High-performance Liquid Chromatography-refractive Index Research Articles

Assessment of mineral and sugar contents of Plectranthus edulis landraces

ABSTRACT The Ethiopian potato [Plectranthus edulis (Vatke) Agnew] is an annual root tuber that is underutilized due to a lack of knowledge about its nutritional value. The plant is known by its local landrace names. Investigating the relatedness, and variation in nutrient composition, among P. edulis landraces is important for selecting the best genotype for breeding. The study was undertaken to examine mineral nutrients and sugars in 5 P. edulis landraces collected from southern Ethiopia. Samples were analyzed for Ca, Mg, Fe, Zn, Cu, Mn, Co, Cr Ni, Cd, Pb, and fructose, glucose, sucrose, and maltose using Inductively Coupled Plasma-Optical Emission Spectrometry and a High-Performance Liquid Chromatography-Refractive Index Detector, respectively. The P. edulis tubers accumulate large levels of macro- and micro-elements. The Mg and Ca were the most abundant macro-nutrients. The Tseka landrace had the highest Mg content. Iron was the most abundant micro-nutrient in P. edulis tubers. There were no Co, Cd, and Pb detected in P. edulis tuber landraces. All P. edulis landraces contained glucose (15.1–21.1 g/100 g) and fructose (3.34–4.10 g/100 g); sucrose and maltose were not detected. Quantities of all parameters were affected by landraces and their concentrations were different. The unnuko landrace had a lower total sugar level than the other 4 types, making it a good choice for diabetics. The P. edulis landraces can provide minerals and sugar for the human diet. Diversity in nutrient content within landraces could be an opportunity to generate improved hybrids.

Paper Waste Management: Extraction of Fermentable Sugar from Lignocellulosic Waste Paper

The utilization of paper on a commercial scale is increasing day by day throughout the world that produces million of tons of paper waste yearly and burdened for landfills. The present study focuses on the exploitation of waste papers (office paper, newspaper and tissue paper) as a cheapest alternative source of energy to extract fermentable sugar by applying chemical and enzymatic pretreatments. The quantification of released sugar was analyzed by spectrophotometer and high performance liquid chromatography refractive index (HPLC-RI) detector. Cellulose (12 FPU/g) and β-glucosidase (12 FPU/g) was found to be effective for the extraction of fermentable sugar from paper waste. The contents of cellulose (C6H10O5)n, hemicellulose (C5H10O5)n and lignin (C9H10O2,C10H12O3,C11H14O4) found in office paper were 40%, 32.5%, 22.5%, in newspaper 46.5%, 30.5% 22.5%, and in tissue paper 62%, 22%, 15.5%, respectively. The percentages of sugar contents assessed in this study were 62% in tissue paper and 46.5% in newspaper and 40% in office papers. Among the three substrates, tissue paper (23.4 mg/mL) released a significant amount of glucose (C6H6O12), whereas newspaper (20.8 mg/mL) and office paper (19.6 mg/mL) released less amount of sugar. This research of acid pre-treatment and enzymatic hydrolysis was an efficient method to improve glucose conversion from lignocellulosic waste. Furthermore, this approach can be proved the first step towards the sustainable production of bioethanol from wastepaper-extracted sugar.

Extraction of soluble sugars from green coffee beans using hot water and quantification by a chromatographic method without an organic solvent

An optimal condition for extraction of soluble sugars from green coffee using water and a validated chromatographic method for its separation and quantification were proposed in this research. An orbital incubator shaker (OIS) and microwave-assisted extraction (MAE) were the 2 techniques used to extract soluble sugars. In such experiments, the variables: sample amount (300, 400, and 500 mg), time (30, 60, and 90 min), and temperature (30, 45, and 60 °C) were tested. The separation of sugars was performed in a chromatographic system (high-performance liquid chromatography refractive index detector [HPLC-RID]), which presented the selectivity for the analytes, a limit of detection of 0.020 g/L, a limit of quantification of 0.0625 g/L, and recovery rates greater than 95%. The repeatability and inter-day precision had low dispersion, RSD < 2.0% and < 3.0%, respectively. Sucrose content ranged from 0.65 to 2.39 g/L using an OIS and from 1.19 to 2.72 g/L by MAE, while glucose and fructose concentration varied from 0.08 to 0.12 g/L using both methods. The OIS technique is preferably indicated for extraction of soluble sugars at the following conditions: 500 mg of grounded green coffee, 90 min, and 60 °C. The proposed method for soluble sugar extraction and quantification may be applied in research laboratories and food industries since it is a low-cost and environment-friendly technique.

Rapid detection of sucrose adulteration in honey using Fourier transform infrared spectroscopy

Honey authenticity is of great importance for commercial and health reasons. The most common adulteration methods are overfeeding of bees with sugar or by adding sucrose. In the study, a Fourier transform infrared spectroscopy based on chemometric method, a partial least squares, for determination of sucrose sirup adulteration of Turkish honeys was improved. For this purpose, honey samples were adulterated with known amounts of sucrose sirup in concentrations ranging between 5.94 and 27.80%. A calibration curve was constructed within 992–923 cm−1 with the regression coefficient of 0.999. Obtained results from the spectra were confirmed by high performance liquid chromatography-refractive index detector and a good correlation was found between the results of the spectroscopic and chromatographic methods. Additionally, some method validation parameters (linearity, repeatability, limit of detection, and quantification) of the spectroscopic method were studied and obtained results were compared with chromatographic method. To prove the usefulness of the method, twenty different honey samples were collected from markets in Turkey and analyzed for sucrose contents. The results indicated that predicted sucrose concentration of honey samples by the spectroscopic method ranged between 4.52 and 15.16% and results were confirmed by the chromatographic method with high correlation (correlation coefficient = 92%). The results of the present indicated showed that adulteration ratio of honey samples by sugar addition can be detected using Fourier transform infrared spectroscopy technique in a short time with high accuracy. In conclusion, this method could be regarded as a new alternative technique for routine analysis in quality control of honey samples.

Hydrodearomatization of Distillates and Heavy Naphtha over a Precious Metal Hydrogenation Catalyst and the Determination of Low Aromatic Content

Because of new regulations on the allowable aromatic content in petroleum products, the saturation of aromatics in distillates and heavy naphtha is gaining more attention. Catalytic hydrodearomatization (HDA) improves the qualities and properties of distillates for health, food, and pharmaceutical applications. In this research, the aromatic content of the four different middle distillate fractions varies from 10 to 12% volume. The goal is to saturate the aromatics to <0.1% volume at pressures, temperatures, and space velocities that are optimized for the lowest operating cost and longest catalyst life. Additionally, this study aims to determine if HDA is more favorable for processing the individual distillate cuts or the comingled products. This study also utilizes a modified high-performance liquid chromatography refractive index (HPLC-RI) method in which an ultraviolet (UV) detector is used to measure low levels of aromatics. A combination of UV and HPLC techniques was able to quantify aromatic levels as low as 0.03% v/v and 0.01% v/v, respectively.

Characterization and differentiation of botanical and geographical origin of selected popular sweet cherry cultivars grown in Greece

Four sweet cherry cultivars grown in northern Greece (Kordia, Regina, Skeena, Mpakirtzeika) were characterized and differentiated according to both botanical and geographical origin (Skeena cultivar). Conventional quality parameter analyses applied to achieve differentiation included: a) conventional quality parameters namely, titratable acidity (TA), pH and total soluble solids (TSS) measurement of mechanical properties-penetration, b) analysis of glucose and fructose (major sugars) using High Performance Liquid Chromatography-Refractive Index Detector (HPLC-RID), c) identification and semi-quantification of volatile compounds using Solid Phase Micro Extraction in combination with Gas Chromatography/Mass Spectrometry (SPME-GC/MS) and d) analysis of minerals using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). Data were analysed using Multivariate Analysis of Variance (MANOVA) and Linear Discriminant Analysis (LDA). Results showed that the combination of volatile compounds-conventional properties-minerals provided the highest correct prediction rate of 97.4% for botanical differentiation while for the geographical differentiation of the Skeena cultivar, conventional parameters and volatile compounds provided a 94.9% and 95.0% correct prediction rate respectively.

Preparation and evaluation of a new type of amino-bonded silica chromatographic stationary phase for the determination of lactose

A method for the separation and determination of lactose and sucrose using high performance liquid chromatography-refractive index (HPLC-RI) detection base on the prepared amino-bonded silica gel chromatographic stationary phase was established. The method was accorded to the content determination method in Pharmacopoeia of the People's Republic of China (2015). Then, a series of chromatographic behaviors of lactose and sucrose such as the retention times, resolutions and peak area stabilities were investigated on three different types of amino-bonded silica columns (300 mm×4.6 mm, 5 μm). The mobile phase was acetonitrile-water (70:30, v/v) with isocratic elution, the flow rate was 1.0 mL/min and the injection volume was 10 μL. The resolution of lactose and sucrose was 3.03 by isopropyl side chain protected amino column, and the peak shape of each target compound was good. The relative standard deviation (RSD) of peak area of lactose was 1.14%, less than 2.0% prescribed in the Pharmacopoeia of the People's Republic of China (2015). Therefore, this method meets the requirements of lactose content determination, and is suitable as the quality control method in the above mentioned Pharmacopoeia.

Development and application of HPLC-RI and HPLC-MS/MS based methods for quantification of residual deoxycholate levels in pneumococcal polysaccharides

The analysis of residual sodium deoxycholate (DOC); a detergent of biological origin used in manufacturing of polysaccharide vaccines is challenging due to complex sample matrices and the lack of suitable methods. Here we report, rapid and sensitive high-performance liquid chromatography-refractive index (HPLC-RI) and tandem mass spectrometry (HPLC-MS/MS) methods for estimation of residual DOC in pneumococcal polysaccharides. For HPLC-RI method, separation was achieved using Luna C18 column and mobile phase compositions of acetonitrile: methanol: 20 mM sodium acetate (60:05:35% v/v). For HPLC-MS/MS method, separation was achieved using a Hypersil BDS C18 column with gradient elution of methanol and water (0.1% formic acid). MS/MS method showed linearity (r2 = 0.997) over the range of 10–320 ng/mL with limits of detection (LOD) and lower limit of quantitation (LOQ) of 3 and 10 ng/mL respectively. Precision (% RSD) and accuracy (% recovery) for both methods were in the range of 0.74–8.29% and 82.33–117.86% respectively. Sample matrices interferences were addressed following novel sample clean-up method based on liquid–liquid extraction. Both methods enabled traceable quantitation of DOC in intermediate and purified pneumococcal polysaccharides of serotypes: 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F.

Rapid Simultaneous Analysis for Three Sugars Fraction in Enzymatic Degradation of Stachyose

The objective of the present study was to develop an effective method of High Performance Liquid Chromatography-Refractive Index Detector (HPLC-RID) for the simultaneous quantification of stachyose, raffinose and galactose in enzymatic degradation of stachyose. The three sugars were separated on an Agilent zorbax carbohydrate column with acetonitrile water (75:25 v/v). The calibration curves of the three standards exhibited good linearity (R 2 >0.9989). The Limits of Detection (LOD) values of the stachyose, raffinose and galactose were 0.137, 0.301 and 0.386 μg/mL, respectively and their Limits of Quantification (LOQ) values were 0.452, 0.993 and 1.25 μg/mL, respectively. The Relative Standard Deviations (RSDs) of the data of the intra- and inter-day experiments were less than 2.68, 2.42 and 3.70%, respectively. The results of the recovery test were 97.71-102.61% with an RSD value of 0.92-2.41%. In conclusion, the results of validation showed that the proposed HPLC-RID method was useful for the quantification of the three sugars in enzymatic degradation of stachyose and similar reaction.

Microwave assisted extraction–solid phase extraction for high-efficient and rapid analysis of monosaccharides in plants

Monosaccharides are the fundamental composition units of saccharides which are a common source of energy for metabolism. An effective and simple method consisting of microwave assisted extraction (MAE), solid phase extraction (SPE) and high performance liquid chromatography-refractive index detector (HPLC-RID) was developed for rapid detection of monosaccharides in plants. The MAE was applied to break down the structure of the plant cells and release the monosaccharides, while the SPE procedure was adopted to purify the extract before analysis. Finally, the HPLC-RID was employed to separate and analyze the monosaccharides with amino column. As a result, the extraction time was reduced to 17min, which was nearly 85 times faster than soxhlet extraction. The recoveries of arabinose, xylose, fructose and glucose were 85.01%, 87.79%, 103.17%, and 101.24%, with excellent relative standard deviations (RSDs) of 1.94%, 1.13%, 0.60% and 1.67%, respectively. The proposed method was demonstrated to be efficient and time-saving, and had been applied to analyze monosaccharides in tobacco and tea successfully.

Analysis of mixtures of fatty acids and fatty alcohols in fermentation broth

Microbial production of fatty acids and fatty alcohols has attracted increasing concerns because of energy crisis and environmental impact of fossil fuels. Therefore, simple and efficient methods for the extraction and quantification of these compounds become necessary. In this study, a high-performance liquid chromatography-refractive index detection (HPLC-RID) method was developed for the simultaneous quantification of fatty acids and fatty alcohols in these samples. The optimum chromatographic conditions are C18 column eluted with methanol:water:acetic acid (90:9.9:0.1, v/v/v); column temperature, 26°C; flow rate, 1.0mL/min. Calibration curves of all selected analytes showed good linearity (r2≥0.9989). The intra-day and inter-day relative standard deviations (RSDs) of the 10 compounds were less than 4.46% and 5.38%, respectively, which indicated that the method had good repeatability and precision. Besides, a method for simultaneous extraction of fatty acids and fatty alcohols from fermentation broth was optimized by orthogonal design. The optimal extraction conditions were as follows: solvent, ethyl acetate; solvent to sample ratio, 0.5:1; rotation speed, 2min at 260rpm; extraction temperature, 10°C. This study provides simple and fast methods to simultaneously extract and quantify fatty acids and fatty alcohols for the first time. It will be useful for the study of microbial production of these products.

Modeling and optimizing of steam pyrolysis of dimethyl formamide by using response surface methodology coupled with Box-Behnken design

A non-catalytic steam pyrolysis of N,N-dimethyl formamide (DMF) was studied in a SS 316 plug flow reactor at atmospheric pressure as a waste solvent minimization method. The reaction products were analyzed by using gas chromatography-thermal conductivity detector and high performance liquid chromatography-refractive index detector. The optimization of process variables namely, temperature, flow rate and water to DMF ratio (v/v) for the maximum conversion of DMF have been studied by response surface methodology (RSM) coupled with a Box-Behnken design. The analysis of variance (ANOVA) data shows the conversion of DMF significantly enhances with temperature. Higher flow rate has adverse effect on DMF conversion, whereas water to DMF ratio (v/v) shows negligible effect on conversion of DMF. With the help of a developed RSM model, the highest conversion of DMF (89.82wt%) was predicted at 1100°C, 0.48ml/min and water:DMF ratio of 1.35 (v/v). Based on the experimental design, eight empirical models were developed those depict the interacting effect of different sets of operating variables on conversion of DMF and yield of seven products. Effect of process variables on yield of CO2 and N2 were studied, individually. Oxidative pyrolysis of DMF was also investigated, which shows significant reduction in the coke formation with increasing H2O2 to DMF mol ratio.

On-line flow injection analysis using gold particle modified carbon electrode amperometric detection for real-time determination of glucose in immobilized enzyme hydrolysate of waste bamboo chopsticks

Dual on-line flow injection analysis (FIA) systems were coupled to an immobilized enzyme reactor. The primary system was detected by amperometric enzyme electrode through a specifically designed three-electrode flow cell and a stopped-flow sampling and injection device to perform the successive real-time determination of glucose in the enzyme hydrolysate of waste bamboo chopsticks fibers. The secondary system was established via high-performance liquid chromatography-refractive index detection for comparison purpose. The novel enzyme electrode was made by chemically bonding both the redox mediator ferrocenecarboxaldehyde and glucose oxidase to a gold-particles modified pencil lead carbon electrode that possessed the widest linear standard calibration concentration range of 0–39.0 mM ( r 2 = 0.9956), a limit of detection of 7.8 μM, fast response time of 5 s, and a detection sensitivity of 2212 nA mM −1. The electrode was usable more than 50 days. The greatest amount of glucose produced from hydrolysis was 0.46 mM at 32 h. Interference from mannose and galactose was 7% and 5%, respectively, that was neglected by their small concentration in hydrolysate. The on-line FIA of amperometric enzyme electrode detection has good analytical precision (RSD 2.2–4.5%) and accuracy (94.4–97.7%) for the real-time glucose determination. The statistical analysis shows that no systematic error exists between the quantitative results of two FIA systems.

Identification of Mosquito Biting Deterrent Constituents From the Indian Folk Remedy Plant Jatropha curcas

An investigation of the Indian folk remedy plant Jatropha curcas L., was performed to identify the constituents responsible for the mosquito biting deterrent activity of the oil. J. curcas seed oil is burned in oil lamps in India and parts of Africa to repel biting insects, primarily mosquitoes. The seed oil was thoroughly analyzed by 1H NMR, 13C NMR, high-performance liquid chromatography-refractive index, and gas chromatography-flame ionization detection to identify the constituents in the oil. Identified constituents, both free fatty acids and triglycerides, were evaluated for activity in Aedes aegypti (L.) (Diptera: Culicidae) biting deterrent assays. Furthermore, an oil condensation trap was used to demonstrate that free fatty acids or triglycerides are partially volatilized during the combustion process. These compounds were found to be responsible for the biting deterrency of the burned oil. Specifically, oleic, palmitic, linoleic, and stearic acids were all active at 25 nmol/cm2 above that of solvent control in Ae. aegypti biting deterrent assays. Oleic, palmitic, and linoleic acids were all more active than stearic acid in the same bioassay. Evaluation of the triglycerides containing each of these fatty acids revealed that tripalmitin, tristearin, trilinolein, and triolein all demonstrated significant activity above a solvent control at 10 microg/cm2, whereas tripalmitin was the most active. Due to literature reports suggesting larvicidal activity of the oil, J. curcas seed oil and its free fatty acid constituents also were evaluated against 1-d-old Ae. aegypti larvae up to 500 ppm. Oleic acid was the only fatty acid having larvicidal activity against 1-d-old Ae. aegypti larvae, with an LD50 of 47.9 ppm.

Complex analytical approach to characterization of the influence of carbon dioxide concentration on carbohydrate composition in Norway spruce needles

Water-soluble non-structural carbohydrates (NSC) in the needles of Norway spruce Picea abies [L.] Karst have been studied by using a combination of several separation techniques, having various detectors, with mass spectrometry. The intent was to find a suitable methodology that enables the characterization and determination of NSC, covering a wide range of molar masses, and being suitable to assess how NCS are influenced by both external conditions, e.g. different carbon dioxide (CO 2) concentrations, light intensity, and by internal conditions such as the needle age. The techniques were liquid–liquid extraction, high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), asymmetrical flow field-flow fractionation (AsFlFFF), electrospray ionization mass spectrometry (ESI-MS), and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). NSC were extracted by a methanol/chloroform/water mixture into the water-rich phase. Application of AsFlFFF and SEC, using refractive index (RI) and multi-angle light scattering (MALS) detectors to the water-rich extracts resulted in three or four main fractions covering molar masses from 10 3 to 10 6 g/mol. Individual fractions collected from SEC were directly subjected to both MALDI and ESI-MS analysis in order to identify NSC. MALDI mass spectra confirmed the presence of hexose oligomers in individual fractions while ESI-MS was used for evaluation of low mass NSC. HPLC–RI was used for quantification of NSC and predominant carbohydrates were found to be fructose, glucose, and sucrose. The changes in their content during seasonal course were studied in detail. HPLC coupled to ESI-MS enabled the identification of low concentration NSC like raffinose that occurred in the needles of autumn samplings. An influence of the increased CO 2 concentration on sucrose and glucose accumulation was observed and it was found that the light intensity as well as the needle age has significant influence on the sucrose content.

A simple high-performance liquid chromatography method for the analysis of glucose, glycerol, and methanol in a bioprocess.

A reliable high-performance liquid chromatography-refractive index detection method for the simultaneous analysis of glucose, methanol, and glycerol in a bioprocess fermentation media using direct injection is presented. The validation studies show a satisfactory selectivity, linearity, accuracy, and recovery of the method. The lowest concentration detectable for glucose and methanol is 3.5 and 6.7 mg/100 mL, respectively. This method could be an attractive choice for the analysis of these compounds not only in fermentation media but also in biomedical and environmental samples.

Complete on-line determination of biopolymer molecular weight via high-performance liquid chromatography coupled to low-angle laser light scattering, ultraviolet, and differential refractive index detection

An optically modified high-performance liquid chromatography refractive index detector was developed to allow complete on-line determinations for biopolymer molecular weights. On-line concentration, refractive index, specific refractive index increment (dn/dc2)mu, and Rayleigh factor were determined under flow injection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrumental system is capable of determining absolute on-line molecular weights. The error and time requirements involved in conventional methodologies for proteins have been reduced. Sample quantities have been reduced from 150 to 200 mg, in conventional off-line methods, to less than 2 mg for on-line FIA and 0.5 mg for on-line SEC, if mass absorptivities (a) are known. Otherwise, the determination of a will be the most sample-demanding step, requiring about 3 mg of the pure protein. On-line measurements of (dn/dc2)mu are in good agreement with traditional off-line values established at Donnan equilibrium (usually within 5%). In addition, this technique provides true injected mass as determined by the UV detector, after chromatographic exposure where losses may occur, which is then used in the calculation of biopolymer molecular weight.