Characterisation of RPLC columns packed with porous sub-2 microm particles

Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.     

A new device to study ex-vivo the effects of extracorporeal photochemotherapy on the immune system

Extracorporeal photochemotherapy (ECP) is a medical procedure effective in the treatment of several different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient's blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes, which are then incubated with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused to the patient. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Although ECP has been shown to have an in-vivo immunomodulatory effect, the mechanisms through which ECP exerts its effect remain elusive. One of the reasons for this incomplete knowledge is the absence of a reliable model for ECP. In order to investigate the effect of ECP on the peripheral immune system, we developed a new device which mimics the complete ECP cycle including blood transit through the cell separator. Peripheral blood samples (50ml) were obtained from volunteers and processed using a peristaltic pump. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and UV-A under the same conditions used for the patients' therapy. Using this strategy we investigated 8-MOP, UV-A and their combined effect on the production of the pro-inflammatory cytokines interferon-gamma (IFN-gamma), interleukine-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) in PBMC with and without polyclonal stimulation. We firstly demonstrated that our device does not affect total red and white blood cell counts. After 8-MOP and UV-A irradiation a significant decrease was observed in both activated CD4(+) and CD8(+) T lymphocytes producing IFN-gamma, IL-2 and TNF-alpha. Our findings are in line with those previously obtained in humans after complete ECP treatment, thus suggesting that our newly developed device is suitable for investigating the mechanism of action of ECP ex-vivo.     

An investigation of peak-broadening effects arising when combining CE with MS

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.     

Experimental analysis of flashback in lean premixed swirling flames: conditions close to flashback

Swirling lean premixed flames are of practical relevance due to their potential for low nitric oxide (NOx) emissions. Unfortunately, these flames have various drawbacks. One critical attribute is the possibility for flashback of the reacting flow into the nozzle. Advanced numerical simulations should be able in the future to predict the transition from stable flames to flashback. For a better understanding of the process itself and for validation of numerical simulation a well-documented generic benchmark experiment is needed. This study presents a burner configuration that has already been studied extensively in the past. By minor geometrical adaptations, and via the possibility to vary the swirl intensity in a controlled way, the transition from stable flames to flashback is now accessible to detailed characterisation using advanced laser diagnostics. In a first part of this study the different states of the flame were classified. In the second part, both a stable and a precessing flame very close to flash back were compared in terms of flow and scalar field. The variation of the swirl intensity on the flame is discussed. Because the flame is strongly influenced by its inflow conditions additional velocity measurements inside the nozzle were carried out. This is of special importance for subsequent numerical simulations to match the experimental conditions. The quantitative investigation of the flame during flashback is subjected to consecutive experiments where planar laser diagnostics at high repetition rates will be exploited.     

An automatic peak finding method for LC‐MS data using Gaussian second derivative filtering

A highly automated procedure for localising and characterising peaks in the chromatographic time domain of LC‐MS data has been developed. The work was initiated by an identified need to facilitate the detection and tracking of chromatographic peaks during method development for the analysis of impurities in pharmaceutical products. The algorithm is mainly based on a digital filter for which the settings are automatically adapted to the data set under study. The procedure was evaluated for synthetic data sets with various S/N levels, peak widths and baseline properties. It was found that even for the worst case tested with S/N=10 and a high variability in the baseline, 94% of the simulated analytical peaks could be detected without producing any false‐positive identifications. Furthermore, the number of correctly estimated peak heights and peak widths falling within a 10% error of the true values were 94 and 91%, respectively. For experimental data sets, peak height, and width estimations were more difficult, but the processed reconstructions showed an excellent agreement with the analytical signals of the raw data, and also a clearly improved visualisation in total ion‐ and base‐peak chromatograms.     

Applications of proton transfer reaction time-of-flight mass spectrometry for the sensitive and rapid real-time detection of solid high explosives

Using recent developments in proton transfer reaction mass spectrometry, proof-of-principle investigations are reported here to illustrate the capabilities of detecting solid explosives in real-time. Two proton transfer reaction time-of-flight mass spectrometers (Ionicon Analytik) have been used in this study. One has an enhanced mass resolution (m/Δm up to 8000) and high sensitivity (～50 cps/ppbv). The second has enhanced sensitivity (～250 cps/ppbv) whilst still retaining high resolution capabilities (m/Δm up to 2000). Both of these instruments have been successfully used to identify solid explosives (RDX, TNT, HMX, PETN and Semtex A) by analyzing the headspace above small quantities of samples at room temperature and from trace quantities not visible to the naked eye placed on surfaces. For the trace measurements a simple pre-concentration and thermal desorption technique was devised and used. Importantly, we demonstrate the unambiguous identification of threat agents in complex chemical environments, where multiple threat agents and interferents may be present, thereby eliminating false positives. This is of considerable benefit to security and for the fight against terrorism.     

Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.     

Reversed Phase Continuous Full Filling CEC–ESI–MS

P. Viberg, P. Spégel and J. Nilsson contributed equally to the presented work.     

Electro- and photoinduced spallation reactions on27Al and51V at intermediate energies

Cross sections for some electro- and photoinduced spallation reactions on²⁷Al and⁵¹V have been measured in the energy region 130–580 MeV with the activation method. The cross sections per photon are compared to Monte-Carlo calculations based on a cascade-evaporation model. The electrodisintegration cross sections are compared to calculations based on the Dalitz formalism for virtual photon spectra.