Separation and characterization of aluminium malate species by ion chromatography

Aluminium malate complexes have a high relevance in biological systems. The anionic species present in an aqueous aluminium malate mixture can be investigated by ion chromatography coupled online with inductively coupled plasma atomic emission spectroscopy. As malic acid is a chiral ligand, the experiments were carried out using the racemic and the enantiopure forms. In both systems four anionic complexes are observable in the model solutions. One of two crystallized and well-characterized anions, [Al(4)Malate(6)](6-) and [Al(4)Malate(4)](2-), serves as a reference anion for the assignment of one of the species occurring in the model solutions. The main species in the enantiopure aluminium malate model with biological relevance over a wide pH range is the [Al(4)Malate(4)](2-) anion. The [Al(4)Malate(6)](6-) anion is not present in the racemic model solution. This anion suffers complete species disintegration after dissolution. The kinetics of the decay is first order and the activation energy of the decomposition is 74 kJ/mol. A retention model for ion chromatography was used for the determination of the effective charges of the species. The effective charge obtained by the retention model was calibrated in relation to nominal charges of the anions by using several well-defined and differently charged anions and anionic aluminium species.     

Hematoporphyrin derivative: experimental evidence for aggregated species

Absorption and fluorescence properties of hematoporphyrin derivative in different solvent systems are investigated. The presence of a large amount of stable aggregates is demonstrated. The existence of these aggregates represents one major difference between hematoporphyrin derivative and hematoporphyrin free base.     

Optical trapping for the characterization of amyloid-beta aggregation kinetics

Alzheimer's disease (AD) is marked by the accumulation of neuronal plaques from insoluble amyloid-beta (Aβ) peptides. Growing evidence for the role of Aβ oligomers in neuronal cell cytotoxicity and pathogenesis has prompted the development of novel techniques to better understand the early stages of aggregation. Near infrared (NIR) optical trapping was applied to characterize the early stages of Aβ aggregation in the presence of a β-sheet intercalating dye, Congo Red (CR), as the fluorescent marker. The integration of fluorescence analysis with NIR optical trapping has provided a new outlook into the first two hours of Aβ aggregation.     

Separation of peptides by pressurized capillary electrochromatography

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresiscan be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

Separation of peptides on a polystyrene resin column

A high-performance liquid chromatographic column of porous spherical polystyrene--divinylbenzene copolymer (Hamilton PRP-1) was found to be useful for reversed-phase resolution of a wide range of peptides. Recoveries and resolution were comparable with those from more widely used alkyl silica-based columns. Tests involving more than 40 peptides, ranging from 2 to 34 residues, and 3 proteins, indicated wide applicability of this column. The retention times of peptides of known composition are predicted. The stability of the resin at high pH permits protocols of separation involving successive chromatograms at widely different pH values, and offers a second dimension to the resolving power of a single column.     

Separation of peptides utilizing ultrahigh-temperature micellar electrokinetic chromatography

The use of ultrahigh column temperatures, up to 110 degrees C, in micellar electrokinetic capillary chromatography was investigated. The number of plates generated per unit time increased from 0.22 to 12.8 plates/s for separations at 15 degrees C and 110 degrees C, respectively. Ultrahigh-temperature micellar electrokinetic capillary chromatography was used for the separation of cyclic undecapeptides (cyclosporins). A minimum resolution of 1.39 was calculated for a critical peak pair at 110 degrees C, which is more than a 50% increase over resolution generated at 40 degrees C. During a run time of more than 90 min at 110 degrees C and at pH 9.3, no sample degradation or solvent boiling was observed.     

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240000 to 460000/m, and the relative standard deviation for t0 and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated.     

Separation of phosphorylated peptides utilizing dual pH- and temperature-responsive chromatography

The phosphorylation of a peptide is considered to be one of the most important post-translational modification reactions that can alter protein function in mammalian cells. To separate and purify, we developed a dual temperature- and pH-responsive chromatography based on terpolymer composed of N-isopropylacrylamide, N,N'-dimethylaminopropylacrylamide and butylmethacrylate. The property of the surface of the terpolymer-grafted stationary phase altered from hydrophilic to hydrophobic, and from changed to non-charged by changes in the temperature and the pH, respectively. In addition, it was possible to appear and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena resulted from changes in the charge and the hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface by controlling the temperature. In the developed environmental-responsive chromatographic system, the ionizable dimethylamino group of N,N'-dimethylaminopropylacrylamide in terpolymer played a key role for the separation. We applied the developed chromatographic system to the separation of phosphorylated compounds, such as phospho-tyrosine, phosphopeptide and oligonucleotides. At a low column temperature, the electrostatic interaction plays a predominant role for retain anionic phosphorylated compounds, because of the strong interaction between the cationic dimethylamino group in the stationary phase and the anionic phosphoric group in the analyte. On the contrary, the hydrophobic interaction became predominant upon increasing the temperature. The results showed that both the electrostatic and the hydrophobic interactions became controllable with a temperature change during the chromatographic process. Dual pH- and temperature-responsive chromatography would be very useful for biomacromolecules separation and purification.     

Separation of polythiolate peptides and metallopeptides by covalent affinity chromatography

Summary Metallothionein (MT) a low-molecular weight polythiolate metallopeptide was isolated quantitatively by covalent affinity chromatography on Sepharose DTNB support of our own synthesis. The protein was prepared from the vital organs of rats exposed to heavy metals (Hg, Cd).Isolation of low-molecular weight polythiolate and thiolate proteins of a small number of SH-groups are reported. Changes in II and III order structure of the protein and aggregation resulting from chaotic formation of disulphide bridges were observed.A mechanism of separation of polythiolate proteins by covalent chromatography, based on the presented data, is suggested.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Open-tubular electrochromatographic characterization of synthetic peptides

The open-tubular electrochromatographic (OT-CEC) migration behavior of a series of peptides, based on a common structural feature, has been characterized using two different types of chemically modified etched capillaries. The organic moieties immobilized onto the capillary inner surface were n-butylphenyl and cholesterol-10-undecenaoate, respectively. The structure-migration behavior of this set of peptides has been studied at several pH values and with methanol at different concentrations as an organic solvent modifier of the buffer electrolyte composition. By comparing the structural properties of the peptides, such as their amino acid sequences, charge-to-mass ratios and intrinsic hydrophobicities to their migrational behavior, the relative contribution of electrophoretic and chromatographic mobility to the overall migration times, elution order, and selectivity has been determined. Moreover, the experimental data provide important insight into procedures that can be used to modulate the separation of peptides in OT-CEC through variation of the composition of the electrolyte buffer as well as via the properties of the bonded organic moiety.     

激光诱导荧光检测微流控芯片毛细管电泳分离多肽的研究

微流控分析系统是分析科学及分析仪器重要的发展前沿,是90年代初发展起来的微分析系统的主要组成部分及目前较为活跃的领域.将微流控分析系统应用于电泳分离,与传统的电泳分离手段相比较而言,具有微型化、可集成化、速度快、进样量小等特点.它的检测方法有多种,其中激光诱导荧光(Laser induced fluorescence,LIF)法因其灵敏度高,成为微流控芯片分析检测目前最广为采用的方法[1].     

Separation and characterization of basic barley seed proteins

Basic proteins in barley starchy endosperm from developing seeds were separated by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as partial extracts were analyzed. Edman degradation sequencing and immunological detection were performed after transfer of separated proteins onto membranes. Only one protein could be analyzed by N-terminal sequencing of blotted and separated proteins from the total extract. Fractionation of extracts was done using cation exchange chromatography, concanavalin A and heparin affinity chromatography. Internal sequences were determined after in-gel cleavage of proteins using trypsin or cyanogen bromide and separation of the fragments by reversed-phase chromatography or in a gel electrophoresis system for peptide separation. This resulted in a new protocol for obtaining internal sequences from proteins separated by 2-D electrophoresis. A total of 16 sequences, including nine internal sequences, were analyzed, permitting the identification of ten proteins, including five that appeared to have a blocked N-terminus. An additional protein was identified using immunological detection. Three protein sequences remained unidentified. Separated proteins were also analyzed with a glycan detection method.     

Separation and analysis of peptides and proteins from Vipera lebetina snake venom

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences.The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α₂-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.     

Separation of iodine species by adsorption chromatography

Based on the different properties of iodide and iodate species in somesorption materials a new chromatographic method was developed to study thespeciation of iodine in a mangrove system. Two sorption materials, aluminaand silica, were investigated and several distribution coefficients for iodideand iodate were determined at different concentrations of NaOH, NaNO₃ and NaHCO₃ solutions. The best separation results wereobtained percolating sea water samples, containing iodine species, througha glass column filled with alumina. The iodide passed through the column afterwashing the column with 0.1M sodium nitrate solution, and the iodate was elutedwith a 0.5M sodium bicarbonate solution.     

Separation of organic and inorganic arsenic species by HPLC-ICP-MS

The HPLC separation of eight anionic, cationic or neutral arsenic species (arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide and tetramethylarsonium ion) on a high-capacity, anion-exchange column (Ion Pac AS 7, Dionex) was studied. The separation was performed during one run with a nitric acid gradient ranging from pH 4–1.3. The influence of sodium dodecyl sulfate (SDS), sodium octyl sulfate (SOS) and 1,2-benzenedisulfonic acid (BDSA) as ion pairing eluent modifiers was investigated. In addition the effect of elevated temperatures (30 to 40 °C) was studied. The best results were obtained at room temperature of 20 °C with 0.05 mM benzenedisulfonic acid as the eluent modifier. The chromatograph was connected to an ICP-MS via a cross-flow nebulizer. Detection limits obtained with the optimized chromatographic separation were 0.16–0.60 μg As L^–1 for different species. The proposed speciation method was applied to the determination of arsenic species in the DORM-2 reference material (Dogfish Muscle) and in aqueous extracts of mushrooms collected on arsenic contaminated ground. Received: 3 August 1998 / Revised: 17 September 1998 / Accepted: 21 September 1998     

Separation and determination of enkephalin-related peptides by capillary electrophoresis with amperometric detection

A simple, reliable, and reproducible method for separation and determination of five enkephalin-related peptides based on CE with amperometric detection (AD) is described in this paper. A potential of 1.0 V was applied to the carbon disk electrode, which was used as a working electrode in this system. At 15 kV of applied voltage, the five compounds were separated within 18 min in a 20 mmol/L phosphate buffer solution (PBS, pH 7.6) including 2.5% methanol v/v. LOD for five enkephalins were ranged from 6.31 to 54.3 nmol/L. The method was applied successfully to determine the five compounds added in the human cerebrospinal fluid (CSF) and the recoveries were in the range of 95.8-98.2%.