Elution Step Research Articles

Mechanistic model of minute virus of mice elution behavior in anion exchange chromatography purification.

This study aimed to propose a methodology for developing a mechanistic model for viral clearance of the minute virus of mice (MVM) on flow-through anion exchange (AEX) chromatography. Protein surface analysis was applied to investigate the possibility of molecular interaction between the recombinant biotherapeutic and MVM. The protein product-free Tris buffers were spiked with MVM, and the MVM elution profile from AEX chromatography was quantitatively analyzed using quantitative polymerase chain reaction (qPCR) for pooled fractions. GoSilico™ Chromatography Modeling Software was applied to develop the mechanistic models for MVM species. For evaluating the visual fit of the developed model, the R2 of intact MVM virions and uncoated capsids between the simulated and measured amount in each fraction are 0.880 and 0.948, respectively. Response surface plots of logarithmic reduction values (LRV) against pH and conductivity in loaded sample were generated to show the range for suitable loaded sample conditions for achieving a good LRV. To evaluate the applicability of the developed MVM elution model to a recombinant biotherapeutic, two demonstrations of AEX chromatography purification were performed with a loaded sample of a model monoclonal antibody. The peaks of the MVM species in the elution step of both runs were accurately simulated by the developed model. In addition, to assess the possibility of molecular interaction between the virus and the target protein significantly affecting the MVM elution behavior, the antibody's surface was evaluated in terms of hydrophobicity/hydrophilicity using surface analysis.

Diagnosis and Biomarker Screening of Endometrial Cancer Enabled by a Versatile Exosome Metabolic Fingerprint Platform.

Exosomes have emerged as a revolutionary tool for liquid biopsy (LB), as they carry specific cargo from cells. Profiling the metabolites of exosomes is crucial for cancer diagnosis and biomarker discovery. Herein, we propose a versatile platform for exosomal metabolite assay of endometrial cancer (EC). The platform is based on a nanostructured composite material comprising gold nanoparticle-coated magnetic COF with aptamer modification (Fe3O4@COF@Au-Apt). The unique design and novel synthesis strategy of Fe3O4@COF@Au-Apt provide the material with a large specific surface area, enabling the efficient and specific isolation of exosomes. The exosomes captured Fe3O4@COF@Au-Apt can be directly used as the laser desorption/ionization mass spectrometry (LDI-MS) matrix for rapid exosomal metabolic patterns. By integrating these functionalities into a single platform, the analytical process is simplified, eliminating the need for additional elution steps and minimizing potential sample loss, resulting in large-scale exosomal metabolic fingerprints. Combining with machine learning algorithms on the metabolic patterns, accurate discrimination between endometrial patients (EGs) and benign controls (CGs) was achieved, and the area under the receiver operating characteristic curve of the blind test cohort was 0.924. Confusion matrix analysis of important metabolic fingerprint features further demonstrates the high accuracy of the proposed approach toward EC diagnosis, with an overall accuracy of 94.1%. Moreover, four metabolites, namely, hydroxychalcone, l-acetylcarnitine, elaidic acid, and glutathione, have been identified as potential biomarkers of EC. These results highlight the great value of the integrated exosome metabolic fingerprint platform in facilitating low-cost and high-throughput characterization of exosomal metabolites for cancer diagnosis and biomarker discovery.

A spatial hue smartphone-based colorimetric detection and discrimination of carmine and carminic acid in food products based on differential adsorptivity

A novel, portable, disposable, affordable, and environmentally friendly paper-based analytical device (PAD) was designed for on-site determination of carmine and carminic acid. This platform utilized paper test strips with a chitosan coating as an adsorption layer, which was characterized using scanning electron microscope, energy-dispersive X-ray analysis, and water contact angle measurement. Carmine and carminic acid could be efficiently adsorbed on chitosan-coated paper test strips, producing distinct colors that could be captured using a smartphone camera without the need for an elution step. Notably, by utilizing the Hue component of the HSL model, it was possible to differentiate between carmine and carminic acid, confirming their presence in a sample. Furthermore, the color saturation intensity changed in a concentration-dependent manner, allowing for the determination of carmine and carminic acid concentrations in the ranges of 200–800 μg/mL and 20–100 μg/mL, respectively. Additionally, the created test strip could be used to measure the percentage of carminic acid in the presence of carmine. The developed PAD enabled the quantification of carmine in various food samples without the need for reagents or complex equipment. The environmental impact of this method was found to be positive based on assessments using GAPI and AGREE tools.

Spectrofluorimetric determination of acetone in water samples with solid-phase extraction and a new benzaldehyde derivative

A new method is described for acetone (C(CH3)2O) determination in water samples. The method is based on the reaction with 4-(dimethylamino)benzaldehyde (DMAB) in dimethyl sulfoxide (DMSO) in slightly basic medium, resulting in a highly fluorescent compound with fluorescent wavelengths undisturbed by other common fluorescent compounds. Experimental conditions were optimized (reagents concentrations, reaction time) to reach optimal sensitivity. For the analysis of aqueous samples, a preconcentration step of acetone by solid-phase extraction (SPE) followed by an elution step in DMSO was optimized using Isolute ENV + columns. A highly satisfactory low detection limit of 0.014 μM (0.8 μg L−1) was achieved by combining these two steps, with a linear range from 0.048 to 5 μM and relative standard deviations between 5.7 % and 6.9 %. The protocol was validated on complex real water samples such as river water and wastewater, and our fluorimetric method with DMAB was in good agreement with the reference LC-UV method with DNPH.

Preparation and evaluation of polymeric ionic liquid functionalized microparticles as the efficient adsorbent for pipette tip solid phase extraction of sulfonamides

Sulfonamides (SAs) residues are commonly found in environmental samples, which is highly concerning for public safety and environmental protection. The detection of SAs is quite important but challenging. In this work, a kind of polymeric ionic liquid functionalized microparticles (PIL-FM) was synthesized via a simple free radical polymerization reaction, and applied as the adsorbent for pipette tip solid phase extraction (PT-SPE) of three SAs from river water, milk powder and honey samples. The adsorption performance of PIL-FM on SAs were evaluated using adsorption kinetics and adsorption isotherms, and the results showed that PIL-FM had the best selectivity for SMZ with the maximum adsorption capacity was as high as 45.05 mg·g−1. The extraction process was optimized and the adsorption mechanism was preliminarily discussed. PIL-FM has the excellent selectivity for SAs mainly because of the strong π-π interaction, and the electrostatic repulsion between them in the elution step also has a positive impact. Under optimal conditions, combined with HPLC, this PT-SPE method had wide linearity (0.2–100 μg·L−1, R2 ≥ 0.9998) and low limits of detection (0.06–0.08 μg·L−1). The intra-day and inter-day repeatability were found to be 3.7 %–4.6 % and 4.5 %–6.2 %, respectively. The suggested PT-SPE method is simple, low cost, rapid, and efficient to extract SAs, and can be applied for the monitoring of SAs residues in the aquatic environment and in food.

One-Tube Nested PCR Coupled with CRISPR-Cas12a for Ultrasensitive Nucleic Acid Testing.

Nucleic acid testing with high sensitivity and specificity is of great importance for accurate disease diagnostics. Here, we developed an in situ one-tube nucleic acid testing assay. In this assay, the target nucleic acid is captured using magnetic silica beads, avoiding an elution step, followed directly by the polymerase chain reaction (PCR) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a detection. This assay achieved visual readout and a sensitivity of 120 copies/mL for detecting SARS-CoV-2. More importantly, the assay demonstrated over 95% sensitivity and 100% specificity compared to the gold standard real-time quantitative PCR (RT-qPCR) test by using 75 SARS-CoV-2 clinical samples. By integrating nested PCR and Cas12a, this all-in-one nucleic acid testing approach enables ultrasensitive, highly specific, and cost-effective diagnosis at community clinics and township hospitals.

A method for suppressing pH excursion during elution in ion-exchange chromatography

A simple method for reducing pH excursion during salt-induced elution in cation exchange chromatography is presented in this paper. Connecting a preequilibrated anion exchange cartridge in series in front of the cation exchange device just prior to the elution step significantly reduced the transient decrease in pH that is typically observed during elution. The working principle of the proposed method is hypothesized. Bind and elute chromatography experiments showed that the suppression of pH excursion during elution of a monoclonal antibody from a cation exchange chromatography device resulted in significantly sharper and narrower eluted protein peaks. Improvement in pH control during elution would not only result in better reproducibility and efficiency in separation but would also ensure greater stability of pH sensitive proteins during their purification using ion exchange chromatography.

Fabrication of an IgG-imprinted monolith micro-solid phase extraction chip for IgG extraction from human plasma

Molecular imprinted polymers (MIPs), also known as synthetic antibodies, offer a novel and promising approach for protein separation at analytical scale. In proteomics research, this approach could capture target high abundant proteins from biological fluids to reduce sample complexity. In this study, we have explored the potential to imprint human immunoglobulin G (IgG) to develop a micro-solid phase extraction chip for selective removal of IgG from human plasma. Imprinting of IgG within the polyhydroxy ethyl methacrylate monolith containing dimethyl amino ethyl methacrylate (DMAEMA) as a functional monomer was successfully achieved and characterized using FT-IR spectroscopy and protein assay kit. 0.1 M tris buffer (pH 7) and UV-polymerization were identified as the ideal buffer and polymerization strategy to imprint IgG within 30 min. Template (IgG) elution using 10 % acetic acid containing 10 % (v/v) SDS as elution buffer was moderately successful (25 % IgG-template free monolith was obtained). Protein shape has influenced its imprinting and elution from the monolith. The protocol developed to imprint one protein cannot be translated to another protein without further modifications. A 10-fold decrease of DMAEMA concentration within the monolith and a sequential elution step has resulted in >90 % IgG-template free monolith. The IgG-template free monolith has displayed an adsorption capacity of ∼ 3.7 mg per g monolith and showed good IgG selectivity as compared to other tested human proteins (human serum albumin, transferrin and fibrinogen). Translation of imprinted monolith protocol to develop a micro-solid phase extraction chip was successful. The chip can be re-used for 5 cycles with >95 % reusability and has selectively captured IgG from human plasma.

Mechanistic modeling of minute virus of mice surrogate removal by anion exchange chromatography in micro scale

Biopharmaceutical products are often produced in Chinese hamster ovary (CHO) cell cultures that are vulnerable to virus infections. Therefore, it is a regulatory requirement that downstream purification steps for biopharmaceuticals can remove viruses from feedstocks. Anion exchange chromatography (AEX) is one of the downstream unit operations that is most frequently used for this purpose and claimed for its capability to remove viruses. However, the impact of various process parameters on virus removal by AEX is still not fully understood. Mechanistic modeling could be a promising way to approach this gap, as these models require comparatively few experiments for calibration. This makes them a valuable tool to improve understanding of viral clearance, especially since virus spiking studies are costly and time consuming.In this study, we present how the virus clearance of a MVM mock virus particle by Q Sepharose FF resin can be described by mechanistic modeling. A lumped kinetic model was combined with a steric mass action model and calibrated at micro scale using three linear gradient experiments and an incremental step elution gradient. The model was subsequently verified for its capability to predict the effect of different sodium chloride concentrations, as well as residence times, on virus clearance and was in good agreement with the LRVs of the verification runs. Overall, models like this could enhance the mechanistic understanding of viral clearance mechanisms and thereby contribute to the development of more efficient and safer biopharmaceutical downstream processes.

A batch screening technique for the calculation of chromatographic separability

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing “empty” capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

High‐throughput in silico workflow for optimization and characterization of multimodal chromatographic processes

AbstractWhile high‐throughput (HT) experimentation and mechanistic modeling have long been employed in chromatographic process development, it remains unclear how these techniques should be used in concert within development workflows. In this work, a process development workflow based on HT experiments and mechanistic modeling was constructed. The integration of HT and modeling approaches offers improved workflow efficiency and speed. This high‐throughput in silico (HT‐IS) workflow was employed to develop a Capto MMC polishing step for mAb aggregate removal. High‐throughput batch isotherm data was first generated over a range of mobile phase conditions and a suite of analytics were employed. Parameters for the extended steric mass action (SMA) isotherm were regressed for the multicomponent system. Model validation was performed using the extended SMA isotherm in concert with the general rate model of chromatography using the CADET modeling software. Here, step elution profiles were predicted for eight RoboColumn runs across a range of ionic strength, pH, and load density. Optimized processes were generated through minimization of a complex objective function based on key process metrics. Processes were evaluated at lab‐scale using two feedstocks, differing in composition. The results confirmed that both processes obtained high monomer yield (>85%) and removed of aggregate species. Column simulations were then carried out to determine sensitivity to a wide range of process inputs. Elution buffer pH was found to be the most critical process parameter, followed by resin ionic capacity. Overall, this study demonstrated the utility of the HT‐IS workflow for rapid process development and characterization.

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Lysis-Free Isolation and Direct Amplification of Pathogenic Bacterial DNA Using Diatom Frustules.

DNA has been implicated as an important biomarker for the diagnosis of bacterial infections. Herein, we developed a streamlined methodology that uses diatom frustules (DFs) to liberate and capture bacterial DNA and allows direct downstream amplification tests without any lysis, washing, or elution steps. Unlike most conventional DNA isolation methods that rely on cell lysis to release bacterial DNA, DFs can trigger the oxidative stress response of bacterial cells to promote bacterial membrane vesicle formation and DNA release by generating reactive oxygen species in aqueous solutions. Due to the hierarchical porous structure, DFs provided high DNA capture efficiency exceeding 80% over a wide range of DNA amounts from 10 pg to 10 ng, making only 10 μg DFs sufficient for each test. Since laborious liquid handling steps are not required, the entire DNA sample preparation process using DFs can be completed within 3 min. The diagnostic use of this DF-based methodology was illustrated, which showed that the DNA of the pathogenic bacteria in serum samples was isolated by DFs and directly detected using polymerase chain reaction (PCR) at concentrations as low as 102 CFU/mL, outperforming the most used approaches based on solid-phase DNA extraction. Furthermore, most of the bacterial cells were still alive after DNA isolation using DFs, providing the possibility of recycling samples for storage and further diagnosis. The proposed DF-based methodology is anticipated to simplify bacterial infection diagnosis and be broadly applied to various medical diagnoses and biological research.

Identification and characterization of rabbit scFv antibodies suitable for immuno-affinity separation of recombinant human kynureninase from Escherichia coli cell lysate

In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.0, thereby facilitating enzyme elution. Subsequently, all four clones were successfully produced and purified, followed by covalent coupling to NHS-activated HiTrap HP columns. While KYNase was specifically adsorbed to all four scFv-immobilized columns and was eluted at pH 4.0, the respective levels of static binding capacity (SBC) and recovery among the four scFv clones were different at this elution pH. That is, the scFv-immobilized columns captured KYNase with SBC ranging from 1.15 to 2.68 mg/cm3-bed with clone R2-47 exhibiting the highest level of SBC, with a ligand utilization of 39.4%. Moreover, using the scFv column of R2-47, 90.7% of the captured human KYNase was recovered in the first elution step at pH 4.0, and approximately 67% of enzymatic activity was retained. In summary, high-purity human KYNase was obtained from the E.coli cell lysate by one-step affinity purification, and 89.7% of KYNase was recovered in the first elution step. The methodology demonstrated in the current study could be applied for the purification and development of various therapeutic proteins.

Abstract 2590: Automating 6-plex chromogenic multiplexing with BOND RX

Abstract Multiplex staining using a sequential approach provides a means of visualizing multiple targets in a single biopsy section, further evaluation paired with digital imaging provides a deeper analysis into the spatial relationship at a cellular level. Leica Biosystems enables researchers to complete proof of concept for sequential multiplex staining methods, on board the BOND RX system. Thus, paired with a compatible reagent workflow and a digital slide imaging offering (Aperio GT 450/Aperio AT2) enhances the possibilities for analysis. As with any assay there is a requirement for user optimization, in particular, marker/chromogen layer selection and the pertinent use of an elution step. Such elution step is specifically important within a sequential stain utilizing the same detection reagents on subsequent layers. The results from the multiple staining rounds show a successful 6-plex fully automated stain, possible without the removal and reapplication of the slide onto the BOND system between staining layers. The marker combinations chosen demonstrate compatibility of different host species (Rabbit & Mouse), cellular compartments and isotypes which are provided by Leica Biosystems as BOND Ready to Use reagents. Overall, chromogenic multiplexing provides a stained image which enables color separation, due to the assignment of each chromogen to a marker with an individual cell type and cellular compartment. Finally, the process outlined avoids the risk of signal quenching commonly associated with fluorescent offerings and negates the requirement for specialized equipment due to the enablement of brightfield primary analysis. Citation Format: Dean Talia, Stacey Lindsay, Sarah Ketting, Rebecca Williamson, Damian Cockfield. Automating 6-plex chromogenic multiplexing with BOND RX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2590.

Mass Spectrometry-Compatible Elution Technique Enables an Improved Mucin-Selective Enrichment Strategy to Probe the Mucinome.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

Click-imprinted furan-modified poly(acrylonitrile-co-divinylbenzene for S-naproxen recognition

Naproxen (NX) drug exists in two R- and S-enantiomers, requiring enantioseparation for effective pharmacological effects. Herein, a new S-NX enantioselective poly(acrylonitrile-co-divinylbenzene) (CPAN)-based polymeric sorbent was successfully synthesized and used to separate the NX racemate. First, low crosslinked CPAN was prepared in microparticles via suspension polymerization. Furan-2-ylmethylhydrazine (Fu-Hyd) was then interacted with the abundant nitrile units. The resulting cationic furan-hydrazidine-functionalized polymeric particles (FUH-P) were loaded with the anionic S-NX, which was then imprinted via post-crosslinking upon treatment with bis-maleimidoethane (BME) dienophile crosslinker that interacted with the conjugate diene system of the already inserted furan units via Diels-Alder cycloaddition reaction. Using acidic elution, the S-NX enantiomers were then freed from the polymer particle and left their enantio-selective receptor-like active sites in the structure of the imprinted polymeric particles (S-NX-P). FTIR, 13C NMR, and XRD techniques were utilized for investigating the obtained polymers. Also, the surface morphologies of the sorbent materials were visualized using SEM. The best conditions for enantio-selective uptake showed that 235 mg/g of S-NX could be adsorbed at pH 7. The enantiomeric separation of the NX racemic mixture was effectively achieved with an enantiomeric excess (ee) of 99% for R-NX upon the initial solution run and 93% for S-NX upon the elution step.

Platinum group metals removal using expanded vermiculite

In this work, a granular expanded vermiculite (GEV) is employed as a sorbent to remove Platinum group metals (PGMs) such as Pt (IV), Rh (III), Ru (III) and Ir (III). Batch adsorption experiments included factors such as metal concentration, pH, GEV dosage and contact time; whereas in a continuous system, the bed height and the desorption process were also investigated. Pseudo-second order was the rate equation that accurately described the adsorption kinetic and adsorption isotherms that were adequately simulated by the Langmuir and Freundlich model. Maximum sorption capacities of GEV in a batch system for Pt (IV), Rh (III), Ru (III) and Ir (III) were 73.01 μg g−1, 157.03 μg g−1, 147.12 μg g−1 and 47.91 μg g−1 at optimal pH values, respectively. Additionally, the adsorption capacities obtained in continuous adsorption experiments for Pt (IV), Rh (III), Ru (III) and Ir (III) were 48.78 μg g−1, 50.11 μg g−1, 51.88 μg g−1 and 37.96 μg g−1. The elution step was accomplished efficiently (71.9-82.5%) using an HCl solution. Hence, the GEV adsorbent could be used as a profitable alternative to remove and recover PGMs from solutions.

Development of an On-Column Trace Enrichment Method for the Determination of Sub-μg/L Bisphenol A in Bottled Water by RP-HPLC with Fluorescent Detection.

A simple extraction-free, on-column trace enrichment liquid chromatographic method for the determination of trace levels of bisphenol A (BPA) in bottled water samples has been developed. It was found possible to determine ng/L (ppt) levels of BPA by the direct introduction of 6 mL of sample water to the HPLC column utilising fluorescence detection (Exλ = 274 nm, Emλ = 314 nm). Following the loading of the sample and the chromatographic focusing of the BPA on the analytical column, a simple switch from the aqueous sample to the isocratic chromatographic elution step of 50% acetonitrile/deionised water was undertaken. Using a BPA concentration of 0.596 μg/L the effect of sample volume was investigated over the range 1.0 to 12 mL. A linear relationship with the sample volume introduced to the HPLC column and the resulting peak height for BPA was found over the entire range investigated (R2 = 0.999). Using a sample volume of 6.0 mL, a well-defined chromatographic peak was recorded for BPA over the concentration range of 0.1 μg/L to 6.25 μg/L (R2 = 0.9998). A limit of detection of 0.058 μg/L for BPA was calculated based on 3 δ. A mean recovery of 100% with an associated %CV of 7.6% (n = 5) was obtained for a bottled spring water sample fortified with 1.25 μg/L BPA. Samples can be processed in under 12 minutes, much faster than that commonly reported for conventional offline extraction and chromatographic-based methods. The results show that the optimised method holds promise for the determination of BPA in such samples.