Non-chromatographic Method Research Articles

Microwave assisted solvent free one pot synthesis of acenaphtho [1,2-d] imidazole derivatives using NH4 + OAc– / NH4 +Cl –

A novel, highly efficient, eco-friendly, and quick microwave-assisted synthesis of 2, 4, 5- substituted imidazoles by one-pot cyclo condensation of acenaphthoquinone, aromatic aldehydes, and ammonium acetate was reported under solvent-free conditions using ammonium chloride as a catalyst with excellent yield. The present methodology offers several advantages, such as mild reaction conditions, easy workup, short reaction time, excellent yield, purification of products by the non-chromatographic method, and reproducibility in large-scale synthesis. Compounds were characterized by IR, 1HNMR, 13C NMR spectroscopy and their melting points were compared with the literature reports.

An efficient, green and micellar catalyzed preparative-scale synthesis of 3,4-Disubstituted isoxazole-5(4H)-ones in the water

The one-pot three-component synthesis of 3,4-Disubstituted isoxazole-5(4H)-ones has been developed using an aqueous micellar medium as a catalyst under milder reaction conditions. The reaction was completed by using 25 mol% of sodium lauryl sulfate in an aqueous medium at room temperature. The reaction employs a green solvent, an eco-friendly reagent, and a non-chromatographic method for purification. The present reaction tolerates several substituted aldehydes and has proved to be efficient along with excellent yield. Moreover, the micellar catalyst was recovered and reused several times without significantly losing its catalytic activity.

Non-chromatographic Method for the Purification of High-Purity Paclitaxel from Taxus chinensis Using Tandem Cavitation Fractional Precipitation

Non-chromatographic Method for the Purification of High-Purity Paclitaxel from Taxus chinensis Using Tandem Cavitation Fractional Precipitation

Optimization of Three Phase Partitioning Purification of Papain from Carica papaya Latex by Response Surface Methodology

Three phase partitioning (TPP), a novel and non-chromatographic method was effectively applied for the extraction and recovery of papain from Carica papaya latex. The effect of change in ammonium sulfate concentration, crude extract to solvent ratio and pH on extraction efficiency was studied, and these parameters were optimized by response surface methodology (RSM) using Box-Behnken Design. The protease was found to be concentrated more at the aqueous phase (AP) with 51.08% ammonium sulfate precipitation, crude extract to tert-butanol ratio of 1:0.78 (v/v), and pH 6.05 resulting 9.242 purification fold and 159.806% activity recovery. Compared to the intermediate phase (IP), the AP demonstrated higher recovery and purification fold. Purified papain showed significantly (p<0.05) greater activity at a temperature of 60°C and pH of 6.0. It also demonstrated stability within the temperature range of 40 to 70°C and pH range of 6.0 to 8.0. Enzyme retained maximum milk clotting activity when stored under freezing (-20°C) for 3 weeks. This study showed that the TPP can be used to extract and purify papain from papaya latex economically and it can also be employed as a vegetable coagulant for cheesemaking.

A simple and high efficiency purification of His-tagged turnip yellow mosaic virus-like particle (TYMV-VLP) by nickel ion affinity precipitation

Virus-like particles (VLPs) is one of the most favourable subjects of study, especially in the field of nanobiotechnology and vaccine development because they possess good immunogenicity and self-adjuvant properties. Conventionally, VLPs can be tagged and purified using affinity chromatography or density gradient ultracentrifugation which is costly and time-consuming. Turnip yellow mosaic virus (TYMV) is a plant virus, where expression of the viral coat protein (TYMVc) in Escherichia coli (E. coli) has been shown to form VLP. In this study, we report a non-chromatographic method for VLP purification using C-terminally His-tagged TYMVc (TYMVcHis6) as a protein model. Firstly, the TYMVcHis6 was cloned and expressed in E. coli. Upon clarification of cell lysate, nickel (II) chloride [NiCl2; 15 µM or equivalent to 0.0000194% (w/v)] was added to precipitate TYMVcHis6. Following centrifugation, the pellet was resuspended in buffer containing 1 mM EDTA to chelate Ni2+, which is then removed via dialysis. A total of 50% of TYMVcHis6 was successfully recovered with purity above 0.90. Later, the purified TYMVcHis6 was analysed with sucrose density ultracentrifugation, dynamic light scattering (DLS), and transmission electron microscopy (TEM) to confirm VLP formation, which is comparable to TYMVcHis6 purified using the standard immobilized metal affinity chromatography (IMAC) column. As the current method omitted the need for IMAC column and beads while significantly reducing the time needed for column washing, nickel affinity precipitation represents a novel method for the purification of VLPs displaying poly-histidine tags (His-tags).

A scalable method to purify reflectins from inclusion bodies

Structural proteins are an attractive inspiration for functional biobased materials. In nature, cephalopods skin colour modulation is related to the dynamic self-assembly of a family of structural proteins known as reflectins. To fully reach their potential as engineered bio-based materials, reflectins need to be produced by biotechnological means. One of the challenges is associated with establishing and optimizing reflectin purification processes to achieve the highest yield and productivity. Here, we studied purification strategies for two reflectin sequences from different organisms which were recombinantly expressed in a bacterial host at laboratory scale. Reflectins purification was then assessed by two chromatographic and one non-chromatographic methods. Methods were compared considering final purity and yield, productivity, cost and sustainability. The non-chromatographic method based on inclusion bodies washing presented the most promising results (protein purity > 90% and purification yields up to 88%). Our results contribute to define bioprocessing strategies to address the vision of biodegradable and sustainable protein-based materials.

Magnetic solid phase extraction as a nonchromatographic method for the quantification of ultratrace inorganic arsenic in rice by inductively coupled plasma-optical emission spectrometry (ICP OES)

An alternative analytical method was developed for the quantification of inorganic arsenic (iAs) in rice by ICP OES. Iron nanoparticles modified with an organophosphorus compound were used as the solid phase for MSPE of iAs from the plant matrix. The MSPE procedure was performed using 4 mL of a buffer solution with pH 4.0, 20 mg of the nanomaterial, and a 15-min extraction time. The total As (tAs) by ICP OES was also quantified using the same MSPE procedure after solubilization of the samples by a block digester. The accuracy of tAs and iAs quantification was verified using CRM NIST 1568b (97 % and 101 % recovery, respectively). The precision (RSD < 15 %) and LOD and LOQ (1.08 and 3.70 µg kg−1, respectively) of the proposed method were satisfactory. The rice samples had tAs contents between 0.090 and 0.295 mg kg−1 and iAs mass fractions between 0.055 and 0.109 mg kg−1.

IsoTag™AAV: an innovative, scalable & non-chromatographic method for streamlined AAV manufacturing.

Demand for gene therapies capable of treating previously inaccessible targets has risen precipitously in the past decade. Adeno-associated viruses (AAVs) are the preferred vector for gene delivery because of their favorable safety profile and tissue tropism, but they have significant manufacturing challenges, with end-to-end yields as low as 10-30%. To combat these low yields, we developed IsoTag™AAV, a novel purification technology for AAV that is a departure from the chromatographic paradigm in downstream processing. This proprietary technology uses a self-scaffolding recombinant protein reagent that can improve manufacturing yields. It enables purification by cost-effective and scalable filtration processes and improves product quality with minimal optimization. Herein, we describe the development of IsoTag™AAV, provide a head-to-head comparison to industry-leading affinity chromatography (evaluation carried out through a joint research project with Capsida Biotherapeutics), and demonstrate how it can reduce cost of goods for a clinical AAV program by 25%.

Ag-TiO2 Nanoparticles-catalyzed Three-component Synthesis of 12-aryl- 8,9,10,12-tetrahydrobenzo[a]-Xanthen-11-ones in Aqueous Medium.

Ag-TiO2 nanoparticles catalyzed synthesis of 12-aryl-8,9,10,12- tetrahydrobenzo[a]-xanthen-11-ones have been enhanced via a three-component one-pot reaction betweenβ-naphthol, several aldehydes and dimedone in H2O at room temperature. Xanthenes are essential intermediates in chemistry owing to their vast difference in biological activity. This process offered significant advantages containing appropriate cost efficiency, low amount of the catalyst, application of low-cost available Ag-TiO2 nanoparticles catalyst, purification of the product by non-chromatographic method, easy process, good atom economy, simple isolation and reusability of nanocatalyst. Ag-TiO2 nanoparticles catalyst shows easy access to Xanthenes with appropriate yields in short reaction time and purity. This nanoparticles catalyst was recycled and recovered by easy filtration and was reused up to five times with only an unimportant loss in its catalytic efficacy. This method achieves to have a numerous scope relating to the difference in the aldehydes. Correspondingly, the attractiveness of this research was that H2O was the only by-product.

Economic purification of recombinant uricase by artificial oil bodies

Rasburicase is an expensive treatment used to control hyperuricemia caused by tumour lysis syndrome (TLS). In this study, a non-chromatographic method was designed based on nano-oil bodies for convenient and economical purification of the recombinant uricase. For this purpose, two chimaeras were synthesized with a different arrangement of the uricase, caleosin and intein fragments. After confirming the protein expression by measuring the uricase activity at 293 nm, purification was conducted through oil-body construction. The results were resolved on the 12% SDS-PAGE gel. Finally, the stability of the oil bodies was examined against different salts, surfactants, temperatures, and pH values. According to our results, the overexpression of uricase–caleosin chimaera under the T7 promoter in Escherichia coli led to the production of soluble protein, which was successfully purified by artificial oil bodies. The active uricase was subsequently released through the self-splicing of intein. Further investigations highlighted the importance of the free C-terminus of caleosin in constructing artificial oil bodies. Moreover, surfactants and low temperature, in contrast to salts, improved the stability of oil bodies. In conclusion, caleosins are an efficient purification tag reducing the cost of purification compared to conventional chromatography methods.Graphical

Application of Design of Experiments (DoE) Approach for the Optimization of Phase-transfer Catalyzed Biginelli Dihydropyrimidinone (DHPM) Synthesis

The conventional Biginelli synthesis is more cumbersome and produces lower yields. Several improved methods are reported in the literature to replace the Biginelli catalyst. The design of biocompatible organic transformation is a major concern and a versatile greener procedure to construct Biginelli analogs is in great demand. Factorial design guided, energy-efficient, and versatile synthesis of 3,4-dihydropyrimidin-2-(1H)-ones (DHPM) was developed. One-factor-at-a time (OFAT) and factorial design (2&lt;sup&gt;3&lt;/sup&gt;) studies were utilized for screening the independent variables. The optimum levels of potential variables (benzyl-n-triethylammonium chloride (BTEAC) and glacial acetic acid) were determined through studies. The factorial design (3&lt;sup&gt;2&lt;/sup&gt;) analysis inferred the use of BTEAC (10.25 mol%) and glacial acetic acid (7.6 ml) as optimal for the 60 min condensation. Thirteen new 3,4- dihydropyrimidine-2-(1H)-one (DHPM) analogs were synthesized using optimized reaction conditions. The quaternary ammonium ion of BTEAC stabilizes the polarization of carbonyl group in aryl aldehydes and enolizable ketone (alkyl acetoacetate) to facilitate the cyclocondensation, in order to produce DHPMs through N-acyliminium ion and Michael adducts formation. The biocompatible strategy, simple product isolation (non-chromatographic method), and good to excellent yields are attractive features of this new protocol. Hence, the newly developed methodology is superior to the literature methods.

N-compounds speciation analysis in environmental samples using ultrasound-assisted solid-liquid extraction and non-chromatographic techniques.

A fast, efficient, and non-chromatographic method was presented in this study for nitrite, nitrate, and p-nitrophenol (N-compounds) extraction and speciation analysis of environmental samples. By applying ultrasound-assisted solid-liquid extraction (USLE), analytes were efficiently extracted from water, soil, or sediment collected in areas of environmental disaster. These analytes were selectively converted to NO(g) through UV photolysis (NO3-), H2O2/UV photocatalysis (PNP), and direct conversion (NO2-). Following conversion, NO(g) was separated from the liquid phase and determined by high-resolution continuum source molecular absorption spectrometry (HR-CS MAS). The LODs obtained were 0.097 ± 0.004mgL-1 for nitrite, 0.119 ± 0.004mgL-1 for nitrate, and 0.090 ± 0.006mgL-1 for p-nitrophenol. On applying this speciation method to environmental samples, concentrations were found to be up to 0.99 ± 0.03mgL-1 (NO2-), 49.80 ± 2.5mgL-1 (NO3-), and 0.10 ± 0.02mgL-1 (PNP). Finally, addition/recovery study of real water, soil, and sediment samples showed 101 ± 2% recovery for NO2-, 100 ± 1% for NO3-, and 96 ± 5% for PNP.

Synthesis of 2, 4 disubstituted 1, 5 benzodiazepines promoted by efficient Silica-Alumina Catalyst

An efficient and environment friendly valuable synthon of 1, 5 benzodiazepine derivatives were synthesized having unique physical and medicinal properties. This reaction is performed by mesoporous binary mixed metal oxide SiO2-Al2O3 which is synthesized by hydrothermal method at high temperature. This mesoporous material is investigated by XRD, SEM, EDS, TEM, TPD and BET surface area. Present method offers several remarkable advantages such as non-toxic, noncorrosive and easy work-up procedure for the purification of product with non-chromatographic method, ecofriendly reaction condition.

An effective non-chromatographic method for the purification of phenanthrolines and related ligands

1,10-Phenanthrolines are widely employed as ligands, but their use on a large scale is constrained by their difficult purification, which usually requires lengthy chromatographic separations. Herein, we describe a purification strategy that takes advantage of the high stability and low solubility of phenanthroline complexes to separate them from the by products of their synthesis. The formation of ZnCl2 complexes was employed, from which the free ligand can be recovered by reaction with aqueous NH3 in a biphasic CH2Cl2/H2O system. The same strategy was also successfully employed to purify related quinolino-guanidine ligands, demonstrating that the procedure is of general applicability.

Non-chromatographic method for separation and determination of Fe, Ni and V porphyrins in crude oil

A method combining ultracentrifugation and cloud point extraction is proposed for the separation and determination of Fe, Ni and V porphyrins in crude oil. An emulsion containing about 200 mg of crude oil, 6% (w v−1) of Triton X-100™, 1.6% (v v−1) of chloroform and 0.1 mol L−1 of HCl was prepared. An aliquot (1.5 mL) of this emulsion was ultracentrifuged to separate the crude oil heavy fraction (asphaltene and particulate matter) and inorganic species, organometallic compounds and porphyrins of Fe, Ni and V remained in the supernatant. Another aliquot (1.5 mL) was heated up to 90 °C for 5 min to separate the porphyrins, both heavy and light organics compounds, light organic and particulate matter by cloud point extraction, only the inorganic and ionic organometallic compounds species remained in the aqueous phase. The supernatant and aqueous phase were analysed by simultaneous graphite furnace atomic absorption spectrometry. The concentration determined in the supernatant was subtracted from that determined in the aqueous phase, providing the quantification of Fe, Ni and V porphyrin in the crude sample. The accuracy of the proposed method was checked using SARA reference method. The elemental determinations in the fractions obtained using the proposed method and the comparison by SARA showed an absence of Fe in the aromatics and resins fractions. The results showed that about 40% of Ni and 70% of V in the crude oil sample are bounded to porphyrins.

Selective Extraction and Determination of Chromium Concentration Using Luffa cylindrica Fibers as Sorbent and Detection by FAAS

In this approach, a nonchromatographic method was developed for selective extraction and determination of Cr(III) concentration in isotonic and energy drinks by FAAS. This new method uses Luffa cylindrica fibers as the solid phase extraction and an off-line flow injection system. Cr(III) and Cr(VI) adsorption behavior onto Luffa fibers allowed the selective separation of Cr(III) in the pH range of 4 to 5. The system variables were optimized through full factorial and Doehlert designs. Under optimal conditions, the detection and quantification limit for Cr(III) was 19.2 µg·L−1 and 63.9 µg·L−1, respectively, with precision below 0.19%. A high tolerance toward interfering ions was observed for the proposed method. Recovery test results obtained for different isotonic and energy drinks samples were higher than 87.6%. The method’s accuracy was confirmed through analysis of certified reference materials, water and sediment river samples (APS-1071 and APS-1066).

Non-chromatographic Speciation of Inorganic Arsenic in Rice by Hydride Generation Inductively Coupled Plasma Optical Emission Spectrometry

Simple, straightforward non-chromatographic method for inorganic arsenic (i-As) determination in rice using species-selective hydride generation (HG) combined with inductively coupled plasma optical emission spectrometry (ICP OES) without prior separation of methylated organoarsenicals (o-As) before measurements was developed and fully validated. i-As [As(III,V)] and o-As (DMA, MMA) species were extracted in aqua regia that oxidized As(III) to As(V), while integrity of both o-As forms was preserved. Arsenic hydrides from i-As were generated in reaction with NaBH4 (1%) in strong acidic conditions (10 mol L−1 HCl) after pre-reduction of As(V) to As(III) in a KI (0.5%)-ascorbic acid (2%)-HCl (3 mol L−1) mixture. Reactivity of As species toward HG under conditions of a rice matrix was investigated in order to improve detectability and selectivity of i-As when o-As coexists. Interferences related to contribution of DMA and MMA to the As signal recorded for i-As were pointed out. Limitations of the method for DMA and matrix effects for MMA were found advantageous to selectively measure i-As by HG-ICP OES. No matrix effects on i-As allowed to use external calibration for its quantification. Detection limit of 0.28 ng g−1 (5.6 ng g−1 in original sample), precision < 5%, and adequate accuracy (96.5–103.9%), verified by the analyte recovery study, were achieved. Applicability of the method was demonstrated by i-As determination in nine brown rice samples. Its concentrations found along with the percentage of i-As to total As (obtained after microwave digestion and HG-ICP OES detection) were similar with these reported for this type of rice.

Inexpensive FIA method to determine trace levels of imazapyr by UV-detection enhanced with electrochemical polarization

A new flow injection analysis method with spectro-electrochemical detection (FIA/SEC) to determine trace levels of imazapyr on water samples is presented. The non-chromatographic method involves the use of a bench photometer, a single-potential potentiostat, a low pressure pumping system and a home-made spectro-electrochemical flow detector (SEC-FD), specifically adapted for the described method and using a stainless steel/electrolytic lead/lead chloride (SS/e-Pb/PbCl2) working electrode. The limit of quantification (LOQ) reached for the optimized work parameters was 0.02 µg/mL, the relative standard deviation (RSD) in the whole range of linear response was less than 2% and a wide linear response range from 0.005 to 6.0 µg/mL was obtained. A standard addition method was used to determine the imazapyr amount in natural waters containing commercial formulation of such herbicide, the Recovery % has values close to 105%. The method performance makes it suitable to analyze the legal imazapyr tolerances established by the U.S. EPA avoiding the use of expensive chromatographic equipments and/or complicated sample preparations.

A simple and sensitive non-chromatographic method for quantification of four arsenic species in rice by hydride generation−atomic fluorescence spectrometry

Herein, a simple, sensitive and effective non-chromatographic analytical method was developed for speciation analysis of four target arsenic (As) species in rice samples. The hydride generation conditions and sample pretreatment techniques were optimized for the determination of arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)]. For optimum results, hydrochloric acid/sodium citrate buffer (1.6 mol L−1 sodium citrate; pH 4.8) was used for the carrier solution and 0.06 mol L−1 citric acid for sample acidification. The concentrations of the four As species were determined by hydride generation-atomic fluorescence spectrometry using a series of linear independent equations corresponding to four different sample pretreatment procedures. The limits of detection of the method were 0.21, 0.52, 0.65 and 0.9 μg kg−1 for As(III), As(V), MMA(V) and DMA(V), respectively. Extraction efficiencies (96–104%) and recoveries of As species in rice (95–100%) were always good, and interconversion among the four As species in the extract did not occur.

Enhanced expression and purification of anti-VEGF nanobody in cucurbit plants

Neutralizing nanobodies against vascular endothelial growth factor (VEGF) and its receptors are the most important types of medicine which are used in cancer therapy strategies. To increase the level of anti-VEGF (Nb42) nanobody accumulation and also to simplify its subsequent purification, we fused codon-optimized Nb42 nanobody to hydrophobin I (HFBI) stabilizing partner, and transiently expressed the fusion gene cassette in cucurbit plants using a zucchini yellow mosaic virus-based viral vector. The successful transcription of the target gene was confirmed with the use of RT-PCR and Real-time PCR analyses. According to the immunoblot assay results the expression level of Nb42-HFBI fusion protein, 22 μg/g fresh leaf tissue, was twofolds higher than the unfused nanobody. Furthermore, the endoplasmic reticulum (ER)-targeted Nb42-HFBI fusion protein was successfully purified from leaf extract using an aqueous-two phase system (ATPS) method. The use of HFBI fusion partner increased the expression level of the Nb42 recombinant protein while simultaneously facilitated its subsequent purification using an inexpensive non-chromatographic method known as ATPS. As a conclusion, the application of ER-targeted hydrophobin fusion strategy with the use of special features of plant viral vectors, can improve the value of plant-based transient expression platform for the production of important proteins with the industrial and therapeutic application.