Elution Of Sample Research Articles

Sensitive Photonic System to Measure Oxidative Potential of Airborne Nanoparticles and ROS Levels in Exhaled Air

A photonic system has been developed that enables sensitive quantitative determination of reactive oxygen species (ROS) – mainly hydrogen peroxide (H2O2) – in aerosol samples such as airborne nanoparticles and exhaled air from patients. The detection principle relies on the amplification of the absorbance under multiple scattering conditions due to optical path lengthening [1,2]. In this study, the presence of cellulose membrane that acts as random medium into the glass optical cell considerably improved the sensitivity of the detection based on colorimetric FOX assay (FeII/orange xylenol). Despite the loss of assay volume (cellulose occupies 75% of cell volume) the limit of detection is enhanced by one order of magnitude reaching the value of 9nM (H2O2 equivalents). Spectral analysis is performed automatically with a periodicity of 5 to 15 s, giving rise to real-time ROS measurements. Moreover, the elution of air sample into the collection chamber via a micro-diffuser (impinger) enables quantitative determination of ROS contained in or generated from airborne samples. As proof-of-concept the photonic ROS detection system was used in the determination of both ROS generated from traffic pollution and ROS contained in the exhaled breath as lung inflammation biomarkers.

Improvements to 232‐thorium, 230‐thorium, and 231‐protactinium analysis in seawater arising from GEOTRACES intercalibration

The GEOTRACES program requires the analysis of large numbers of seawater samples for 232Th, 230Th, and 231Pa. During the GEOTRACES international intercalibration exercise, we encountered unexpected difficulties with recovery and contamination of these isotopes, 232Th in particular. Experiments were carried out to identify the source of these issues, leading to a more streamlined and efficient procedure. The two particular problems that we identified and corrected were (1) frits in columns supplied by Bio‐Rad Laboratories caused loss of Th during column chemistry and (2) new batches of AG1‐X8 resin supplied by Bio‐Rad Laboratories released more than 100 pg of 232Th during elution of sample. To improve yields and blanks, we implemented a series of changes including switching to Eichrom anion exchange resin (100‐200 µm mesh) and Environmental Express columns. All Th and Pa samples were analyzed on a Neptune multi‐collector inductively‐coupled‐plasma mass spectrometer (MC‐ICP‐MS) using peak hopping of 230Th and 229Th on the central SEM, with either 232Th, 236U (or both) used to monitor for beam intensity. We used in‐house laboratory standards to check for machine reproducibility, and the GEOTRACES intercalibration standard to check for accuracy. Over a 1‐y period, the 2 s.d. reproducibility on the GEOTRACES SW STD 2010‐1 was 2.5% for 230Th, 1.8% for 232Th, and 4% for 231Pa. The lessons learned during this intercalibration process will be of value to those analyzing U‐Th‐Pa and rare earth elements as part of the GEOTRACES program as well as those using U‐series elements in other applications that require high yields and low blanks, such as geochronology.

Flocked swabs combined with platforms to inoculate and streak specimens: a pre-analytical workstation for microbiology automation

Introduction. The bacteriology processes almost all the clinical specimens through manually performed cultures and therefore needs to evolve toward automated systems.We report our experience on the benefits of combination between liquid-based flocked swab transport systems (Copan Innovation) and platforms of plating and automatic streaking of agar plates.The main innovation is that, for the first time, the automation can be performed not only for urine and other few liquids materials, but also to most of other biological samples. Methods. Our considerations are based on more than one year of experience on the instrument InocuLAB® (Dynacon Inc.) in two Italian Hospitals, supplemented by theoretical knowledge of other marketed in Italy instruments.The three instruments currently available in Italy (A: InocuLAB® Copan, B:WASP® Copan and C: Previ-Isola bioMerieux®) can inoculate and streak liquid or eluated specimens from liquidbased flocked swab transport systems on any agar plate.The main differences are: streaking of already deposited material on plate (only A and B); choice among various protocols of streak and inoculate (only A and B); automatic opening and recapping of the specimen containers (only A and B); automatic choice of the plates (B: 9 columns and C: 5 columns); partition of the streaked plates in groups (B: up to 4 group s and C: up to 3); forced use of disposables devices (only instrument C). No instrument present problems of carryover and the quality of plated specimens is excellent. Conclusions. Although significantly different, all the platforms combined with flocked swabs represent a pre-analytical workstation for microbiology automation (inoculating, streaking, and bar-coding of liquid specimens). The main advantages can be seen in the operator’s production time, in safety and in quality of streak.The almost complete elution of the biological sample in liquid-based flocked swab transport systems is a standard base which allows to perform many diagnostic tests, including quantitative streaking for quantitative evaluation and multiple culture for additional investigation.

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.

Analysis of human blood serum using the off-line coupling of capillary isoelectric focusing to matrix-assisted laser desorption/ionization time of flight mass spectrometry

Off-line coupling of capillary IEF (CIEF) with matrix-assisted laser desorption/ionization mass spectrometry was utilized for the analysis of human blood serum. Serum proteins were initially separated by CIEF, and fractions of the isoelectric separation were eluted sequentially to a MALDI-TOF MS sample target. During pressure elution of the CIEF sample, voltage was maintained across the capillary system utilizing a sheath flow arrangement to minimize band broadening induced by the laminar flow field. Both pI and mass information were obtained from the complex biological sample, similar to traditional 2-DE techniques, and the platform was faster (hours versus days), more automatable, and simpler than 2-DE. The volume of raw sample present in the actual analysis was approximately 100 nL, making this technique well suited for very rare specimens. Additionally, the speed and simplicity of the technology make it an attractive technique for performing initial comparative analyses of complex samples.

Rapid sequential heart-cut multidimensional gas chromatographic analysis

A method is described which allows the sequential fast analysis of heart-cuts taken during the first column elution of an essential oil sample in a multidimensional gas chromatography experiment. In this investigation, a relatively short, narrow bore capillary column is used in the second dimension, with fast cryogenic modulation permitting rapid delivery of cryofocussed heart-cuts into the second column of the multidimensional arrangement. In this implementation, the total analysis time on the second column is of the order of 30 s, which is less than the duration of the sampled heart-cuts (1 min), with peak theoretical efficiencies of about 7000 m −1. Thus, the method should allow on-line heart-cutting of as many discrete heart-cuts as one may require from the first dimension chromatogram, and so two-dimensional separation can be achieved for almost the whole sample in one single analysis without the need for repeat injections or cycling of the oven temperature. The method is demonstrated by the transfer and rapid re-injection of 1 min heart-cuts taken from an initial separation stage of peppermint essential oil. The cryofocussing and fast analysis step is shown to increase signal response by up to 40-fold. Total peak capacity in the described system is measured to be of the order of 1800, with capacities of about 20–30 for each 30 s second dimension analysis. By increasing the frequency of sampling to remove excessive (unused) time to develop the 2 D separation, it should be possible to achieve peak capacities of 5000 or more baseline resolved peaks.

Purification of Cajanus cajan Root Lectin and Its Interaction with Rhizobial Lipopolysaccharide as Studied by Different Spectroscopic Techniques

A lectin present in roots of Cajanus cajan seedlings was isolated and purified by affinity chromatography. Sugar specificity assayed by hemagglutination-inhibition activity indicated that lectin belongs to glucose/mannose-specific group. The root lectin was found to be mannose-specific from the second day onwards as it was reconfirmed by specific elution of different days' sample from mannose agarose matrix. The maximum interaction of lectin with goat IgM was obtained in 10-day-old sample, indicating the highest crude lectin content. Lectin (total amount of eluted protein) from different days soil sample showed a maximum amount in 10-day-old sample. For further studies, the lectin has been isolated from the roots of 10-day C. cajan seedlings and purified on mannose–CL agarose column by affinity chromatography. Lectin was found to be a dimer of 18.5-kDa subunit as revealed by SDS–PAGE. Tryptophan quenching fluorescence was studied for C. cajan root lectin. Secondary structure of C. cajan root lectin as studied by circular dichroism was found to be a typical β-pleated sheet structure. The interaction of purified root lectin with C. cajan-specific rhizobial lipopolysaccharide and its inhibition by specific and nonspecific sugars was demonstrated by fluorescence and circular dichroism. Results discussed in this paper were studied for the first time by different spectroscopic methods, suggesting that C. cajan root lectin–lipopolysaccharide interaction is specific.

An anion exchange technique for separation of Na and K for neutron activation analysis of W-Ti alloy

A radiochemical neutro activation analysis procedure has been developed for the determination of sodium and potassium at parts per billion levels in high purity tungsten/titanium alloy material. The procedure involves the use of an anion exchange separation for purifying sodium and potassium activities from the alloy’s matrix activities,46Sc and187W. In addition, the use of two sequential sample loadings on the resin column prior to elution of the matrix activities has been investigated. Irradiation of 200 mg of the alloy results in a sample of nearly 1 Ci which must be handled in a remote manipulation cell. After acid dissolution of the sample, the sample solution in 1M HF was loaded onto the column. Elution of the sodium and potassium was accomplished using 40 ml of 1M HF. Similar loading and elution of the second sample was followed by the elution of matrix activities with a solution of 1M HF+6M HCl. Extensive tracer experimentation was employed to establish the possibility of retaining the matrix activities on the column while a second sample solution was processed, thus greatly increasing the throughput in terms of the number of samples analyzed per day. The detection limits of Na and K using the method developed are 4.0 ppb and 200 ppb, respectively.

Multiple Peaks in HPLC of Proteins: Bovine Serum Albumin Eluted in a Reversed-Phase System

Elution of a commercial sample of bovine serum albumin (BSA) in a reversed-phase (RP) HPLC system at room temperature gives a distorted peak. If a shallow gradient is used during elution a split peak is observed. The nature of the several parts of this multiple peak is studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography (SEC), amino acid analysis, re-elution in RP-HPLC of collected fractions, capillary electrophoresis (CE), and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS). This study demonstrates that the split peak of BSA observed in these chromatographic conditions is due to the monomer, dimer and other aggregates existing in the commercial sample of the BSA used. Moreover, it is proved that typical RP-chromatographic conditions do not cause aggregation of BSA.

The interactions of porcine and ovine, serum and colostral immunoglobulins with staphylococcal Protein A.

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produce by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG1, IgG2, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG1 from most ewes possessed a low affinity for Protein A whereas ovine IgG2 generally possessed a high affinity; 100% of the IgG2 in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.

Biospecific chromatography of chymosin on quinonated Sepharose and its application to enzyme content determination in rennets

SummaryAn insoluble benzoquinone derivative, obtained by reacting benzoquinone with Sepharose 4B and subsequent deactivation with ethanolamine, selectively absorbed chymosin from a mixture of milk-clotting enzymes and other proteins such as in rennets. This selective absorption has been used for developing a purification method and for quantitatively estimating the chymosin content of commercial rennets. Thus, from a powdered calf rennet, it was possible to obtain in one step an 8-fold purified chymosin free of bovine pepsins. The proposed analytical test involves 2 milk-clotting activity measurements and a chromatographic elution of the sample on a quinonated Sepharose column. It can be applied directly to commercial rennets, because it does not require pre-dialysis of the sample against the elution buffer. Furthermore, a combination of quinonated Sepharose and DEAE-cellulose chromatography gave a method for isolation of bovine pepsin I from rennets.