Separation of Proteins by Electrophoretic Affinity Chromatography

A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed. Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the central compartments. The presence of an electric field accelerated the migration of proteins inside the gel matrix during adsorption and desorption processes. This led to the increase of the overall speed of separation. The present study was focused on the effect of the strength of the electric field on adsorption and desorption processes.

High Performance Affinity Chromatography of Antithrombin III Based on Monodisperse Poly (glycidyl methacrylate) Beads

A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were used as the affinity support. With the water-soluble carbodiimide, heparin was linked covalently to amino-PGMA-beads, which was prepared by amination of PGMA. The adsorbent obtained exhibits high binding activity to antithrombin III (ATIII), good resolution and excellent mechanical properties and can be used under high flow rate.

On-column Refolding of Diphtheria Toxin Variant CRM197 by Different Metal-Chelating Affinity Chromatography Matrices

We have previously shown that the diphtheria toxin variant CRM 197 （cross-reacting material 197） can be overexpressed in Escherichia coli at high levels, yielding insoluble aggregates, which were solubilized using urea. This study reports a comparison of three matrices suitable for the purification and refolding of recombinant CRM197 by metal-chelating affinity chromatography, Moreover, we show that refolded CRM197 features enzymatic activity.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.

PREPARATION OF CHITOSAN COATED METAL AFFINITY CHROMATOGRAPHY ADSORBENT

A new and an inexpensive adsorbent of chitosan coated silica for immobilized metal affinity chromatography (IMIC) was studied. After a double coating, the chitosan coated on silica beads could be up to 53. 4 mg/g silica beads.When pH＞3. 8, the metal ligand Cu2+ was chelated on the coated chitosan witha bound capacity of 14. 6 mg/g chitosan without introducing iminodiacetic acid(IDA).

Quantitative analysis of hepatoma-specific α-fetoprotein(HS-AFP) by a new mini-column affinity chromatography and its clinical value in diagnosis of hepatocellular carcinoma

Objective： To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein （HS- AFP） for diagnosis of hepatocellular carcinoma （HCC）. Methods： HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin （LCA）-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay （RIA）. Results： Circulating AFP was separated into three peaks （AFP-1, AFP-2, and AFP-3） by LCA-affinity chromatography. Dunng the elution course, the AFP-1 and AFP-2 could be eluted with TE buffer. HSAFP （AFP-3） from sera of HCC patients was eluted clearly on the LCA-sepharose gel mini-column with a solution containing α-methyI-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity （92.7%） and specificity （88.2%）. Conclusion： The new assay for circulating HS-AFP analysis is more sensitive, repeatable, and convenient. Its clinical application would be useful to early diagnosis of HCC.

Isolation of Human Antibodies Against Hepatitis E Virus From Phage Display Library by Immobilized Metal Affinity Chromatography

Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography （1MAC）. Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus （HEV） from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

Separation of Recombinant Geranylgeranyl Diphosphate Synthase of Deinococcus radiodurans from Expressed Strain Cell Homogenate by Immobilized Metal Affinity Chromatography on a Characterized Monolithic Cryogel Column

Geranylgeranyl diphosphate synthase （GGPPS） plays a key role in the biosynthesis of antioxidative carotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinantGGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2＋-iminodiacetic acid （IDA）-cryogel. The properties of the Cu2＋-IDA-cryogel were characterized using capillary-based mathematical model and experi- mental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The porosity of the present Cu2＋-IDA-cryogel is 90.4% and the water permeability is 5.04x10-12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore （capillary） size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40x 10-4μm. s-1 by the Cu2＋-IDA-cryogel bed. High-purity GGPPS （about 91.4%） is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and ef- fective atmroach to isolate GGPPS from cell homoenate of engineering, strains.

Affinity Purification of Insulin by Peptide-Ligand Affinity Chromatography

The affinity heptapeptide(HWWWPAS)for insulin,selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column.The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation.It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography.Nearly 40 mg of insulin could be purified with the 1-mL affinity column.The results revealed the high specificity and capacity of the affinity column for insulin purification.Moreover,based on the analysis of the amino acids in the peptide sequence,shorter peptides were designed and synthesized for insulin chromatography.As a result,HWWPS was found to be a good alternative to HWWWPAS,while the other two peptides with three or four amino acids showed weak affinity for insulin.The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.

IMMOBILIZED CIBACRON BLUE F3G-A ON CROSSLINKED POLY(VINYL ALCOHOL) FOR AFFINITY CHROMATOGRAPHY

Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, Cibacron Blue F3G-A was immobilized, through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N(-Cibacron Blue F3G-A, which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by treating macroporous poly(vinyl alcohol) with excess epichlorohydrin in the presence of NaOH in dimethyl sulfoxide. The macroporous poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinked poly(vinyl acetate), which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The Cibacron Blue F3G-A-immobilized poly(vinyl alcohol) was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.

Adsorption and Step Elution of Urokinase Using Affinity Chromatography-Comparison of Data with Rate Model Simulation

A non-equilibrium chromatographic rate model was employed to simulate the affinity chromatography of urokinase. The chromatography process was developed to a yield of high purity product of urokinase from crude materials. The affinity gel used in the process was prepared by an epichlorohydrin-activation method using epichlorohydrin activated Sepharose 4B as a matrix and p-aminobenzamidine as a ligand. The chromatographic process were numerically simulated and analyzed with the aid of VERSE-LC computer simulator. Considering the basic principles, rate model with the back mixing in column inlet was utilized in simulating and studying the effect of the column inlet pattern on other parameters. Comparison of the simulation results with the experimental data showed that the rate model can be used to describe the affinity chromatography of urokinase in a fixed bed column with satisfactory accuracy.

Determination of 14 Organophosphorus Pesticide Residues in Mutton by Gel Permeation Chromatography-Gas Chromatography-Mass Spectrometry(GPC-GC-MS)

[Objectives]This study was conducted to purify mutton samples by gel permeation chromatography(GPC).[Methods]Fourteen organophosphorus pesticide residues in samples were qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry(GC-MS)in selective ion scanning mode(SIM).[Results]The organophosphorus pesticide standard solutions showed good linearity in the mass concentration range of 0.1-10.0μg/ml with correlation coefficients(r)not lower than 0.999,and the detection limits(S=3 N)ranged from 0.01 to 0.05 mg/kg.The average recovery values were in the range of 80.2%-99.7%,with relative standard deviations(RSDs,n=3)in the range of 1.8%-6.3%,at the addition levels of 0.5,1.0 and 2.0 mg/kg.[Conclusions]The method is simple,sensitive and accurate,and can be used for the determination of organophosphorus pesticide residues in mutton.

Boronic Acids as Ligands for Affinity Chromatography

A review on the principles and applications of boronic acids as affinity ligands for the chromatographic separation of carbohydrates,nucleic acid components,glycoproteins,and other small biomolecules.The mechanisms of interactions between boronate ligands and analytes are described.Various boronate ligands and supports are discussed.Examples of the use of boronate affinity chromatography for separation of each class of analytes are presented.

Using frontal affinity chromatography to study how silver binds with particulates

Frontal affinity chromatography was applied to characterizing the mechanism of binding of silver with sediment particulates collected from Lake Ontario, Canada. The results showed that there was one major binding site for Ag+ in the particulates. The binding capacity ranges from 6.06 to 1.01 μ·mol·g-1, and the binding constant (lgK) from 6.23 to 7.43 M-1 in 0.005 M ion strength at pH=3-7. The binding capacity and affinity constant were found to be pH-dependent. It is suggested that the particulate surface site where silver was bound was the anionic base. This study would be helpful for better understanding of the fundamental environmental chemistry of silver in sediments.

Simultaneous purification of minor components in natural products using twin-column recycling chromatography with a step solvent gradient

The isolation of minor components from complex natural product matrices presents a significant challenge in the field of purification science due to their low concentrations and the presence of structurally similar compounds.This study introduces an optimized twin-column recycling chromatography method for the efficient and simultaneous purification of these elusive constituents.By introducing water at a small flowing rate between the twin columns,a step solvent gradient is created,by which the leading edge of concentration band would migrate at a slower rate than the trailing edge as it flowing from the upstream to downstream column.Hence,the band broadening is counterbalanced,resulting in an enrichment effect for those minor components in separation process.Herein,two target substances,which showed similar peak position in high performance liquid chromatography(HPLC)and did not exceed 1.8%in crude paclitaxel were selected as target compounds for separation.By using the twin-column recycling chromatography with a step solvent gradient,a successful purification was achieved in getting the two with the purity almost 100%.We suggest this method is suitable for the separation of most components in natural produces,which shows higher precision and recovery rate compared with the common lab-operated separation ways for natural products(thin-layer chromatography and prep-HPLC).

Comprehensive analysis of phenolic composition and antioxidant mechanisms in Gymnema sylvestre extracts using LC-MS and column chromatography

Recent studies have highlighted the potential of plant extracts as therapeutic agents for managing oxidative stress and related disorders.This study aims to elucidate the phenolic composition and antioxidant properties of Gymnema sylvestre extracts.Ethanolic reflux extraction followed by column chromatography was employed to isolate phenolic compounds.The total phenolic and flavonoid contents were quantified using the Folin–Ciocalteu and aluminum chloride colorimetric methods,respectively.Antioxidant activities were assessed by DPPH,ABTS scavenging assays and the ferric reducing antioxidant power(FRAP)assay.High-Performance Liquid Chromatography(HPLC)with a C18 column and Thermo TSQ Quantum Access Max(LC-MS)were used to determine the levels of gymnemic acid and identify other potential phenolic compounds.The analysis revealed significant antioxidant activities in the fractions.Fraction A showed the highest DPPH and ABTS scavenging activities,and Fraction C demonstrated the highest ferric reducing power.LC-MS analysis identified several phenolic compounds,indicating that these are major contributors to the antioxidant efficacy of the extract.This study provides a detailed phenolic profile and confirms the strong antioxidant potential of Gymnema sylvestre leaf extract,supporting its therapeutic use and further investigation.

Determination of Benzo[a]pyrene in Edible Oil by High Performance Liquid Chromatography-Fluorescence Detector (HPLC-FL)

In this study, an optimized high performance liquid chromatography-fluorescence detector (HPLC-FL) method for the determination of benzo[a]pyrene in edible oil was established. HPLC was performed with Thermo Fisher Scientific C18 column (250 mm×4.6 mm, 5 μm) as the chromatographic column and acetonitrile and water as the mobile phase, and the excitation wavelength and emission wavelength of fluorescence detector were 286 and 430 nm, respectively. The response was high, and the linear range was 0.5-10.0 ng/ml. The lowest limit of detection was 0.11 ng/ml, and the average recovery was 92.5%. This method is suitable for quantitative analysis of benzo[a]pyrene content in edible oil.

Separation and analysis of six fractions in low temperature coal tar by column chromatography

The low temperature coal tar(CT)is taken as the raw material,and the extraction and column chromatography are used for detailed and accurate characterization in this paper.The n-heptane soluble fraction(CT-HS)and insoluble fraction(CT-HI)were obtained by n-heptane Soxhlet extraction.The extraction rate of CT-HS reached 92.79%(mass),which indicated that there are few heavy compounds in it.Further,different solvents(methylbenzene,benzene,ethyl acetate,methylbenzene-ethanol)were used to elute CT-HS by chromatographic column to obtain five fractions(saturates,aromatics,heteroatoms,phenolics and resins,named CT-SA,CT-AR,CT-HE,CT-PH,CT-RE,respectively).The yields of CTSA,CT-AR,CT-HE,CT-PH,CT-RE are 42.12%,10.43%,2.19%,9.50%and 6.63%(mass),respectively.Gas chromatography-mass spectrometry analysis of eluting components show that alkanes are the main components in CT,followed by polycyclic aromatics,and the corresponding fractions are CT-SA and CT-AR,respectively.The relative content of aliphatics in CT-SA is 76.93%,and the relative content of aromatics in CT-AR is 75.05%.This separation technology effectively separates and enriches different components in CT,and the activation energy required for the pyrolysis process of a single eluting fraction is lower than that of CT,which is expected to provide an important reference for the separation,analysis and conversion of complex oil products such as coal-oil co-processing products,coal tar and other complex heavy carbon oil products.

AFFINITY CHROMATOGRAPHY PURIFICATION OF UROKINASE WITH EPICHLOROHYDRIN ACTIVATED AGAROSE MATRIX

1 INTRODUCTIONIn literature,most matrices of affinity chromatography for urokinase(EC 3.4.99.26)purification were prepared by cyanogen bromide activation.However,theseadsorbents usually suffered from the drawback of leakage of the ligand,particularly inalkaline medium,because of the instability of the isourea linkage between the ligandand the spacer or agarose.Moreover,the positively charged imido group of theN-substituted isourea derivative and the hydrophobicity of the spacers might promotenonspecific adsorption.On the contrary,the adsorbents prepared by the method

Efficient and selective extraction of uranium from seawater based on a novel pulsed liquid chromatography radionuclide separation method

The novel pulsed liquid chromatography radionuclide separation method presented here provides a new and promising strategy for the extraction of uranium from seawater.In this study,a new chromatographic separation method was proposed,and a pulsed nuclide automated separation device was developed,alongside a new chromatographic column.The length of this chromatographic column was 10 m,with an internal warp of 3 mm and a packing size of 1 mm,while the total separation units of the column reached 12,250.The most favorable conditions for the separation of nuclides were then obtained through optimizing the separation conditions of the device:Sample pH in the column=2,sample injection flow rate=5.698 mL/min,chromatographic column heating temperature=60℃.Separation experiments were also carried out for uranium,europium,and sodium ions in mixed solutions;uranium and sodium ions in water samples from the Ganjiang River;and uranium,sodium,and magnesium ions from seawater samples.The separation factors between the different nuclei were then calculated based on the experimental data,and a formula for the separation level was derived.The experimental results showed that the separation factor in the mixed solution of uranium and europium(1:1)was 1.088,while achieving the initial separation of uranium and europium theoretically required a 47-stage separation.Considering the separation factor of 1.50for the uranium and sodium ions in water samples from the Ganjiang River,achieving the initial separation of uranium and sodium ions would have theoretically required at least a 21-stage separation.Furthermore,for the seawater sample separation experiments,the separation factor of uranium and sodium ions was 1.2885;therefore,more than 28 stages of sample separation would be required to achieve uranium extraction from seawater.The novel pulsed liquid chromatography method proposed in this study was innovative in terms of uranium separation and enrichment,while expanding the possibilities of extracting uranium from seawater through chromatography.