Column Chromatography Experiments Research Articles

Identification and Toxicity Profiling of Column Fractions of Ethanol Leaf Extract of Ziziphus mauritiana

Different plants may contain varying amounts of phytochemicals and also different degrees of toxicity. This study aimed to evaluate the phytochemicals present in the most active column fraction of ethanol leaf extract of the Ziziphus mauritiana plant and their toxic effects using brine shrimp lethality assay and animal model. Crude ethanol extract was obtained by maceration while fraction was achieved using a column chromatography experiment. Toxicity was evaluated with brine shrimp lethality assay and albino rat’s models while characterization was achieved with liquid chromatography-mass spectrophotometry. Ziziphus mauritiana leaves revealed 42 fractions pooled into seven fractions. Fraction three (3) was the most toxic with the brine shrimp lethality assay of (31.48ug/ml) and its toxicological evaluation revealed an adverse effect on the hematological parameter, biochemical indices, and histo-architecture of the liver and kidney of the experimental model studied. LCMS analysis of the most toxic fraction revealed the presence of Antirrhinoside, Lucidumol A, Apigenin 7-glucuronide-4'-(6''-malonylglucoside), Dioscoreside C, Camellioside D, and others which have been reported for various pharmacological effects including adverse effects, The mode of toxicity may be synergistic, individual, or antagonistic which may explain the moderate toxicity observed in animal model. Administration of these fractions may lead to toxicity despite their potential.

Transformation of a Classic Column and Thin Layer Chromatography Experiment into a Multioutcome, Discovery-Based Activity

A multioutcome, discovery-based column (CC) and thin layer chromatography (TLC) experiment has been designed for introductory organic chemistry. Multiple outcomes were established using commercially available two- and three-component analgesic medications based on acetaminophen, acetylsalicylic acid (aspirin), and ibuprofen. A series of 11 different medications were identified as unknown analgesic mixtures that can be separated using chromatography. The objective of this experiment is to have students use CC to separate an unknown analgesic medication into its individual components, and TLC to evaluate the quality of their separation and to identify the brand name and composition of their medication. This discovery-based experimental procedure requires students to obtain an individual data set for their assigned unknown analgesic-based mixture and then apply their problem-solving skills toward their TLC results to successfully distinguish the composition of their mixture. Student feedback provided evidence that students acquired lab skills related to chromatography, while also improving their understanding and confidence toward CC and TLC, and enhancing their problem-solving and critical thinking abilities. Further, students felt that this discovery-based activity provided a more realistic demonstration of how chromatographic techniques are utilized in the lab, and presented them in a fun and engaging manner. The capability to differentiate the unknown analgesic mixture ensures that students not only develop lab skills for CC and TLC, but also acquire other pedagogical gains common to inquiry-based learning.

Quinone Synthesis and a Visual Introduction to Column Chromatography: An Undergraduate Experiment

An introductory normal phase column chromatography experiment is described and evaluated for second year undergraduate chemistry laboratory class. The experiment involves a nucleophilic substitution yielding a crude mixture of reactant and one product, both of which are brightly colored and contrasting, creating a clearly visual separation when purified using column chromatography. A student survey was conducted which showed that 75.4% of students (n = 53, approximately 50% of year group) agreed or strongly agreed that the experiment worked well for them, and 88.7% agreed or strongly agreed that the experiment was enjoyable. Every student was also assessed in the lab on their practical technique, with the year group achieving an average of 72.1% in this assessment.

Effect of adding dodecanol as modifier to N,N,N′,N′-tetra-n-hexyl-3,6-dithiaoctane-1,8-diamide silica-based adsorbent on the adsorption behaviors of platinum-group metals and other metals from simulated high-level liquid waste

Abstract To adsorb and separate platinum-group metals efficiently from simulated high-level liquid waste, two adsorbents, N,N,N′,N′-tetra-n-hexyl-3,6-dithiaoctane-1,8-diamide (THDTODA)/SiO2-P and (THDTODA + dodecanol)/SiO2-P, were prepared by impregnation of THDTODA with or without dodecanol into macroporous silica/styrene–divinylbenzene copolymer composite particles SiO2-P. The effect of the addition of dodecanol to THDTODA fixed silica-based adsorbents on the separation of platinum-group metals and other metals was evaluated by batch adsorption and chromatographic separation experiments. THDTODA/SiO2-P adsorbed Ru(III) and Rh(III) more than (THDTODA + dodecanol)/SiO2-P did in concentrated HNO3 solution. From the calculated thermodynamic parameters, dodecanol was considered to have little effect on the temperature dependence of the adsorptions of Ru(III), Zr(IV), Mo(VI), and Re(VII) onto the THDTODA adsorbents. Furthermore, in the results of column chromatography experiments, the effects of dodecanol addition on the separation properties were observed for Ru(III), Zr(IV), and Mo(VI), but little effect was observed for Pd(II) and Re(VII).

Simultaneous Determination of 78 Compounds of Rhodiola rosea Extract by Supercritical CO2-Extraction and HPLC-ESI-MS/MS Spectrometry.

The plant Rhodiola rosea L. of family Crassulaceae was extracted using the supercritical CO2-extraction method. Several experimental conditions were investigated in the pressure range of 200–500 bar, with the used volume of cosolvent ethanol in the amount of 1% in the liquid phase at a temperature in the range of 31–70°C. The most effective extraction conditions are pressure 350 bar and temperature 60°C. The extracts were analyzed by HPLC with MS/MS identification. 78 target analytes were isolated from Rhodiola rosea (Russia) using a series of column chromatography and mass spectrometry experiments. The results of the analysis showed a spectrum of the main active ingredients Rh. rosea: salidroside, rhodiolosides (B and C), rhodiosin, luteolin, catechin, quercetin, quercitrin, herbacetin, sacranoside A, vimalin, and others. In addition to the reported metabolites, 29 metabolites were newly annotated in Rh. rosea. There were flavonols: dihydroquercetin, acacetin, mearnsetin, and taxifolin-O-pentoside; flavones: apigenin-O-hexoside derivative, tricetin trimethyl ether 7-O-hexosyl-hexoside, tricin 7-O-glucoronyl-O-hexoside, tricin O-pentoside, and tricin-O-dihexoside; flavanones: eriodictyol-7-O-glucoside; flavan-3-ols: gallocatechin, hydroxycinnamic acid caffeoylmalic acid, and di-O-caffeoylquinic acid; coumarins: esculetin; esculin: fraxin; and lignans: hinokinin, pinoresinol, L-ascorbic acid, glucaric acid, palmitic acid, and linolenic acid. The results of supercritical CO2-extraction from roots and rhizomes of Rh. rosea, in particular, indicate that the extract contained all biologically active components of the plant, as well as inert mixtures of extracted compositions.

ANTIFUNGAL ACTIVITY OF CRUDE EXTRACTS OF Euphorbia tirucalli; ISOLATION AND CHARACTERIZATION OF ONE OF ITS BIOACTIVE PRINCIPLES TIRUCALLOL

Antifungal properties of extracts from Euphorbia tirucalli against some wood decay fungi were studied. E. tirucalli fresh sample was harvested, cleansed and chipped into chips of 3 x 2 cm length and breadth. Chipped sample (846.3 g) was macerated in 1000 mL of ethyl acetate, n hexane and methanol solvents, respectively. Column chromatography experiment on E. tirucalli was carried out and antifungal screening was done according to standard method for 7 days to observe zones of inhibition of fungi growth. Broth dilution method was adopted to determine the Minimum Inhibition Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of E. tirucalli extracts and fraction. Results show that Fibroporia vaillantii, Fomitopsis pinicola, Gloeophyllum sepiarium, Rhizopus sp. and Serpula lacrymans were sensitive to ethyl acetate, n’ hexane and methanol E. tirucalli extracts at concentration of 200 mg/mL with zone of inhibition of 18-26 mm and methanol extract had the highest value of 26 mm. The fraction had zone of inhibition of between 18 and 20 mm which are not significantly different (p<0.05) from 29-35 mm obtained from antibiotics. The MIC of 10 mg/mL and MFC of 20 mg/mL were recorded for extracts against the test fungi while MIC of 50 µg/mL and MFC 200 µg/mL were noted for fraction (Et15), respectively. Fraction (Et15) obtained from E. tirucalli was characterised as tirucallol compound. In conclusion, E. tirucalli extracts and fraction have proved to be effective in the control of wood decay fungi and may serve as control of diseases caused by the test fungi

Setting Up an Educational Column Chromatography Experiment from Home

In light of the COVID-19 pandemic situation, the separation of the primary colors of ink markers and food coloring was performed using a simplified column chromatography setup at home to accomplish the educational outcome of the course. The virtual class instruction, for the students enrolled in the Organic Techniques Laboratory course, allows the understanding of the basic principles of chromatography in a way similar to that in the face-to-face experience. The materials proposed for conducting the experiment are readily available at home or easily found in a drugstore or in a grocery store. A wide variety of results were observed where the separation of at least two primary colors was achieved by 80% of the students.

Evaluation of polybenzimidazole-based polymers for the removal of uranium, thorium and palladium from aqueous medium.

Four types of polybenzimidazole (PBI)-based polymers (m-PBI, p-PBI, pyridine-based m-PBI and alkylated m-PBI) have been prepared and characterized. Extraction behaviour of heavy metal ions, viz. U(VI), Th(IV) and Pd(II), with these polymers was investigated. Distribution ratios for the extraction of these metal ions were measured as a function of nitric acid concentration. Extraction data reveal that, in general, p-PBI exhibits a higher distribution ratio for U(VI), Th(IV) and Pd(II) compared with the other polymeric resins evaluated in the present study. Column chromatography experiments were carried out with a solution of U(VI), Th(IV) and Pd(II) in dilute nitric acid media using columns packed with m- and p-PBI polymeric material for understanding the sorption and elution behaviour. The p-PBI-based resin has shown higher palladium sorption capacity (1.8 mmol g−1). The studies also established that p-PBI resin is a potential candidate material for the recovery of U(VI) and Th(IV) (capacity 0.22 mmol g−1 and 0.13 mmol g−1) from an aqueous stream, e.g. mine water samples.

Water on hydrophobic surfaces: Mechanistic modeling of hydrophobic interaction chromatography

Mechanistic models are successfully used for protein purification process development as shown for ion-exchange column chromatography (IEX). Modeling and simulation of hydrophobic interaction chromatography (HIC) in the column mode has been seldom reported. As a combination of these two techniques is often encountered in biopharmaceutical purification steps, accurate modeling of protein adsorption in HIC is a core issue for applying holistic model-based process development, especially in the light of the Quality by Design (QbD) approach.In this work, a new mechanistic isotherm model for HIC is derived by consideration of an equilibrium between well-ordered water molecules and bulk-like ordered water molecules on the hydrophobic surfaces of protein and ligand.The model's capability of describing column chromatography experiments is demonstrated with glucose oxidase, bovine serum albumin (BSA), and lysozyme on Capto™ Phenyl (high sub) as model system. After model calibration from chromatograms of bind-and-elute experiments, results were validated with batch isotherms and prediction of further gradient elution chromatograms.

Column Chromatography To Obtain Organic Cation Sorption Isotherms.

Column chromatography was evaluated as a method to obtain organic cation sorption isotherms for environmental solids while using the peak skewness to identify the linear range of the sorption isotherm. Custom packed HPLC columns and standard batch sorption techniques were used to intercompare sorption isotherms and solid-water sorption coefficients (Kd) for four organic cations (benzylamine, 2,4-dichlorobenzylamine, phenyltrimethylammonium, oxytetracycline) with two aluminosilicate clay minerals and one soil. A comparison of Freundlich isotherm parameters revealed isotherm linearity or nonlinearity was not significantly different between column chromatography and traditional batch experiments. Importantly, skewness (a metric of eluting peak symmetry) analysis of eluting peaks can establish isotherm linearity, thereby enabling a less labor intensive means to generate the extensive data sets of linear Kd values required for the development of predictive sorption models. Our findings clearly show that column chromatography can reproduce sorption measures from conventional batch experiments with the benefit of lower labor-intensity, faster analysis times, and allow for consistent sorption measures across laboratories with distinct chromatography instrumentation.

Effect of nanolayering of calcium salts of phosphoric acid ester monomers on the durability of resin-dentin bonds

To investigate the contribution of nanolayering on resin-dentin bond durability, two phosphoric acid ester resin monomers, 10-methacryloyloxy-decyl-dihydrogen-phosphate (10-MDP) or its analog, methacryloyloxy-penta-propyleneglycol-dihydrogen-phosphate (MDA), were examined for their affinity for mineralized dentin powder in a column chromatography setup. Hydroxyapatite (HA) powder was dispersed in experimental primers consisting of 10-MDP or MDA solvated in ethanol/water and examined with FTIR, 31P MAS-NMR and XPS. Light-curable 10-MDP or MDA primers were used for bonding to dentin, and examined after 24h or one-year of water-aging by TEM for evidence of nanolayering, and for microtensile bond strength evaluation. Primer-bonded dentin was examined by thin-film XRD to identify short-range order peaks characteristic of nanolayering of resin monomer-Ca salts. Although 10-MDP had better affinity for mineralized dentin than MDA, both monomers completely eluted from the mineralized dentin powder column using ethanol-water as mobile phase, indicating that the adsorption processes were reversible. This finding was supported by chemoanalytic data. XRD of 10-MDP-bonded dentin showed three diffraction peaks hat were absent from MDA-bonded dentin. Nanolayering was identified by TEM in 10-MDP-bonded dentin, but not in MDA-bonded dentin. Significant drop in bond strength (in MPa) was observed for both groups after one-year of water-aging compared with 24-h: 10-MDP group from 48.3±6.3 to 37.4±4.6; MDA group from 50.7±5.0 to 35.7±3.8 (P<0.05), with no significant difference between the two groups at the same time-point. Because both functional monomer-primed, resin-bonded dentin exhibited similar bond strength decline after water-aging, presence of nanolayering is unlikely to contribute to the overall resin-dentin bond durability. Statement of SignificanceThe durability of resin-dentin bonds in 10-MDP containing self-etching adhesives has been anecdotally attributed to the presence of nanolayering of 10-MDP-calcium salts in the resin-dentin interface. Results of the present work indicate that such a claim cannot be justified. Complete elution of the phosphoric acid ester monomer from mineralized dentin powder in the column chromatography experiments using ethanol-water mobile phase to simulate the solvent mixture employed in most 10-MDP-containing dentin adhesives further challenges the previously proposed adhesion-decalcification concept that utilizes chemical bonding of phosphoric acid ester monomers to apatite as a bonding mechanism in 10-MDP containing dentin adhesives.

Model-based design and experimental validation of simulated moving bed reactor for production of glycol ether ester

This work proposes a practical and systematic model-based approach to identify the optimal operating conditions for a simulated moving bed reactor (SMBR). The SMBR operation is applied to an industrial case study for the continuous production of a solvent, propylene glycol methyl ether acetate (DOWANOL™ PMA), which is produced through an acid-catalyzed esterification reaction of 1-methoxy-2-propanol and acetic acid.The model-based approach is demonstrated by lab-scale SMBR experiments. A multi-objective optimization problem was formulated for developing an SMBR process to maximize the production rate of PMA and the conversion of the esterification reaction simultaneously. In this study, this optimization problem is solved using the epsilon-constrained method and a Pareto plot is presented. The solutions that corresponded to three different values of conversion, 70%, 80%, and 85%, are experimentally validated. The SMBR model that was developed from batch kinetic and single column chromatography experiments demonstrates reasonable agreement with the experimental results. Furthermore, the SMBR experimental data was used to correct the parameters in the model. A validation study at a higher conversion of 95% demonstrates improved predictability of the corrected parameters.

Synthesis of 1,1-Diphenylethylene (DPE): The Marriage of a Grignard Reaction and a Column Chromatography Experiment

The synthesis of 1,1-diphenylethylene (DPE) via a Grignard reaction, followed by an acid-catalyzed dehydration reaction, yields a mixture of compounds. DPE is a high boiling liquid that cannot be purified using simple distillation. However, it is easily separated from the more polar starting material and intermediate alcohol using both thin layer and column chromatography. Second-semester organic chemistry laboratory students learn the techniques of running reactions under anhydrous conditions and of column chromatography. In addition, the experiment demonstrates clearly the relationship between thin layer and column chromatography.

High-throughput process development of purification alternatives for the protein avidin.

With an increased number of applications in the field of the avidin-biotin technology, the resulting demand for highly-purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high-throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion-exchange chromatography. In a high-throughput format, process parameters for aqueous two-phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed-mode column chromatography experiments were performed. The HTPD strategy was complemented by a high-throughput tandem high-performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two-phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two-phase extraction and precipitation results were largely confirmed in larger scale with scale-up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed-mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task.

Effects of resin ligand density on yield and impurity clearance in preparative cation exchange chromatography. II. Process characterization

Ion exchange resins are key raw materials in biopharmaceutical manufacturing processes, and variability in ligand density has the potential to compromise process robustness if not controlled within appropriate ranges. In this study, yield and impurity clearance were evaluated for several preparative cation exchange chromatography steps using SP Sepharose™ Fast Flow resins at the high, low, and center of the commercial ligand density specification range. This was accomplished using a design of experiments (DoE) approach coupled to high-throughput screening in 96-well plate format, as well as column chromatography experiments with gradient elution. Results of the DoE study indicated that ligand density variation within the commercial specification range of SP Sepharose™ Fast Flow had no effect on yield, HCP clearance, aggregate clearance, or distribution of charge variants. However, results from the column experiments showed that ligand density has the potential to influence protein elution profiles which can lead to small changes in impurity clearance in some cases.

Preparation of Poly(Hydroxamic Acid) for Separation of Zr/Y, Sr System

Poly(hydroxamic acid) resin (PHA) was prepared by modification of polyacrylamide (PAAm) prepared through -irradiation technique and used for the first time in separation of Zr(IV) from Y(III) and Sr(II) as a simulation mode for the purification of89Zr from its parents (Y or Sr). The adsorption behaviors of zirconium, yttrium, and strontium on the prepared PHA resin in different media, namely, hydrochloric acid, acetate buffer, and citrate buffer were studied as a function of pH. In addition, in cation-exchange column chromatography experiments using PHA, three different eluants, namely, mol/L HCl, acetate buffer pH 3.5, and 2 mol/L HCl, were employed for elution of Zr(IV), Y(III) and Sr(II), respectively, where Zr(IV), Y(III) and Sr(II) were eluted in amounts of 80%, 99.9%, and 100%, respectively. The purification process of Y(III) from Zr(IV) was carried out using regenerated PHA.

Ion-exchange chromatography using citrate buffer, EDTA and α-HIBA for improving the separation of no-carrier-added 139Ce from La2O3 target irradiated with 14.5 MeV protons

The adsorption behaviour of La/Ce system on Dowex 50W-X8 in different media, namely, nitric acid, acetate buffer and citrate buffer was studied as a function of the concentration of nitric acid and buffer pH. In addition, in cation-exchange column chromatography experiments, three different eluants, namely, citrate buffer of pH 5.5, 0.1 M EDTA and 0.2 M α-HIBA, were employed for separation of Ce(III) from La(III). The optimum conditions for improvement of radiochemical separation of no-carrier-added 139Ce from proton irradiated lanthanum were applied using the most suitable chelating agent 0.2 M α-HIBA. The purification of 139Ce from macro amount of La(III) was done using two columns in a sequence. The target was prepared by pressing. The production of high radionuclidic and chemical purity 139Ce via irradiation of lanthanum oxide target at MGC-20 cyclotron of proton energy 14.5 MeV was described. The experimental yield was found to be 200 kBq/μA h.

Chiral layered molecular spaces with amino acids

Novel chiral layered molecular space L-(+)-leucine amidepropylamidephenylsilica (L-(+)-Leu-APAPhS) and D-(−)-alanine amidepropylamidephenylsilica (D-(−)-Ala-APAPhS) were synthesized through grafting L-leucine tert-butyl ester (L-Leu-OtBu) and D-alanine tert-butyl ester (D-Ala-OtBu), in layered carboxylpropylamidephenylsilica (CPAPhS), which was developed in our previous research, with regular carboxyl groups, and the tert-butyl ester as protective groups of carboxyls in amino acids was cleaved with dilute trifluoroacetic acid (TFA). The improved XRD response indicated that the two dimensional layered structure had been retained in L-Leu-APAPhS and D-Ala-APAPhS. The chiral behaviors of layered molecular spaces CPAPhS, L-(+)-Leu-APAPhS and D-(−)-Ala-APAPhS were investigated. DL-Alanine tert-butyl ester (DL-Ala-OtBu) was grafted in the achiral layered molecular space CPAPhS as enantiomer pair, even if 70% L-Ala-OtBu and 30% D-Ala-OtBu existed in the reaction solution. L-(+)-Leu-APAPhS and D-(−)-Ala-APAPhS represented better chiral selectivity for amino acids both in chiral selective reactions and chiral column chromatography experiments, and exhibited the potential of chiral layered molecular space with an amino acid structure for chiral molecular recognition and selection processes. The formation of L-Leu-APAPhS-L-Ala-OMe also exhibited potential for that multi-amino acid (peptide) can be grafted in CPAPhS to form layered molecular spaces with peptides.

A Computer-Interfaced Drop Counter as an Inexpensive Fraction Collector for Column Chromatography

A computer-interfaced drop counter is described that serves as an inexpensive alternative to a fraction collector for column chromatography experiments. Undergraduate biochemistry laboratories frequently do not have the budget to purchase fraction collectors. Protocols that call for the manual measurement of fraction volumes as well as the manual application of buffer to the column do not provide students with the skills required to function in a professional setting. In this experiment, students use a column outfitted with Luer fittings to set up an automatic buffer-feeding system and also monitor fraction collection using a drop counter. Students have the opportunity to learn troubleshooting techniques for both methods. To illustrate this system, a sample of typical student data from an experiment in which the molecular weight of myoglobin is determined by gel filtration chromatography is shown. The system described can be used for any column chromatography experiment, including preparative gel filtration chromatography, ion exchange chromatography, and affinity chromatography.

Effects of cross-linking and spacer groups on beta-cyclodextrin bonded liquid chromatographic separation

Mesoporous glass beads and 5 μm silica particles were modified with beta-cyclodextrin ( Β-CD) by means of directly bonding, linking spacer group, and cross-linking agent. The selectivities of the Β-CD modified silica particles were measured by simple column chromatography to separate a model mixture of 1-and 2-hydroxy-naphthalene (naphthols) and ortho, meta, and para-xylene. In the packed column chromatography experiments, two major controlling factors (inclusion complex formation effect and steric hindrance effect of the analytes) on the separations were observed. The elusion orders of the Β-CD directly bonded glass beads were meta-, para-, ortho-xylene and 1-naphthol, 2-naphthol. The phenomena of Β-CD pore blocking and narrowing by spacer groups and cross-linking agent were observed. The spacer group and cross-linking agent decreased inclusion complex formation of 2-naphthol.