Gradient Column Research Articles

Pharmacological Effects of a Fraction of the Methanolic Extract of <i>Prosopis africana</i> Fruit (Guill and Perr ) Taub

The pharmacological effects of some fractions of the Prosopis africana fruits (MEPAF) were investigated. The fractions were obtained by subjecting the MEPAF to accelerated gradient column and Thin-Layer chromatography (TLC). Nine (9) fractions were obtained based on their retention factor (R ). One of the fractions, f fraction G was selected for further studies based on its better local anaesthetic activity using the guinea pig wheal test. The MEPAF did not show any measurable effect on the ocular reflexes and isolated frog rectus abdominis muscles but showed both concentration and timedependent inhibitions of the intrinsic peristaltic contractions of the rabbit jejunum. It also induced concentration-dependent inhibitions of the contractions induced by acetylcholine (2.5 ig/ml) on isolated rabbit jejunum. These inhibitions are thought to be antimuscarinic or atropine-like, since the contractions of the jejunal segment is believed to be through muscarinic receptors and could be blocked by atropine and related compounds . Phytochemical spot tests of the fraction G revealed the presence of alkaloids, carbohydrates, saponins, tannins, sterol, terpenes but not flavonoids, polyuronoids and reducing sugar. These findings give credence to the folkloric use of Propsopis africana in the treatment of body pain, toothache and dysentery.

Magnetic resonance imaging indicators of blood-brain barrier and brain water changes in young rats with kaolin-induced hydrocephalus

BackgroundHydrocephalus is associated with enlargement of cerebral ventricles. We hypothesized that magnetic resonance (MR) imaging parameters known to be influenced by tissue water content would change in parallel with ventricle size in young rats and that changes in blood-brain barrier (BBB) permeability would be detected.MethodsHydrocephalus was induced by injection of kaolin into the cisterna magna of 4-week-old rats, which were studied 1 or 3 weeks later. MR was used to measure longitudinal and transverse relaxation times (T1 and T2) and apparent diffusion coefficients in several regions. Brain tissue water content was measured by the wet-dry weight method, and tissue density was measured in Percoll gradient columns. BBB permeability was measured by quantitative imaging of changes on T1-weighted images following injection of gadolinium diethylenetriamine penta-acetate (Gd-DTPA) tracer and microscopically by detection of fluorescent dextran conjugates.ResultsIn nonhydrocephalic rats, water content decreased progressively from age 3 to 7 weeks. T1 and T2 and apparent diffusion coefficients did not exhibit parallel changes and there was no evidence of BBB permeability to tracers. The cerebral ventricles enlarged progressively in the weeks following kaolin injection. In hydrocephalic rats, the dorsal cortex was more dense and the white matter less so, indicating that the increased water content was largely confined to white matter. Hydrocephalus was associated with transient elevation of T1 in gray and white matter and persistent elevation of T2 in white matter. Changes in the apparent diffusion coefficients were significant only in white matter. Ventricle size correlated significantly with dorsal water content, T1, T2, and apparent diffusion coefficients. MR imaging showed evidence of Gd-DTPA leakage in periventricular tissue foci but not diffusely. These correlated with microscopic leak of larger dextran tracers.ConclusionsMR characteristics cannot be used as direct surrogates for water content in the immature rat model of hydrocephalus, probably because they are also influenced by other changes in tissue composition that occur during brain maturation. There is no evidence for widespread persistent opening of BBB as a consequence of hydrocephalus in young rats. However, increase in focal BBB permeability suggests that periventricular blood vessels may be disrupted.

Covalent Grafting of Polydimethylsiloxane over Surface-Modified Alumina Nanoparticles

The synthesis of ``smart structured'' conducting polymers and the fabrication of devices using them are important areas of research. However, conducting polymeric materials that are used in devices are susceptible to degradation due to oxygen and moisture. Thus, protection of such devices to ensure long-term stability is always desirable. Polymer nanocomposites are promising materials for the encapsulation of such devices. Therefore, it is important to develop suitable polymer nanocomposites as encapsulation materials to protect such devices. This work presents a technique based on grafting between surface-decorated gamma-alumina nanoparticles and polymer to make nanocomposites that can be used for the encapsulation of devices. Alumina was functionalized with allyltrimethoxysilane and used to conjugate polymer molecules (hydride-terminated polydimethylsiloxane) through a platinum-catalyzed hydrosilylation reaction. Fourier transform infrared spectroscopy, X-ray-photoelectron spectroscopy, and Raman spectroscopy were used to characterize the surface chemistry of the nanoparticles after surface modification. The grafting density of alkene groups on the surface of the modified nanoparticles was calculated using CHN and thermogravimetric analyses. The thermal stability of the composites was also evaluated using thermogravimetric analysis. The nanoindentation technique was used to analyze the mechanical characteristics of the composites. The densities of the composites were evaluated using a density gradient column, and the morphology of the composites was evaluated by scanning electron microscopy. All of our studies reveal that the composites have good thermal stability and mechanical flexibility and, thus, can potentially be used for the encapsulation of organic photovoltaic devices.

Changes in egg buoyancy during development and its effects on the vertical distribution of anchovy eggs

Small pelagic fish populations exhibit reproductive strategies resulting from past natural selection pressure, by which certain traits become more or less common in a population, allowing them to adapt and become better suited to certain habitats. One such adaptation is the buoyancy of eggs, which is observed as density changes during development. This is an important issue in fisheries and modeling science, as it affects the vertical distribution of eggs and, therefore, egg transport. Recently, individual-based models for anchovies in the Mediterranean have focused on developing adequate biological algorithms to simulate realistic spatial variations of eggs and larvae. Some models that include movements of particles according to Stokes’ law also assume a constant value of egg density during egg development. However, field observations show differences in the vertical distribution of eggs when egg density during development is considered. We address the problem of egg density and its vertical distribution within a biological context. In Mediterranean waters, the incubation time for anchovy eggs during peak spawning is approximately 48–70h; during these first hours, egg density has an influence on the horizontal and vertical trajectories of eggs, as well as their routes and hatching zones. In this study, we introduce an algorithm describing the egg density of European anchovy eggs throughout development. Egg density measurements were carried out in a density gradient column (DGC). We fitted a polynomial model that estimated egg density, as a function of time from fertilization and that was dependent on temperature. Simulations to study the vertical transport of eggs in the Mediterranean were carried out using ICHTHYOP/MARS3D. The vertical distribution of pelagic eggs was determined by a set of interacting biological and physical parameters related to eggs (density, diameter) and ambient seawater (density, viscosity, turbulence), respectively. The egg buoyancy model introduced here was validated and will provide insight for the design of anchovy egg surveys, as the vertical position of the eggs in the water column during development can be inferred by the hydrographic structure of seawater.

89 SURVIVAL OF CAT EPIDIDYMAL SPERM AFTER TEMPORARY COOL STORAGE OR CRYOPRESERVATION IN DEFINED EXTENDERS

A general objective of our studies on cat sperm is to enhance methods for both short- (+4°C) and long-term (–196°C) cryostorage, with particular focuses on improving compatibility with sex sorting and conforming to regulations for international shipment. Here, our specific aims were to a) determine the ability of cat sperm to survive during temporary cool storage in defined extenders (Exp. 1), and b) compare sperm survival after cryopreservation in the optimal defined extender v. TEST buffered extender + 2% egg yolk (TYB, Exp. 2). Testes from local veterinary clinics were transported in HEPES saline. Epididymides were dissected in HEPES 199 medium (He199), repeatedly sliced, and held at 37°C for ∼20 min. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL He199 and centrifuged for 5 min at 250 × g. In Exp. 1 (5 replicates), aliquots of the sperm pellet were extended in either of 2 defined extenders, Bioxcell® (BXC; IMV, Minneapolis, MN, USA) or HypoThermosol®-FSR (HTS; BioLife Solutions Inc., Bothell, WA, USA) or in TYB. Motility (Mot, Hamilton Thorne Sperm Analysis System CEROS 12, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at days 0, 1, 2, and 3 (Exp. 1), or after cooling (4°C) and post-thawing (p.t.), after 0 and 3 h incubation at 37°C (Exp. 2). In Exp. 2 (10 replicates), the sperm pellet was extended in BXC or TYB and gradually cooled to 4°C. Then, BXC or TYB + 12% glycerol was added (1:1) using a modified fixed osmolarity method (1995 Hum. Reprod. 10, 1109). Samples were loaded into 0.25-mL straws and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed in air (∼22°C) for 5 s and immersed in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps, centrifuged at 800 × g for 5 min, and pellets resuspended in He199. In Exp. 1, sperm in TYB, BXC, and HTS maintained 93, 69, and 56%, respectively, of initial motility (71%) after 3 days at 4°C (TYB > BXC and HTS; P < 0.05, 1-way ANOVA). Initially, 75 and 86% of sperm had membrane integrity and intact acrosomes, respectively. At 72 h, ∼80% of membrane intact sperm retained integrity in the two defined extenders v. nearly 90% in TYB (P > 0.05). At 24 h, all groups had high percentages of sperm with intact acrosomes (87 to 93%), but at 72 h, there was a difference between HTS (96%) and BXC (79%; P < 0.05). In Exp. 2 (Table 1), motility in TYB and BXC at 0 h p.t. was 77 and 70% of pre-freeze values – 77% (TYB) and 73% (BXC), respectively. Motility at 3 h p.t. was similar (BXC = 35% v. TYB = 37%). Membrane integrity and acrosomal status at 3 h p.t. ranged from 60% (BXC) to 72% (TYB) and from 65% (BXC) to 68% (TYB) of pre-freeze values, respectively. At 3 h p.t. M.I. of sperm in TYB was higher (P < 0.05) than in BXC. In summary, we have shown that cat epididymal sperm can be stored temporarily and cryopreserved successfully in a defined extender without animal proteins. Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

Optimisation of ultra‐performance LC conditions using response surface methodology for rapid separation and quantitative determination of phenolic compounds in Artemisia minor

A method that couples rapid, sensitive, reproducible and accurate ultra-performance LC (UPLC) with quadrupole-TOF-MS was established for the first simultaneous qualitative and quantitative analysis of phenolic compounds in Artemisia minor. Box-Behnken designs (BBDs) were applied as an effective tool to optimise major parameters that influence the resolution of UPLC, including three gradient steps and column temperature. Under optimal UPLC conditions, a total of 23 phenolic compounds in the crude methanol extracts of A. minor were well separated on a Waters Acquity UPLC BEH C(18) column (100×2.1 mm, 1.7 μm particle size) within 16.5 min, and the compounds were unequivocally or tentatively identified via comparisons with authentic standards and literature. In this study, a total of six major phenolic compounds were quantified in A. minor and the method was validated to be sensitive, precise and accurate within the LOD from 1.24 to 5.27 μg/mL, and the overall intra- and inter-day variations in detection were less than 3.76%. The recovery of the method ranged from 97.9 to 103.8% with RSDs that were less than 5.8%. These results demonstrate that this approach has the potential for quality control of A. minor and other Tibetan herbal medicines.

Predicting Retention Time Shifts Associated with Variation of the Gradient Slope in Peptide RP-HPLC

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.993-0.996 (compared to a theoretical value of ~1.00). The observed retention time shifts associated with variations of the gradient slope can be predicted a priori using the approach described here. The proposed model is based on our findings for a set of synthetic species (Vu, H.; Spicer, V.; Gotfrid, A.; Krokhin, O. V. J. Chromatogr., A, 2010, 1217, 489-497), which postulate that slopes S can be predicted taking into account simultaneously peptide length, charge, and hydrophobicity. Here we extend this approach using an extensive set of real tryptic peptides. We developed the procedure to accurately measure S-values in nano-RP HPLC MS experiments and introduced sequence-specific corrections for a more accurate prediction of the slopes S. A correlation of ~0.95 R(2)-value between the predicted and experimental S-values was demonstrated. Predicting S-values and calculating the expected retention time shifts when the physical parameters of separation like gradient slope are altered will facilitate a more accurate application of peptide retention prediction protocols, aid in the transfer of scheduled MRM (SRM) procedures between LC systems, and increase the efficiency of interlaboratory data collection and comparison.

ChemInform Abstract: Isolation and Purification of Proteoglycans

Purification of a protein typically involves development of a quantitative assay to track protein integrity (e.g. enzyme activity) during subsequent isolation steps. The generalized procedure involves choosing the source of the protein, defining extraction conditions, developing bulk purification methods followed by refined, more selective methods. The purification of proteoglycans is often complicated by a) limited source quantities, b) necessity of chaotropic solvents for efficient extraction, c) their large molecular size and d) lack of defined functions to enable purity (i.e. activity, conformation) to be assessed. Because the usual goal of proteoglycan purification is physical characterization (intact molecular weight, core protein and glycosaminoglycan class and size), the problems of a suitable assay and/or native conformation are avoided. The ‘assay’ for tracking proteoglycan isolation typically utilizes uronic acid content or radiolabel incorporation as a marker. Once extracted from their cellular/extracellular environment, proteoglycans can be isolated by density gradient centrifugation and/or column chromatography techniques. Recent advances in the composition of chromatographic supports have enabled the application of ion-exchange, gel permeation, hydrophobic interaction and affinity chromatography resins using efficient high-pressure liquid chromatography to proteoglycan purification.

PENGARUH ESCHERICHIA COLI DAN GRANULOSIT TERHADAP KADAR REACTIVE OXYGEN SPECIES SECARA IN VITRO

In humans, male genital tract infection has been recognized as one of the causes of infertility. Indicators of the occurrence of genital tract infection are the presence of bacteria during semen culture (bacteriospermia) and the finding of leucocyte of more than 1 million/ml semen (leucotytospermia). Escherichia coli (E. coli) is the most common cause of prostatitis and epididymitis. The objective of this study was to determine the effects of E. coli and granulocytes on Reactive Oxygen Species (ROS) level in vitro. This study comprised of two experiments. In the experiment 1, sperm was incubated with E. coli, and the experiment 2, the sperm was incubated with granulocyte. In those experiments, ROS levels were observed. Spermatozoa were obtained from donor with normal spermatozoa according to WHO (1999). Escherichia coli was obtained by culturing the semen of infertile males. Granulocytes were obtained from donors’ blood. Sperm preparation was made by using Percoll gradient column method. Granulocyte isolation used Histopaque 1077 and 1119. ROS level was detected by means of chemiluminescence method with beta counter device. The result of this study showed that in vitro E. coli had the effect on ROS level, both stimulated by peroxidase (p = 0.000) and PMA (p = 0.006). Granulocyte had effect on ROS level. In peroxidase-stimulated ROS level, there was the effect between spermatozoa and granulocyte-incubated sperm (p = 0.000), granulocyte-incubated sperm and granulocyte (p = 0.002), and sperm and granulocyte (p = 0.000). In PMA-stimulated ROS level, there was effect between sperm and granulocyte-incubated sperm (p = 0.000), sperm and granulocyte (p = 0.000), granulocyte-incubated sperm and granulocyte (p = 0.000). In conclusion, under in vitro experiment, sperm incubated with E. coli and sperm incubated with granulocyte had the effect on the level of the ROS.

Probing the kinetic performance limits for ion chromatography. II. Gradient conditions for small ions

A gradient kinetic plot method is used for theoretical characterisation of the performance of polymeric particulate anion exchange columns for gradient separations of small inorganic anions. The method employed requires only information obtained from a series of isocratic column performance measurements and in silico predictions of retention time and peak width under gradient conditions. Results obtained under practically constrained conditions provide parameters for the generation of high peak capacities and rapid peak production for fast analysis to be determined. Using this prediction method, a maximum theoretical peak capacity of 84 could be used to achieve separation of 26 components using a 120 min gradient ( R s > 1). This approach provides a highly convenient tool for development of both mono- and multidimensional ion chromatography (IC) methodologies as it yields comprehensive understanding of the influence of gradient slope, analysis time, column length and temperature upon kinetically optimised gradient performance.

Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7μm sorbent

Separation by hydrophilic interaction chromatography (HILIC) with fluorescence detection utilizing a sub-2 microm glycan column for the separation of 2-aminobenzamide (2-AB) labeled N-linked glycans is described. The HILIC column packed with a 1.7 microm amide sorbent improves the peak capacity compared to a 3.0 microm HILIC column by a similar degree as observed in reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results indicated that the optimal peak capacity was achieved at flow rate 0.2-0.5 mL/min. HILIC method transfer guidelines were shown to further enhance the resolution of glycans by changing initial gradient conditions, flow rate, column temperature, and different column lengths. Additionally, excellent resolution can be achieved in the separation of 2-AB labeled glycans released from fetuin, RNase B, and human IgG with a rapid analysis time.

112 EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION

Our purpose was to examine the effect of egg yolk concentration (EY; 2, 5, or 10%) on in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST-buffered extender (TYB). Testes were transported in HEPES saline; epididymes were dissected in HEPES 199 medium (He199) and repeatedly sliced. The sperm suspension was filtered (40 μ), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 g for 20 min. Aliquots of the sperm pellet were extended in TYB containing 2, 5, or 10% EY. After cooling to 4°C, samples were diluted 1:1 with TYB containing 2, 5, or 10% EY + 12% glycerol in 4 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (-80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (˜22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122 centrifuged at 200g for 10 min, and pellets resuspended in He199. Motility (Mot, phase contrast, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at 0 h, after gradual cooling to 4°C and after freezing at 0 and 3 h post-thaw (37°C). Ten replicates were done. Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in TYB + 2% egg yolk or HSPM (no egg yolk) in droplets (1 million sperm/mL) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured until blastocyst development was evaluated (Day 8). There were no treatment differences at any time or temperature point for the 3 sperm characteristics evaluated (one-way ANOVA; P > 0.05). As shown in the Table 1, at 0 h post-thawing, sperm in each group retained ˜70% of their initial pre-freeze motility. After 3 h of post-thaw incubation, motility decreased to ˜50% of the pre-freeze value. Cooling to 4°C did not affect membrane integrity or acrosomal status, but post-thaw values were reduced by 30-35% as compared with pre-freeze. Cleavage frequency and blastocyst development of 284 IVM oocytes after IVF using sperm frozen in TYB + 2% EY and HSPM were 53 v. 52% and 42 v. 38%, respectively (P > 0.05). In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in a cryoprotectant solution containing minimal egg yolk (2%). Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

A gradient search and column generation approach for the build–pack planning problem with approved vendor matrices and stochastic demand

We study a production planning problem of product assembly with random demand, where the customers choose their preferred suppliers for pairs of inter-dependent components through the approved vendor matrix. The problem is to develop production plans that minimise the expected total shortage and holding costs while observing the matrix restrictions and limited component supplies. We provide a mathematical programming formulation of the problem with a large number of decision variables, whose cost function is the solution of a parametric stochastic transportation problem. We present a gradient-based interior-point approach to solve this problem where the gradient is estimated by the shadow price from the solution of such a transportation problem. A column generation scheme is integrated into the approach to handle the large problem issue. Computational results show that our algorithm significantly improves the computational time when compared with the approach without column generation. In addition, we also discuss some extensions of the basic problem to the multi-period rolling horizon case.

Evaluation of The Peak Capacity of Various RP-Columns for Small Molecule Compounds in Gradient Elution

Peak capacity is the commonly used measure of separation efficiency in gradient elution. This study focuses on the effect of column characteristics (particle size and column length) and operating parameters (gradient time and flow rate) on the peak capacity for small molecule compounds in gradient elution. The goal of this study is to develop a practical strategy to maximize the separation efficiency (i.e., peak capacity) under different constraints (analysis time or pressure limit). Using both experimental data and theoretical modeling, the current study reveals that the peak capacity increases with both gradient time and column length in a non-linear fashion. Marginal peak capacity is proposed to characterize the non-linear increase of peak capacity over the gradient time and column length. This study also attempts to understand the maximum peak capacity achievable under certain pressure limits using Neue’s peak capacity model. The results of this study provide a better understanding of the UPLC technology, and can also help to develop practical strategies to maximize the separation efficiency in gradient elution to meet the separation needs.

A comparative study on natural fibre density measurement

This is a study for the comparison of existing test methods for measuring the density of natural fibres to be used as reinforcement in biocomposite materials. Normally, any such method is evaluated by referring to its accuracy, cost, speed of test result (i.e. how quick a method is to give the result), and whether it is convenient and safe to use. Through literature review, we identified five such methods: (1) diameter and linear density, (2) Archimedes, (3) helium pycnometry, (4) gradient column and (5) liquid pycnometry. The same sample (oilseed flax fibres) was tried using these methods to measure its density. As a result of the study, two methods are recommended: Archimedes (with the use of canola oil as immersion fluid) and the helium pycnometry. The former was found to be simple, quick to give the result and incur lower cost, and therefore promising for general use in measuring the density of natural fibre, but the latter, although capable of providing more repeatable results, be at a much higher cost.

Enrichment of human X- and Y-chromosome bearing spermatozoa by density gradient centrifugation.

Human spermatozoa (120--180 Mill/0.6 ml Tyrode solution) were laid on the top of a continous density gradient column consisting of 4--16% bovine serum albumin. The columns were then centifuged at 1500--1600 rpm (200-240 g) at 20 degrees C or 4 degrees C for 15 min. By this procedure the spermatozoa sedimented within the tubes of normal distribution curves. The mean percentages of y-spermatozoa gradually decreased from 62,3 +/- 6,1% (max. 70%) in the upper fractions to 42,4 +/- 3,8% (min. 37%) in the bottom fractions. These results could be verified by inseminations of rabbit does with spermatozoa separated by the same technique.

Critical considerations for the accelerated ageing of high-density polyethylene potable water materials

Accelerated ageing conditions with chlorinated water were identified that minimize variations in solution chemistry and water sorption, and that also enable the interpretation of HDPE physical and chemical changes during 20 week (3884 h) immersion periods. Of the ten conditions tested, three conditions with an alkalinity concentration of 50 ppm as CaCO 3 at pH 6.5 and at 23 and 37 °C performed best. These three conditions exhibited stable pH, free available chlorine, and alkalinity concentration during 20 weeks of HDPE immersion with 72 h changes of ageing solution. HDPE was periodically characterized using differential scanning calorimetry, thermogravimetric analysis, tensile analysis, a density gradient column, moisture analysis, and optical and infrared spectroscopy. Formation of surface carbonyl bonds and gradual reductions in oxidation induction time were detected, as well as water sorption into HDPE. Ageing solution and water sorption monitoring recommendations from this work should be incorporated into accelerated ageing protocols and considered when characterizing aged PE drinking water pipe.

Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/×/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-μm particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.

Protamine ratio and the level of histone retention in sperm selected from a density gradient preparation

Fertile males express two forms of sperm nuclear proteins, protamine 1 (P1) and protamine 2 (P2), in roughly equal quantities, whereas some infertile men have been shown to have a reduction in protamine content and an increase in the level of histones retained in mature sperm. In this study, we assessed histone and protamine levels in spermatozoa isolated from different layers of a density gradient centrifugation column to evaluate the nuclear protein content of the sperm population selected. Protamine levels were measured using acid gel electrophoresis and immunofluorescence, and the percentage of cells retaining histones was evaluated using aniline staining and immunofluorescence. Our data suggests that there is an inverse correlation between P1/P2 ratio and the level of histone expression in the different layers of the density gradient. Paradoxically, the 90% layer had a lower P1/P2 ratio, which corresponded with an increase in histone expression. It is concluded that although the sperm population selected in the 90% layer of the density gradient columns had a lower P1/P2 ratio, it was yet similar to the P1/P2 ratio observed in previously screened fertile donors.

Palladium‐catalyzed electroless plating of gold on latex particle surfaces

AbstractGold can be deposited onto a latex particle surface via the growth of metal islands with the electroless plating method. A new method is proposed for the electroless plating of gold on the surface of poly(styrene‐co‐vinylimidazole) latex particles, which is catalyzed by palladium present on the latex particle surfaces. The palladium ions are anchored to the latex particle surfaces by the formation of a palladium–imidazole complex, and palladium nanoparticles are nucleated by a reductant. These palladium islands act as catalytic sites, so gold is preferentially deposited onto the latex particle surfaces. Transmission electron microscopy, X‐ray photoelectron spectroscopy, and sucrose density gradient column results indicate that the palladium is associated with the imidazole‐functionalized latex particles. Different gold loading levels and reductant types were explored. Latex particles were partially encapsulated by finely dispersed gold nanoparticles less than 2 nm in diameter or by gold islands with sizes ranging from 10 to 100 nm up to a gold loading level of 3.1 mg of Au/m2 of latex. However, using higher gold loading levels led to uncontrollable electroless plating of gold because gold reduction in the water phase became very dominant even in the presence of catalytic palladium on the latex particle surface. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009