Lysozyme refolding at high concentration by dilution and size—exclusion chromatography

This study of renaturation by dilution and size exclusion chromatogra phy (SEC) addition of urea to improve yield as well as the initial and final pro tein concentrations showed that although urea decreased the rate of lysozyme ref o lding, it could suppress protein aggregation to sustain the pathway of correct r efolding at high protein concentration; and that there existed an optimum urea c oncentration in renaturation buffer. Under the above conditions, lysozyme was su ccessfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. τ>2(t_R2 -t_R1 ), the efficiency was increased by 25% and the renaturation buffe r could be recycled for SEC refolding in continuous operation of downstream proc ess.

COMPOSITION DETERMINATION OF BINARY POLYMER MIXTURES BY SIZE EXCLUSION CHROMATOGRAPHY WITH LIGHT SCATTERING DETECTION

Base on the principle of absolute quantification of size exclusion chromatography （SEC）, a light scattering （LS） detector coupled with a concentration detector （refractive index detector） is utilized to determine the compositions of complicated binary mixtures. A theoretical analysis predicts that the response factors for both LS and RI detectors are linear functions with the composition of any specified polymer mixtures in the binary polymer mixtures. Two pairs of complicated binary mixtures were used to test the theory mentioned in the present paper, and the experimental results show an excellent accordance with the theory.

Preparation and Application of Silica-based Perfusion Packing of Size Exclusion Chromatography for Quantitation of Polyacrylamide in Enhanced Oil Recovery Systems

The synthesis and characterization of a novel macroporous silica derived size exclusion chromatography （SEC） packing for quantitative analysis of high molecular weight （MW） polyacrylamide （PAM） are presented. Using this packing, a fast, sensitive and reproducible approach for quantitation of super high-MW PAM in demanding enhanced oil recovery （EOR） waters was developed and the effect of synthesis parameters on the properties of resultant materials was investigated. These parameters include salt addition, reaction temperature and duration, activation condition of functional groups on the silica surface, as well as the reaction cycles required for optimal silica modification. Moreover, SEC analysis conditions, such as mobile phase composition, flow rate, detection and sample preparation, were also explored and an optimal analysis protocol was developed. Under this optimized SEC analysis conditions, the synthesized macroporous materials proved satisfactory for quantification of PAM with average MW up to 22 million Daltons. An SEC analysis required less than few minutes with a detection limit of 1 ng, a linear response range of 0.1 to 75 mg/L with squared R value of 0.99 and reproducibility better than 9.2% RSD （relative standard deviation）. The analysis of PAM in highly saline oilfield production water containing interfering high MW polymeric surfactants indicated the recovery ranges from 92.5% to 110.1% for 1.0 mg/L PAM and 94.2% to 103.8% for 50 mg/L PAM solution. This study presented for the ftrst time that the reliable quantization of high MW PAM in highly demanding EOR waters can be achieved by SEC.

Characterization of blueberry exosome-like nanoparticles and miRNAs with potential cross-kingdom human gene targets

Edible plant derived exosome-like nanoparticles(ELNs)have been shown to have multiple nutraceutical functions.However,the diversity of plant materials makes the plant derived ELN study challenging.More efforts are still needed to explore the feasible isolation methods of edible plant derived ELNs and the possible roles of food-derived ELNs in improving human health.In this study,a size exclusion chromatography based method was compared with the traditional ultracentrifugation method to isolate blueberry derived ELNs(B-ELNs),and the miRNA profile of B-ELNs was analyzed by high-throughput sequencing.A total of 36 miRNAs were found to be enriched in B-ELNs compared with berry tissue,and their potential cross-kingdom human gene targets were further predicted.Results showed that size exclusion chromatography was effective for B-ELN isolation.The most abundant miRNAs in B-ELNs mainly belonged to the miR166 family and miR396 family.Target gene prediction indicated that B-ELNs could potentially regulate pathways related to the human digestive system,immune system and infectious diseases.

Simultaneous determination of dissolved organic nitrogen, nitrite, nitrate and ammonia using size exclusion chromatography coupled with nitrogen detector

Accurate quantification of dissolved organic nitrogen(DON) has been a challenge due to the cumulative analytical errors in the conventional method via subtracting dissolved inorganic nitrogen species(DIN) from total dissolved nitrogen(TDN). Size exclusion chromatography coupled with an organic nitrogen detector(SEC-OND) has been developed as a direct method for quantification and characterization of DON. However, the applications of SECOND method still subject to poor separations between DON and DIN species and unsatisfied N recoveries of macromolecules. In this study, we packed a series of SEC columns with different lengths and resin materials for separation of different N species and designed an independent vacuum ultraviolet(VUV) oxidation device for complete oxidation converting N species to nitrate. To guarantee sufficient N recoveries, the operation conditions were optimized as oxidation time ≥ 30 min, injection mass(sample concentration × injection volume) < 1000 μL × mg-N/L for macromolecular proteins, and neutral p H mobile eluent. The dissolved O_(2)concentration in SEC mobile phase determined the upper limit of VUV oxidation at a specific oxidation time. Compared to conventional HW50S column(20 × 250 mm),HW40S column(20 × 350 mm) with mobile phase comprising of 1.5 g/L Na2HPO_(4)·2H_(2)O + 2.5g/L KH_(2)PO_(4)(p H = 6.85) could achieve a better separation of DON, nitrite, nitrate, and ammonia. When applied to river water, lake water, wastewater effluent, groundwater, and landfill leachate, the SEC-OND method could quantify DON as well as DIN species accurately and conveniently even the DIN/TDN ratio reached 0.98.

Measurement of total phenolic content and antioxidant activity of aerial parts of medicinal plant Coronopus didymus

Objective: To evaluate the total phenolic content and compare the antioxidant activity of various solvent extracts and fractions from the aerial parts of Coronopus didymus through various assays. Methods: Total phenolic content was determined using the Folin-Ciocalteu assay and the in vitro antioxidant activity of a number of different extracts was investigated in a dose-dependent manner with three different methods: the 2,2-diphenyl-1-picrylhydrazyl(DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(ABTS) and ferric reducing antioxidant power(FRAP) assays. A flavone was isolated from the most active ethanolic extract with high antioxidant activity using size exclusion chromatography. IC_(50) values were calculated for the DPPH and ABTS methods. The FRAP activity was assessed in terms of μM Fe(II) equivalent. Results: The phenolic content was found to be highest in the ethanol extract(CDA Et; 47.8 mM GAE) and the lowest in the dichloromethane extract(CDA DCM; 3.13 mM GAE). The ethanol extract showed high radical scavenging activity towards DPPH and ABTS radicals with IC_(50) values of(7.80 × 10~2) and(4.32 × 10~2) μg/m L, respectively. The most active ethanol extract had a FRAP value of 1 921.7 μM Fe(Ⅱ) equivalent. The isolated flavone F10C(5,7,4'-trihydroxy-3'-methoxy flavone) was far more effective for scavenging free radicals in the DPPH and ABTS assays with IC_(50) of 43.8 and 0.08 μg/m L, than the standard trolox, with IC_(50) values of 97.5 and 21.1 μg/m L, respectively. In addition, the flavone F10C and the standard ascorbic acid had FRAP values of 1 621.7 and 16 038.0 μM Fe(Ⅱ) equivalents, respectively. Conclusions: The total phenolic content of extracts in decreasing order is ethanol extract(CDA Et)> acetone extract(CDA ACE)> phenolic extract(CDA MW)> n-hexane extract(CDA nHX)> chloroform extract(CDA CHL)> dichloromethane extract(CDA DCM). The ordering of extracts in terms of antioxidant activity from highest to lowest is CDA Et> CDA MW> CDA DCM> CDA CHL> CDA ACE> CDA nHX in DPPH, ABTS and FRAP assays. A significant relationship is found between antioxidant potential and total phenolic content, suggesting that phenolic compounds are the major contributors to the antioxidant activity of Coronopus didymus.

Determination of sodium hyaluronate in pharmaceutical formulations by HPLC–UV

A liquid chromatography （HPLC） method with UV detection was developed for determination of sodium hyaluronate in pharmaceutical formulation. Sodium hyaluronate is a polymer of disaccharides, composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternating 13-1, 4 and D-l, 3 glycosidic bonds. Being a polymer compound it lacks a UV absorbing chromophore. In the absence of a UV absorbing chromophore and highly polar nature of compound, the analysis becomes a major challenge. To overcome these problems a novel method for the determination of sodium hyaluronate was developed and validated based on size exclusion liquid chromatography （SEC） with UV detection. An isocratic mobile phase consisting of buffer 0.05 M potassium dihydrogen phosphate, pH adjusted to 7.0 using potassium hydroxide （10%） was used. Chromatography was carried out at 25 ~C on a BioSep SEC $2000, 300 mm ~ 7.8 mm column. The detection was carded out using variable wavelength UV-vis detector set at 205 nm. The compounds were eluted isocratically at a steady flow rate of 1.0 mL/min. Sodium hyaluronate retention time was about 4.9 min with an asymmetry factor of 1.93. A calibration curve was obtained from 1 to 38 g/mL （r〉0.9998）. Within-day % RSD was 1.0 and between-day % RSD was 1.10. Specificity/selectivity experiments revealed the absence of interference from excipients, recovery from spiked samples for sodium hyaluronate was 99-102. The developed method was applied to the determination of sodium hyaluronate in pharmaceutical drug substance and product.

CHARACTERIZATION OF MOLECULAR MASS OF SIX WATER-SOLUBLE POLYSACCHARIDE -PROTEIN COMPLEXES FROM GANODERMA TSUGAE MYCELIUM

Six water-soluble polysaccharide-protein complexes coded as GM1, GM2, GM3, GM4, GM5 and GM6 wereisolated from the mycelium of Ganoderma tsugae by extracting with 0.2 mol/L phosphate buffer solution at 25, 40 and80℃, water at 120℃, 0.5 mol/L aqueous NaOH solution at 25 and 65℃, consecutively. Their chemical components wereanalyzed by using IR, GC, HPLC and ^(13)C-NMR, and some new results were obtained. The four samples GM1, GM2, GM3and GM4 are heteropolysaccharide-prote in complexes, in which, α- (1→3) linked D-glucose is the major monosaccharidewhile galactose, mannose and ribose are the secondary ones. GM5 and GM6 are β-(1→3)-D-glucan-protein complexes. Theprotein content increased from 32% to 69% with the progress of isolation. Weight-average molecu1ar mass M_w and theintrinsic viscosity [η] of the GM samples in 0.5 mol/L aqueous NaCl solution at 25℃ were measured systematically by laserlight scartering (LLS), size exclusion chromatography (SEC) combined with LLS, and viscometry. The M_w of GM1 to GM6are 35.5, 46.8, 58.9, 41.6, 3.3 and 22.0×10~4, respectively. The conformation and molecular mass of the two fractions of sample GM5 were characterized satisfactorily by SEC-LLS without further fractionation.

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography(SEC).It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride(GuHCl)or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase.Although the oxidized and reduced glutathione(GSSG and GSH)were employed in the oxidized mobile phase,the reduced-denatured insulin still could not be renatured.However,in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed,the reduced-denatured insulin can be refolded with SEC,and the aggregation of denatured insulin can be diminished by urea.In addition,the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase.The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely.The results were further tested with reversed-phase liquid chromatography(RPLC)and hydrophobic interaction chromatography(HIC).

Rapid Purification of Enhanced Green Fluorescent Protein from Escherichia coli

As an excellent reporter molecule, enhanced green fluorescent protein (eGFP) was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was devel- oped for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic pu- rity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of ex- citation and emission maxima, indicating that the purification procedure did not influence the construct and fluo- rescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.

Synthesis of Monodisperse Poly(chloromethylstyrene divinylbenzene)Particle and Its Applications

Monodisperse poly (chloromethylstyrene divinylbenzene) particles were prepared by a two-step swelling method. It was demonstrated that the packings have comparative advantages for biopolymer separation with high column efficiency, low interstitial volume and low back pressure to the packings prepared with suspension polymerization method. Using size exclusionchromatography and weak ion exchange chromatography as examples, it was shown that these packings could be modified chemically more easily than poly (styrene divinylbenzene) packings.

STUDIES ON MOLECULAR WEIGHT DISTRIBUTION OF CARBON FIBER POLYMER PRECURSORS SYNTHESIZED USING MIXED SOLVENTS

The molecular weight distributions were estimated for carbon fiber polymer precursors such as poly（acrylonitrile-co-itaconic acid） synthesized by semi batch solution polymerization in mixed solvents media with the azonitrile compounds as initiator under the different ratios of solvent and non solvent from 0.75 to 2.5 in weight. The copolymer was characterized by using Fourier transform infrared spectroscopy （FTIR） and nuclear magnetic resonance （1H-NMR） analyses. The molecular weight distributions were evaluated by Mv/Mn ratios estimated from viscosity and osmotic measurements, and Mw/Mn estimated from size exclusion chromatography. The molecular weight distributions of these polymers as determined from M, JMn and Mw/Mn are 2.9 to 3,2 and 2.0 to 2.5 respectively. The molecular distributions were close to a narrow distribution of 2.0 when the solvent/non-solvent ratio was varied between 1.4 and 2.0. Intrinsic viscosity [η] as a function of molecular weight of poly（acrylonitrile-co-itaconic acid） was evaluated by means of low angle laser light scattering with size exclusion chromatography （SEC-LALLS） and viscometry with SEC （SEC-VISCO）. The relationship between [η] and Mw for poly（acrylonitrile-co-itaconic acid） in DMF at 50℃ was [q] = 1.1×10-5 Mw0.79, where [η] is obtained in dL/g.

Simultaneous size characterization and mass quantification of the in vivo core-biocorona structure and dissolved species of silver nanoparticles

Size characterization of silver nanoparticles with biomolecule corona（AgNP@BCs） and mass quantification of various silver species in organisms are essential for understanding the in vivo transformation of Ag NPs. Herein, we report a versatile method that allows simultaneous determination of the size of AgNP@BCs and mass concentration of various silver species in rat liver. Both particulate and ionic silver were extracted in their original forms from the organs by alkaline digestion, and analyzed by size exclusion chromatography combined with inductively coupled plasma mass spectrometry（SEC-ICP-MS）. While the silver mass concentrations were quantified by ICP-MS with a detection limit of 0.1 μg/g, the effective diameter of AgNP@BCs was determined based on the retention time in SEC separation with size discrimination of 0.6-3.3 nm. More importantly, we found that the BC thickness of AgNP@BCs is core size independent, and a linear correlation was found between the effective diameter and core diameter of AgNP@BCs in extracted tissues, which was used to calibrate the core diameter with standard deviations in the range of 0.2-1.1 nm. The utility of this strategy was demonstrated through application to rat livers in vivo. Our method is powerful for investigating the transformation mechanism of Ag NPs in vivo.

Selective Suspension in Aqueous Sodium Dodecyl Sulfate According to Electronic Structure Type Allows Simple Separation of Metallic from Semiconducting Single-Walled Carbon Nanotubes

Both density gradient centrifugation and gel electrophoresis have been reported to allow high throughput separation of metallic from semiconducting single-walled carbon nanotubes(SWNTs)when using aqueous sodium dodecyl sulphate(SDS)suspensions.We show here that both methods rely on an initial dispersion-by-sonication step,which is already selective with respect to electronic structure type.The corresponding aqueous SDS“starting”suspensions obtained after sonication and purifi cation by simple centrifugation(70,000 g,1 h)contain semiconducting SWNTs primarily in the form of small bundles whereas metallic SWNTs are predominantly suspended as individual tubes.Density gradient centrifugation then separates the bundles from the individual tubes on the basis of differences in their overall buoyant densities.Gel electrophoresis separates the longer bundles from the shorter individual tubes on the basis of their different mobilities.We also demonstrate that such starting suspensions can be fractionated according to electronic structure type by even simpler techniques such as size exclusion chromatography or gel fi ltration,thus opening the way for simple scale-up.

Prediction of DOM removal of low specific UV absorbance surface waters using HPSEC combined with peak fitting

High performance size exclusion chromatography （HPSEC） is used in water quality research primarily to determine the molecular weight distribution of the dissolved organic matter （DOM）, but by applying peak fitting to the chromatogram, this technique can also be used as a tool to model and predict DOM removal. Six low specific UV absorbance （SUVA） source waters were treated using coagulation with alum and both the source and treated water samples were analysed using HPSEC. By comparing the molecular weight profiles of the source and treated waters, it was established that several DOM components were not effectively removed by alum coagulation even after high dosage alum treatment. A peak-fitting technique was applied based on the concept of linking the character （molecular weight profile） of the recalcitrant organics in the treated water with those of the source water. This was then applied to predict DOM treatability by determining the areas of the peaks which were assigned to removable organics from the source water molecular weight profile after peak fitting, and this technique quantified the removable and non-removable organics. The prediction was compared with the actual dissolved organic carbon （DOC） removal determined from jar testing and showed good agreement, with variance between 2% and 10%. This confirmed that this prediction approach, which was originally developed for high SUVA waters, can also be applied successfully to predict DOC removal in low SUVA waters.

Characterization of natural organic matter in water for optimizing water treatment and minimizing disinfection by-product formation

Introduction Natural organic matter（NOM）present in source water has significant impact on water treatment processes and on the quality of drinking water.NOM is a complex mixture of diverse groups of organic compounds,humic and fulvic acids,proteins,peptides,carbohydrates,and heterogeneous materials

Effect of environmental factors on the complexation of iron and humic acid

A method of size exclusion chromatography coupled with ultraviolet spectrophotometry and off-line graphite furnace atomic absorption spectrometry was developed to assess the complexation properties of iron（Fe） and humic acid（HA） in a water environment. The factors affecting the complexation of Fe and HA, such as ionic strength, pH, temperature and UV radiation, were investigated. The Fe–HA complex residence time was also studied. Experimental results showed that pH could influence the deprotonation of HA and hydrolysis of Fe, and thus affected the complexation of Fe and HA. The complexation was greatly disrupted by the presence of NaCl. Temperature had some influence on the complexation. The yield of Fe–HA complexes showed a small decrease at high levels of UV radiation, but the effect of UV radiation on Fe–HA complex formation at natural levels could be neglected. It took about 10 hr for the complexation to reach equilibrium, and the Fe–HA complex residence time was about 20 hr.Complexation of Fe and HA reached a maximum level under the conditions of pH 6, very low ionic strength, in the dark and at a water temperature of about 25°C, for 10 hr. It was suggested that the Fe–HA complex could form mainly in freshwater bodies and reach high levels in the warm season with mild sunlight radiation. With changing environmental parameters, such as at lower temperature in winter or higher pH and ionic strength in an estuary, the concentration of the Fe–HA complex would decrease.

Water chemistry influences on long-term dissolution kinetics of CdSe/ZnS quantum dots

Widespread usage of engineered metallic quantum dots(QDs)within consumer products has evoked a need to assess their fate within environmental systems.QDs are mixed-metal nanocrystals that often include Cd2+which poses a health risk as a nanocrystal or when leached into water.The goal of this work is to study the long-term metal cation leaching behavior and the factors affecting the dissolution processes of mercaptopropionic acid(MPA)capped CdSe/ZnS QDs in aphotic conditions.QD suspensions were prepared in different water conditions,and release of Zn2+and Cd2+cations were monitored over time by size exclusion chromatography-inductively coupled plasm a-mass spectrometry.In most conditions with dissolved 02 present,the ZnS shell degraded fairly rapidly over^1 week,while some of the CdSe core remained up to 80 days.Additional MPA,Zn2+,and Cd2+temporarily delayed dissolution,indicating a moderate role for capping agent detachment and mineral solubility.The presence of H2 O2 and the ligand ethylenediaminetetraacetate accelerated dissolution,while NOM had no kinetic effect.No dissolution of CdSe core was observed when 02 was absent or when QDs formed aggregates at higher concentrations with 02 present.The shrinking particle model with product layer diffusion control best describes Zn2+and Cd2+dissolution kinetics.The longevity of QDs in their nanocrystal form appears to be partly controlled by environmental conditions,with anoxic,aphotic environments preserving the core mineral phase,and oxidants or complexing ligands promoting shell and core mineral dissolution.

INVESTIGATION ON THE RESPONSE FACTORS OF CONCENTRATION DETECTORS WITHIN SEC PROCESS

The response factors of refractive index（RI） and ultraviolet（UV） detectors of size exclusion chromatography （SEC） defined as the ratio of area of output signal to the mass of injected sample are studied and analyzed by using five narrowly distributed polystyrene（PS） standard samples with known molar masses.It is found that the individual response factor for a given sample varies with the concentration of the injected solution within a limited range bounded by an upper and a lower limiting response factor values.This variation reveals the conformational change of the polymer chains with the concentration of the injected solution.The dynamic contact concentrations c_s of the PS samples derived from the response factor data are in good accordance with those reported earlier by other methods.The physical meanings of the signals of the two detectors are further analyzed and theoretically formulated.The solvation of the polymer chain and the conformation changes play an important role in these detecting systems.Both of the solvation number of the structural repeating unit and the extra embedded solvent due to cluster forming in higher concentrations could be deduced from the variation of response factor with the concentration of the injected solution.