A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

Analysis of trace elements in air particulate matters by non-suppressed ion chromatography

The application of non-suppressed ion chromatography for monitoring of trace elements in air particulate matter was studied in the present investigation. The results indicate that the use of microwave acid digestion method is superior in comparison with the conventional thermal acid digestion method as it leads to higher recovery, better reproducibility, lower volatility loss, better protection against environmental contamination and much less digestion time (5 minutes vs. 24 hours). The use of eluent as extractant is shown to reduce the water dip problem in the chro-matogram. The addition of chelating agent in the eluent coupled with UV detection is shown to provide satisfactory chromatographic separation and good sensitivity for the analysis of transition metals present in the air particulate matter. Using the U.S. National Bureau of Standards Reference Material 1648 Urban Particulate Matter as standard for checking, the analytical procedure is shown to give good recovery and reproducibility for the detection of the following cations and anions in air particulate matter: Fe2 Cu, Mn, Pb, Zn, Mg, Na, HN4+, Cl-, NO3- and SO42-. Field test was also performed to check the applicability of the method and the results obtained were discussed in the present paper.

Separation and purification of Yb_(2)O_(3) by ion exchange chromatography and preparation of ultra-high purity Yb_(2)O_(3)

Ultra-high purity Yb_(2)O_(3) is the critical material of many high-tech materials such as laser glass and fiber,in which impurities seriously affect the laser color quality,intensity and power.In order to reduce the influence of impurities on the properties of laser materials,the purification process of Yb_(2)O_(3) was studied by comparing two kinds of resins(RT-1 and RS-1)using improved ion-exchange chromatography(IEC)method.In this study,through the synergistic improvement of resin structure and eluting system,the environmental pollution caused by ammonia water in the traditional IEC method was reduced,and the requirements of high temperature and pressure were cut.The ion exchange behavior and impurity removal mechanism in the resin column during the loading and eluting process were compared and analyzed.The experimental results show that RS-1 resin is all superior to RT-1resin in elements selectivity,ion exchange capacity and impurities removal rate.After separation and purification by IEC with RS-1 resin,the total removal rate of rare earth impurities was 77.59%and that of non-rare earth impurities was 95.86%when Yb recovery was more than 70%,both higher than that of RT-1 resin(73.26%and 83.18%).This indicates that the improved IEC method is very effective in separating and removing different metal impurities from Yb_(2)O_(3).The pilot test results of IEC method separating and purifying Yb_(2)O_(3) with RS-1 resin show that the purity of Yb_(2)O_(3) can be increased from 99.9929%to 99.9997%by IEC method.It has exhibited huge potential of preparing ultra-high purity Yb_(2)O_(3),especially the deep removal of non-rare earth impurities.

Impact of Hydrogen Ion Concentration on Amino Acids Composition of Macadamia Protein: Approached Using Cation-Exchange Chromatography

In the present context, the objective of this study was to synthesize and analyze the content of AA of macadamia protein and the impact of hydrogen ion concentration (pH) on AA composition. The determination of AA mainly by cation-exchange chromatography was also investigated. Reproducible and reliable techniques for quantification and identification of AA usually require derivatization. However, techniques such as AA analyzer are composed of cation-exchange chromatography and other components can sideline the derivatization with significant accuracy. The present analysis revealed a higher concentration of essential amino acids especially acidic AA, Glu and Asp and basic AA, Arg than other AA in macadamia protein. The study constitutes first report of use of bubble chart for evaluation of AA and explaination of AAS. The results may elaborate that the degradation of AA of macadamia protein for extraction of pH 11 is caused by the impact of pH. Moreover, the nutritional values of AA present in macadamia protein could change for the better by adjusting pH of extraction.

Research Progress on the Separation of Alkaloids from Chinese Medicines by Column Chromatography

Alkaloids have a variety of bioactivities and great development value in the fields of pharmaceuticals, cosmetics and health food. Column chromatography is a common method for preparing alkaloids. In this paper, the research status of the separation and purification of alkaloids from Chinese medicines by column chromatography is reviewed, and the factors that influence the refining of alkaloids via a macroporous adsorption resin, ion exchange resin and silica gel are summarized. The thermodynamic and kinetic modeling methods for the static adsorption of adsorbents are also reviewed in this paper. It is suggested that the modeling method of the column chromatography process be deeply studied to establish a more stringent quality control method for sampling liquid and to strengthen the online detection of the chromatography process to improve the refining effect of alkaloids.

Opportunities and challenges of high-pressure ion exchange chromatography for nuclide separation and enrichment

With the rapid development of the nuclear industry,more-stringent requirements are proposed for highlevel radioactive waste liquid treatment and the enrichment of isotope products.High-pressure ion exchange chromatography has been widely accepted for the fine separation of elements and nuclides due to its advantages,such as high efficiency,environmental friendliness,ease of operation,and feasibility for large-scale industrial applications.Here,we summarized the evolution of high-pressure ion exchange chromatography and the relevant research progress in ion exchange equilibrium and related separation technology.The prospects for application of high-pressure ion exchange chromatography to rare earth elements,actinide elements and isotope separation were discussed.High-pressure ion exchange chromatography represents a promising strategy for the extraction of rare earth elements and actinide elements from high-level radioactive waste liquid,as well as being an effective method for the automated production of high purity isotope products with great environmental benefits.

Implications from protein uptake kinetics onto dextran-grafted Sepharose FF coupled with ion exchange and affinity ligands

Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups, such as dextran-grafted and poly(ethylenimine)-modified Sepharose gels. However, it is unclear if the "chain delivery" occurs on affinity adsorption with specific interactions. This work is designed to address this issue. A dextran-grafted Sepharose gel was prepared, and then the matrix was modified using diethylaminoethyl, a typical ion-exchange group, or octapeptide(FYCHWQDE), an affinity ligand for human immunoglobulin G(h Ig G) to prepare ion-exchange or affinity adsorbents, respectively.Results of h Ig G adsorption showed that the uptake rate represented by the effective diffusivity of h Ig G onto the dextran-grafted ion exchangers was obviously enhanced by the dextran grafting, indicating the presence of"chain delivery" of the bound proteins on the charged groups on the dextran chains. By contrast, the effective diffusivity of h Ig G changed little as ligand density increased on the dextran-grafted FYCHWQDE adsorbents.Their adsorption capacities decreased and effective diffusivities were not accelerated by the dextran grafting.Thus, this work clarified that grafted dextran could not accelerate h Ig G uptake rate on the affinity resins, or in other words, chain delivery did not occur on the specific interaction-based affinity adsorption.

Retention model of protein for mixed-mode interaction mechanism in ion exchange and hydrophobic interaction chromatography

A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a 'U' shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.

Purification of L-Lysine in Simulated Moving Bed and Fixed-Bed Chromatography

L-Lysine was produced by a microbial process utilizing a Corynebacterium glutamicum （ATCC 21799） strain. L-Lysine was purified from the cultivated medium by fixed-bed and simulated moving bed （SMB） chromatography. The separation conditions including pH, eluent concentration and Lys+ and Lys2+ adsorption isotherms were studied in batch adsorption. The column capacity, eluent flow rate and eluent concentration have been studied in fixed-bed chromatography. Maximum purification rate of lysine was obtained as 0.066 （g/（g·h）） （per gram resin and per hour） at an eluent flow rate of 10 （mL/min） in fixed-bed chromatography. The results obtained from SMB were 0.11 （g/（g·h）） for L-lysine purification rate and 96% for L-lysine recovery.

A reversed phase/hydrophilic interaction/ion exchange mixed-mode stationary phase for liquid chromatography

A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epoxy reactions with 1,4-butanedioldiglycidyl ether and tertiary amines (N,Ndimethyldecylamine, DMDA). Its retention mechanism was found to follow RP/HILIC/IEX mixed-mode.The stop-flow test revealed that TMSP had good compatibility with 100% aqueous mobile phase. It demonstrated effective separation towards several kinds of compounds or drug molecules and their counterions within a single run.

A novel composite stationary phase composed of polystyrene/divinybenzene beads and quaternized nanodiamond for anion exchange chromatography

An approach for preparation of a novel composite anion exchanger composed of polystyrene/divinylbenzene (PS/DVB) beads and quaternized nanodiamods (QND) were proposed. Oxidized nanodiamonds (OND) were quaternized by the condensation polymerization between methylamine (MA) and 1,4-butanediol diglycidyl ether (BDDE), which were characterized by Fourier transform infrared (FTIR) spectra, X-ray phtoelectron spectroscopy (XPS), thermogravimetric analysis (TGA). QND with layers of cationic polyelectrolyte was attached onto the surface of sulfonated PS/DVB beads electrostatically. Subsequently, hyperbranched reaction of QND agglomerated on the PS/DVB bead surface was performed by the alternate reaction between MA and BDDE to increase the exchange capacity. The composite anion exchanger showed good stability in organic solvent and a wide pH range.The surface of these microspheres was characterized by scanning electron microscopy. In addition, ion exchange selectivity and separation efficiency of the anion exchangers were assessed using the mixtures of anions (F,Cl,NO_2,Br,NO_3,HPO_4~2 and SO_4~2) with carbonate/bicarbonate as eluent, and the anion exchanger with high exchange capacity could be used to analyze chloride in aqueous solution with high concentration of fluoride. This work explores the potential of nanodiamods as an agglomerated material for ion chromatography stationary phases for the separation of inorganic anions.

Protein adsorption onto diethylaminoethyl dextran modified anion exchanger:Effect of ionic strength and column behavior

Our previous studies on bovine serum albumin （BSA） adsorption to diethylaminoethyl dextran （DEAE dextran, DexD, grafting-ligand） and DEAE （D, surface-ligand） modified Sepharose FF resins found that all the grafted resins （FF-DexD and FF-D-DexD） exhibited extremely fast uptake rate （effective diffusivity, De, De/Do 〉 1.4）, which was six times greater than the ungrafted resins （De/Do 〈 0.3）. In this work, the influence of ionic strength （IS） on 6 typical DEAE dextran-grafted resins was investigated. Bath adsorption equilibria and kinetics, breakthrough, and linear gradient elution experiments were conducted. Commercial DEAE Sepharose FF was used for comparison. It is found that protein adsorption capacities on DEAE dextran-FF resins and the commercial resin decreased with increasing IS, but DEAE dextran-FF resins exhibited much higher capacity sensitivity to salt concentration. Besides, steeper decrease of adsorption capacities could be obtained at higher graftingligand or surface-ligand density. It is worth noting that the facilitating role of surface-ligand to the ＂chain delivery＂ effect was weakened after adding salt, leading to the less improvement in uptake rate by increasing surface-ligand density at higher IS. Although the uptake rates of the DEAE dextran-FF resins increased first and then decreased with increasing fS, they kept the extremely high level of De values （De/Do 〉 1.1 ） at the their working/binding IS range. Moreover, the DEAE dextran-FF resin displayed much higher adsorption capacities and De values than commercial ungrafted resin in their working condition. Furthermore, the column results of DEAE dextran-FF resins presented higher dynamic binding capacities than and similar elution ISs with DEAE Sepharose FF to achieve similar （or even higher） recoveries suggest the excellent chromatographic column performance of the DEAE dextran-FF resins. Finally, both high recovery and purity of BSA and γ-globulin could be easily achieved using the typical DEAE dextran-FF column, FF-D60-DexD160, to separate their binary mixtures, by step gradient elution. The research has provided new insights into the practical application of the series of DEAE-dextran grafted resins in protein chromatography and proved their superiority.

Elution Behaviour of Monocarboxylic Acids on a Cation-exchange Resin Column

The retention mechanism of monocarboxylic acids on a cation-exchange resin column was investigated. It was assumed that both Donnan membrane equilibrium and adsorption equilibrium were involved in the chromatographic process. On the basis of the proposed mechanism, an equation was derived for correlating distribution coefficient, Kd, dissociation constant, Aa, and adsorption equilibrium constant, K, of the analyzed acid. By this approach, retention data for some aliphatic acids under different operating conditions were predicted. Results are reasonably in agreement with experiment.

Study on Separation of Phycoerythrin by Q-Sepharose Fast Flow

[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and flow rate were optimized to determine the optimal separation condition, under which the scale-up process was verified. [Result] The optimal condition for separation of phycoerythrin by using Q Sepharose FF resin was investigated： 30 ml of laver extract was loaded to the Q Sepharose FF column with a bed volume of 8 ml; subsequently, the column was stepwise eluted with 0-0.10-0.35-1.00 mol/L NaCI solution （pH 6.0） at a constant flow rate of 1 ml/min; the elution peak under 0.35 mol/L NaCI solution was collected, and the recovery rate and purity coefficient （A565/A280） of phycoerythrin were determined as 44.3 and 1.15, respectively. Based on the established process, 75 ml of phycoerythrin extract was loaded to the Q Sepharose FF column with a bed volume of 20 ml for separation, while no significant variation was observed in the separation result. [Conclusion] Phycoerythrin can be well separated from laver extract by using Q Sepharose FF resin and the process is feasible for scale-up.

Purification and characterization of acetylcholinesterase from brain tissues of Oreochromis aurea and its application in environmental pesticide monitoring

Acetylcholinesterase （ACHE） plays an important role in enzyme-based detection of pesticides in the environment. In this paper, ACHE from the Triton X-100 extract of brain tissues of Oreochromis aurea was purified by （NH4）2SO4 fractional precipitation, Sephadex G-100 gel filtration, and DEAE-cellulose ion exchange chromatography. Certain biochemical characterizations of the purified enzyme and inhibition of pesticides on the enzyme were also studied. The specific activity of this purified enzyme was 20.628 U/mg protein, fold of purification was 139, and recovery was 22.1%. The optimal temperature of this enzyme was between 35-40 ℃, and optimal pH was between 7.5-8.0. The Michaelis constant （Kin） for acetylthiocholine iodide was 0.183 mmol/L. The enzyme activity was inhibited by excess substrate, and optimal substrate concentration was 6 mmol/L. Four pesticides （dichlorvos, phoxim, triazophos, and methomyl） exhibited strong inhibitions on this enzyme with IC50 less than 5 μg/mL. This study suggests that Oreochromis aurea （tilapia） could be a good enzyme source for pesticide monitoring in water environments.

Purification and Stability of an Antithrombus Enzyme from Bacillus subtilis

An antithrombus enzyme (ATE) was precipitated by (NH4)2SO4 or ethanol from supernatant of Bacillus subtilis culture broth then purified using ion exchange chromatography on CM-sepharose fast flow. The effects of ionic strength and pH value on protein adsorption, the gradient elution at different flow rates and step elution were examined respectively. The recovery yield of the optimised process was 74.5% with a purification factor 8.1. The ATE molecular weight was estimated as 30ku by SDS-PAGE. The experimental results showed that the enzyme was stable in the range of pH 7 to pH11, and temperature 25℃ to 37℃.

Amino Acid Composition of Tightly Bound Exopolymeric Substances Produced by Corrosion-Related Bacteria in Presence of Mild Steel

The composition of tightly bound exopolymeric substances (EPS) obtained from biofilm and plan- ktonic cell subpopulations of Stenotrophomonas maltophilia 5426 UKM was analyzed to study an impact of mild steel presence in the cultivation medium. The overall protein production was found to increase in the presence of a steel coupon. Some amino acids such as lysine, valine, iso-leucine, tyrosine and phenylalanine were found to be secreted in higher amounts in the presence of mild steel, while its absence leads to an increase of arginine, asparagine, serine, glutamine, proline, glycine, cysteine, leucine, methionine production levels. Biofilm cells EPS contained more arginine, glycine, cysteine, valine, methionine and leucine, comparing to EPS of planktonic cells. The chang- es of tightly bound EPS aggregation around the cells induced in the presence of steel coupons were revealed by transmission electron microscopy. The results suggested the transition of S. maltophilia 5426 UKM cells from planktonic to biofilm lifestyle to be a complex process involving more than a single step.

Effects of Cross Linking on the Chromatographic Nitrogen Isotope Separation

The effects of cross linking with porous cation exchange resin were studied for nitrogen isotope separation. The displacement chromatography was conducted by the resin with cross linking rang from 20% to 40%. A sharp adsorbed ammonium band was maintained for each operation. Enriched 15N isotopes with 0.93% were obtained by 20% cross linking resin and two meters chromatographic operation which started from the natural abundance of ammonium molecule. The effect of cross linking percentage on the height equivalent to a theoretical plate (HETP) was evaluated in the present work and the HETP value is proportional to the cross linking percentage. HETP value of 0.036 cm was obtained at the present system by using 20% cross linking resin.

Isolation and Identification of Ectromelia Virus from Suspected Mice

Choleragenoid was obtained in pure form by ultra-filteration and fractionation on cationexchange resin-phospho-cellulose column. The choleragenoid was highly pure as judged by the electrophoresis of isoelectric focusing,immunization and SDS-gel electrophoresis.The results of test are thesame as that of the standard choleragenoid.