Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (> 16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).

Optimization of conditions for cell cultivation of Porphyra haitanensis conchocelis in a bubble-column bioreactor

Process conditions for cell cultures derived from conchocelis of female red macroalga Porphyra haitanensis were optimized in an illuminated 0.3-l bubble-column photobioreactor, using CO2 in air as the sole carbon source during a 20-day cultivation period. It reached the highest growth rate when the initial cell density was 700 mg l(-1)(dry weight), the optional aeration rate was 1.2 v/v/min, inorganic nitrate concentration was 15 mM and inorganic phosphate concentration was 0.6 mM. This is the first reported bioreactor cultivation study of cell cultures derived from conchocelis of Porphyra haitanensis.

海洋藻类功能性蛋白质的纯化、鉴定及其应用的研究

使用膨化柱和离子交换或羟基磷灰石柱层析相结合的方法，分别从多管藻、坛紫菜及钝顶螺旋藻中分离纯化了R-藻红蛋白溶液和C-藻蓝蛋白。光谱检测及电泳分析结果证明完全符合经典的藻胆蛋白纯度标准。彭化床最突出的优点是克服了常规分离方法堵塞色谱柱的难题，纯化速度快、产量高、不需要常规色谱方法所要求的填料的平衡及粗提液的预处理，仅需一步操作就可以得到满足一般食品添加剂纯度要求的藻胆蛋白，极大地简化了后续的纯化程序，减少了分离纯化的步骤和时间，而其产率及纯度均高于常规的藻胆蛋白分离方法。这同时也降低了藻胆蛋白分离纯化的成本。 本文通过戊二醛或环氧氯丙烷交联的方法，合成了四种壳聚糖-氨基酸共聚小球。并选取吸附性好的戊二醛交联孔球和戊二醛交联微球系统测定了其对R-藻红蛋白和C-藻蓝蛋白的吸附和缓释性能。 纯化了藓羽藻中与其细胞器团聚密切相关的一种凝集素并进行了部分性质的鉴定。N端前15个氨基酸序列及LC-ESI-MS质谱分析结果证明此凝集素属于一种新的蛋白质族。实验证明，凝血活性与细胞器团聚活性并不完全依赖于此凝集素分子相同的结构域。 通过异双功能试剂SPDP处理藓羽藻凝集素使之衍生化，DTT处理R-PE在其分子内引入外源巯基，然后将活化的R-藻红蛋白与凝集素进行交联反应。交联产物经凝胶过滤纯化并检测，但电泳及荧光显微镜检测结果并不能证明交联探针的成功制备。

Application of 2-(11H-benzo[a] carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

Determination of aliphatic amines from soil and wastewater of a paper mill by pre-column derivatization using HPLC and tandem mass spectrometry (HPLC-MS/MS)

A pre-column derivatization method for the sensitive determination of aliphatic amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by HPLC with fluorescence detection and APCI/NIS identification in positive-ion mode has been developed. The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by the 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent, BCEOC, that could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M + H](+) with APCI/MS in positive-ion mode. The collision induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 264.1, m/z 246.0 and m/z 218.1, corresponding to the cleavages of CH2CH2O-CO, CH2CH2-OCO, and N-CH2CH2O bonds. Studies on derivatization conditions demonstrated that excellent derivatization yields close to 100% were observed with a 3 to 4-fold molar reagent excess in acetonitrile solvent, in the presence of borate buffer (pH 9.0) at 40 degrees C for 10 min. In addition, the detection responses for BCEOC derivatives were compared with those obtained with CEOC and FMOC as labeling reagents. The ratios I-BCEOC/I-CEOC and I-BCEOC/I-FMOC were, respectively, 1.40-2.76 and 1.36-2.92 for fluorescence responses (here, I was the relative fluorescence intensity). Separation of the amine derivatives had been optimized on an Eclipse XDB-C-8 column. Detection limits calculated from an 0.10 pmol injection, at a signal-to-noise ratio of 3, were 18.65-38.82 fmol (injection volume 10 mu L for fluorescence detection. The relative standard deviations for intraday determination (n = 6) of standard amine derivatives (50 pmol) were 0.0063-0.037% for retention times and 3.36-6.93% for peak areas. The mean intra-and inter-assay precision for all amines were <5.4% and 5.8%, respectively. The recoveries of amines ranged from 96 to 113%. Excellent linear responses were observed with correlation coefficients of >0.9994. The established method provided a simple and highly sensitive technique for the quantitative analysis of trace amounts of aliphatic amines from biological and natural environmental samples.

Pre-column derivatization of amines with 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate followed by LC-fluorescence and LC-APCI-MS

A pre-column derivatization method for the sensitive determination of amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate (BCIC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives is carried out by high performance liquid chromatography/atmospheric pressure chemical ionization (LC-APCl-MS-MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent is replaced by 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl functional group, which results in a sensitive fluorescence derivatizing reagent BCIC-Cl. BCIC-Cl can easily and quickly label amines. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography and show an intense protonated molecular ion corresponding m/z [MH](+) under APCl in positive-ion mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 260 corresponding to the cleavage of CH2-OCO bond. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3 to 4-fold molar reagent excess. In addition, the detection responses for BCIC derivatives are compared with those obtained using CEOC and FMOC as derivatization reagents. The ratios of l(BCIC)/l(CEOC) and l(BCIC)/l(FMOC) are, respectively, 1.23-3.14 and 1.25-3.08 for fluorescent (FL) responses (here, l is relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C-8 column. Detection limits are calculated from 1.0 pmol injection, at a signal-to-noise ratio of 3, are 10.6-37.8 fmol. The mean interday accuracy ranges from 94 to 105% for fluorescence detection with the largest mean %CV < 7.5. The mean interday precision for all standards is < 6.0% of the expected concentration. Excellent linear responses are observed with coefficients of > 0.9997.

A novel silica-based column packing for reversed-phase HPLC of basic compounds

A novel bonded phase for reversed-phase HPLC was synthesized in two steps. Octylamine was first reacted with beta-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (beta -ECTS) and then the intermediate product was coupled onto porous silica. The prepared packing was characterized by elemental analysis, solid-state C-13 NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes and methanol-water as binary mobile phase. The results showed that the stationary phase has excellent chromatographic properties and is resistant to hydrolysis between pH = 2 similar to 8. It can be used efficiently for the separation of basic compounds.

A gas chromatographic analysis of light hydrocarbons on a column packed with modified silica gel

A one-meter long column packed with silica gel is used to separate light hydrocarbons. The silica gel has been modified with several kinds of gas chromatography stationary phases. Among these, PEG 2000 shows fairly good effect when using 80-100 meshes silica gel for the separation of mixture of methane, ethane, ethylene, acetylene, propane, propylene and n-, i-butane. The different behavior of silica gel between batch to batch is also found. When silica gel is coated with a small amount of Al2O3 prepared with sol-gel method, better resolution has been observed on a 2-meter column compared with the non-modified silica gel.

Capillary electrochromatography using a strong cation-exchange column with a dynamically modified cationic surfactant

A novel mode of capillary electrochromatography (CEC), called dynamically modified strong cation-exchange CEC (DMSCX-CEC), is described in this paper. A column packed with a strong cation-exchange (SCX) packing material was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX packing material, and the resulting hydrophobic layer on this packing was used as the stationary phase. Using the dynamically modified SCX column, neutral solutes were separated with the CEC mode. The highest number of theoretical plates obtained was about 190 000/m, and the relative standard deviations (RSD's) for migration times and capacity factors of alkylbenzenes were less than 1.0% and 2.0% for five consecutive runs, respectively. The effects of CTAB and methanol concentrations and the pH value of the mobile phase on the electroosmotic flow and the separation mechanism were investigated. Excellent simultaneous separation of the basic and neutral solutes in DMSCX-CEC with a high-pH mobile phase was obtained, A mixture containing the acidic, basic, and neutral compounds was well separated in this mode with a low-pH mobile phase; however, peak tailing for basic compounds was observed in this mobile phase.

Alkaline treatment of the cellulose fiber affecting membrane column behaviour for high-performance immunoaffinity chromatography

The original cellulose fibers and those treated by alkaline solution were both used to prepare the acrylic membranes. The two kinds of membranes were packed into the columns for high-performance immunoaffinity chromatography by the immobilization of protein A on them. It was observed that the alkaline treatment of the cellulose fiber decreased the pressure resistance of the membrane to the mobile phases and greatly increased the accessible volume to the proteins, but affected the adsorption capacity of human IgG on the protein A membrane columns less. There is little difference between those two kinds of membranes on the adsorption capacities of HIgG, which means that the alkaline treatment of the cellulose fiber only significantly changes the void volume inter-membrane, and the porosity and surface area of membrane less. Alkaline treatment of the cellulose fiber reduced the membrane-column efficiency significantly. Some typical examples for the immunoaffinity analysis of IgG from human and dog plasma on the protein A membrane columns are illustrated. Copyright (C) 2000 John Wiley & Sons, Ltd.

Determination of alcohols by high-performance liquid chromatography with fluorimetric detection after pre-column derivatisation with carbazole-9-N-acetic acid and chromatographic behaviour of alcoholic derivatives

Alcohols were derivatised to their carbazole-9-N-acetic acid (CRA) esters with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC . HCl) as the dehydrating agent. Studies on derivatisation conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives. The retention behaviour of alcohol derivatives was investigated by varying mobile phase compositions (ACN-water and MeOH-water). The parameters from the equation log k'=A-BX were evaluated by retention data of derivatives using an isocratic elution with different mobile phases. The results indicated that the parameters derived allowed computation of retention factors in good agreement with experiments. At the same time, a general equation was derived that makes possible predictions of partition coefficient in binary mobile phases with different proportions of organic solvent to water based on some simple regression analysis. The LC separation for the derivatised alcohols containing higher carbon alcohols showed good reproducibility on a reversed-phase C-18 column with gradient elution. The detection limits (excitation at 335 nm, emission at 360 nm) for derivatised alcohols (signal-to-noise ratio=3:1) were in the range of 0.1-0.4 pg per injection. (C) 2001 Elsevier Science B.V. All rights reserved.

Estimation of soil organic partition coefficients: from retention factors measured by soil column chromatography with water as eluent

The retention factors (k) of 104 hydrophobic organic chemicals (HOCs) were measured in soil column chromatography (SCC) over columns filled with three naturally occurring reference soils and eluted with Milli-Q water. A novel method for the estimation of soil organic partition coefficient (K-oc) was developed based on correlations with k in soil/water systems. Strong log K-oc versus log k correlations (r>0.96) were found. The estimated K-oc values were in accordance with the literature values with a maximum deviation of less than 0.4 log units. All estimated K-oc values from three soils were consistent with each other. The SCC approach is promising for fast screening of a large number of chemicals in their environmental applications. (C) 2002 Elsevier Science B.V. All rights reserved.

Influence of methanol on retention of hydrophobic organic chemicals in soil leaching column chromatography

The influence of methanol in methanol-water mixed eluents on the capacity factor (P), an important parameter which could depict leaching potential of hydrophobic organic chemicals (HOCs) in soil leaching column chromatography (SLCC), was investigated. Two reference soils, GSE 17201 obtained from Bayer Landwirtschaftszentrum, Monheim, Germany and SP 14696 from LUFA, Spencer, Germany, were used as packing materials in soil columns, and isocratic elution with methanol-water mixtures at different volume fractions of methanol (phi) were tested. Shortterm exposure of the column (packed with the GSE 17201 soil) to the eluents increased solute retention by a certain (23% log-unit) degree evaluated through a correlation with the retention on the same soil column but unpreconditioned by methanol-containing eluents. Long-term exposure of soil columns to the eluents did not influence the solute retention. A log-linear equation, log k' = log k'(w) - Sphi, could well and generally describe the retention of HOCs in SLCC. For the compounds of homologous series, logk'(w), had good linear relationship with S, indicating the hydrophobic partition mechanism existing in the retention process. (C) 2002 Elsevier Science Ltd. All rights reserved.

Linear solvation energy relationships regarding sorption and retention properties of hydrophobic organic compounds in soil leaching column chromatography

The capacity factors of a series of hydrophobic organic compounds (HOCs) were measured in soil leaching column chromatography (SLCC) on a soil column, and in reversed-phase liquid chromatography on a C-18 column with different volumetric fractions (phi) of methanol in methanol-water mixtures. A general equation of linear solvation energy relationships, log(XYZ) = XYZ(0) + mV(1)/100 + spi* + bbeta(m) + aalpha(m), was applied to analyze capacity factors (k'), soil organic partition coefficients (K-oc) and octanol-water partition coefficients (P). The analyses exhibited high accuracy. The chief solute factors that control log K-oc, log P, and log k' (on soil and on C-18) are the solute size (V-1/100) and hydrogen-bond basicity (beta(m)). Less important solute factors are the dipolarity/polarizability (pi*) and hydrogen-bond acidity (alpha(m)). Log k' on soil and log K-oc have similar signs in four fitting coefficients (m, s, b and a) and similar ratios (m:s:b:a), while log k' on C-18 and log P have similar signs in coefficients (m, s, b and a) and similar ratios (m:s:b:a). Consequently, log k' values on C-18 have good correlations with log P (r > 0.97), while log k' values on soil have good correlations with log K-oc (r > 0.98). Two K-oc estimation methods were developed, one through solute solvatochromic parameters, and the other through correlations with k' on soil. For HOCs, a linear relationship between logarithmic capacity factor and methanol composition in methanol-water mixtures could also be derived in SLCC. (C) 2002 Elsevier Science Ltd. All rights reserved.