Elution Agent Research Articles

AbFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC–MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the anti-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry.

Recovery of uranium, thorium and zirconium from allanite by extraction chromatography using impregnated chromosorb

Chromosorb 102 impregnated with Cyanex 923 was used as a stationary phase in the extraction chromatographic separation of lanthanides, U(VI), Th(IV) and Zr(IV). Batch studies were carried out to investigate the uptake behaviour of these metal ions, along with few other metal ions. The effect of different parameters like equilibration time, concentration of acid, metal ions and extractant has been studied. The distribution data has been used to develop suitable eluting agents. The sorbing and desorbing capacity of the column for U(VI), Th(IV) and Zr(IV) was determined. Results indicate an insignificant change in the efficiency of the column up to 10 such cycles. The practical utility of the column has been demonstrated by the recovery of around 95% of U(VI), Th(IV) and Zr(IV) from allanite sample with a purity of around 97%±2%.

Elution Efficiency of Herbicides Polluted Soil Samples

Simulated soil samples, quartz sands contaminated with 2,4-D, MCPA and dicamba, are separated using the method of ethanol elusion. The experimental results show that the elution temperature, the elution method and the purity of the eluting agent have obvious influence on separation efficiency. In addition, the elution velocity and the pollutant loading of soil have less effect. The impacts of these factors on the separation efficiency of 2,4-D is higher than that of MCPA and dicamba. With the increment of temperature, the separation efficiencies of three herbicides increase rapidly when the elution temperature is below 293 K, while the separation efficiencies increase slowly when the elution temperature is above 293 K. With the reduction of purity of elution agent, the downtrend of separation efficiency in the range of 95~90% or 90~85% is steeper than that of 100~95%. Compared with oft-repeated stationary method, continuous flowing method shows the advantages for three pollutants.

Gelatin-Immobilised Poly(hydroxyethyl methacrylate) Cryogel for Affinity Purification of Fibronectin

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10-200μm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30mg/ml, while much higher adsorption values, up to 38mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92% of the adsorbed fibronectin was eluted using 2M urea containing 1M NaCl as elution agent. The adsorption-elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.

Smart surface for elution of protein-protein bound particles: nanonewton dielectrophoretic forces using atomic layer deposited oxides.

By increasing the strength of the negative dielectrophoresis force, we demonstrated a significantly improved electrokinetic actuation and switching microsystem that can be used to elute specifically bound beads from the surface. In this work using atomic layer deposition we deposited a pinhole free nanometer-scale thin film oxide as a protective layer to prevent electrodes from corrosion, when applying high voltages (>20 V(pp)) at the electrodes. Then, by exciting the electrodes at high frequency, we capacitively coupled the electrodes to the buffer in order to avoid electric field degradation and, hence, reduction in dielectrophoresis force due to the presence of the insulating oxide layer. To illustrate the functionality of our system, we demonstrated 100% detachment of anti-IgG and IgG bound beads (which is on the same order of magnitude in strength as typical antibody-antigen interactions) from the surface, upon applying the improved negative dielectrophoresis force. The significantly enhanced switching performance presented in this work shows orders of magnitude of improvement in on-to-off ratio and switching response time, without any need for chemical eluting agents, as compared to the previous work. The promising results from this work vindicates that the functionality of this singleplexed platform can be extended to perform a multiplexed bead-based assay where in a single channel an array of proteins are patterned each targeting a different antigen or protein.

Separation of Sensitized and Non-Sensitized RBCs: Sephadex-Based Cell-Affinity Adsorbents

IntroductionIn transfusion medicine, antibodies that cause RBCs positive DATs, may interfere with patients' phenotyping. Traditionally, these antibodies were removed using various antibody elution methodologies. However, the elution agents and conditions used have been only partially successful; and no one method is superior. The purpose of this study was to develop a general and efficient method to separate non-sensitized from sensitized RBCs using Sephadex-based cell-affinity adsorbents.MethodsFirst, we coupled Sephadex support with Staphylococcal Protein G (SpG) with or without NHS. Then we simulated clinical conditions by mixing differe∏nt ratios of sensitized and non-sensitized RBCs in vitro. Sensitized cells were prepared by mixing antibody with corresponding antigen-positive RBCs. Finally, we checked the sensitization status of absorbed RBCs after absorption with modified Sephadex support.ResultsThe number of sensitized RBCs bound to Sephadex-based cell-affinity adsorbents is approximately 5×108 RBCs/mL support. Activated Sephadex could separate sensitized from non-sensitized RBCs. Conclusion Sephadex-based cell-affinity adsorbents with an NHS spacer arm have bigger capacity for binding RBCs than unmodified Sephadex. The Sephadex-based cell-affinity adsorbents readily separate non-sensitized RBCs from sensitized RBCs, thus providing a new strategy to type the blood for transfused patients.

Equilibrium and Thermodynamic Studies of Zinc Adsorption on Expanded Vermiculite

Because of industrial growth, large amounts of effluents are generated, which need to be treated. Among the micropollutants usually present in industrial effluents, heavy metals are especially dangerous to aquatic environment because they are bioaccumulated along the food chain. This work aims to study the removal of zinc ions in aqueous solution by adsorbing them onto expanded vermiculite. In addition, equilibrium and thermodynamics studies were carried out to verify the spontaneity and the characteristics of the process. Langmuir, Freundlich and Dubinin-Radushkevich's models were applied to the data obtained from isotherms at temperatures of 273, 293, 313 and 333 K. Furthermore, experiments in static system were carried out to choose a suitable eluent to this process, to find the metal concentration and identify a method for effective recovery of the adsorbent. This adsorption process was found to be spontaneous, favourable, endothermic and physical. The best eluting agent was ethylenediaminetetraacetic...

Study on Macroporous Resin Separation and Purification of Total Flavonoids of Plantago Process

To study the macroporous resin separation and purification of total flavonoids of Plantago optimum conditions, the static adsorption and desorption experiments by filtering out the best macroporous resin, and plantain extract for the assessment of total flavonoid content indicators in the single-factor test based on the orthogonal design, the amount of resin in the hole, eluent concentration, alcohol consumption and other factors for the study to optimize the AB-8 macroporous resin separation and purification of total flavonoids plantain best process conditions. The results show that: the effect of various factors affecting the size of the order of separation and purification has to: eluent concentration> ethanol consumption> macroporous resin usage, optimization of the optimum conditions: resin dosage is 1 times the amount of crude drug, the use of eluent volume fraction of 40% ethanol, elution agent is 12.5 times the amount of crude drug. Verify the conditions in this test, three repeated experiments were similar, the average flavonoid content 0.2127%, the transfer rate of 90.57%, indicating that the scientific and rational and stable process conditions can be effectively used plantain purified flavonoids.

Reversed-Phase Column Extractive Separation of Gd(III) with Poly[dibenzo-18-crown-6

A poly[dibenzo-18-crown-6] exhibits good chemical stability, reusability, and faster rate equilibrium for the separation of Gd(III). Both uptake and stripping of metal ions were rapid, indicating a better accessibility of the complexing sites. The proposed method has been applied for chromatographic separation of Gd(III) by using picric acid as medium and poly[dibenzo-18-crown-6] as stationary phase. The influences of picric acid concentration, different eluting agents, and so forth, were discussed and the optimum conditions were established. The breakthrough capacity of poly[dibenzo-18-crown-6] for Gd(III) was 0.572±0.01 mmolg-1 of crown polymer. The proposed method has been applied to sequential chromatographic separation of their binary and multicomponent mixtures. Gd(III) has been determined from real samples with good analytical reliability.

Determination of C5–C12 perfluoroalkyl carboxylic acids in river water samples in the Czech Republic by GC–MS after SPE preconcentration

A method employing solid phase extraction followed by gas chromatography–mass spectrometry with negative chemical ionization has been developed for determination of ultratrace concentrations of perfluoroalkyl carboxylic acids in river water. The effects of the experimental parameters, such as the pH, additions of NaCl and an ion-pairing agent (tetraethylammonium bromide) and the kind of the elution agent, on the efficiency of the test acid extraction have been studied. The analyte extraction recoveries and the limits of detection and determination have been found. The method developed has been tested on determinations of perfluoroalkyl carboxylic acids in the waters of the two largest Czech rivers, Vltava and Labe (Elbe). The best extraction results have been attained without any alteration of the sample pH, with an addition of tetrabutylammonium bromide (a concentration of 50μgmL−1 in the sample) and using methanol as the elution agent. Under these conditions, the recoveries of the test acids in the spiked real samples are within ranges from 60% to 104% and 53% to 111% for analyte concentration levels of 1.40ngmL−1 and 0.14ngmL−1, respectively, depending on the lengths of the perfluorinated chains of the acids. In general, the recovery decreases with increasing length of this chain. The method developed exhibits very low limits of detection and determination and the results are fully comparable with those obtained when using more expensive HPLC–MS/MS instrumentation. Typical values amount to tenths to tens of pgmL−1 and units to one hundred pgmL−1 for the limits of detection and determination, respectively; the measuring sensitivity increases with increasing length of the analyte chain. The analyzes of real samples from the Vltava and Elbe rivers have demonstrated that the results obtained are similar to the values published for contamination of the Elbe and other rivers in western and central Europe. The concentrations determined are of the order of units to tens of pgmL−1 and the C8–C10 acids occur most often.

Readily prepared resin-bound 2-pyridinethiol and its application for removal of mercury ions

2-Pyridinethiol attached to commercially available monosize chloromethylated polystyrene was used for selective removal of Hg(II) from aqueous solutions containing different amounts of Hg(II) (10–100 ppm). The adsorption rate was high at the beginning of the reaction and then equilibrium was reached in about 10 min. The maximum Hg(II) adsorption capacity of this novel and stable resin was about 55.94 mg/g of the dry polymer. The Hg(II) adsorption ability increased with increasing pH, in the range where the solubility of the Hg(II) was not affected by pH. More than 95% of the adsorbed Hg(II) was desorbed in 15 min using 1 M HNO3 as an elution agent. The regeneration of this resin by strong acid was feasible and desorption ratio was very high (up to 96%).

Adsorption of ytterbium (III) from aqueous solution by SQD–85 resin

Adsorption and desorption behavior of Yb (III) by SQD-85 resin was investigated by various chemical methods and IR spectrometry. The adsorption capacity of SQD–85 resin for Yb (III) was studied as a function of solution pH, initial concentration of Yb(III), temperature and contact time. The optimal pH for the adsorption was 5.50 in the HAc-NaAc system, and the maximum adsorption capacity was estimated to be 347.6 mg/g at 308 K. The isotherms adsorption data fit well with Langmuir model. The adsorption kinetics data are in agreement with pseudo-second-order model. Thermodynamic parameters indicate that Yb (III) adsorption by SQD–85 resin is endothermic and spontaneous in nature. Thomas model is reasonably accurate in predicting experimental column results. The dynamic desorption rate of Yb(III) can increase to 97.3% when the elution agent is 1.0 mol/L HCl. These results suggest that Yb(III) in aqueous solution can be removed and recovered by SQD–85 resin efficiently.

Isolation of Lysozyme from Chicken Egg White Using Polyacrylamide-based Cation-exchange Cryogel

An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer (pH 7.8) containing different concentrations of NaCl is used as elution agent. The corresponding breakthrough characteristics and elution behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The maximum purity of the obtained lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of high-purity lysozyme from chicken egg white.

Use of pyrocatechol violet modified sodium dodecyl sulfate coated on alumina for separation and preconcentration of uranium(VI)

A sensitive and selective solid phase extraction procedure for the determination of trace of uranium(VI) has been developed. An alumina-sodium dodecyl sulfate coated on with pyrocatechol violet was used for preconcentration and determination of uranyl ions by spectrophotometry method using Arsenazo III reagent. Sorbed ions were quantitatively eluted using 5 mL of 0.25 mol L−1 HNO3. The effects of parameters such as pH, amount of alumina, amount of ligand, flow rate, type and concentration of elution agent were examined. The capacity of the sorbent for U(VI) was found to be 0.92 mmol g−1. The relative standard deviation was 1.28% for 10 replicate determinations of U(VI) ion in a solution with a concentration of 1.0 μg mL−1. The practical applicability of the developed sorbent was examined using synthetic and real samples such as standard reference material 2709 (San Joaquin Soil) and 2711 (Montana Soil).

Study on the Purification of Longan Seed Flavonoids by Macro-Porous Resin

The weight of longan seed is seventy percent weight of fresh longan, and it is a kinds of biology resource available. The methods and conditions of absorption and desorption of longan seed flavonoids by macro-porous resin were studied. The facts of pH, temperature, concentration of longan seed flavonoids leaching solution which affect adsorption performances were researched, and the effects of kinds of elution agent and its dosage on ratio of extract were discussed. The results showed that macro-porous resin had remarkable ability in absorbing longan seed flavonoids. In optimum conditions, the kinds of flavonoids of elution solution with 50％ ethanol was determined by colour reaction, and the results were that the kinds of flavonoids of Longan seed including flavonones and flavonol.

Simultaneous ion exchange recovery of platinum and rhodium from chloride solutions

This work focuses on the sorption recovery of platinum (II, IV) and rhodium (III) simultaneously present in chloride solutions, freshly prepared and stored over 3 months, on commercial anion exchangers with different physical and chemical structure. The sorption was carried out from solutions with 0.001–4.0 mol/L HCl. The initial platinum and rhodium concentrations in contacting solutions were 0.25–2.5 mmol/L. Sorption and kinetic properties of the chosen anion exchangers were investigated and the basic parameters of exchange capacity, recovery, distribution coefficients, separation factors, process rate, diffusion coefficients and half-exchange times were calculated. It is shown that anion exchangers investigated possess high sorption ability to platinum and rhodium chloride complexes, which does not deteriorate in case of stored solutions. Desorption of platinum and rhodium from the resins investigated was carried out with hydrochloric acid (2 mol/L), thiourea (1 mol/L) in sulfuric acid (2 mol/L) or in potassium hydroxide (2 mol/L) as well as by ammonium thiocyanate (2 mol/L). It was shown that complete separation of platinum and rhodium can be carried out with 2 mol/L HCl on anion exchanger Purolite S 985, whereas 2 mol/L NH 4 SCN as an elution agent leads to complete separation of noble metals on anion exchangers Purolite S 985, Purolite A 500 and AM-2B.

Readily prepared polystyrene-bound pyridine-2,6-dicarboxylate and its application for removal of mercury ions

Commercially available chloromethylated polystyrene (cross-linked with 2% divinylbenzene) was used to prepare novel and stable polymer supported pyridine-2,6-dicarboxylate. This polymer was found to accelerate the selective removal of Hg(II) from aqueous solutions containing different amounts of Hg(II) (10-100 ppm). Adsorption rate was high at the beginning and then equilibrium was reached in about 25 min. Maximum Hg(II) adsorption capacity of the resin was about 41.5 mg/g of the dry polymer. The Hg(II) adsorption ability increased with the increase in pH, in the range where the solubility of the Hg(II) was not affected by pH. More than 95% of the adsorbed Hg(II) was desorbed in 15 min by using 0.1 M HNO3 as an elution agent. The regeneration of this resin by strong acid was feasible and desorption ratio was very high (up to 96%).

Use of Phosphate Rock for the Removal of Ni2+ from Aqueous Solutions: Kinetic and Thermodynamics Studies

A study was carried out in batch conditions to examine the removal of nickel ions from an aqueous solution by phosphate rock. The effect of different sorption parameters, such as initial metal concentration, equilibration time, solution pH, and temperature on the amount of Ni2+ sorbed was studied and discussed. The sorption process followed pseudo-second-order kinetics with necessary time of 2h to reach equilibrium. The maximum removal obtained is at initial pH around 8. Nickel uptake was quantitatively evaluated using the Langmuir and Dubinin–Kaganer–Radushkevich model. The Langmuir adsorption isotherm constant corresponding to adsorption capacity, Q0, was found to be 7.63mg∕g. The possibility of metal recovery was investigated using several eluting agents. The desorbed amount of nickel decreased continuously with increasing pH, and increased with increasing Ca2+ concentration in leaching solution.

Elucidating the Mechanisms of Assembly and Subunit Interaction of the Cellulose Synthase Complex of Arabidopsis Secondary Cell Walls

Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.

Optimization of adsorption conditions of papain on dye affinity membrane using response surface methodology

The adsorption of papain on Reactive Blue 4 dye–ligand affinity membrane was investigated in a batch system. The combined effects of operating parameters such as initial pH, temperature, and initial papain concentration on the adsorption were analyzed using response surface methodology. The optimum adsorption conditions were determined as initial pH 7.05, temperature 39 °C, and initial papain concentration 11.0 mg/ml. At optimum conditions, the adsorption capacity of dye–ligand affinity membrane for papain was found to be 27.85 mg/g after 120 min adsorption. The papain was purified 34.6-fold in a single step determined by fast protein liquid chromatography. More than 85% of the adsorbed papain was desorbed using 1.0 M NaCl at pH 9.0 as the elution agent. The purification process showed that the dye–ligand immobilized composite membrane gave good separation of papain from aqueous solution.