Isolation of cytochrome P-450 components from marmoset liver microsomes by high-performance liquid chromatography.

A fast protein liquid chromatographic (FPLC) system with pre-packed and laboratory-packed columns was used for the analytical and preparative isolation of marmoset monkey cytochrome P-450 (P450) and NADPH-P450-reductase. Chromatographic separations also allowed the recovery of cytochrome b5, NADH-b5-reductase and epoxide hydratase. Cholate-solubilized liver microsomes from phenobarbital-induced marmosets were crudely purified on 8-aminooctyl-Sepharose or 6-aminohexyl-Sepharose and then fractionated into several isoenzyme groups using hydroxyapatite. Further purification on Mono S or CM-Sepharose and finally on phenyl-Superose, phenyl-Sepharose or octyl-Sepharose yielded a P450 fraction which was apparently homogeneous as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the automated Phast system using silver staining. Removal of excess of non-ionic detergent was effected by hydroxyapatite columns, and this was compared with other methods. For the isolation of P450 isoenzymes from untreated marmosets, Mono Q columns were employed and yielded at least two highly purified forms. NADPH-P450-reductase was recovered from the 8-aminooctyl-Sepharose column or crudely fractionated on DEAE-Sepharose Fast Flow. Subsequent purification via 2',5'-ADP-Sepharose and Superose 12 chromatography resulted in a homogeneous preparation.     

Gold recovery onto poly(acrylamide-allylthiourea) hydrogels synthesized by treating with gamma radiation

Poly(acrylamide-1-allyl-2-thiourea) hydrogels, Poly(AAm-ATU), were synthesized by gamma irradiation using a ⁶⁰Co γ source at different irradiation dose rates and in a monomer mixture with different 1-allyl-2-thiourea contents. These hydrogels were used for the specific gold recovery from single and competitive media. It was observed that the gold adsorption capacity onto the hydrogels was high at low pHs and reached a maximum value at pH 0.5. It was found that the adsorption capacity of the hydrogels for gold ions in acidic media around pH 0.5 was high and about 940 mg g⁻¹ dry hydrogel. Adsorption of these hydrogels for gold ions was found to be very fast and also these hydrogels were showed extremely high selectivity to the gold ions in acidic media even when the concentrations of the other metal ions were extremely higher than that of the gold. Because of the high specificity of these hydrogels to gold ions compared with the other metal ions at low pHs, all matrix effects could be easily eliminated by adsorbing gold ions onto the hydrogels at around pH 0.5 and desorbing into 0.8 M thiourea in 3.0 M HCl. The swellability of the synthesized hydrogels varied with irradiation dose rates and increased at high irradiation dose rates. The minimum swellability of the hydrogels was found to be at least 1000% which made it attractive for gold to penetrate into the hydrogels and react with all the functional groups in the interior surface of the hydrogels.     

High-performance hydroxyapatite chromatography of integral membrane proteins and water-soluble proteins in complex with sodium dodecyl sulphate.

Integral membrane proteins from human erythrocytes were fractionated in the presence of sodium dodecyl sulphate (SDS) on four types of high-performance hydroxyapatite columns. A column of 2-microns sintered hydroxyapatite beads from Asahi Optical (Tokyo, Japan) gave the best resolution. With this column, glycophorin was eluted early in a gradient of increasing sodium phosphate buffer concentration, the glucose transporter was eluted later in two zones, one of which contained this protein alone, and the anion transporter was eluted last. Water-soluble proteins applied in complex with SDS also separated reasonably well upon elution. The water-soluble proteins and the membrane proteins were all eluted mainly in the order of increasing polypeptide length, but with considerable individual variation. SDS-polypeptide complexes are probably adsorbed onto hydroxyapatite by the interaction of positively charged amino acid side groups with phosphate ions (at P-sites) and of negatively charged amino acid side groups and polypeptide-bound dodecyl sulphate anions with calcium ions (at C-sites). As a rule, the number of charged side groups and dodecyl sulphate anions, and thus the number of binding sites, increases with the polypeptide chain length, which explains the general order of release of the polypeptides.     

Bacteriophage and impurity carryover and total organic carbon release during extended protein A chromatography

In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na₂SO₄ with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance.     

Interaction of DNA with hydroxyapatite. Studies on the effect of the phosphate concentration of the column equilibration and washing buffer

The ability of hydroxyapatite to bind DNA effectively in phosphate solutions used for column equilibration, sample loading and column washing has been examined. It was demonstrated that substantial amounts of DNA (up to 40%) were eluted in the washing buffer when the phosphate concentration in the lysing solution or urea-phosphate used for column equilibration, sample loading and column washing was 0.24 M. A reduction in the phosphate concentration from 0.24 to 0.15 M in urea-phosphate solution led to almost 100% binding, whereas a similar reduction in the lysing solution did not. A modified method for loading and eluting DNA from hydroxyapatite columns is presented.     

A selective lon-exchange separation and spectrophotometric determination of traces of copper in silicate rocks

Summary A combined cation-exchange separation-spectrophotometric procedure has been worked out for the accurate determination of traces of copper in silicate rocks. Silicates are opened up with sulfuric and hydrofluoric acids. The residue is taken up into a 0.5 M hydrochloric acid −0.05 M oxalic acid −1% hydrogen peroxide solution and loaded on a strongly acidic cation-exchange resin column. Polyvalent ions including ferric ions do not adsorb on the column, while copper (II) retains together with divalent metal ions as well as aluminum (III). Copper (II) can selectively be eluted by a small volume of 0.05 M thiosulfate solution. This fraction is sufficiently pure to allow a direct spectrophotometric determination of copper with Na-DDTC without the addition of tartrate and EDTA as masking agents. Quantitative results are quoted for the determination of copper in international standard rocks of the Geological Survey of Japan (GSJ) and the U.S. Geological Survey (USGS).     

Purification and characterization of transglutaminases from the genital tract of the male rat.

In the genital tract of the male rat two different forms of the enzyme transglutaminase (TGase) could be identified and characterized. The coagulating gland and the dorsal prostate secrete a glycosylated and acylated TGase with a molecular weight of 65,000 dalton and pI value of 8.7. This secretory form was purified to homogeneity using preparative isoelectric focusing and gel filtration on a Superdex 200 column. Running fast protein liquid chromatographic gel filtration on a Superose 12 column in the presence of calcium ions, high-molecular-weight aggregates were physically formed which could only be eluted using drastic conditions (0.1 M sodium hydroxide). In the presence of 10 mM EDTA this tendency to aggregate was greatly diminished. Utilizing a Superdex 200 column for gel filtration, the secretory TGase was even eluted as a monomeric protein. Testicular TGase was isolated by ion-exchange fast protein liquid chromatography on a Mono Q and by gel filtration on a Superdex 200 column. This enzyme represents a tissue-type TGase with a molecular weight of 82,000 dalton and pI value of 5.25. Hydrophobic interaction chromatography on a phenyl-Superose column showed no further enrichment of the GTP-binding form of transglutaminase.     

Chemical separation of enriched cadmium target from copper backing in cyclotron production of radioisotope 111In

A radiochemical purification procedure was developed for the separation of enriched cadmium (¹¹¹Cd and ¹¹²Cd) from natural copper that used as backing; and was based upon the chromatographic adsorption. The separation of copper from cadmium was studied in this work. The ions were selectively separated from aqueous solution. Ion-exchange chromatography was employed as a column (1.5 cm i.d. and 15 cm length) with AG1-X8 resin (chloride form, 100–200 mesh) and a flow rate of 1–2 ml/min throughout the separation. 6 M HCl media was used for the adsorption of Cd and Cu on the resin. Then, Cu was eluted by 2 M HCl and Cd by 100 ml 0.5 M HNO₃. The amount of Cu and Cd ions in the final solution (0.5 M HNO₃) were measured by pulse polarographic method and the concentration of Cu was found to be <0.1 ppm. The Cd was quantitatively recovered and the recovery yield from ion-exchange chromatography was greater than 96 %.     

Determination of tin in marine materials by using hydride generation-high resolution inductively coulped plasma mass spectrometry

A hydride generation system combined with high-resolution inductively coulped plasma mass spectrometry was used to determine tin in marine materials. The optimization conditions for determination of tin in this system are 0.015 M of sulfuric acid solution as medium, 0.2% (w/v) of sodium tetrahydroborate(III) in 0.015 M of sodium hydroxide solution as a reductant and argon as the carrier gas at a flow rate of 1.1 l min(-1). In order to remove the interferences from transition element ions, a strongly basic anion exchanger was used in this method. Tin was converted to its chlorostannate with 2 M hydrochloric acid followed by passing to an anion exchanger. The tin absorbed on the column was then eluted with 1 M nitric acid. Under the optimized conditions, the detection limit of the method was 12 ng l(-1) without using the anion column as preconcentration method. The results obtained using this method were in good agreement with the certified values of marine standard reference materials. The recoveries for the method when applied to determine trace tin in river water were 95-115%.     

在线固相萃取-离子色谱法测定4种芳环磺酸盐中的硫酸根离子

建立一种在线固相萃取-离子色谱测定4种芳环磺酸盐中硫酸根离子含量的新方法。将自装填的多孔石墨化碳固相萃取柱应用于离子色谱系统,对样品进行在线前处理。样品经过多孔石墨化碳固相萃取柱基体消除后进入收集环,通过阀切换方式使待测硫酸根离子转入阴离子分析柱和检测系统。固相萃取流路用1.5 mmol/L碳酸钠以0.8 mL/min的流速对基体在线富集,进样量为20μL,分析柱为SH-AC-3(250 mm×4.0 mm)+SH-AG-3(50 mm×4.0 mm)色谱柱,柱温为35℃,在6 mmol/L碳酸钠-4 mmol/L碳酸氢钠条件下等度洗脱,流速为0.8 mL/min。结果表明:硫酸根离子在0.50~20.00 mg/L范围内呈良好的线性关系,线性相关系数为0.998 3,保留时间、峰高和峰面积的相对标准偏差均在0.28%~2.86%之间,方法检出限为0.010 6 mg/L,回收率为91.01%~109.3%,具有良好的线性关系和重复性。整个在线分析过程在25 min之内完成。该方法进样量少、快速、高效。     

Enrichment of complexing analytes on a copper-loaded silica surface and on-line coupling with high-performance liquid chromatography, using L-tryptophan and L-tyrosine as model compounds

A chemically modified silica (bisdithiocarbamate) strongly binds copper and has a loading capacity of 0.5 mmol g-1 for the copper ion. This new metal-loaded silica, packed in a short stainless-steel pre-column, is highly selective for the enrichment of two amino acids chosen as model compounds. On-line coupling of this pre-column with an analytical RP-18 column allowed the separation and detection of L-tryptophan and L-tyrosine at parts per billion levels in aqueous media with good accuracy. Separation was achieved by ion-pair chromatography and the solutes were detected by fluorescence. The ions generally present in natural aqueous media did not interfere.     

Terbium hybrid particles with spherical shape as luminescent probe for detection of Cu2+ and Fe3+ in water

A novel green emissive terbium inorganic-polymeric hybrid particle was designed and this material could detect cations in water. Polyvinyl alcohol as an amphiphilic surfactant rendered the powders dispersible in water with regular round shape (10-20 μm). Interestingly, we noticed that not only Cu(2+) (detection limit 10(-4)M) but also Fe(3+) (detection limit 10(-4) M) can give rise to emission quenching to this target material in comparison with K(+), Na(+), Fe(2+), Mn(2+), Pd(2+), Cd(2+) and Co(2+) (10(-3) mol L(-1)). We regarded that the coordination interactions between ligand and metal ions resulted in these quenching processes. Additionally, it was found that the sensing material can be repeatedly used at least 5 cycles. More importantly, this novel material demonstrated higher thermal-stability in aqueous media than pure silica hybrid material.     

Effect of thiocyanate counterion condensation on poly(allylamine hydrochloride) chains on the buildup and permeability of polystyrenesulfonate/polyallylamine polyelectrolyte multilayers

In this study, we investigate the buildup of PEI-(PSS-PAH)(n) polyelectrolyte multilayers at pH 7.4 in the presence of either NaCl or NaSCN as a supporting electrolyte. It appears that in the presence of increasing thiocyanate concentrations (from 0.1 to 0.5 M), the thickness increment, obtained from optical waveguide lightmode spectroscopy experiments, increases whereas it stays practically constant for increasing sodium chloride concentrations (between 0.1 and 0.5 M). The hydration of the films differs also markedly between both electrolyte solutions. The differences in the construction of the polyelectrolyte multilayers in the presence of both supporting electrolytes are rationalized in terms of strong SCN(-) condensation on the PAH chains. The occurrence of this ion condensation is indirectly demonstrated by means of zeta potential measurements and directly demonstrated by means of attenuated total internal reflection infrared spectroscopy on the multilayer films. Moreover when the films are built up in the presence of SCN(-), these ions are only slowly exchanged by the Cl(-) ions introduced in the bulk. Conversely the thick films obtained from 0.5 M NaSCN solutions do not deswell when the buffer solution is replaced by a 0.5 M NaCl containing buffer. The permeability of the films constructed in the presence of both sodium salts is also studied by means of cyclic voltametry and is found to be markedly different in the case of films made from five bilayers at 0.5 M salt concentration. This difference is due to the different morphology and porosity of the films constructed in the presence of 0.5 M NaCl and 0.5 M NaSCN.     

Structure refinements of Pb2+ ion-exchanged apatites by x-ray powder pattern-fitting

The crystal structures of Pb²⁺ ion-exchanged hydroxyapatite (OHAp), chlorapatite (ClAp), and fluorapatite (FAp) in aqueous solutions with low pH value of 3.0 or 4.0 have been investigated by X-ray powder pattern-fitting methods. The site occupancy factors of Pb atoms for the M1 (column site) and M2 sites were determined to be 0.72 and 0.77 for Pb_7.5Ca_2.5OHAp, 0.69 and 0.86 for Pb_7.9Ca_2.1ClAp, and 0.60 and 0.52 for Pb_5.5Ca_4.5FAp, respectively. These results imply that Ca²⁺ ions in calcium hydroxyapatite are exchanged for Pb²⁺ ions in acidic aqueous solution regardless of whether they occupy M1 or M2 sites.     

Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Analysis of sodium polycarboxylates in detergents by size exclusion chromatography

Summary This paper describes the quantitative analysis and preparative isolation of sodium polycarboxylates in detergents by means of gel permeation chromatography. An analytical monitoring method separates the polymers from other low molecular detergent ingredients within 10 minutes. There is no separation of the various molecular weight polycarboxylate macromolecules themselves. They elute from the column as a single narrow peak at the exclusion volume. A second preparative gel filtration method allows isolation of polycarboxylates in amounts necessary for further characterization. Appropriate sample pretreatments and possible interferences are discussed.     

The influence of complexing agents on the effectiveness of electrochemical masking with anionic surfactants in anodic stripping voltammetry

The influence of sodium dodecyl sulphate, sodium dodecyl sulphonate and sodium stearinate on the anodic stripping peaks of Tl(I), Pb(II), Cd(II), Cu(II) and In(III) was investigated. The supporting electrolytes were 0.5M sodium sulphate solution, 0.2M citrate solution (pH 3.7, 4.6 and 7.3), 0.5M tartrate solution (pH 4.4) and 0.1M solution of EDTA (pH 4.4). The composition of complex compounds forming in a solution under experimental conditions was defined. The conditions of ion reduction of metals on hanging mercury electrode during the electrolytical deposition were investigated. The investigation included an analysis of voltammetric curves of the metal ions. The obtained results suggest that "electrochemical masking" is much stronger in electrolytes containing a complexing agent than in the sodium sulphate solution. The influence of the complexing agent may not be explained in terms of the interaction between the form of the complex and the charge of the adsorbed surfactant particles; rather the complexing process is connected with indirect inhibition, i.e., by decreasing the rate of charge transfer reaction.     

Simultaneous purification of the neuroproteins synapsin I and synaptophysin.

A procedure for the simultaneous purification of synapsin I and synaptophysin from calf brain was developed. Demyelinated membranes were extracted with 2% Triton X-100 and 2 M KCl. The extracted proteins were separated by weak cation-exchange chromatography on carboxymethyl-Sepharose Fast Flow. Synaptophysin was finally purified by preparative sodium dodecyl sulphate-polyacrylamine gel electrophoresis and synapsin I by affinity chromatography using a calmodulin-Sepharose column. The recovery obtained was 40 micrograms/g in brain for synaptophysin and 25 micrograms/g in brain for synapsin I.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.