Eluate Fractions Research Articles

Enrichment of secoiridoid glycosides from Gentiana rigescens extracts in amino acid-based deep eutectic solvents via macroporous resin adsorption

The simultaneous enrichment of the secoiridoid glycosides gentiopicroside, loganic acid, swertiamaroside, and sweroside from amino acid-based deep eutectic solvent (AADES) extracts of Gentiana rigescens roots and rhizomes was developed. To screen suitable resins, the performance and characterization of 11 widely used macroporous resins for the adsorption and desorption of target secoiridoid glycosides were considered. Compared with the other resins, the H103 resin was found to have greater adsorption and desorption capacities for gentiopicroside, loganic acid, swertiamaroside, and sweroside. Therefore, this resin was chosen as the best resin for secoiridoid glycosides enrichment. In a column packed with H103 resin, the dynamic adsorption and desorption factors for the target secoiridoid glycosides were investigated and optimized. After dynamic testing on the H103 packed column, the optimal operating conditions were determined as follows: a sample volume of 12 bed volumes (BVs) and a loading flow rate of 2 BV/h were used, the volume of deionized water used for the aqueous washing process was 8 BV, and the aqueous washing flow rate was 3 BV/h. Ethanol with a volume fraction of 10–90 % was used as the eluent in the ethanol desorption process with a gradient of 10 %. Each ethanol volume fraction was used for 2 BV, with a flow rate of 3 BV/h. The eluent fractions containing 40–80 % ethanol were collected, combined, and concentrated, followed by vacuum drying to obtain the final product. The purities of gentiopicroside, loganic acid, swertiamaroside, and sweroside in the final product were 58.11, 13.73, 3.86, and 2.77 %, respectively, with corresponding recovery rates of 74.09, 72.19, 77.50, and 76.45 %, respectively. Additionally, the reuse of AADES proved to be effective.

Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes.

Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.

Evaluation of hemostatic, anti-inflammatory, wound healing, skin irritation and allergy, and antimicrobial properties of active fraction from the ethanol extract of Chromolaena odorata (L.) R.M. King & H. Rob

Ethnopharmacological relevanceChromolaenaodorata (L.) R.M. King & H. Rob, a perennial herb, has been traditionally utilized as a herbal remedy for treating leech bites, soft tissue wounds, burn wounds, skin infections, and dento-alveolitis in tropical and subtropical regions. Aim of the studyThe present study was to analyze the active fraction of C. odorata ethanol extract and investigate its hemostatic, anti-inflammatory, wound healing, and antimicrobial properties. Additionally, the safety of the active fraction as an external preparation was assessed through skin irritation and allergy tests. Materials and methodsThe leaves and stems of C. odorata were initially extracted with ethanol, followed by purification through AB-8 macroporous adsorption resin column chromatography to yield different fractions. These fractions were then screened for hemostatic activity in mice and rabbits to identify the active fraction. Subsequently, the hemostatic effect of the active fraction was assessed through the bleeding time of the rabbit ear artery in vivo and the coagulant time of rabbit blood in vitro. The anti-inflammatory activity of the active fraction was tested on mice ear edema induced by xylene and rat paw edema induced by carrageenin. Furthermore, the active fraction's promotion effect on wound healing was evaluated using a rat skin injury model, and skin safety tests were conducted on rabbits and guinea pigs. Lastly, antimicrobial activities against two Gram-positive bacteria (G+, Staphylococcus aureus and S. epidermidis) and three Gram-negative bacteria (G−, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) were determined using the plate dilution method. ResultsThe ethanol extract of C. odorata leaves and stems was fractionated into 30%, 60%, and 90% ethanol eluate fractions. These fractions demonstrated hemostatic activity, with the 30% ethanol eluate fraction (30% EEF) showing the strongest effect, significantly reducing bleeding time (P < 0.05). A concentration of 1.0 g/mL of the 30% EEF accelerated cutaneous wound healing in rats on the 3rd, 6th, and 9th day post-operation, with the healing effect increasing over time. No irritation or allergy reactions were observed in rabbits and guinea pigs exposed to the 30% EEF. Additionally, the 30% EEF exhibited mild inhibitory effect on mice ear and rat paw edema, as well as antimicrobial activity against tested bacteria, with varying minimal inhibitory concentration (MIC) values. ConclusionsThe 30% EEF demonstrated a clear hemostatic effect on rabbit bleeding time, a slight inhibitory effect on mice ear edema and rat paw edema, significant wound healing activity in rats, and no observed irritation or allergic reactions. Antibacterial activity was observed against certain clinically isolated bacteria, particularly the G− bacteria. This study lays the groundwork for the potential development and application of C. odorata in wound treatment.

Exploring canine olfactory generalization using odor profile fractions from native crude oils.

Canines are used by both government agencies and industries for their keen olfactory capability as well as selectivity, reliability, versatility, and speed. Within the last decade, canines have been used for the detection of on-shore crude oil. They were previously shown to find these deposits with high accuracy, providing increased confidence with little risk to oil spill response survey teams. In order to efficiently train canines, it is important to understand the odorants or groups of odorants that such canines use when locating subsurface crude oil deposits, as well as track how the odorant profile changes as the crude oil undergoes degradation. In this study, headspace solid phase microextraction (HS-SPME) was used in combination with gas chromatography-mass spectrometry (GC-MS) to extract and separate odorants from the headspace of various crude oils. After, eluent fractions of the crude oil odor profile were separated and collected onto sorbent materials, which were then used as canine testing probes in a series of trials. These probes, along with negative and positive controls were presented to three previously-trained and operational crude oil detection canines. Three eluent fractions of both fresh and weathered samples were presented, resulting in a 100% response rate from the canines on all three fractions from both the fresh and weathered samples. These results indicated that canines are capable of detecting crude oil from any fraction of the odor profile demonstrating the potential of the canines to generalize across a variety of crude oils and stages of weathering.

Recovery of Succinic Acid-Rich Fraction from the Juice of Chaenomeles japonica (Thunb.) Lindl. Ex Spach by Ion Exchange Fractionation Resin

Abstract Succinic acid (SA) is one of the most demanded and widely used constituents for producing various healthcare and skincare products, biodegradable polymers, and food and feed additives. The present study was undertaken to establish the protocol for selective recovery of SA from a complex matrix of Japanese quince (JQ) (Chaenomeles japonica) fruit using ion-exchange fractionation resin DIAION™ UBK550 with negatively charged functional groups cross-linked with a styrene-divinylbenzene matrix. A second SA purification method was used for data comparison using either Supel™-Select HLB or Strata-X columns packed with a hydrophilic modified sty-rene-based polymer. The analysis of SA accomplished by multiple reaction monitoring (MRM) using triple quadrupole mass selective detection (HPLC-ESI-TQ-MS/MS) revealed the presence of SA in the JQ juice without pretreatment, in the amount of 0.202 mg·100 ml–1. The use of sty-rene-based polymer Strata-X sorbent for purification of JQ juice led to an increase of 11.0% in the concentration of SA compared with the initial concentration of SA in JQ juice. On the other hand, after purification of JQ juice with Supel™-Select HLB, the concentration of desorbed SA in the eluate fraction was 7.8% lower compared to the initial concentration. The use of ion-exchange fractionation resin DIAION™ UBK550 for purification and concentration of JQ juice ensured up to 152.3 mg of SA in 100 ml–1 of syrup-like brown substance, which was much (754.4-fold) higher than that observed in JQ juice itself. All three sorbents ensured the complete removal of monoand disaccharides from the juice, as no signs of mono- and disaccharides were detected in the eluate fractions.

Chromatographic Profiles and Determination of Total Flavonoid Content of the Ethyl Acetate Fraction of Kalangkala Leaves (Litsea Angulata Blum) Using Uv-Vis Spectrophotometry

Background: Kalangkala is a fruit plant typical of Kalimantan and belongs to the species of the genus Litsea which is thought to have potential as a natural antioxidant. Empirically, some people in South Kalimantan use the kalangkala plant, especially the seeds of the fruit, to treat boils. One of the compounds that have antioxidant and antibacterial content is flavonoids.Objectives: To determine the thin layer chromatography profile of the total flavonoid compound of the ethyl acetate fraction and to calculate the total flavonoid content of the ethyl acetate fraction of the leaf extract of Kalangkala (Litsea angulata Blum).Method: Thin Layer Chromatography Profile and Determination of Total Flavonoid Content of the Ethyl Acetate Fraction of Kalangkala Leaf Extract using the UV-Vis Spectrophotometry method. The data were then analyzed qualitatively and quantitatively.Results: The chromatographic profile of the total flavonoids of the ethyl acetate fraction of Kalangkala leaf eluent that was most optimal was Ethyl acetate : n-Hexane (3:7) because it showed the most stains, namely 5 stains. And in the uv-vis spectrophotometry method, the total flavonoid content of the ethyl acetate fraction of Kalangkala leaves (Litsea angulata Blum) was 0.9 mg QE/g.Conclusion: Kalangkala leaf extract on the TLC profile using ethyl acetate : n-Hexane (3:7) showed a total of 5 stains. And the value of the total flavonoid content of the ethyl acetate fraction of kalangkala leaves was 0.9 mg QE/g.

Surface engineered functional biomaterials for hazardous pollutants removal from aqueous environment

The issue of water contamination by heavy metal ions as highly persistent pollutants with harmful influence primarily on biological systems, even in trace levels, has become a great environmental concern globally. Therefore, there is a need for the use of highly sensitive techniques or preconcentration methods for the removal of heavy metal ions at trace levels. Thus, this research investigates a novel approach by examining the possibility of using pomegranate (Punica granatum) peel layered material for the simultaneous preconcentration of seven heavy metal ions; Cd(II), Co(II), Cr(III), Cu(II), Mn(II), Ni(II) and Pb(II) from aqueous solution and three river water samples. The quantification of the heavy metals was performed by the means of FAAS technique. The characterization of biomaterial was performed by SEM/EDS, FTIR analysis and pHpzc determination before and after the remediation process. The reusability study as well as the influence of interfering ions (Ca, K, Mg, Na and Zn) were evaluated. The conditions of preconcentration by the column method included the optimization of solution pH (5), flow rate (1.5 mL/min), a dose of biosorbent (200 mg), type of the eluent (1 mol/L HNO3), sample volume (100 mL) and sorbent fraction (<0.25 mm). The biosorbent capacity ranged from 4.45 to 57.70 μmol/g for the investigated heavy metals. The practical relevance of this study is further extended by novel data regarding adsorbent cost analysis (17.49 $/mol). The Punica granatum sorbent represents a highly effective and economical biosorbent for the preconcentration of heavy metal ions for possible application in industrial sectors.

A Comparative Study of the Dynamic Fractionation of Rare-Earth Elements in Soils Using a Rotating Coiled Column and a Microcolumn

Rare earth elements (REEs) are currently used in fertilizers, but their behavior in the soil–plant system remains poorly studied. The assessment of the binding of REEs to various organomineral phases of soils remains an important task. Using soddy-podzolic soil and typical chernozem as examples, we performed a comparative study of the dynamic fractionation of REEs in a rotating coiled column (RCC) and a microcolumn (MC). We isolated the exchangeable fraction, specifically adsorbed fraction, and fractions bound to manganese oxides, bound to organic matter, and bound to amorphous and weakly crystallized iron and aluminum oxides using, 0.05 M Ca(NO3)2, 0.43 M CH3COOH, and a 0.1 M NH2OH·HCl solution (pH 3.6), a 0.1 M K4P2O7 solution (pH 11.0), and a 0.1 M (NH4)2C2O4 solution (pH 3.2), respectively. Concentrations of elements in the initial samples and eluate fractions were determined by atomic emission spectrometry and inductively coupled plasma mass spectrometry. The results suggest that the main extractable REE form (up to 40% of the total content) is provided by organometallic complexes extracted with a 0.1 M K4P2O7 solution. For chernozem (soils with a high content of organic matter), fractionation in the RCC and MC yielded comparable results. For soddy-podzolic soil, some differences were observed in the isolation of the first three fractions: exchangeable, specifically adsorbed, and bound to manganese oxides. Both RCC and MC can be successfully used for the dynamic fractionation of REE in soils; however, it is preferable to use an MC in analyzing many samples as a simpler and more affordable device.

High-throughput capillary electrophoresis analysis of biopharmaceuticals utilizing sequential injections.

The complexity of biotherapeutic products implies an ever-increasing list of product quality attributes that need to be monitored and characterized. In addition, the growing interest in implementing process analytical technology in biopharmaceutical production has further increased the testing burden, together with the need for rapid testing that can facilitate real-time or near-real-time decision-making. Capillary electrophoresis (CE) has made a place in biopharmaceutical analysis but is regarded as a low-throughput method, with the instrument dead time constituting more than 80% of the total time of analysis. In this study, the dead time of CE was utilized to analyse 3mAb samples in a single-CE run. This approach resulted in an up to 77% reduction in the total analysis time and increased the productivity by up to 300%, compared to traditional single CE-ultraviolet runs, without compromising resolution or relative peak areas. Additionally, good method reproducibility was observed. The compatibility of the method has been demonstrated with protein A eluate and cation exchange chromatography fractions. We, thus, propose that sequential injections can be applied for fast and robust CE analysis of biopharmaceuticals.

The Release of Non-Extractable Ferulic Acid from Cereal By-Products by Enzyme-Assisted Hydrolysis for Possible Utilization in Green Synthesis of Silver Nanoparticles.

The present work was undertaken to elucidate the potential contribution of biosynthetically produced ferulic acid (FA) via enzymatic hydrolysis (EH) of rye bran (RB) to the formation of silver nanoparticles (AgNPs) during green synthesis. An analytical approach accomplished by multiple reaction monitoring (MRM) using triple quadrupole mass selective detection (HPLC-ESI-TQ-MS/MS) of the obtained hydrolysate revealed a relative abundance of two isomeric forms of FA, i.e., trans-FA (t-FA) and trans-iso-FA (t-iso-FA). Further analysis utilizing high-performance liquid chromatography with refractive index (HPLC-RID) detection confirmed the effectiveness of RB EH, indicating the presence of cellulose and hemicellulose degradation products in the hydrolysate, i.e., xylose, arabinose, and glucose. The purification process by solid-phase extraction with styrene-divinylbenzene-based reversed-phase sorbent ensured up to 116.02 and 126.21 mg g−1 of t-FA and t-iso-FA in the final eluate fraction, respectively. In the green synthesis of AgNPs using synthetic t-FA, the formation of NPs with an average size of 56.8 nm was confirmed by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) techniques. The inclusion of polyvinylpyrrolidone (PVP-40) in the composition of NPs during synthesis favorably affected the morphological features, i.e., the size and shape of AgNPs, in which as big as 22.4 nm NPs were engineered. Meanwhile, nearly homogeneous round-shaped AgNPs with an average size of 16.5 nm were engineered using biosynthetically produced a mixture of t-FA and t-iso-FA and PVP-40 as a capping agent. The antimicrobial activity of AgNPs against Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa, E. coli, Enterococcus faecalis, Bacillus subtilis, and Staphylococcus aureus was confirmed by the disk diffusion method and additionally supported by values of minimum inhibitory (MIC) and bactericidal (MBC) concentrations. Given the need to reduce problems of environmental pollution with cereal processing by-products, this study demonstrated a technological solution of RB rational use in the sustainable production of AgNPs during green synthesis. The AgNPs can be considered as active pharmaceutical ingredients (APIs) to be used for developing new antimicrobial agents and modifying therapies in treating multi-drug resistant (MDR) pathogens.

Screening of acetylcholinesterase inhibitory and antioxidant active compounds from Terminalia chebula fruits by spectrum‐effect relationship and liquid chromatography‒mass spectrometry analysis

Screening and identification of active components from traditional Chinese medicines is rather challenging due to the diversity and complexity of chemical components. Herein, a comprehensive strategy based on a spectrum-effect relationship model and LC-MS analysis was developed to screen active components from Terminalia chebula fruits. The water extract of T. chebula fruits was subjected to macroporous resin column and then eluted successively with water and 30%, 50%, 70%, and 95% ethanol. The 30% ethanol eluate fractions of eighteen batches from T. chebula fruits were used for the spectrum-effect relationship study. The IC50 values for acetylcholinesterase inhibitory and 2,2-diphenyl-1-picrylhydrazyl scavenging activities were measured, LC fingerprints were established, and 15 common peaks were specified. The spectrum-effect relationship between common peaks and IC50 values was investigated by principal component analysis, gray relational analysis, partial least square and multiple linear regression. The 30% ethanol eluate fraction was further characterized by LC-MS analysis. The chromatographic peaks (Peaks 1, 2, 3, 5, 12, 14, 15) making great contributions to the efficacy were screened through a spectrum-effect relationship model, and sixteen components were further identified. The results suggested that the proposed strategy is simple and effective for acquiring active components from a complex matrix.

Determination of polypeptide antibiotics in animal tissues using liquid chromatography tandem mass spectrometry based on in-line molecularly imprinted solid-phase extraction

Effective purification and enrichment of polypeptide antibiotics in animal tissues is always a challenge, due to the co-extraction of other endogenous peptides which usually interfere their final determination. In this study, a molecularly imprinted column was prepared by packing polymyxin E-imprinted particles into a 100 mm × 4.6 mm i.d. HPLC column. The as-prepared imprinted columns were able to tolerate 100% aqueous phase and exhibited good stability and high column efficiency. Polypeptides antibiotics with similar molecular size or spatial structure to polymyxin E were well retained by the imprinted column, suggesting class selectivity. After optimization of mobile phase conditions of imprinted column, polypeptide antibiotics in animal tissue extracts were enriched and cleaned up by in-line molecularly imprinted solid-phase extraction, allowing the screening of target analytes in complex samples at low concentration levels by UV detection. Eluate fraction from the imprinted column was collected, and further dried and re-dissolved with methanol-0.5% formic acid aqueous solution (80:20, v/v) for final LC-MS/MS analysis. Analysis was accomplished using multiple reaction monitoring (MRM) in positive electrospray ionization mode and analytes quantified using the matrix-matched external calibration curves. The results showed high correlation coefficients for target analytes in the linear range of 2 ∼ 200 μg kg−1. At four different concentration levels (limit of quantification, 50, 100 and 200 μg kg−1), recoveries of four polypeptide antibiotics in swine, cattle and chicken muscles ranged from 66.7 to 94.5% with relative standard deviations lower than 16.0%. The limits of detection (LOD) were 2.0 ∼ 4.0 μg/kg, depending upon the analyte and sample. Compared with a conventional pretreatment method, the imprinted column was able to remove more impurities and to significantly reduce matrix effects, allowing the accurate analysis of polypeptide antibiotics.

Screening and identification of α-glucosidase inhibitors from Cyclocarya paliurus leaves by ultrafiltration coupled with liquid chromatography-mass spectrometry and molecular docking

Cyclocarya paliurus, as an important edible and medicinal product, has shown a good prospect in the prevention of diabetes mellitus (DM). However, it is unclear which active compounds derived from C. paliurus play a significant role in inhibiting α-glucosidase activity. In present study, affinity-based screening assay was developed to screen and identify potential α-glucosidase inhibitors from C. paliurus leaves based on affinity ultrafiltration coupled with ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) and molecular docking. After being enriched by D-101 macroporous resin, five eluent fractions with different polarity were obtained and their inhibitory activities on α-glucosidase were evaluated by an enzyme inhibition assay in vitro. The result showed that 70% ethanol fraction of C. paliurus leaves exhibited remarkable α-glucosidase inhibitory activity with the IC50 value of 17.81 µg/mL. The 70% ethanol fraction was incubated with α-glucosidase and then active compounds would form enzyme-inhibitor complexes. The complexes could be separated from inactive components by the interception ability of ultrafiltration membrane under centrifugation. A total of 36 active compounds were screened from C. paliurus leaves and the chemical structures were further characterized by UPLC-QTOF-MS/MS. Furthermore, molecular docking was performed to investigate possible inhibitory mechanisms between active compounds and α-glucosidase. The docking result showed that cyclocarioside I, pterocaryoside B, arjunolic acid, cyclocarioside Z5, cypaliuruside D and cyclocarioside N could be embedded well into the active pocket of α-glucosidase, and had significant affinity interactions with critical amino acid residues by forming hydrogen bonds, hydrophobic interactions and van der Waals, and affinity energies ranged from -9.3 to -6.7 kJ/mol. The results indicated that the developed method is rapid and effective for high throughput screening of potential α-glucosidase inhibitors from complex mixtures. Moreover, C. paliurus exhibited a remarkable inhibitory activity on α-glucosidase, making it a promising candidate for the prevention of DM.

α-Glucosidase inhibitors screening from Cyclocarya paliurus based on spectrum-effect relationship and UPLC-MS/MS.

Cyclocarya paliurus is an edible and medicinal plant exhibiting significant hypoglycemic effect. However, its active components are still unclear and need further elucidation. In this research, the active components of the leaves of C. paliurus responsible for the α-glucosidase inhibitory activity were screened and identified based on a spectrum-effect relationship study in combination with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis. The 70% ethanol eluate fraction of the leaves of C. paliurus with the strongest α-glucosidase inhibitory activity was obtained after extraction and purification with macroporous resin. Their chromatographic fingerprints (15 batches) were established by UPLC analysis and 32 common peaks were specified by similarity analysis. Their IC50 values for α-glucosidase inhibition were measured by an enzymatic reaction. Several multivariate statistical analysis methods including hierarchical cluster analysis, principal component analysis, partial least square analysis and gray relational analysis were applied to explore the spectrum-effect relationship between common peaks and IC50 values, and the chromatographic peaks making a large contribution to efficacy were screened out. To further elucidate the active components of leaves of C. paliurus, the 70% ethanol eluate fraction was characterized by UPLC-MS/MS analysis, and 10 compounds were identified. This study provides a valuable reference for further research and development of hypoglycemic active components of C. paliurus.

Purification Technology of Polysaccharides from Lophatherum gracile Brongn. by Macroporous Resin Adsorption and Its Effect on Athletic Endurance of Mice

The purified technological parameters of polysaccharide extract from Lophatherum gracile Brongn was optimized, and its effect on athletic endurance was observed. The best type of macroporous resin was selected through performance comparison of static adsorption and elution experiments, then the effects of sample solution pH, sample concentration, loading speed, sample volume, volume fraction of eluent, dosage of eluent and flow rate of elution on the dynamic purification effect were analyzed. Meanwhile, purified product on the influence of athletic endurance of mice was investigated by loading swimming test and related biochemical index detection. The results showed that the optimum purification process parameters were as follows: The initial concentration of sample solution was 5 mg/mL, the sample volume was 60 mL with loading speed of 2.0 mL/min, the pH value of sample solution was 5.0, and the volume fraction of ethanol was 80%, the eluent volume was 160 mL with flow rate of 1.0 mL/min. The purity of polysaccharide in product increased from 16.39% to 57.35%. Compared with the blank control group, the purified product groups at middle and high doses significantly prolonged exhaustive swimming time (P<0.05, P<0.01), enhanced activity of lactic dehydrogenase(P<0.05, P<0.01), decreased lactic acid and urea nitrogen content (P<0.05, P<0.01) after exercise. Therefore, polysaccharide of Lophatherum gracile Brongn. could preferably improve athletic endurance in body.

Identification and Quantification of Technetium Species in Hanford Waste Tank AN-102.

Technetium-99 (Tc), a high yield fission product generated in nuclear reactors, is one of the most difficult contaminants to address at the U.S. Department of Energy Hanford, Savannah River, and other sites. In strongly alkaline solutions typifying Hanford tank waste, Tc exists as pertechnetate (TcO4-) (oxidation state VII) as well as in reduced forms (oxidation state < VII), collectively known as non-pertechnetate (non-TcO4-) species. Designing strategies for effective Tc management, including separation and immobilization, necessitates understanding the molecular structure of the non-TcO4- species and their identification in actual tank waste samples. Identification of non-TcO4- species would facilitate the development of new treatment technologies effective for dissimilar Tc species. Toward this objective, a spectroscopic library of the Tc(I) [fac-Tc(CO)3]+ and Tc(II, IV, V, VII) compounds was generated and applied to the characterization of the actual Hanford AN-102 tank waste supernatant, which was processed to adjust Na concentration to ∼5.6 M and remove 137Cs by spherical resorcinol-formaldehyde (sRF) ion-exchange resin. Post 137Cs removal, the cesium-loaded sRF column was eluted with 0.45 M HNO3. As-received AN-102, Cs-depleted effluent, and sRF eluate fractions were comprehensively characterized for chemical composition and speciation of Tc using 99Tc nuclear magnetic resonance spectroscopy and X-ray absorption spectroscopy. It was demonstrated for the first time that non-TcO4- Tc present in the AN-102 tank waste is composed of several low-valent Tc species, including the Tc(I) [fac-Tc(CO)3]+ and Tc(IV) compounds. This is the first demonstration of multiple non-TcO4- species co-existing in the Hanford tank waste, highlighting their importance for the waste processing.

Batch chromatography with recycle lag. II—Physical realization and experimental validation

This is the second of a two-part study in which we explore the concept of batch chromatography with recycle lag, concluding with the design, construction, and experimental validation of a prototype—an eluate recycling device (ERD)—that embodies the physical realization of this concept. The ERD implements an approximate “first in, first out” method of organizing and manipulating the to-be-recycled fractions of eluate collected from the chromatography column, where the oldest (first) amount fluid, or ‘head’ of the fraction, is the first to exit and be recycled back to the column. Moreover, the apparatus is simple to set up in particular in view of large-scale applications. Here we detail the construction of the ERD and assembly of a setup to interconnect the ERD and a chromatography column. Through the coordinated operation of two-way valves and two-position six-port switching valves it is possible to implement a diverse set of configurations or operating modes interconnecting the chromatography column and the ERD. The setup is validated experimentally with success using the separation of a nucleoside mixture by reversed phase chromatography as a model problem. It is also shown that by redesigning the fluid distributor using 3D printing technology the ERD performance can be improved.

Batch chromatography with recycle lag. I—Concept and design

This is the first of a two-part study in which we explore the concept of batch chromatography with recycle lag, concluding with the design, construction, and experimental validation of a prototype that embodies the physical realization of this concept. Moreover, the apparatus is simple to set up in particular in view of large-scale applications. Here the theory behind batch chromatography with recycle lag is revisited and extended, with emphasis on the mathematical formulation and procedure for deriving the single-column batch analogue of any variant of multicolumn simulated countercurrent chromatography. By resorting to selected examples, namely GE Healthcare Bio-science’s three-column periodic countercurrent chromatography, Novasep’s sequential multicolumn chromatography, and a few hypothetical multicolumn processes, we discuss how the theory can be operationalized. Finally, we conclude by describing the design of a device or apparatus—an eluate recycling device (ERD)—to physically realize the proposed concept. The ERD implements an approximate “first in, first out” method for organizing and manipulating the to-be-recycled fractions of eluate collected from the chromatography column, where the oldest (first) amount fluid, or ‘head’ of the fraction, is the first to exit and be recycled to the column.

Geldanamycin Alters Abundance and Composition of Low Molecular Weight Proteins and Multiprotein Complexes as Observed by Size Exclusion Column Chromatography

Size exclusion chromatography (SEC) is a commonly used and minimally disruptive liquid chromatography‐based technique that separates unbound cytosolic proteins and multiprotein complexes according to their perceived molecular weights. The purpose of this project was to develop a method using SEC to partially purify and isolate biomolecular complexes, including those containing the essential molecular chaperone protein hsp90, in the presence and absence of the hsp90 inhibitor geldanamycin. Using an automated SEC program and S‐200 column, unbound proteins and protein complexes of differing molecular weights were isolated from reticulocyte lysate incubated in the presence and absence of geldanamycin. In addition to hsp90 eluting in a complex of ~250–300 kDa, consistent with it being part of a multiprotein chaperone machinery, other predominant protein elution peaks corresponded to ~26 kDa and ~16 kDa. Interestingly, incubating cell lysate with geldanamycin depleted nearly all of the ~26 kDa peak, indicating an unexpected hsp90 effect. Continued examination of eluate fractions from + and − geldanamycin‐incubated lysates has allowed us to further characterize these geldanamycin‐sensitive low molecular weight proteins as we examine the multitude of intracellular hsp90 functional activities.Support or Funding InformationFunding was provided by the Seattle University College of Science &amp; Engineering undergraduate research program.

Use of a phosphopeptide as a ligand to purify phospholipase A2 from the venom of Crotalus durisuss terrificus by affinity chromatography

The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.