High-performance Size-exclusion Chromatography System Research Articles

Removal Behavior of Protein-like Dissolved Organic Matter During Different Water Treatment Processes in Full-Scale Drinking Water Treatment Plants

Protein-like dissolved organic matter (pDOM), which is ubiquitous in natural waters, is a critical precursor of nitrogenous disinfection byproducts. Recently, the control and elimination of pDOM have been a growing concern during drinking water treatment processes. In this study, a high-performance size exclusion chromatography system coupled with photo-diode array, fluorescence detector, and online organic carbon detector (HPSEC-PDA/FLD/OCD) was used to determine the removal behaviors of different-sized pDOM from two full-scale drinking water treatment plants (DWTPs). Coagulation and activated carbon adsorption were selected for bench-scale experiments to further assess the removal behavior of pDOM during conventional water treatment processes. The results showed that different-sized pDOM fractions exhibited different removal characteristics. Pre-oxidation can effectively remove some tyrosine-like and tryptophan-like components with high MW, and as the oxidization effect was enhanced, more high MW fractions decomposed into low MW ones. Conversely, some aliphatic pDOM fractions in high MW (e.g., aliphatic proteins) were not subject to pre-oxidation removal. The coagulation-sedimentation unit was efficient in removing high MW fractions, specifically tryptophan-like fractions. Additionally, some pDOM components may be released during coagulation. pDOM with low MW and high hydrophobicity were easily removed during activated carbon filtration. However, long-term operation of the activated carbon filter may breed microorganisms, resulting in the partial release of pDOM fractions. Moreover, UV disinfection processes promoted the degradation of low MW pDOM components. Due to the complex water quality and uncontrollable microbial activities, the aforementioned water treatment units did not exhibit a synergistic effect on pDOM removal. In comparison with humic-like substances, pDOM was susceptible to water quality changes, and its removal was limited in the surveyed DWTPs. Therefore, DWTPs must strengthen pDOM monitoring in influent and effluent and adjust the operating parameters of different treatment units in a timely manner. Moreover, the combination of advanced water treatment processes, such as ozone-biological activated carbon process and nanofiltration, should also be considered to strictly control pDOM component removal.

Inhibitory Effects of Pectic Polysaccharide Isolated from Diospyros kaki Leaves on Tumor Cell Angiogenesis via VEGF and MMP-9 Regulation.

Persimmon leaves are an attractive source of phytochemicals with potential health benefits. However, there are only a few reports on the chemical properties and biological activity of the polysaccharide fractions (PLE-I–III) of persimmon leaves. We evaluated the angiogenesis-inhibiting ability of pectic-polysaccharides. The molecular weight of PLEs was determined using a high-performance size-exclusion chromatography system. Tube formation assay of human umbilical vein endothelial cells (HUVECs) was performed using Matrigel-coated 96-well plates. Matrix metalloproteinase (MMP-9), vascular endothelial growth factor (VEGF), PI3K, Akt, and p38 phosphorylation levels were determined using Western blotting; VEGF and MMP-9 transcript levels were measured using SYBR Green qRT-PCR. PLE-I–III significantly inhibited HUVEC tube formation at 12.5 and 25 μg/mL. Among them, PLE-II showed the strongest anti-tube formation activity, and the mRNA/protein expression of angiogenesis-related factors (VEGF/MMP-9) was significantly reduced by PLE-II. PLE-II also suppressed the phosphorylation of PI3K/AKT and p38, JNK, and NF-κB p65 in HUVECs. These results suggest that the polysaccharide PLE-II isolated from persimmon leaves inhibited VEGF and MMP-9 expression in HUVECs via regulation of PI3K/AKT, p38, JNK, and NF-κB p65 signaling pathways.

Low molecular weight dextran production by Leuconostoc mesenteroides strains: Optimization of a new culture medium and the rheological assessments

Strains belonging to genus Leuoconostoc mesenteroides are able to produce dextran exo-polysaccharide. In this study, MRS broth and milk permeate were used for growth of three strains (NRRL B-512F, NCIB 8023 and NRRL B-512) of L. mesenteroides and the growth rate, dextran production, and its rheological properties were studied. Evaluation of molecular weight by high-performance size exclusion chromatography system showed that dextran produced in milk permeate had a lower molecular weight (<10000 Dalton) compared to the dextran produced in MRS broth (<40000 Dalton). The viscosity of dextran solution (2%) produced in MRS was 0.005–0.01 Pa.s while in the synthetic medium it was <0.005 Pa.s. Also in our study maximum ratio of growth factor and dextran yield belonged to NRRL B-512F, which was subjected to optimization of fermentation condition for dextran production. For optimization, response surface methodology by using sucrose, milk permeate and yeast extract in 5 levels were used. According to experiments, optimum concentrations for sucrose, milk permeate, and yeast extract were found to be 20, 15, and 15 (g/l), respectively.

Molecular Weight Determination of Heparin and Dermatan Sulfate by Size Exclusion Chromatography with a Triple Detector Array

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.

Preliminary Study of Lake Dissolved Organic Matter in Light of Nanoscale Supramolecular Assembly

Large-scale preparative high-performance size-exclusion chromatography (HPSEC) was performed to separate different molecular size fractions in milligram quantities from strongly colored dissolved organic matter (DOM) of a freshwater using a very mild conjugate acid-base pair (10 mM acetic acid-sodium acetate solution at pH 7.0 with an ionic strength of 6 x 10(-5)) as the mobile phase. The homogeneity-uniformity of different molecular size fractions in relation to their combined original mixture was verified by an analytical HPSEC system. In addition to molecular size distribution and basic spectroscopic characteristics, Fourier transform infrared spectroscopy was applied to specify structural features for different size fractions. The results demonstrate clearly that only a very small amount of conjugate organic acid-base pair is required to generate a powerful resolution for a DOM mixture, and very strong treatments with organic acids are not necessarily needed to reach a better SEC resolution. Most essential is the combined outcome of different HPSEC experiments and determined structural functionalities which indicate that almost all original DOM solutes are aggregated mixtures consisting of different associations possessing various molecular size ranges, which can be separated from their integrated whole as nearly homogeneous and uniform species. In summary, the present study strongly speaks for the need to direct the research of natural dissolved and colloidal organic carbon more strongly toward a nanoscale study of supramolecular assemblies. More precise knowledge about the primary, secondary, and tertiary structure of dissolved DOM constituents has its essential function, e.g., for environmental protection and utilization of surface waters.

β‐Glucanase Activity and Molecular Weight of β‐Glucans in Barley After Various Treatments

ABSTRACTβ‐Glucanase activity interferes with molecular characterization of mixed‐linkage (1→3)(1→4)‐β‐d‐glucans (β‐glucans). Reductions in β‐glucanase activity were determined after barley cvs. Azhul, Waxbar, and Baronesse were treated with autoclaving (120°C, 45 min), calcium chloride (0.05M, 1 hr), 70% ethanol (80°C, 4 hr), hydrochloric acid (0.1N, 1 hr), oven heating (120 and 140°C, 40 min), sodium hydroxide (0.0025M, 1 hr), and 5% trichloroacetic acid (TCA) (40°C, 1 hr). High‐performance size‐exclusion chromatography (HPSEC) of α‐amylase‐treated aqueous extracts was used to demonstrate the effects of treatments on the molecular weights of β‐glucans. The HPSEC system included multiple‐angle, laser light scattering, refractive index, and fluorescence detectors. β‐Glucanase activities, ranging from 52 to 65 U/kg of barley, were reduced by autoclaving (50–75%), hot alcohol (67–76%), oven heating (40–96%), CaCl2 (75–95%), NaOH (76–89%), and TCA (92–96%). Some malt β‐glucanase activity remained after most treatments. HCl and TCA treatments reduced extraction and molecular weights of β‐glucans. Weight‐average molecular weights (Mw) for β‐glucans extracted with water at 23°C were low (most &lt;8 × 105). Base treatment (pH 9) and extraction at 100°C for 2.5 hr resulted in the greatest extraction of β‐glucans and highest Mw. As a result, the conditions seem appropriate for measurement of physical characteristics of β‐glucans in cereal products.

Calibration of a size-exclusion chromatography system using fractions with defined amylopectin unit chains

A high-performance size-exclusion chromatography (HPSEC) system was successfully calibrated using fractions of debranched amylopectin unit chains, obtained by gel filtration, and with the average chain length of the fractions determined by high-performance anion-exchange chromatography. As a comparison, calibration of the HPSEC system was also performed using maltoheptaose and pullulan standards as reference substances. The relationships between the degree of polymerization and the retention time for the defined amylopectin unit chain fractions and the commercial standards, i.e. maltoheptaose and pullulans, were very similar, indicating similar elution behaviour.

Molecular size distribution and spectroscopic properties of aquatic humic substances

The number- and weight-averaged molecular weights of different isolated humic fractions and unfractionated organic matter from natural water samples were measured using high-performance size-exclusion chromatography (HPSEC). The chromatograms obtained, with sodium acetate as the mobile phase on a TSK G3000SW high-speed column, provided illustrative information about the nature of the dissolved organic matter that was not available with phosphate buffer eluents. The calibration of the column system merely with protein standards generated misleading information about the molecular weight and size distribution of humic solutes. Instead, the present results support the utilization of polystyrene-sulphonates (PSS) either alone or together with certain other model compounds for the calibration of HPSEC in studying molecular weights of humic solutes. The data obtained with the HPSEC system indicate that the average molecular weight of humic solutes is distinctly lower than is generally assumed. Besides, aquatic humic solutes also contain some very large-sized though minor constituents, leading to a fairly polydisperse mixture. Number-averaged molecular weights obtained using the HPSEC system were 4–5-fold greater than those from vapour-pressure osmometry. Bulk spectroscopic properties such as molar absorptivity at 280 nm and the quotient, E 2 E 3 , correlated strongly with the total aromaticity and averaged molecular weights of all the humic solutes. This observation suggests that bulk spectroscopic properties can be applied, as a first approximation, for estimating the size of humic solutes and their aromaticity in natural surface waters.

Molecular Weight Patterns of Nägeli Amylodextrins

AbstractThis research was designed to assess the utility of a four‐column high performance size‐exclusion chromatography (HPSEC) system to characterize starch‐based carbohydrates of different sizes. Corn starches with varying amounts of amylose were treated with 16% sulfuric acid to create Nägeli amylodextrins. During treatment, sub‐samples were taken at various intervals from 0–100 days. The washed, dried sub‐samples were chromatographically analyzed using four Shodex lonpak columns linked in series. Chromatograms showed the progressive depolymerization of starch. Pullulan molecular weight standards were used to estimate amylodextrin molecular weights (MW). Number‐average MWs of amylodextrins decreased as the original starches' amylopectin content decreased. A single HPSEC system can be effectively used to characterize carbohydrates ranging in size from starch to amylodextrins; and to monitor the acid (or enzyme) depolymerization of starches.