytes. The method was successfully used in the analysis of hawthorn and hawthorn piece and the assay results were satisfactory. The text was submitted by the authors in English.     

Cationic poly(amidoamine) dendrimers as additives for capillary electroseparation and detection of proteins

We examine the influence of cationic poly(amidoamine) (PAMAM) dendrimers on capillary electroseparation–UV analysis of proteins. PAMAMs adsorbing to the capillary surface suppressed the wall‐adsorption of proteins; meanwhile, PAMAMs added to the buffer exhibited selectivity toward proteins. Presence of 3×10^?4 g/mL PAMAM generation one (G 1.0) in 30 mM phosphate, at pH 2.6, rendered significant enhancement in separation efficiency; the merged peaks of myoglobin and trypsin inhibitor were separated. Moreover, the protein–dendrimer interactions changed the inherent UV absorbance profiles of proteins. UV–Vis study showed that the absorbance of cytochrome C and transferrin increased at the detection wavelength of 214 nm; their detection sensitivity enhanced by 2.44 and 2.01‐folds, respectively, with addition of 5×10^?4 g/mL PAMAM G 1.0.     

Chiral Determination of Salbutamol, Salmeterol and Atenolol by Two-Dimensional LC–LC: Application to Urine Samples

A heart-cut two-dimensional high-performance liquid chromatography method for enantiomeric determination of salbutamol, salmeterol and atenolol in urine is presented. It involves the use of two separations in a liquid chromatography?Cliquid chromatography achiral?Cchiral coupling. Target compounds were previously separated in a primary column (Kinetex? HILIC, 2.6???m, 150?×?2.1?mm I.D.) with a mixture of MeOH:ACN:ammonium acetate buffer (5?mM, pH 6) 90:5:5 (v/v/v) as mobile phase at a flow rate of 0.40?mL?min^?1. Enantiomeric separation was carried out by transferring peak of each compound through a switching valve to a vancomycin chiral column (Chirobiotic? V, 2.6???m, 150?×?2.1?mm I.D.) using MeOH:ammonium acetate buffer (2?mM, pH 4) 97:3 (v/v) as mobile phase at a flow rate of 0.50?mL?min^?1. Ultraviolet detection was done at 227?nm. The method was applied to determine target analytes in urine samples after enzymatic hydrolysis with ??-glucuronidase from Helix pomatia, followed by a solid-phase extraction procedure using Isolute^? HCX mixed-mode cartridges. Extraction recoveries ranged from 82 to 90?% in urine samples. Detection limits were 0.091?C0.095???g for each enantiomer of atenolol and between 0.058 and 0.076 and 0.18?C0.14???g for enantiomers of salbutamol and salmeterol, respectively (3?mL of urine). Linearity ranges were between 0.5 and 10???g?mL^?1. Intraday and interday reproducibilities of enantiomeric ratio and enantiomeric fraction, expressed as relative standard deviation, were between 1.9 and 9.0?%. The optimized method was successfully applied to the analysis of urine samples obtained from excretion studies in volunteers and in freeze-dried urine samples, containing urinary components with MW?<?10,000 and components with MW?>?10,000, spiked with different amounts of studied drugs.     

Flotation Separation of Lead with Sodium Nitrate‐Potassium Iodide‐Cetyltrimethyl Ammonium Bromide System

The flotation separation behavior of lead with Sodium Nitrate‐Potassium Iodide‐Cetyltrimethyl Ammonium Bromide system and the conditions for the separation of lead with other metal ions are studied in this research. With 0.1 M potassium iodide, 1.0 × 10^?2 M Cetyltrimethyl Ammonium Bromide and 1.0 g/10 mL of sodium nitrate, Pb(II) can form an ion‐association complex (PbI₄^2?) (CTMAB⁺)₂ and be separated completely from Zn(II), Fe(III), Co(II), Ni(II), Mn(II) and Al(III) by flotation at pH = 1.0–3.0.