Aminex HPX-87H Research Articles

Valorisation of date fruits by-products for the production of biopolymer polyhydroxybutyrate (PHB) using the bacterial strain Bacillus paramycoides

The aim of this research was to study the valorization of the date fruit by-product by conversion of date syrup into biopolymer polyhydroxybutyrate (PHB), based on the metabolic capacity of the bacterial strain Bacillus paramycoides to accumulate PHB from date syrup. Total reducing sugars in date syrup was essayed using 3',5-dinitrosalicylic acid (DNS) and HPLC methods. Bacillus paramycoides was isolated from soil of the botanic garden of Skikda university, Algeria. The accumulated PHB was extracted using chloroform. It was quantified as crotonic acid in concentrated sulfuric acid (H2SO4) by spectrophotometry at 300nm. Date syrup is characterized by high levels of total sugars (79.66 g/L) with 31.86 g/L of total reducing sugars. PHB accumulation reached its maximum (104.3 ug/mL) after 96 h of incubation at pH 7 and temperature 37°C using Tryptophane as the nitrogen source and acid pretreated syrup at a concentration of 8%. HPLC analysis on Aminex HPX-87H showed that the produced PHB from date syrup is characterized by a chromatogram peak with a retention time at 22.5 min.

Potential use of <i>Bacillus paramycoides</i> for the production of the biopolymer polyhydroxybutyrate from leftover carob fruit agro-waste

This study was designed to investigate, at a laboratory scale, the possibility of valorizing the leftover carob fruits to produce the eco-friendly biopolymer polyhydroxybutyrate (PHB) by using the bacterial strain Bacillus paramycoides, which has been isolated from the botanical garden of Skikda University in Algeria. The PHB production was tested under various conditions: a pH of 3–8, temperature range of 30–44 °C, carob extracted molasses concentration of 2–8% v/v, an incubation time of 24–96 h and an agitation speed of 150–300 rpm. The effects of different nitrogen sources and carob extracted molasses treatment types were also investigated. The PHB concentration was determined quantitatively as crotonic acid by measuring the absorbance at 300 nm. Cell growth was quantified by measuring the density of the culture at 600 nm. The presence of PHB was confirmed by applying high-performance liquid chromatography (HPLC) using an Aminex HPX-87H and implementing gas chromatography analysis. The best yield of PHB synthesis was obtained by using 6% v/v of 5 M H2SO4 treated with carob molasses as a carbon source, with peptone as a nitrogen source; incubation was conducted at 37 °C for 96 h at an agitation speed of 300 rpm (114.95 mg/L). The HPLC analysis confirmed the synthesis of PHB by B. paramycoides to have a chromatogram retention time of 22.5 min. Carob waste was successfully valorized to PHB.

Electrochemical Reduction of Carbon Dioxide on Lanthanum Based Transition Metal Oxide Electrocatalysts

Among the various methods available for reduction of CO2, the electrochemical reduction method stands out due to it being capable of producing industrially important fuels, organic chemicals and pure oxygen. Such process takes place in an electrochemical cell, where carbon dioxide is reduced to at room temperature and atmospheric pressure, using only water, carbon dioxide and electricity at specific electrochemical potentials. Here, electrocatalysts have an important role to play, for enhancing the kinetics of the process and also for rendering the formation of desired products feasible at some selected potentials. In the present research, we are exploring the usage of La-based transition metal (TM) oxides, crystallizing in perovskite structure, as electrocatalysts. The synthesis of these metal oxides is cost effective and tuning the performance via doping is facile. Furthermore, these perovskites are stable in stoichiometric, as well as non-stoichiometric, forms and due to the basicity of La exhibits affinity for CO2. Accordingly, here we have investigated the efficiency and effectiveness of different La-based TM oxides, as electrocatalysts, towards the formation of industrially relevant products upon reduction of CO2. It may be mentioned here that, in order to enhance the efficiency of the electrocatalysts, as well as the mass transport of CO2 to the surface of the electrocatalysts, we have designed a new electrolysis cell where gas diffusion electrode (GDE) containing the electrocatalysts is used as working electrode. All the La-based TM oxides (i.e., LaCoO3, LaCrO3, LaFeO3, LaMnO3, LaNiO3) were synthesized via citrate solution-combustion route and calcined in air atmosphere at 850 oC for 6 h, with the heating rate being 10 oC/minute. Phase evolution of the TM oxides before and after usage, as electrocatalysts, were checked using X-ray diffraction (XRD; EMPYREAN PANalytical diffractometer) having Cu Kα radiation and scanning at a rate of 1 o/min between 10 to 90o. Inorganic Crystal Structure Database was used for the phase analysis of the powder catalysts. The composition of the perovskite catalysts (in terms of the ratios of the metals) were quantitatively analyzed by energy dispersive spectroscopy (EDS; QUANTA 200). Morphologies of the oxide particles were observed using scanning electron microscopy (SEM) JEOL JSM-7600F. The specific surface area of the perovskite catalysts were measured via BET using Micromeritics 3 FLEX Surface Characterization. The electrochemical CO2 reduction reaction was performed in an electrolysis cell via chronoamperometry using Biologic potentiostat (Model VSP 300). The reduction reactions were carried out on gas diffusion electrode (GDE) using basic aqueous electrolyte. The applied potentials were -1.1 V, -1.3 V, -1.5 V, -1.7 V, -1.9 V and -2.1 V vs. Hg/HgO, as reference electrode. The chronoamperometry experiments were performed for 3 h at each potential. The current densities obtained during the electrochemical CO2 reduction experiments were obtained for all the La-based TM oxide electrocatalysts used, viz., LaCoO3, LaCrO3, LaFeO3, LaMnO3, LaNiO3. The liquid products present in the electrolyte after the experiments were analyzed by high performance liquid chromatography (HPLC) using Agilent 1260 infinity series model with Aminex HPX-87H column. The gaseous products were analyzed by gas chromatography - thermal conductivity detector (GC-TCD) using Nucon 5700 gas chromatography. XRD scans recorded with the as-synthesized and calcined La-based TM oxides indicate that the oxides were phase pure (as shown in Fig. 1). The crystal structure was established as perovskite. The composition of the as-synthesized and calcined perovskite electrocatalysts, as determined using EDS, were in good agreement with the target compositions. The current densities were fairly stable during the 3 h long experiments for all the potentials studied; with an example being shown in Fig. 2. All the electrocatalysts produced formate (HCOO-), acetate (CH3COO-) and hydrogen as the products of electrochemical CO2 reduction. Furthermore, ethanol (C2H5OH) and 1-propanol (C3H7OH) were also formed on LaCoO3, with LaMnO3 being able to produce 1-propanol (C3H7OH) as one of the products. Hence, as per the present set of results in the context of the formation of desired products, LaCoO3 and LaMnO3 seem to be more promising among the La-TM-oxide electrocatalysts studied here. Keywords: CO2 reduction, transition metal oxide, electrocatalyst, chronoamperometry, reaction product. Figure 1

High Temperature Electrooxidation of Glycerol on Nickel

This work involves the long term electrooxidation of glycerol in a custom autoclave setup. Experiments are usually performed over approximately 48 hours. The custom autoclave setup was adapted from work done by Thomas Holm [1,2]. Within the autoclave a typical three electrode cell is built, wherein all glass pieces have been replaced with alkaline stable counterparts. This is largely done with Teflon, however a Nafion membrane is used to separate the working and reference electrode volumes from the bulk volume at the counter electrode to avoid contamination at the reference, and to keep the oxidation volume small. A small oxidation volume leads to higher concentrations of products, allowing them to be more easily detected using HPLC.Analysis of the oxidized products is performed via HPLC using an Aminex HPX-87H column. Using these techniques we have been able to show that the temperature at which electrooxidation occurs significantly alters the selectivity of products, as well as causing some new products to form (See Figure 1). Calibration curves have been made up for each of these products using known standards, and so we are able to quantitatively determine how much of each product is formed. However, the carbonate peak is not quantitative, and so carbonate was separately analysed by gravimetric or titrimetric methods.With this information we have been attempting to carry out a carbon balance between products and reactants to ensure all products has been accounted for. Though some reasonable values have been attained for this, problems arise when an electron balance is attempted. Much more oxidation is carried out than is expected from the number of electrons used during the experiment, and so we can conclude that some chemical interactions are occurring in solution. It is likely that some products are reacting with the alkaline solution, perhaps through chemical disproproportionation reactions. The existance of such reactions can be confirmed for glyceraldehyde, which has been shown to exist on a short time scale using PM-IRRAS [3] even though no glyceraldehyde is detected by HPLC. We find that HPLC glyceraldehyde standards decompose into other compounds that we see here. Work is ongoing to establish the identity and distribution of products as a function of the reaction conditions. This research was conducted as part of the Engineered Nickel Catalysts for Electrochemical Clean Energy project administered from Queen’s University and supported by Grant No. RGPNM 477963-2015 under the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Frontiers Program. 1. T. Holm, P.K. Dahlstrøm, S. Sunde, D. A. Harrington, F. Seland, ECS Trans, 75, 1055, (2016).2. T. Holm, P.K. Dahlstrøm, O.S. Burheim, S. Sunde, D.A. Harrington, and F. Seland, Electrochim. Acta., 222, 1792 (2016).3. M.S.E. Houache, E. Cossar, S. Ntais, E.A. Baranova, J. Power Sources, on the web, doi:10.1016/j.jpowsour.2017.08.089. Figure 1

Method for the Determination of Benzene Metabolite t,t-Muconic Acid in Urine by HPLC-UV with an Ion Exclusion Column

The benzene metabolite trans,trans-muconic acid (tt-MA) is widely used as a biological indicator of exposure to this xenobiotic. An analytical method was developed and validated for the determination of urinary tt-MA using solid phase extraction onto a Strata SAX cartridge. The analysis was performed by HPLC with an Aminex HPX-87H ion exclusion column and a UV diode array detector. The sample preparation conditions were optimized using a 24−1 factorial design. Comparison of the slopes of standard analytical curves prepared in aqueous solution and urine showed that the biological matrix suppressed the tt-MA signal by around 50%, so the analytical curves were prepared with tt-MA standard at low concentrations in pooled urine. The analytical curves in the range of 5–500 μg·L−1 showed determination coefficients values (R2) > 0.99 for tt-MA standard in water and R2 > 0.98 for tt-MA standards in pooled urine. The coefficients of variation obtained using seven replicates were lower than 3.6%, and recoveries of tt-MA from solutions containing 5, 25, 50 and 100 μg·L−1 of the analyte were in the range 85%–90%, demonstrating the satisfactory precision and accuracy of the method. The limits of detection and quantification were 0.11 and 0.36 µg ·L−1, respectively. The benefits of this new method developed is the possibility of complete chromatographic peak separation for the determination of tt-MA at baseline, without matrix components’ interference as normally found in the C18 column. This is the first time that this chromatographic column has been used for the analysis of tt-MA in urine.

Simultaneous analysis of carbohydrates and organic acids by HPLC-DAD-RI for monitoring goat's milk yogurts fermentation

During yogurt manufacture, the lactose fermentation and organic acid production can be used to monitor the fermentation process by starter cultures and probiotic bacteria. In the present work, a simple, sensitive and reproducible high-performance liquid chromatography with dual detectors, diode array detector and refractive index was validated by simultaneous analysis of carbohydrates and organic acids in goat milk yogurts. In addition, pH and bacterial analysis were performed. Separation of all the compounds was performed on an Aminex HPX-87H column (300×7.8mm, 9µm) utilizing a 3mmolL−1 sulfuric acid aqueous mobile phase under isocratic conditions. Lactose, glucose, galactose, citric, lactic and formic acids were used to evaluate the following performance parameters: selectivity, linearity, precision, limit of detection (LOD), limit of quantification (LOQ), decision limits (CCα), detection capabilities (CCβ), recovery and robustness. For the method application a six goat milk yogurts were elaborated: natural, probiotic, prebiotic, symbiotic, cupuassu fruit pulp, and probiotic with cupuassu fruit pulp. The validated method presented an excellent selectivity with no significant matrix effect, and a broad linear study range with coefficients of determination higher than 0.995. The relative standard deviation was lower than 10% under repeatability and within-laboratory reproducibility conditions for the studied analytes. The LOD of the method was defined from 0.001 to 0.003µgg−1, and the LOQ from 0.003 to 0.013µgg−1. The CCα was ranged from 0.032 to 0.943µgg−1, and the CCβ from 0.053 to 1.604µgg−1. The obtained recovery values were from 78% to 119%. In addition, the method exhibited an appropriate robustness for all parameter evaluated. Base in our data, it was concluded that the performance parameters demonstrated total method adequacy for the detection and quantification of carbohydrates and organic acids in goat milk yogurts. The application of the method was successfully applied to monitoring different goat milk yogurts during fermentation.

Fermentative biohydrogen production using hemicellulose fractions: Analytical validation for C5 and C6-sugars, acids and inhibitors by HPLC

Hemicellulose fractions derived from lignocellulosic biomass acid pretreatments have been considered promising substrates for H2 production via anaerobic fermentation. In this context, the objective of this study is to develop a high-performance liquid chromatography (HPLC) methodology for the simultaneous determination of glucose, xylose, arabinose, acetic acid, butyric acid, 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde (furfural) in the complex matrices used for the H2 production from hemicellulose fractions. HPLC analyses were performed using two columns under different conditions: the Aminex HPX-87H column (mobile phase consisted of 0.005 mol L−1 H2SO4 solution at a flow rate of 0.7 mL min−1 and temperature of 60 °C) and the Rezex RFQ Fast acid (mobile phase consisted of 0.005 mol L−1 H2SO4 solution at a flow rate of 1.0 mL min−1 and temperature of 80 °C). Acids (acetic and butyric acids) and inhibitors (HMF and furfural) were analyzed by UV–Vis detection at 210 nm, and C6 and C5-sugars (glucose, xylose and arabinose) were analyzed using a refractive index detector (RI). Some validation parameters, such as selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and robustness were successfully evaluated. The monitoring of fermentative media for H2 production from different hemicellulose fractions was successfully performed using the proposed methodologies. The acid-pretreated samples from corn cob, cocoa husk, sugarcane straw and bagasse substrates presented a range of xylose contents from 84.85 to 222.98 mmol L−1, which resulted in a maximum production of 2.99 mol H2/mol total carbohydrate.

Part I: the influence of serial repitching ofSaccharomyces pastorianuson the uptake dynamics of metal ions and fermentable carbohydrates during the fermentation of barley and gluten-free buckwheat and quinoa wort

Recently, research has been focusing on the use of alternative raw materials for brewing purposes and gluten-free beer-like beverages from malted buckwheat and quinoa are of commercial interest. A common commercial process involves the serial repitching of the yeast biomass, but this has not been described using buckwheat and quinoa wort fermentations. Our research studies (Parts I–III) explored the serial repitching of the yeast strain Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat and quinoa fermentation medium. The present paper focuses on the fermentation performance and the uptake dynamics of metal ions and fermentable carbohydrates. Both pseudocereals showed high variations in all of the attributes examined during successive fermentations. In buckwheat the differences between successive fermentations were similar to those observed with barley, whereas differences in quinoa varied quite significantly from those observed with barley and showed a directional trend, suggesting a general weakening of the yeast from the sixth successive fermentation onward. In particular, the assimilation of the fermentable carbohydrates lessened and metal ion uptake appeared poorly controlled. It was concluded that buckwheat showed good potential for serial repitching of S. pastorianus TUM 34/70, whereas serial repitching of a quinoa wort appeared to be limited to five or six fermentations. Copyright © 2015 The Institute of Brewing & Distilling

Does size matter? Separations on guard columns for fast sample analysis applied to bioenergy research.

BackgroundIncreasing sample throughput is needed when large numbers of samples have to be processed. In chromatography, one strategy is to reduce column length for decreased analysis time. Therefore, the feasibility of analyzing samples simply on a guard column was explored using refractive index and ultraviolet detection. Results from the guard columns were compared to the analyses using the standard 300 mm Aminex HPX-87H column which is widely applied to the analysis of samples from many biotechnology- and bioenergy-related experiments such as biomass conversions or fermentations.ResultsThe 50 mm Rezex RFQ Fast Acid H+ guard column was able to separate the most common fermentation products (ethanol, acetone, iso- and n-butanol) and promising precursors (furfural and 5-hydroxymethylfurfural) of biofuels and value-added chemicals. Compound profiles in fermentation samples were analyzed with similar accuracy compared to results using the 300 mm column. However, separation of glucose and xylose was not achieved. Nevertheless, it was possible to monitor the consumption of one of the two sugars during fermentation if the other one was absent or remained constant over the course of the experiment. If correct peak integration and interference subtraction was applied, concentration profiles from enzymatic digestibility experiments and even more complex samples (e.g. acetone-butanol-ethanol (ABE) fermentation) were reliably obtained. With the 50 mm guard column, samples were analyzed up to ten-times faster compared to the 300 mm column. A further decrease in analysis time was achieved by using the 30 mm Micro Guard Cation H guard column. This column is especially suitable for the rapid analysis of compounds with long elution times on the standard 300 mm column, such as biofuel-related alcohols (e.g., n-butanol, n-hexanol) and furan- and tetrahydrofuran-type molecules.ConclusionApplied to a suitable set of samples, separations on a guard column can give rapid and sufficiently accurate information on compound changes over the course of an experiment. Therefore, it is an inexpensive and ideal tool for processing a large amount of samples, such as in screening or discovery experiments, where detecting relative changes is often sufficient to identify promising candidates for further analysis.

Determination of ultra-trace organic acids in Masson pine (Pinus massoniana L.) by accelerated solvent extraction and liquid chromatography-tandem mass spectrometry.

An accelerated solvent extraction (ASE)-solid-phase extraction (SPE)-liquid chromatography with electrospray ionization-tandem mass spectrometry (ASE-SPE-LC-ESI-MS/MS) methodology was developed for the extraction, cleanup and quantification of ultra-trace organic acids in Masson pine (Pinus massoniana L.) tissues. The separation was carried out on a Bio-Rad Aminex HPX-87H sulfonic column with an eluent containing 5 mmol L(-1) H₂SO₄ at a flow rate of 0.5 mL min(-1). A linear ion trap mass spectrometer equipped with electrospray ionization (ESI) source was operated in negative ion mode, and the six organic acids were eluted within 20 min. ASE extraction, SPE cleanup and LC-ESI-MS/MS analysis conditions were optimized to obtain reliable information about plant organic acid composition. Selective reaction monitoring (SRM) was employed for quantitative measurement. Intra-day precisions averaged 6.7%, and inter-day precisions were 2.1-10.7% for organic acid measurements in the pine samples. External standard calibration curves were linear over the range of 16.5-5000 ng L(-1), and detection limits based on a signal-to-noise ratio of three were at 0.5-5.0 ng L(-1). The results obtained showed the sensibility of the method was better than that of previously described HPLC methodology, and had no significant matrix effect. The proposed ASE-SPE-LC-ESI-MS/MS method is sensitive and reliable for the determination of ultra-trace organic acids in plant samples, despite the presence of the particularly complex matrix.

Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose in tubers of Solanum tuberosum Group Phureja.

A High Performance Liquid Chromatography (HPLC) method was developed and validated to quantify sucrose (non-reducing sugar), glucose, and fructose (reducing sugars) in raw tubers of Solanum tuberosum Group Phureja. Chromatographic analysis was performed using an AMINEX HPX 87H column, at 18 °C, linked to a refraction index detector, at 35 °C. The eluent was 10mM sulfuric acid. The conditions established for the method provided an optimum separation of sugars, citric acid, and malic acid, with resolution values higher or equal to one. Among the four sugar extraction methods tested, the double 50% (v/v) aqueous methanol extraction gave the highest level of analytes. Recovery of this extraction method ranged between 94.14 and 99.77%. The HPLC method was validated for repeatability, reproducibility, linearity, and limits of detection, and quantification. Relative standard deviation was found to be lower than five, when testing repeatability and reproducibility, which is suitable considering a range of acceptability from 5.3 to 7.3. Additionally, the regression analyses supported the method linearity in a range of quantification from 3 to 100 mg/L with regression coefficients values greater than 0.998 for the three analytes. Limits of detection were 3.0 mg/L for the three sugars and limits of quantification were 2.0 mg/L for sucrose and 3.0 mg/L for glucose and fructose. Four Colombian commercial cultivars (Criolla Guaneña, Criolla Paisa, Criolla Galeras, and Criolla Colombia) and five landrace accessions from the Colombian Core Collection of Group Phureja were grown in the district of Usme (Bogotá) fields to analyze their sugar contents. Sucrose, glucose, and fructose contents were found ranging from 0.93 to 3.11 g/100 g tuber dried weight (DW), from 0.25 to 4.53 g/100 g tuber DW, and from 0.10 to 1.49 g/100 g tuber DW, respectively. Therefore, a high range in the variability of sugar contents was found among genotypes. However, the variability was low among technical replicates of the same genotype, revealing an accurate quantification of sugars in Group Phureja. This method can be used to assess the amount of reducing and non-reducing sugars accumulation in potato germplasm.

Effects of different nitrogen sources on the production of Hyaluronic acid by Streptococcus

Hyaluronic acid (HA) is a linear polysaccharide with high molecular weight composed of disaccharide units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). HA is naturally present in vertebrate organisms, as well as in bacteria. HA can be obtained commercially through three routes: human umbilical cords, rooster combs, and strains of group C Streptococcus .T his is an atural polysaccharide with extensive range of applications in the medical, pharmaceutical and cosmetics. Due to the viscoelastic and hydrophilic, the HA products and derivatives present high aggregated value ranging from US$ 2000 to 60000 Kg -1 , depending on their applications. The most frequently used bacteria in the industrial production of HA are Lancefield group A and C streptococci. These bacteria are nutritionally fastidious microorganisms which require complex nutrients due to their limited ability to synthesize specific aminoacids and B-vitamins. Additionally, there is the nutritional requirement with respect to organic nitrogen, which also supplies a large portion of carbon for their cellular biosynthesis. Recent studies seek alternatives to allow the cost of production of HA using agricultural derivatives and industrial waste. In this context, the objective of this study was to evaluate the effect of nitrogen sources yeast extract, soy protein, whey protein and corn steep liquor in the production of HA. Fermentations were carried out in 125 mL erlenmeyer flasks containing 25 mL culture medium. The culture medium comprised in ( gL -1 ): glucose, 30; nitrogen source (yeast extract, soy protein, whey protein or corn steep liquor), 30; K2HPO4 ,2 .5, NaCl, 2,0 and MgSO4 ,1 .5. The inoculum was 10% (v/v) and the fermentations occurred at 100 rpm, 37°C and pH 8.0 for 24 hours. The fermented medium was centrifuged and the cell free supernatant was treated with ethanol for the precipitation of HA which quantified using a colorimetric reagent Carbazole. The concentration of lactic acid, acetic acid and formic acid were also analyzed in a system of high performance liquid chromatography with IR detector, column Aminex HPX 87H at 60°C and a solution of H2SO4 0.005 mol L -1 as the mobile phase a flow rate of 0.7 mL min -1 . The results performed in triplicate were compared by the Tukey test at 5% probability level (p < 0.05). The highest production of HA, 0.534 g L -1 was obtained when using yeast extract as nitrogen source. Subsequently, experiments which resulted in a better yield of the polymer are those containing soybean protein (0.192 g.L -1 ) and whey protein ( 0.063 g.L -1 ). In medium containing corn steep liquor, no microbial growth or production of HA ocurred. The decreased production of HA was directly followed by a reduction of the production of lactic acid and acetic acid.

Simultaneous determination of organic acids and saccharides in lactic acid fermentation broth from biomass using high performance liquid chromatography

Abstract: A high performance liquid chromatographic method for the simultaneous determination of organic acids and saccharides in lactic acid fermentation broth from biomass was developed. A Bio-Rad Aminex HPX-87H column was used at 55 degrees C. The mobile phase was 5 mmol/L sulfuric acid solution at a flow rate of 0.6 mL/min. The samples were detected by a refractive index detector (RID). The results showed that six organic acids and three saccharides in fermentation broth were completely separated and determined in 17 min. The linear correlation coefficients were above 0.999 8 in the range of 0.15-5.19 g/L. Under the optimized conditions, the recoveries of the organic acids and saccharides in Rhizopus oryzae fermentation broth at two spiked levels were in the range of 96.91%-103.11% with the relative standard deviations (RSDs, n = 6) of 0.81%-4.61%. This method is fast and accurate for the quantitative analysis of the organic acids and saccharides in microbial fermentation broths.

Determination of Heparin in Plasma by HPLC Coupled with Resonance Light Scattering Detection

High performance liquid chromatography coupled with resonance light scattering detection was developed for separation and determination of heparin in plasma. A good chromatographic separation was achieved using an aminex HPX-87H column (300 mm × 7.8 mm, 9 μm) and a mobile phase of 5.0 mmol L−1 H2SO4 at the flow rate of 0.5 mL min−1. The enhanced resonance light scattering signals were derived from a large aggregate formation between heparin and cetyltrimethylammonium bromide used as the molecular recognition probe. The parameters of the post-column reaction (pH, concentration and flow rate of the reagent, length of the reaction coil, and temperature) were optimized. The limit of detection for heparin in plasma was 0.2 mg L−1. The method has been applied to the determination of heparin in dialysis patient plasmas.

Simultaneous Quantification of Carbohydrates, Alcohols, and Toxic Components in a Bio-Based Medium Using Dual-Detection HPLC Analysis

Bio-based media, derived from cellulosic biomass depolymerisation or bioconversion processes, are composed of several chemicals and biochemicals. In this study, the main components of a hemicellulosic-based medium were analyzed using a dual detection HPLC method to separate and determine concentrations of the major monosaccharides (glucose, xylose, and arabinose), alcoholic and acidic components (ethanol, xylitol, and acetic acid) and furanic compounds (furfural and hydroxymethylfurfural (HMF)). The analyses, which employed a single stationary phase (Aminex HPX-87H) and two detection methods, were carried out under isocratic conditions and involved mobile phases consisting of 5 mM sulfuric acid and acetonitrile in different mix ratios from 0 to 0.061 mole fraction of acetonitrile. Based on the analysis run time and the chromatogram quality, the optimum condition was determined for the simultaneous quantification of the components.

Analysis of Glyoxal and Related Substances by Means of High-Performance Liquid Chromatography with Refractive Index Detection

A simple and rapid method is described for the analysis of glyoxal and related substances by high-performance liquid chromatography with a refractive index detector. The following chromatographic conditions were adopted: Aminex HPX-87H column, mobile phase consisting of 0.01N H2SO4, flow rate of 0.8 mL/min and temperature of 65°C. The application of the analytical technique developed in this study demonstrated that the aqueous reaction mixture produced by the oxidation of acetaldehyde with HNO3 was composed of glyoxal, acetaldehyde, acetic acid, formic acid, glyoxylic acid, oxalic acid, butanedione and glycolic acid. The method was validated by evaluating analytical parameters such as linearity, limits of detection and quantification, precision, recovery and robustness. The proposed methodology was successfully applied to the production of glyoxal.

Corrigendum to “Enhancement of the selectivity to dihydroxyacetone in glycerol oxidation using gold nanoparticles supported on carbon nanotubes” [CATCOM 16/1 (2011) 64–69

Inthe paper mentioned above, recently publishedin Catalysis Com-munication [1], we reported about the utilization of carbon nanotubes(MWCNT) as gold support for the catalytic oxidation of glycerol underbasic conditions. According to the paper, this catalyst would lead to ahigher selectivity to dihydroxyacetone (60%) when compared withAu/AC catalysts (18%).The quantitative analysis of the mixture was carried out by highperformance liquid chromatography (HLPC) and the compoundswere identified by comparison with standard samples. Reactant andproducts were separated with an ion exclusion column (Alltech OA1000), which is commonly used in this type of work [2–4].Dihydroxyacetone(DIHA)isareactionproductmentionedinthelit-erature by different groups even under basic conditions [4–8].Underourexperimentalconditions,theretentiontimeofthiscompoundcoin-cided exactly with one of the peaks obtained from the HPLC analysisof the reaction mixture. Therefore, in the chromatogram, this peakwas identified as DIHA. Moreover, the selectivity obtained with goldsupportedonactivatedcarbonwassimilartothatreportedbyotherau-thors, which apparently confirmed our results [9].Unfortunately, we found recently that formic acid (FORMA) was er-roneously identified as dihydroxyacetone at the time the article waspublished. This was due to the fact that both compounds have thesame retention time in the column mentioned above. Some tests werecarried out using another column (BIO-RAD Aminex HPX-87H) wherepartial separation of standard sampl es of formic acid and dihydroxyace-tone was achieved. The analysis of the reaction mixtures in this columnallowedustoconcludethattheproductinitiallyidenti fiedasdihydroxy-acetonewasinfactformicacid.Thislattercompoundisnotwidelymen-tioned as a product of glycerol oxidation, especially when conventionalcarbon materials are used as support. However, an exception wasreported recently by Prati et al. [10], when testing gold supported oncarbon nanofibers, which lead to selectivities to FORMA as high as 30%.The origin of formic acid is still under discussion. This compound isprobably obtained via the base catalyzed degradation of dihydroxyace-tone[11].Therefore,webelievethatthehighformicacidselectivityob-served in this work is still due to the peculiar porous characteristic ofmulti-walled carbon nanotubes, which may favor the initial formationof DIHA, as mentioned both in our article [1] and in the work of Pratietal.[10].Accordingly,wesuggestthatglycerolcanbeinitiallyoxidizedto DIHA; this compound acts as an intermediate prone to degradationdue to the highly basic medium used, and a fraction could be readilyconverted into FORMA, whereas the remaining is further oxidized, viaglyceraldehyde, in agreement with Fig. 1 of reference [1].Consequently, some of the results and conclusions in [1] must bemodified. Table 1 summarizes the most relevant changes.In conclusion, under basic conditions, gold supported on activatedcarbon is preferable in order to obtain a high selectivity towardsglyceric acid, product of commercial interest, and no dihydroxyace-tone is formed.References

Ion-Exclusion Chromatography Determination of Organic Acid in Uridine 5'-Monophosphate Fermentation Broth

Simultaneous determination of organic acids using ion-exclusion liquid chromatography and ultraviolet detection is described. The chromatographic conditions are optimized when an Aminex HPX-87H column (300 × 7.8 mm) is employed, with a solution of 3 mmol/L sulfuric acid as eluent, a flow rate of 0.4 mL/min and a column temperature of 60°C. Eight organic acids (including orotic acid, α-ketoglutaric acid, citric acid, pyruvic acid, malic acid, succinic acid, lactic acid and acetic acid) and one nucleotide are successfully quantified. The calibration curves for these analytes are linear, with correlation coefficients exceeding 0.999. The average recovery of organic acids is in the range of 97.6% ∼ 103.1%, and the relative standard deviation is in the range of 0.037% ∼ 0.38%. The method is subsequently applied to obtain organic acid profiles of uridine 5'-monophosphate culture broth fermented from orotic acid by Saccharomyces cerevisiae. These data demonstrate the quantitative accuracy for nucleotide fermentation mixtures, and suggest that the method may also be applicable to other biological samples.

Quantification of glucose, xylose, arabinose, furfural, and HMF in corncob hydrolysate by HPLC-PDA–ELSD

Lignocellulose and other carbohydrates are being studied extensively as potential renewable carbon sources for liquid biofuels and other valuable chemicals. In the present study, a simple, sensitive, selective, and reliable HPLC method using a photodiode array (PDA) detector and an evaporative light scattering detector (ELSD) was developed for the simultaneous determination of important sugars (d(+)-cellobiose, glucose, xylose, and arabinose), furfural and 5-hydroxymethylfurfural (5-HMF) in lignocellulose hydrolysate. The analysis was carried out on an Aminex HPX-87H column (250mm×4.6mm, 5μm particle size). Ultra-pure water with 0.00035M H2SO4 was used as the mobile phase with a flow rate of 0.6mL/min. The temperature of the ELSD drift tube was kept at 50°C, the carrier gas pressure was 350kPa, and the gain was set at 7. Furfural and 5-HMF were quantified on a PDA detector at 275nm and 284nm, respectively. The sugar concentrations were determined by ELSD. This method was validated for accuracy and precision. The regression equation revealed a good linear relationship (r2=0.9986±0.0012) within the test ranges. The method showed good reproducibility for the quantification of six analytes in corncob hydrolysate, with intra- and inter-day variations less than 1.12%. This method is also convenient because it allows the rapid analysis of the primary products of biomass hydrolysis and carbohydrate degradation.

Optimisation of an HPLC method for the simultaneous quantification of the major sugars and organic acids in grapevine berries

A high performance liquid chromatographic method was developed to profile major sugars and organic acids in grapevine berries. Sugars and organic acids in grapevine berries were extracted by chloroform/polyvinylpolypyrrolidone purification. The extracts were chromatographed on an Aminex HPX-87H ion-exchange HPLC column with 5mM sulphuric acid as mobile phase. Chromatography was visualised via a diode array detector combined with a refractive index detector. The analysis was calibrated using external standard calibration and a novel equation was used to calculate the concentrations of malic acid and fructose from unresolved separation. For the method to be utilised for analysing a large numbers of berry samples, each sample was directly injected after sample extraction and the extraction step was downscaled to allow the use of small amounts of sample material. The concentrations of sugars and organic acids in grapevine berry samples were normalised to the internal standard concentrations obtained after extraction of an internal standard mixture. The analysis method exhibits a good precision and a high analyte recovery from samples spiked with the standard mixture and is suitable for the profiling of major sugars and organic acids in grapevine berry samples at different stages of berry development. This is the first report on the combined profiling of the major sugars and organic acids in grapevine berries using milligram amounts of plant material with direct injection after sample extraction.