Karin E. Markides

Chromatographic alignment by warping and dynamic programming as a pre-processing tool for PARAFAC modelling of liquid chromatography-mass spectrometry data.

Solutes analysed with LC-MS are characterised by their retention times and mass spectra, and quantified by the intensities measured. This highly selective information can be extracted by multiway modelling. However, for full use and interpretability it is necessary that the assumptions made for the model are valid. For PARAFAC modelling, the assumption is a trilinear data structure. With LC-MS, several factors, e.g. non-linear detector response and ionisation suppression may introduce deviations from trilinearity. The single largest problem, however, is the retention time shifts not related to the true sample variations. In this paper, a time warping algorithm for alignment of LC-MS data in the chromatographic direction has been examined. Several refinements have been implemented and the features are demonstrated for both simulated and real data. With moderate time shifts present in the data, pre-processing with this algorithm yields approximately trilinear data for which reasonable models can be made.

Studies of signal suppression in liquid chromatography-electrospray ionization mass spectrometry using volatile ion-pairing reagents

Volatile ion-pairing reagents are useful due to their compatibility with liquid chromatography-electrospray ionization (ESI) mass spectrometry. In this study trifluoroacetic acid, heptafluorobutanoic acid and perfluoroheptanoic acid were used as ion-pairing reagents. The signal intensities of eight amine analytes were measured in the presence of these fluorinated carboxylic acids and compared with the signal intensity when using an ion-pair free formic acid-ammonium formate buffer. It was shown that the ESI signal from most of the studied analytes decreased about 30-80% when the fluorinated carboxylic acids were added to the mobile phase at useful concentrations. The use of these acids in ion-pair chromatography was also compared to the more conventional sodium heptane sulphonate additive. It was found that the chromatographic performance was comparable. Finally, the long-term performance of the ESI interface and the chemical background caused by these fluorinated reagents were examined. No degradation of the ESI interface performance could be seen for over 24 h of continuous infusion.

Novel polyamine coating providing non-covalent deactivation and reversed electroosmotic flow of fused-silica capillaries for capillary electrophoresis

A new polycationic coating for use in capillary electrophoresis has been developed that enables chemical modification of fused-silica capillary surfaces for analysis of compounds like basic proteins. The cationic polyamine, containing short aliphatic blocks of combined 2 and 3-carbon length, was physically adsorbed onto the negatively charged fused-silica surface through ionic interaction by flushing the capillary with a polyamine solution, followed by a self-stabilization step. The polyamine coated capillaries generated an anodal electroosmotic flow that was independent of pH in the investigated range of pH 4-8. The capillary performance was demonstrated by fast separations of basic proteins with peak efficiencies in the range of 265,000-584,000 plates.

Matched filtering with background suppression for improved quality of base peak chromatograms and mass spectra in liquid chromatography - mass spectrometry

A time domain filter that combines the properties of matched filtering and two-fold differentiation is presented. The filter coefficients are given by the second derivative of a Gaussian model peak, controlled by the setting of two parameters related to the chromatographic system. The fundamental characteristics of the filter were derived, and its applicability demonstrated for real liquid chromatography–mass spectrometry (LC–MS) data. The filter is primarily intended as a fast pre-processing step, for a mass chromatogram with 320 scans over 700 mass channels the computation time was 0.6 s on a standard PC. Base peak chromatograms with improved peak detection capability and mass spectra useful for compound identification were obtained with filtered data. The most significant effect of the described filter is background reduction due to the differentiation, which in combination with the matched filter can be performed with maintained or even improved signal-to-noise ratio.

Quantitation of tryptophan, kynurenine and kynurenic acid in human plasma by capillary liquid chromatography - electrospray ionization tandem mass spectrometry

Concentrations of tryptophan and its metabolites in plasma are of great interest in determining proper diagnosis and medication of several neurological diseases like, for example, Alzheimers disease. A method of standard addition was developed to determine total level of tryptophan and two of its metabolites, kynurenine and kynurenic acid, in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry. Plasma samples were simply deproteinized by addition of diluted perchloric acid. Samples were then mixed with trichloroacetic acid and injected onto a capillary column. Analytes were separated by a fast gradient elution of the injected samples. Detection was performed by sheathless electrospray tandem mass spectrometry in the multiple reaction monitoring mode. Linear calibration curves were obtained for spiked plasma sample with up to 100% of the expected analytes concentrations. The determined concentrations were well within ranges previously reported (i.e., 6 nM-95 microM) and limit of detections were around 3 nM for each analyte.

Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry

One feature of complex lipids is that many subtypes of these molecules exist as a diverse mixture in a biological sample. Qualitative and quantitative analysis of these closely related molecules require sensitive and specific analytical methods to detect intact phospholipids (PL) and sphingomyelin (SM) species and to differentiate between them. Conventional analytical methods require laborious procedures including separation by column, argentation thin-layer chromatography or liquid chromatography (LC) after pre- or post-column derivatization. In the present work, a method based on reversed phase capillary LC coupled on-line to electrospray ionization mass spectrometry (LC/ESI/MS) has been developed to gather tools for lipidomic studies, i.e. the profiling of complex mixtures of lipids in small amounts of various cells and tissues. The LC/MS system used consisted of an LC pump in an isocratic elution, a reversed phase capillary column and a single quadrupole mass spectrometer operated in the positive ion mode. A successful separation of phosphatidylcholine (PC) and SM molecular species was obtained with a minimum detectable quantity (MDQ) in the low fmol range injected on column. The method was applied to human brain extracts. Furthermore, the extraction efficiencies of the traditional Folch method and pressurized fluid extraction (PFE) were compared using the human brain. It was found that the intensity of the PC and SM molecular species extracted by PFE is two times that of Folch.

Capillary supercritical fluid chromatography combined with atmospheric pressure chemical ionisation mass spectrometry for the investigation of photoproduct formation in the sunscreen absorber 2-ethylhexyl-p-methoxycinnamate

The photoproducts formed on ultraviolet irradiation of the sunscreen absorber trans-2-ethylhexyl-p-methoxycinnamate, were separated and characterised using a new combined technique, capillary supercritical fluid chromatography-atmospheric pressure chemical ionisation mass spectrometry. Using this technique the commercially available trans-isomer was found to photoisomerise on irradiation at wavelengths greater than 300 nm. Photodimers were also separated and identified, and indicate that the sunscreen absorber can undergo [2+2] cycloaddition reactions with itself.

Shape selectivity in liquid and gas chromatography: Polymeric octadecylsilane (C18) and liquid crystalline stationary phases

SummaryOctadecylsilane (C18) stationary phases for liquid chromatography (LC), which are prepared by polymeric rather than monomeric phase synthesis, exhibit shape selectivity for isomeric polycyclic aromatic hydrocarbons (PAH) that is similar to the shape selectivity observed for liquid crystalline phases in gas chromatography (GC). Relative retention measurements for several sets of isomeric PAH on a polymeric C18 stationary phase (reversed-phase LC) and liquid-crystalline polysiloxane stationary phase (capillary GC) were compared and correlated with the simple length-to-breadth shape descriptor of the solute. Similar retention behavior was observed for both chromatographic systems. In fact, anomalies in elution order relative to length-to-breadth ratios were generally found to be consistent in both chromatographic systems and could often be ascribed to secondary shape factors (i.e., planarity of the molecule).

Using miniature diode array NIR spectrometers for analysing wood chips and bark samples in motion

The chemical and physical properties of the wood chips entering the Kraft pulping process are of high interest for many different reasons. The most important one is the possibility to establish an effective and dynamic forward process control. The same is valid for one of the by‐products, bark, which is burned in a solid fuel furnace for energy recovery. In the present study, NIR (near‐infrared) reflectance measurements with an inexpensive silicon diode array spectrometer operating in the 800–1100 nm range are evaluated as a tool for on‐line analysis of wood chips and bark transported on a conveyor belt moving at 1 m s−1. The objective of this work was to investigate the determination of water content, extractive content, Klason lignin, carbohydrate monomers and size distribution of wood chips together with water content, wood content and coarseness of bark. All these parameters could be measured with the exception of the carbohydrates, for which it was only possible to satisfactorily measure the sum of pentoses (arabinose + xylose) and the sum of hexoses (glucose + galactose + mannose). FWEFA (fixed window evolving factor analysis) was used for automatic identification of outliers. Copyright

Metallized sheathless electrospray emitters for use in capillary electrophoresis orthogonal time-of-flight mass spectrometry

In microscale separations, such as capillary electrophoresis and -liquid chromatography, the liquid flow rates are in the order of nanoliters per second. If such flow rates are to be interfaced with a mass spectrometer (MS) using electrospray (ES) ionization, without loss of separation efficiency, each fraction of the analyte zone must remain undisturbed by the high voltage contact necessary for ES. One design that accomplishes this is the pure sheathless approach, where a thin, vapor deposited metal film covers the outside of the electrospray emitter tip. This thesis describes the development of such sheathless emitters. The lifetimes of polymer embedded gold (“fairy dust”) or graphite (“black dust”) emitters were shown to by far exceed those of previously used conductive films. In addition, the production of emitters with these coatings was substantially simplified. The increase in durability was found to be due to enhanced resistance towards the electrochemical processes associated with ES. In analogy, the reasons for the limited durability of previously used methods were correlated with their tendency to oxidize, or be mechanically removed, during electrochemical reactions. Electrochemical processes associated with the electrospray potential were also found to seriously disturb analyses in which porous graphitic carbon was used as the separation medium. A proper choice of grounding point locations eliminated these disturbances.At last, the differences regarding analytical performance of several sheathless interface configurations, used in capillary liquid chromatography, were examined. The best performance was obtained when a pure sheathless emitter with a conductive layer of polyimide and graphite was coupled to the LC column through a Teflon sleeve.