Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.     

Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate-mediated protease that cleaves factor H

Dermatan sulfate mediates the blood coagulation cascade by binding to heparin cofactor II and potentiating the antithrombin activity. In order to explore another function of dermatan sulfate, a dermatan sulfate affinity column was prepared from biotinylated dermatan sulfate and Streptavidin Sepharose. When human plasma was applied on the dermatan sulfate column, factor H was bound and cleaved. The cleavage products, a 30-kDa N-terminal fragment and a 120-kDa fragment, were eluted from the column with 500 mM NaCl and detected after Western blotting with anti-factor H. The bond between the tandem arginine residues in the sixth domain of factor H was cleaved. When purified factor H was applied on the column, the factor H was not cleaved and was recovered from the column as an intact 150-kDa fraction. The finding that dermatan sulfate-mediated cleavage of factor H was inhibited by (p-amidinophenyl) methanesulfonyl fluoride, but not N-ethylmaleimide or EDTA, indicates that a serine protease in the plasma was activated on the dermatan sulfate column and factor H was cleaved without intervention of the plasma protease inhibitors. Amidase activity was detected in the effluent from the dermatan sulfate column but was abolished by pretreatment of the plasma with dermatan sulfate. Therefore, dermatan sulfate participates in the activation of a protease as well as having the protease inhibitory action.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

Suspended rice particles for cultivation of Monascus purpureus in a tower-type bioreactor

 Cultivation of Monascus purpureus (CCRC 31615) for the production of natural pigments was investigated. Traditionally, Monascus species were grown on rice by solid-state culture. For large-scale cultivation, solid-state cultures were associated with some problems such as contamination and scale-up. By using submerged cultures with rice particles, a stirred-tank fermentor was not suitable for submerged cultures as the impeller tended to break the particles into small pieces. A conventional bubble column was also unsuitable as its mixing capability was poor. In the present study, a modified bubble column with wire-mesh draft tubes was employed for the cultivation of M. purpureus. The proposed column had a shorter mixing time and a higher oxygen transfer rate relative to the conventional bubble column. The production of pigments using the proposed column was up to 80% higher than that achieved using the conventional bubble column. Received: 21 July 1999 / Received revision: 8 November 1999 / Accepted: 19 November 1999     

Plasma transport of 5-hydroxytryptamine-3H. I. Nature and classification of indole derivatives after injection of 5-hydroxytryptamine-3H]

After a single i.p. injection of tritiated-5-hydroxytryptamine to young, old or stressed rats, the blood plasma was filtered through Sephadex-G 25 column. Two peaks of radioactivity were obtained. One was excluded from the column and eluted together with plasma proteins, the other was retained on the column and eluted as free indoles. The radioactivity bound to plasma proteins was identified as 5-hydroxyindole acetic acid. The free radioactivity was identified as 5-hydroxytryptamine.     

A novel method for the isolation and purification of protoplasts from friable,embryogenic corn (Zea Mays L.) callus

A method is described for the isolation of protoplasts from rapidly-growing, friable embryogenic and organogenic cell cultures of corn. A Sepharose 6MB cyanogen-bromide-activated macrobead column coupled with Cellulase RS was used to separate contaminating cells from protoplasts. The column consists of layering 1.5 cm of the coupled-macrobeads into a 2.2-cm diameter column. Contamination of protoplasts by cells possessing partial or complete walls was reduced from 25% to near zero after a single passage through the column. The column was capable of retaining in excess of 30 million cells and recovering 99% cell-free preparations from culture material consisting of less than 1% protoplasts. Coupled-macrobeads were easily recovered, washed free of cells and stored for repeated use. Corn protoplasts appeared undamaged by the column and rapeseed (Brassica napus) protoplasts which were passed through the column have divided and formed colonies in culture. Uncoupled macrobeads were not as efficient as coupled macrobeads in reducing cellular contamination.     

Application of a semipermeable surface column for the determination of amoxicillin in human blood serum

A high-performance liquid chromatography column-switching system for the automated determination of amoxicillin in human serum was developed as a more efficient alternative for the already existing systems with off-line sample pretreatment. The column-switching system consists of a semipermeable surface (SPS) column and an analytical reversed-phase (RP) C18 column. After centrifuging, pure serum samples were injected into the column-switching system. Clean-up, with regard to removal of proteins, was performed on the SPS column. The fraction containing amoxicillin was concentrated on the analytical RP-C18 column. Finally, chromatography and detection were performed with the RP-C18 column using UV detection at 234 nm. The total analysis time was 15 min. The method has proven to be reliable and to be more time- and resource-efficient compared to previously used methods with off-line sample clean-up. It is now used in bioavailability studies for the development of new amoxicillin formulations.     

Effect of Iron Bioavailability on Dissolved Hydrogen Concentrations During Microbial Iron Reduction

Dissolved hydrogen (H2) concentrations have been shown to correlate with specific terminal electron accepting processes (TEAPs) in aquifers. The research presented herein examined the effect of iron bioavailability on H2 concentrations during iron reduction in flow-through column experiments filled with soil obtained from the uncontaminated background area of the Field Research Center (FRC), Oak Ridge, TN and amended with acetate as the electron donor. The first column experiment measured H2 concentrations over 500 days of column operation that fluctuated within a substantial range around an average of 3.9 nM. Iron reduction was determined to be the dominant electron accepting process. AQDS (9,10-anthraquinone-2,6-disulfonic acid) was then used to determine if H2 concentrations during iron reduction were related to iron bioavailability. For this purpose, a 100-day flow-through column experiment was conducted that compared the effect of AQDS on iron reduction and subsequent H2 concentrations using two columns in parallel. Both columns were packed with FRC soil and inoculated with Geobacter sulfurreducens but only one was supplied with AQDS. The addition of AQDS increased the rate of iron reduction in the flow-through column and slightly decreased the steady-state H2 concentrations from an average of 4.0 nM for the column without AQDS to 2.0 nM for the column with AQDS. The results of this study therefore show that H2 can be used as an indicator to monitor rate and bioavailability changes during microbial iron reduction.     

固相萃取-气相色谱法测定水和废水中四种氯苯类有机污染物

研究了用固相萃取技术结合氢火焰气相色谱法测定水和废水中四种氯苯类有机污染物。通过实验比较Sdex C18,Sep-Park Vac Silica,Bond Elut CARBON,Bond Elut SI和Bond Elut PLEXA五种SPE小柱对四种氯苯类的萃取效率,发现Dionex的Sdex C18柱有很好的回收率。系统研究了最佳萃取条件,甲醇洗脱体积为4.0 mL,且洗脱液不可通过氮吹浓缩;四种氯苯类化合物除氯苯外穿透体积都在1.0 L以上,而氯苯的穿透体积为300 mL,表明Sdex C18柱对二氯苯和三氯苯有很强的吸附性。在最佳萃取和测定条件下,方法线性范围为20.0~400μg/L,检出限为0.383~0.635μg/L,完全满足日常环境监测分析要求。     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Comparative androecium morphogenesis ofSicyos angulatus andSechium edule (Cucurbitaceae)

“Androecium” ofSicyos angulatus andSechium edule is unique in having a solid central column below a head portion with thecae. Its morphogenesis was examined for the two species. The developmental course is composed of two distinct successive phases; (1) establishment of stamen primordia and (2) uplift of the stamen primordia caused by development of a central column below them. In the first phase, there is a difference between the two species; inSicyos angulatus, two bithecal and one monothecal stamen primordia are formed by congenital fusion among preformed five protrsions, whilst inSechium edule, three or four monothecal stamen primordia are formed without fusion. The central column is later produced by intercalary growth in a region below the stamen primordia in both species. Concomitant with central column development, the center of the floral primordium, which was surrounded by the early formed stamen primordia, is raised up to the top of the central column. The central column could be interpreted as a receptacular column, and not as congenitally fused stamen filaments, as currently believed. The “androecium” of the both species is considered an androecium complex, which consists of the stamens and a receptacular column.     

Fibrous stationary phase in capillary electrochromatography

Capillary electrochromatography (CEC) using fibrous cellulose acetate (CA) stationary phase was investigated. The advantage of this fiber-packed column is relatively easy preparation process compared with other conventional CEC columns, such as particle-packed and wall-coated capillaries. CA fibers are manually packed into a capillary with two guide liners and fixed with a frit at the column inlet. The separation characteristics of this column were investigated using n-alkyl p-hydroxybenzoates (parabens) as the sample probe. It has been demonstrated that the use of a short column length and a specially designed tee-connector as the injection device should make the separation performance and efficiency much higher on the fiber-packed columns. Sufficient separation between methyl and n-butylparabens is obtained on the 5-cm-packed column and linear relationships between the injection time and the peak area are observed. Bubble formation is not encountered during the analysis.     

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column

This study examined the impact of fouling with yeast homogenate on capacity and breakthrough performance of an ion exchange packed bed column. Column performance was assessed by analysis of breakthrough curves obtained with BSA as a test protein. The overall impact of fouling on breakthrough performance depended heavily on the level of clarification of the feed stream. Challenging the column with particulate-free homogenate caused no change in column performance. Loading successive small volumes of poorly clarified homogenate, interspersed with frequent column salt washes, did not alter significantly the column capacity. By contrast, when the column was challenged with an equivalent cumulative volume of poorly clarified homogenate, dynamic binding capacity decreased significantly and changes in breakthrough curves suggested increased intraparticle and external mass transfer limitations. These changes were ascribed to deposition of solid particulates in void spaces in the bed and colloidal contaminants in the bead pores.     

Reversible interference of Fe³(+) with monoclonal antibody analysis in cation exchange columns

The effect of Fe3(+) treatment on weak cation exchange column chromatography was demonstrated for monoclonal antibody (MAb) analysis. Fe3(+)-exposed columns showed lowered relative peak areas of the parent MAb peak as well as both acidic and basic variant peaks that could lead to erroneous conclusions. Accurate measurement of relative amounts of variants to the parent MAb is essential for demonstrating the safety and efficacy of therapeutic molecules such as MAbs. Complete reversal of the compromised MAb analysis performance was observed after washing the column with chelating agents, confirming that metal contamination was responsible for the compromised column performance.     

Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms

Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified. A thermostable ß-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme. After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column. The isolated ß-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins. The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme. After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250). The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass. The optimum temperature of both enzymes was approximately 75°C.     

花卉柱式无土栽培

于玻璃温室内的160 m2面积上,以干湿交替型盆钵组装起高200 cm, 直径15 cm的立柱77根,然后组成立柱“树林”并种花,25个科的共53种草花用同一营养系统管理,生长都良好,立柱“树林”象是花的“森林”,显示了良好的生态关系。在此基础上挑选不同种盆花组成不同情趣的家庭阳台花柱。阳台花柱具若干优点：新型盆钵具良好的水、气、肥协调关系,适合多种花卉生长;柱高任选,便手提携带;柱体能环绕中轴旋转使植物受光均匀;花柱底部是具中岛结构式底盆,起蓄水和稳定作用,还便于多根立柱串联扩大栽培量;有人工和自动浇灌两种系统,省工省时且干净卫生。     

Cometabolic degradation of chlorinated aromatic compounds

The degradation of chlorobenzene was investigated with the specially chosen strain Methylocystis sp. GB 14 DSM 12955, using 23 ml headspace vials and in a soil column filled with quaternary aquifer material from a depth of 20 m. A long-term experiment was carried out in this column, situated in a mobile test unit at a contaminated location in Bitterfeld (Germany). Groundwater polluted by chlorobenzene was continuously fed through the column, through which a mixture comprising 4% CH(4) and 96% air was bubbled. Chlorobenzene was oxidized by up to 80% under pure culture conditions in the model experiments and was completely degraded under the mixed culture conditions of the column experiments. Over a period of 4 months, the stability of the biological system was monitored regularly by analyzing the sMMO activity as well as by classical microbiological and molecular biological methods.     

Determination of phosphatidylcholine and disaturated phosphatidylcholine content in lung surfactant by high performance liquid chromatography

A rapid isocratic method for determining the total phosphatidylcholine and disaturated phosphatidylcholine levels in lung surfactant preparations by high performance liquid chromatography (HPLC) is described. The analysis was performed on a 3.9 x 300 mm mu-Porasil column with detection by refractive index. The lipids were eluted with a solvent system of chloroform-acetonitrile-methanol-water-85% phosphoric acid 650:650:500:130:2 (v/v/v/v/v). A 4.6 x 30 mm silica guard column was used in place of an injector loop which served as a sample concentrator and purifier. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol, all known components of lung surfactants, were eluted from the loop column and were prevented from reaching the analytical column. Sphingomyelin and lysophosphatidylcholine elute later than the phosphatidylcholines on the analytical column. The method was developed so that phosphatidylcholines elute as a single peak regardless of the fatty acid chain length (C12-C20). When the sample was first oxidized with a potassium permanganate-potassium metaperiodate solution, and potentially interfering oxidation products were removed by extraction into a basic aqueous phase, then only the disaturated phosphatidylcholines were analyzed.     

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale.