Stepwise Elution Research Articles

Chiral separation of catechin and epicatechin by reversed phase high-performance liquid chromatography with β-cyclodextrin stepwise and linear gradient elution modes

Catechin and epicatechin were enantioseparated by high-performance liquid chromatography (HPLC) with a phenyl column and aqueous mobile phases containing 0.05% (w/v) and 0.6% (w/v) of β-cyclodextrin for catechin and epicatechin, respectively. β-Cyclodextrin was found to be scarcely retained on a phenyl column. Consequently, it was suggested that catechin, which was eluted earlier than epicatechin, formed more stable inclusion complex with β-cyclodextrin than epicatechin and earlier eluted enantiomers, (−)-catechin and (+)-epicatechin, formed more stable diastereomer complexes with β-cyclodextrin than the respective enantiomers. This was confirmed by β-cyclodextrin-modified micellar electrokinetic chromatography and Benesi−Hildebrand plots by fluorescence spectrophotometry. Effect of sugars (D-sucrose, D-glucose, and D-fructose) on the epimerization of (+)-catechin and (+)-epicatechin by heating was investigated by HPLC with a β-cyclodextrin stepwise elution mode, in which two kinds of aqueous eluents containing different concentrations of β-cyclodextrin were used by turns. The epimerization of the two enantiomers was suppressed only when D-fructose was added. Separation of ten kinds of catechins including catechin and epicatechin enantiomers was investigated by a β-cyclodextrin linear gradient HPLC elution mode without using organic solvents, where two kinds of aqueous eluents containing different concentrations of β-cyclodextrin were used with changing their ratio gradually. These catechins in a green tea infusion could be separated successfully by this method.

Adsorption and separation of flavonoid aglycones and flavonol aglycones by using Zr(IV) immobilized collagen fiber adsorbent as column packing material

Flavonoid aglycones and flavonol aglycones, are difficult to be separated due to their similar structures and limited hydroxyls. Herein, Zr(IV) immobilized collagen fiber (ZCF) was prepared by immobilizing Zr(IV) on glutaraldehyde cross-linked collagen fiber (GCF) to separate apigenin and kaempferol, the representative compounds of flavonoid aglycones and flavonol aglycones. TG, DSC, SEM and element mapping were applied to characterize ZCF. The static adsorption suggested that when the amount of immobilized Zr(IV) reached only 0.3 mmol/g, the adsorption difference between apigenin and kaempferol on ZCF was highly improved compared with GCF without Zr(IV). The adsorption rate of apigenin increased from 2.21% to 14.31%, while that of kaempferol significantly increased from 22.35% to 87.17%. In addition, the spectroscopy (UV–Vis, FT-IR) were applied to analyze the binding sites of flavonoids with Zr(IV), indicating that the strong adsorption of ZCF to kaempferol may be related to the 3-hydroxyl on the C-ring, while apigenin only can conduct a weak binding with ZCF through 5-hydroxyl on the A-ring. In ZCF column chromatography separation, apigenin and kaempferol could be separated effectively with purities of 94.02% and 89.23% by stepwise elution of 50% aqueous ethanol followed by 50% aqueous ethanol containing 0.005 mol/L citric acid. In separation on ZCF and GCF combined columns, the purities of apigenin and kaempferol reached 95.59% and 92.56%, respectively, and the trace amount of Zr(IV) shedding from ZCF was completely adsorbed by GCF avoiding contaminating the target components. In addition, the effective separation of apigenin and kaempferol in apium graveolens (celery) extract by ZCF column was also achieved. The above results indicated ZCF could selectively adsorb flavonol aglycones with 3-hydroxyl on the C-ring from the mixture with flavonoid aglycones. This investigation provides a novel column chromatography packing material for the fast separation of flavonoid aglycones and flavonol aglycones.

Evaluation of inhibitor activity of bacterial sialidase from Clostridium perfringens against Newcastle disease virus in the cell culture model using chicken embryo fibroblast.

Objective:The Newcastle disease virus (NDV) is an infectious disease that causes very high economic losses due to decreased livestock production and poultry deaths. The vaccine’s ineffectiveness due to mutation of the genetic structure of the virus impacts obstacles in controlling the disease, especially in some endemic areas. This study aimed to provide an alternative treatment for NDV infection by observing the viral replication inhibitor activity of Clostridium perfringens sialidase in primary chicken embryo fibroblast (CEF) cells.Materials and Methods:The virus was adapted in CEF monolayer cells, then collected thrice using the freeze–thaw method and stored at −20°C for the next step in the challenge procedure. C. perfringens crude sialidase was obtained, but it was further purified via stepwise elution in ion exchange using Q Sepharose® Fast Flow and affinity chromatography with oxamic acid agarose. The purified sialidase was tested for its toxicity, ability to breakdown sialic acid, stopping viral replication, and how treated cells expressed their genes.Results:According to this study, purified C. perfringens sialidase at dosages of 187.5, 93.75, and 46.87 mU effectively hydrolyzes CEF cells’ sialic acid and significantly inhibits viral replication on the treated cells. However, sialidase dosages of 375 and 750 mU affected the viability of monolayer CEF cells. Interestingly, downregulation of toll-like receptor (TLR)3 and TLR7 (p < 0.05) in the sialidase-treated group indicates viral endocytosis failure.Conclusions:By stopping endocytosis and viral replication in host cells, sialidase from C. perfringens can be used as an alternative preventive treatment for NDV infection.

Recovery and separation of Mo(VI) and Re(VII) from Mo-Re bearing solution by gallic acid-modified cellulose microspheres

• MCGA effectively separated Mo(VI) and Re(VII) with β Mo/Re 5.23–735.53. • MCGA could selectively recover Mo(VI) in column experiment. • The different eluents can achieve the stepwise elution of Mo(VI) and Re(VII). • The adsorption capacity showed a negligible decrease after 5 cycles. • The ion species and force was the primary reason for selective adsorption. It is significant to develop an effective adsorbent for the separation of Mo(VI) and Re(VII) owing to congenital isomorphism. Herein, gallic acid-functionalized microcrystalline cellulose microspheres (MCGA) were synthesized via radiation technology. Batch and column experiments were systematically performed to evaluate the separation behaviors for Mo(VI) and Re(VII). It was discovered that MCGA showed higher adsorption capacity and stronger affinity for Mo(VI) than Re(VII) under the tested condition. The separation of Mo(VI) and Re(VII) could be easily achieved by MCGA, exhibiting a separation factor (β Mo/Re ) in the range of 5.23–735.53. Significantly, the dynamic column experiment revealed that the MCGA could selectively recover Mo(VI) from a simulated leaching solution of molybdenite. In addition, different eluents can achieve the stepwise elution of Mo(VI) and Re(VII). With increasing temperature, Mo(VI) and Re(VII) could be more easily separation from the solution. Ultimately, the capture of Mo(VI) by MCGA was attributed to the formation of hydrogen bond and complexes between the adjacent phenolic hydroxyl group and metal ions, while Re(VII) was mainly associated with MCGA by hydrogen bond. Moreover, the excellent adsorption properties indicated that MCGA is a candidate for separation of Mo(VI) and Re(VII) from Mo-Re bearing solution.

ZIC-cHILIC-Based StageTip for Simultaneous Glycopeptide Enrichment and Fractionation toward Large-Scale N-Sialoglycoproteomics.

Alterations of protein glycosylation are closely related with pathophysiological regulation. Due to the structural macro- and microheterogeneity, low stoichiometry, and low ionization efficiency of glycopeptides, high-performance tools to enrich glycopeptides, especially the negatively charged and labile sialoglycopeptides, are essential to enhance the identification of the underexplored glycoproteome. Here, we present the first implementation of zwitterionic hydrophilic interaction chromatography with the exposed choline group (ZIC-cHILIC) in StageTip for simultaneous enrichment and fractionation of intact glycopeptides. In a model study using lung cancer cells, early elution by a high percentage of acetonitrile prominently prefilters nonglycopeptides, facilitating high enrichment specificity for glycopeptides (92-96%) and sialoglycopeptides (77-89%) in the subsequent hydrophilic fractions. The stepwise elution shows a high glycopeptide fractionation efficiency by a <10% overlap of glycopeptides between adjacent fractions. Most importantly, the ZIC-cHILIC stepwise strategy demonstrated good reproducibility (>80% in triplicate analysis) as well as superior coverage of 4.6- to 12.0-fold and 2.1- to 35.6-fold more glycopeptides and sialoglycopeptides compared to conventional TiO2 and ZIC-HILIC, respectively. To the best of our knowledge, the result with 2742 sialoglycopeptides among 7367 unique glycopeptides and 166 glycans from 2434 N-glycosites of 1118 glycoproteins (Byonic score > 100) provides one of the deepest glycoproteomic profiles in single-cell type. Without the immunoprecipitation step, the large-scale glycoproteomic atlas also reveals site-specific glycosylation of many druggable receptor proteins, such as EGFR, MET, ERBB2, ERBB3, AXL, and IGF1R. The demonstrated high enrichment specificity and identification depth show that stepwise ZIC-cHILIC is an efficient method to explore the under-represented sialoglycoproteome.

Comparing the relative robustness of anion exchange and the corresponding mixed-mode chromatography on removing a weakly-bound byproduct: A case study of bispecific antibody purification

A certain impurity may bind weaker or tighter to a particular type of chromatography column (e.g., ion exchange, hydrophobic interaction or mixed-mode) than the target protein, and this forms the basis for separation. For impurities that bind weaker, they can be removed by an appropriate pre-elution wash. However, we previously showed that wash-enabled impurity clearance is usually sensitive to loading density, causing poor robustness of the process. In this work, with a bispecific antibody case study, we compared the relative robustness of anion exchange and the corresponding mixed-mode chromatography, which mediates both anion exchange and hydrophobic interactions, on removing a weakly-bound byproduct by wash. It was learned that under a fixed appropriate wash condition, the latter achieves consistent byproduct clearance and good yield over a much wider range of loading density than the former. As wide loading density range is highly desirable for large-scale manufacturing, the above finding suggests that for a chromatography step that employs stepwise elution and relies on pre-elution wash for removing weakly-bound impurities, mixed-mode chromatography could be a better choice than the corresponding monomode ion exchange chromatography.

Synthetisation of an affinity matrix (Procainamide Sepharose Cl-6b) for brain cholinesterase purification and separation source from Monopterus albus

Affinity chromatography for acetylcholinesterase; AChE namely Procainamide Sepharose CL-6B was synthesised through the coupling method between soluble procainamide hydrochloride immobilised by a cross-linked agarose size exclusion, sepharose CL-6B. 1, 4-butanediol diglycidyl ether plays a role in building up a productive and rigid connecting of a biospecific ligand (Procainamide hydrochloride) to an insoluble matrix (Sephacryl CL-6B). Local freshwater eel brain was extracted and centrifuge at high speed. The supernatant was collected, and seven different volumes were separately loaded on to the column for isocratic purification where 12 fractions were collected at the end of elution stage. This study shows 1000 μL of extractant; considered as the maximum volume to load onto the column. Lastly, a stepwise elution was performed with five different concentrations of NaCl, and each of 1 mL fraction was collected then assay for determination of enzyme activity and protein content. The data shows AChE was successfully purified with percentage recovery of 38 % after 21 purification fold. Kinetic study strengthens the data where the efficient coefficient ratio of ATCi was much higher compared to PTCi and BTCi at 3.03, 2.67 and 1.52 Vmax⋅Km −1, respectively, prove that the collected fraction predominantly contained with AChE, which is a targeted enzyme to be used as a sensitive biosensor to detect the presence of carbamate and organophosphate contamination in the environment.

Fluorimetric determination of the enantiomers of vigabatrin, an antiepileptic drug, by reversed-phase HPLC with a novel diastereomer derivatization reagent.

Herein, determination of an antiepileptic drug, (±)-vigabatrin (VB), was performed by reversed-phase HPLC with fluorimetric detection using a newly designed and synthesized fluorescence derivatization reagent, 2,5-dioxopyrrolidin-1-yl (4-{[(2-nitrophenyl)sulfonyl]oxy}-6-(3-oxomorpholino)quinoline-2-carbonyl)prolinate [Ns-MOK-(R)- or (S)-Pro-OSu]. During the derivatization of VB with Ns-MOK-(R)-Pro-OSu at 60°C, the nosyl (Ns) group, which was introduced to protect a phenolic hydroxy group, was released within 30 min to produce MOK-(R)-Pro-VB, which was detected fluorimetrically at 448 nm with an excitation wavelength of 333 nm. The VB enantiomers were separated on an octadecylsilica (ODS) column with a resolution value of 5.57, because Ns-MOK-(R)-Pro-OSu bears an optically active D-proline structure. A complete separation of MOK-(R)-Pro-(R)- and -(S)-VB enantiomers was achieved on the ODS column within 40 min using stepwise gradient elution, and the detection limits were ~0.80 and 0.37 pmol on the column, respectively. The proposed HPLC with fluorimetric detection method was successfully used for determining VB enantiomers in VB-spiked human serum following solid-phase extraction with an anion-exchange cartridge.

Preparation of pure lower-order myo-Inositol phosphates on laboratory scale for physiological and enzymatic studies

A simple method for the preparative production of lower-order myo-inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single myo-inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the myo-inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order myo-inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order myo-inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order myo-inositol phosphates. In a single approach 2–3 mg of pure myo-inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order myo-inositol phosphates. The purified myo-inositol phosphate isomers were virtually free of other myo-inositol phosphate esters and could be used for enzymatic and physiological studies.

Efficient Separation of Phytochemicals from Muehlenbeckia volcanica (Benth.) Endl. by Polarity-Stepwise Elution Counter-Current Chromatography and Their Antioxidant, Antiglycation, and Aldose Reductase Inhibition Potentials.

Muehlenbeckia volcanica (Benth.) Endl. (M. volcanica), native to South America, is a traditional Peruvian medicinal plant that has multi-therapeutic properties; however, no phytochemicals have been identified from it yet. In this study, a five-step polarity-stepwise elution counter-current chromatography (CCC) was developed using methanol/water (1:5, v/v) as the stationary phase and different ratios of n-hexane, ethyl acetate, and n-butanol as mobile phases to separate the compounds from the 70% methanol extract of M. volcanica, by which six compounds with a wide range of polarities were separated in a single run of CCC and were identified as gallic acid, protocatechuic acid, 4,4′-dihydroxy-3,3′-imino-di-benzoic acid, rutin, quercitrin, and quercetin. Then, two compounds from the fractions of stepwise elution CCC were separated using conventional high-speed CCC, pH-zone-refining CCC, and preparative high-performance liquid chromatography, and identified as shikimic acid and miquelianin. These compounds are reported from M. volcanica for the first time. Notably, except for shikimic acid, all other compounds showed anti-diabetic potentials via antioxidant, antiglycation, and aldose reductase inhibition. The results suggest that the polarity-stepwise elution CCC can be used to efficiently separate or fractionate compounds with a wide range of polarities from natural products. Moreover, M. volcanica and its bioactive compounds are potent anti-diabetic agents.

Simultaneous Quantification of κ-Carrageenan Oligosaccharides of DP 3, 5 and 7 by LC-MS/MS: Application to an in vitro Absorption Study

Carrageenans are widely utilized in many commercial applications such as the food and pharmaceutical industry, due to their excellent functional properties. In this study, a sensitive LC-MS/MS method was developed to determine κ-3, κ-5, and κ-7 carrageenan oligosaccharides simultaneously. Optimum MRM transitions for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were (645.079→565.111, [M-H]−), (515.137→474.946, [M-2H]2−), and (471.484→445, [M-3H]3−), respectively. Chromatographic separation was performed on an Amide column coupled with a guard column operated at 60 °C under stepwise gradient elution. The linearity of the LC-MS/MS method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides, evaluated over the concentration range of 0.10–20.0 µmol L−1, was excellent. The precisions of the method for κ-3, κ-5, and κ-7 carrageenan oligosaccharides were from 0.91% to 9.66%, and the inter-day precisions were from 0.92% to 10.5%. Validation of the LC-MS/MS method indicated that the method was precise and in line with the CFDA guidance. This method has been successfully applied to an in vitro absorption study.

Simultaneous determination of FL118 and W34 in rat Blood by LC-MS/MS: Application to pharmacokinetic studies.

W34 is a prodrug of FL118, and it can be converted to FL118 via a hydrolysis reaction. In this report, a highly sensitive LC-MS/MS method using a C18 column was validated and used for the simultaneous determination of W34 and FL118 in rat blood. A stepwise gradient elution with 0.1% formic acid in water and acetonitrile was employed. The assays were linear over a concentration range of 0.50-50.0 ng/ml for both W34 and FL118. The accuracy of the validation method ranged from 89.74 to 98.94% for W34 and from 88.61 to 94.60% for FL118. The precision was within 7.15% for W34 and 9.63% for FL118. Extraction recoveries of W34 were 94.56-100.49 and 87.67-106.32% for FL118. No significant matrix effects for both W34 and FL118 were observed in blood. The assay has been successfully applied to biological samples obtained from a stability and pharmacokinetic study of W34 and FL118.

Adsorption and desorption characteristics of ginsenosides from Panax ginseng C. A. Meyer on middle-pressure chromatogram isolated gels.

Four types of middle-pressure chromatogram isolated gels are evaluated for adsorption or desorption characteristics of ginsenosides from Panax ginseng. Among them, SP207SS and SP2MGS were selected for dynamic investigations based on their static adsorption or desorption capacity of total ginsenoside. Their adsorption kinetics was better explained by pseudosecond-order model and isotherms were preferably fitted to Langmuir model. Dynamic breakthrough experiments indicated an optimum sample loading speed of 4bed volume/h for either SP207SS or SP2MGS. Desorption speed was determined to be 2 bed volume/h according to desorption amount of total ginsenoside in their effluents. Eight ginsenosides were identified and quantified by high performance liquid chromatography-triple quadropole-mass spectrometry in total ginsenoside extract and different fractions during stepwise dynamic elution. For SP207SS, 27.62% of loaded ginsenosides was detected in 40% ethanol fraction, while 59.12% of them were found in 60% ethanol fraction. As on SP2MGS, the number went to 53.71 and 44.43%, respectively. Recovery rate of ginsenosides were calculated to 78.65% for SP207SS and 89.53% for SP2MGS, respectively. Intriguingly, content of Rg1 and Re in 40% ethanol fraction from SP207SS became 20.1 and 18.6 times higher than that in total ginsenoside extract by one-step elution, which could be leveraged for the facile enrichment of these two ginsenosides from natural sources.

Investigation of eluted characteristics of fulvic acids using differential spectroscopy combined with Gaussian deconvolution and spectral indices.

The characteristics of subfractions of soil fulvic acid (FA3, FA5, FA7, FA9, and FA13) using stepwise elution from XAD-8 resin with pyrophosphate buffers were investigated by differential absorption spectroscopy (DAS) and differential fluorescence spectroscopy (DFS) combined with mathematical deconvolution and spectral indices. The log-transformed absorbance spectra (LTAS) exhibited three regions for both acidic-buffer-eluted subfractions (AESF) and neutral-buffer-eluted subfraction (NESF) and four regions for basic-buffer-eluted subfractions (BESF) according to the differences in spectral slopes. The DAS spectra of FA subfractions were closely fitted with seven Gaussian bands with maxima location at 199.66, 216.18 ± 1.50, 246.20 ± 3.85, 285.22 ± 7.26, 345.64 ± 5.30, 389.27, and 307.37nm, respectively (R2 > 0.993). The content of salicylic-like and carboxyl groups in FA subfractions decreased, while the phenolic chromophore increased with elution sequence. From the 11 spectral indices, AESF had greater molecular weight, condensation, polymerization, hydroxyl radical production, humification degree, and terrigenous contribution, as well as contained more conjugated aromatic structures and less N-containing groups than NESF and BESF. The humification degree and humic characters of FA subfractions were closely associated to the aromaticity, molecular condensation, and DOM-metal-bound functional groups. The proper separation of FA into subfractions is beneficial for reducing its complexity and heterogeneity, which helps us to further explore its chemical properties and interactions with various contaminants in soil environments. Graphical abstract.

Purification and Assays of Tachylectin-5.

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008μg/mL for tachylectin-5A and 0.077-0.27μg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08μg/mL for tachylectin-5A and 0.05-0.11μg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4μg/mL for tachylectin-5A and 15.1-26.8μg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Stepwise elution by high-speed counter-current chromatography combined with a modified macroporous resin to isolate and purify antioxidant phenolics from discarded jackfruit (Artocarpusheterophyllus Lam.) peels.

Combined with modified macroporous resin (MR), high-speed counter-current chromatography (HSCCC) was developed to separate and purify the antioxidant phenolics from waste jackfruit peels. First, the 4,4'-bis(chloromethyl)-1,1'-biphenyl was used to modify the commercially available adsorbent resin XAD-4 in order to enrich the antioxidant phenolics. Second, the resultant phenolics-rich fractions were further purified by HSCCC. In the separation process, the petroleum ether-ethyl acetate-methanol-0.1% acetic acid (5 : 5 : 3 : 7, v/v) upper phase was employed as an immobile phase. Afterwards, stepwise elution using petroleum the ether-ethyl acetate-methanol-0.1% acetic acid (5 : 5 : 3 : 7, v/v) lower phase was carried out during the initial 3.5 h, whereas the petroleum ether-ethyl acetate-methanol-0.1% acetic acid (5 : 5 : 6 : 4, v/v) lower phase was adopted for stepwise elution during the later 2.0 h. Four phenolics were obtained from 179 mg of the phenolics-rich fraction in a single run, including 41 mg chlorogenic, 27 mg gallic acid, 33 mg quercetin, and 29 mg catechin. 13C NMR, 1H NMR, and ESI-MS were employed to identify their structures.

Preparative Separation of Alkaloids from Stem of Euchresta tubulosa Dunn. by High-Speed Counter-Current Chromatography Using Stepwise Elution.

Euchresta tubulosa Dunn. is a Chinese herbal medicine with biological activity, but there are few studies on its components at present. Alkaloids in the stem of Euchresta tubulosa Dunn. were isolated and purified by high-speed counter-current chromatography (HSCCC) using stepwise elution. First of all, liquid-liquid extraction (methylene chloride-methanol-water, 5:1:4, v/v) was used for the preliminary enrichment. According to the partition coefficient (K) of a target compound in a series of different two-phase solvents, the final result was that carbon tetrachloride-methylene chloride-methanol-water (2:3:3:2, v/v) (1) and methylene chloride-methanol-water (5:3:2, v/v) (2) were suitable for the HSCCC using stepwise elution. As a result, the purity was all higher than 93% and matrine (1), oxymatrine (2), N-formyl cytisine (3), and N-acetyl cytisine (4) can be eluted at one time by this mode. Cytisine-type alkaloids were isolated for the first time in this plant. Finally, the applicability of the mode was verified.

Characteristics of Pyrolysis Oil as Renewable Source of Chemical Materials and Alternative Fuel from the Sewage Sludge Treatment

This paper describes the present applied research focused on pyrolysis oil as one of the three products of sewage sludge (SS) thermal treatment processing—microwave assisted pyrolysis (MAP). Specifically, the full-scale MAP unit with 3 kW magnetron and with the frequency of 2.45 GHz at low pressure conditions is in operation at AdMaS Research Centre, Brno University of Technology (AdMaS BUT). The aim of this paper is to present a chemical characterization of obtained pyrolysis oil together with the suitable analytical determination methods. Detailed characterization of pyrolysis oil from SS was realized by precipitation in n-pentane, fractionation of pentane-soluble part by column chromatography on activated silica by stepwise elution and analysis of obtained fractions by comprehensive orthogonal two-dimensional gas chromatography with time-of-flight mass spectrometric detection. Substances from several groups have been identified at the pyrolysis oil from SS sample: alkanes (110 compounds), alkenes and alkynes (184 compounds), cycloalkanes (104 compounds), aromatic and polyaromatic hydrocarbons (646 compounds), further lower numbers of alcohols, aldehydes, esters, carboxylic acids and heterocycles. The paper also presents a comparison of the above mentioned used two chromatographic methods and imply a basic ecotoxicological test—small algal screening.

Purification of therapeutic adenoviruses using laterally-fed membrane chromatography

Viruses are an emerging class of biotherapeutics with the potential to treat cancer, genetic, cardiovascular and other diseases. Their commercial success depends on cost-effective purification processes. Membrane chromatography processes offer great potential for virus purification, however conventional devices are known to suffer from issues related to resolution and sample dilution. Laterally-fed membrane chromatography (LFMC) overcomes these issues by minimizing the dead-volume and establishing a uniform flow distribution pattern within the device. While the efficiency of LFMC has been previously demonstrated for protein purification studies, here we present a direct comparison of the performance of a LFMC device and a conventional radial flow device (both containing 1 mL of the same strong anion-exchange membrane) for the purification of adenoviruses. Using a gradient elution strategy, a 74% virus recovery with 4% of residual amount of total protein and 1% residual amount of total DNA was obtained using the LFMC device; for the radial flow device, only 20% virus recovery was achieved for a similar level of purity. Using a stepwise elution strategy, close to 100% virus recovery with 10% residual protein and DNA was achieved with both devices. However, the product was at least 50% more dilute with the radial flow device. Overall, this study demonstrates the considerable benefits of using LFMC over conventional membrane chromatography devices for purifying virus-based biotherapeutics.

Development of a Quantitative Determination Procedure for Polyphenolic Compounds in Medicinal Plant Raw Material and Medicinal Herbal Preparations Using TLC with Stepwise Elution

A cost-effective and rapid procedure to identify and quantify polyphenolic biologically active compounds of plant origin (with gallic acid, tannin, and quercetin as examples) using high-performance thin-layer chromatography (HPTLC) was developed. The optimal TLC conditions were chosen experimentally and justified theoretically with quantitative interpretation of the HPTLC data on a PC. The proposed method was tested using medicinal plant raw materials such as nettle leaves, common oak bark, and sea buckthorn fruit. The procedure could be used for quality control of drug substances, single-component and complex preparations, plant materials, biologically active additives, premixes, food items, and cosmetics.