Current Chromatography - Volume 3, Issue 2, 2016

Background: The currently emerging utilization of biomass for chemicals on an industrial scale is linked to the use of available plant materials, especially of wood and other lignocelluloses. The chemical analysis of biomass for process development and control can be considerably difficult due to the diversity of its constituents, which can cause interferences with the analysis of the target analyte. The capabilities of thin layer chromatography are especially suited for samples that contain a large portion of interfering components, both for quick qualitative screenings and the quantitative analysis of individual components. Methods: A review of the existing literature on the analysis of wood derived compounds by thin layer chromatography was compiled. An introduction to the general capabilities of thin layer chromatography was added for readers unacquainted to the technique. Results: Thin layer chromatography has been applied to all wood components: carbohydrates, phenols, lignans and extractives. The analytical aims comprised amongst others the identification of wood and pitch components, changes in wood composition over time or after treatments, and the quantification of individual compounds. Conclusion: Thin layer chromatography is a very suitable tool for the analysis of wood derived biomass due to its robustness and the ease of detection and hyphenation. It has been used to achieve challenging analytical tasks and can be a well-founded choice to investigate wood derived compounds.

Background: Brewer’s spent grain (BSG) is an important by-product of the brewing process. It is suitable for food and biotechnological applications. The chemical composition of BSG varies depending on many factors. Little is known about low molecular weight extractives which can potentially be used as valuable phytochemicals with nutraceutical and pharmaceutical significance. Objective: The lipid fraction of BSG was characterized in order to evaluate its potential as a source of valuable chemicals and to find potential biomarkers of spoilage. Methods: The lyophilized biomass was extracted by accelerated solvent extraction. The extract was subjected to methylation and silylation. The lyophilized biomass was also subjected to mild alkaline saponification. The comparison of mass spectra obtained using the silylating agent N,O-bis- (trimethylsilyl) trifluoroacetamide and its deuterium-labelled form proved of special significance for structural identification. Results: The majority of fatty acids (~85%) were bound in lipids; free fatty acids accounted for ~15%. The fatty acid pattern revealed typical even-over-odd predominance. An array of 3-hydroxy and cyclopropanoic fatty acids which have not been reported in BSG thus far, along with cisvaccenic acid, are promising candidates for biomarkers of bacterial contamination of BSG. These compounds could be unambiguously identified by adduct formation using dimethyl disulfide. Sterol and fatty alcohol patterns were very similar to those of barley. A series of alkylresorcinols and hydroxyalkyl- methoxy benzenediols could be detected, which have not been known to occur in BSG up to now. Conclusions: Lipidic constituents in BSG contain valuable phytochemicals with nutraceutical and pharmaceutical significance. Certain lipidic surrogates might serve as biomarkers for bacterial and fungal contamination of brewer’s spent grain.

Background: Solid-phase microextraction (SPME) is a simple and rapid extraction method that was introduced by Pawliszyn. During the ages significant developments were made in fiber types in order to overcome the traditional SPME fibers limitations. Objective: In this project, a new application of a solid sorbent was introduced for head-space SPME (HS-SPME) determination of phenolic compounds. In this sorbent, MWCNTs were covalently functionalized and were attached chemically to the PDMS by the sol-gel method. Moreover, ionic liquids were used as porogenic agents in order to increase the fiber porosity. Method: HS-SPME mode was used for analytes determination. The most important affecting parameters on method’s efficiency were investigated and optimized. Results: At the optimized conditions, linear ranges were from 0.002-200 ng mL-1 with limits of detection (LODs) from 0.0005 to 0.005 ng mL-1 and limits of quantitation (LOQs) between 0.002-0.02 ng mL-1. Two different relative standard deviations (RSDs) were defined and calculated at three different concentrations (0.05, 2 and 100 ng mL-1). The first RSDs were for one fiber (repeatability) that were from 4.6 up to 6.7%. The secondary RSDs were between different fibers (reproducibility) that were from 5.7 to 7.8%. Conclusion: The prepared fiber showed high thermal stability (over than 320 °C) with good lifespan (more than 210 times). Eventually, the phenolic compounds were determined in the real urine samples with this method. According to our results, the proposed method showed acceptable relative recovery percentages between 90.7 to 102.1% for the spiked urine samples at 0.5 ng mL-1.

Background: Organochlorine pesticides (OCPs) have been used worldwide in agriculture because of their low prices and effectiveness against insects. However, their rates of chemical and biological degradation are slow. OCPs are still widely detected in the environment and in organisms. Therefore, it is necessary to develop sensitive and accurate methods to investigate the amounts of OCPs in environmental samples. Objective: We present herein two liquid phase microextraction methods, namely shaker-assisted (SADLLME) and surfactant-assisted dispersive liquid liquid microextraction (SDLLME), for the determination of organochlorine pestcides in water samples. Method: Gas chromatography coupled with an electron capture detector (GC-ECD) was employed. Various factors that affect extraction efficiency, such as the type and volume of the extraction solvent, salt addition, the type and volume of surfactant solution in SDLLME, and the effect of shaking time in SADLLME, have been evaluated. Results: Under the optimized conditions, the enrichment factors ranged from 948 to 2241 for SADLLME and from 695 to 1656 for SDLLME. The linear range was from 7 to 5000 ng L-1 and the limit of detection (LOD) ranged from 1.4 to 3.1 ng L-1 for SADLLME. The linear range was from 5 to 1000 ng L-1 and the limit of detection (LOD) ranged from 1.6 to 2.1 ng L-1 for SDLLME. The absolute recoveries and relative recoveries were 19.8-49.3% and 80.8-110% for river water, 19.6-47.8% and 76.9-116% for lake water, and 15.3-46.9% and 77.5-102% for irrigating water, by SADLLME and SDLLME respectively. Conclusion: These methods, employing minimal amounts of extraction solvent and surfactant solution, could be used to accomplish efficient extraction in 3 min and achieved high enrichment factors. For the analysis of environmental samples, they were both successfully applied to the preconcentration of trace OCPs in water samples.

Background: A variety of pharmacological effects have been attributed to ursolic acid, however, its hydrophobicity has made clinical application difficult. In this sense, the development of nanoparticles containing ursolic acid may represent an improvement in drug solubility. Objective: In this study, a simple HPLC method with PDA detection was developed and validated for the quantitative determination of ursolic acid in poly(lactic acid) (PLA) nanoparticles. Method: The method was validated as per ICH guidelines considering the parameters linearity, limits of detection and quantification, specificity, accuracy, precision and robustness. The mobile phase employed was a mixture of acetonitrile and water (90:10, v/v) under isocratic elution with a flow rate of 1 mL/min. Ursolic acid was detected at 203 nm. Results: The method proved to be linear in the range of 10 – 100 μg/mL and the limits of detection and quantitation were 0.50 and 1.53 μg/mL, respectively. Precision (intra-day and inter-day) presented relative standard deviation below 2%. Accuracy was assessed by the recovery test of ursolic acid from nanoparticles. Robustness was demonstrated altering the mobile phase and flow rate. Specificity showed no interference from components of formulation or degradation products from light exposure. Conclusion: The method was successfully employed to determine the content of ursolic acid in PLA nanoparticles.

Background: Aluminum is a toxic metal and is widespread in nature. Therefore, evaluation of aluminum level in environmental and biological fluids has received considerable attention. Among instrumental analytical techniques used for determination of aluminum, HPLC has advantages of selective separation and wide linear dynamic range. However, most of the HPLC methods reported for the analysis of aluminum involve pre-column derivatization with chromogenic chelating reagents and separation of aluminum with the mobile phases free from the reagents, which need to maintain the experimental conditions such as temperature and flow rate of the mobile phase strictly constant in order to obtain reliable results. Methods: In this study a reversed-phase ion-pair HPLC method for determination of trace amounts of aluminum was developed using 5-sulfoquinoline-8-ol (HQS) as the derivatization reagent for fluorometric detection. In order to suppress the appearance of system peaks on the chromatograms, the solutions injected into the HPLC column were prepared so that the compositions of the solutions were the same as that of the mobile phase except only for aluminum originating from the samples. Results: The calibration curve of aluminum standard solutions was linear over the range 0.05-10 μg L-1 with a correlation coefficient, r2, of 0.9997. The limit of detection defined as three times the standard deviation of the blank signal was found to be 18 ng L-1 (0.67 nM). The aluminum concentrations in certified reference materials of river water determined by this method were in excellent agreement with the certified values. This method was also successfully applied for the determination of aluminum in urine and glucose parenteral solution as well as river water and tap water samples. Conclusion: HPLC determination of trace amounts of aluminum in environmental and biological fluids was performed with high accuracy and precision by suppressing the fluctuation of the baseline and detecting the Al-HQS complex at equilibrium of the complexation reaction.

Background: Separation of proteins in countercurrent chromatography requires the use of polymer phase systems to avoid denaturation of proteins by organic solvent. The polymer phase systems has a high viscosity and low interfacial tension and requires spiral column and modification of tubing shapes for achieving a satisfactory separation. In the past, various tubing shapes were made using a pair of pliers but it requires tedious work. Objective: The aim of this paper is to make a simple tool to modify the separation column quickly with high accuracy and reproducibility. Method: A simple tool is introduced which can modify the shape of tubing to enhance the partition efficiency in high-speed countercurrent chromatography. It consists of a pair of interlocking identical gears, each coaxially holding a pressing wheel to intermittently compress plastic tubing in 0 – 10 mm length at every 1 cm interval. Results: The performance of the processed tubing is examined in protein separation with 1.6 mm ID PTFE tubing intermittently pressed in 3 mm and 10 mm width both at 10 mm intervals at various flow rates and revolution speeds. A series of experiments was performed with a polymer phase system composed of polyethylene glycol and dibasic potassium phosphate each at 12.5% (w/w) in deionized water using three protein samples. Overall results clearly demonstrate that the compressed tubing can yield substantially higher peak resolution than the non-processed tubing. Conclusions: The simple tubing modifier is very useful for separation of proteins with high-speed countercurrent chromatography.