This chapter outlines the practical aspects of capillary Supercritical Fluid Chromatography (SFC) with emphasis on mobile phases, column technology, instrumentation, and operating conditions. Ammonia has great potential as a polar mobile phase, but work still must be done to develop instrumentation and column materials that are resistant to this fluid. Knowledge about column materials, dimensions, stationary phases, and methods for column preparation is important for using and treating such columns properly and for optimizing the performance of supercritical fluid chromatographic systems. Recently, several complete SFC systems have been introduced by instrument companies and are now commercially available. One of the limitations of cooled injection systems is that the components that are introduced onto the column must be soluble in the liquid solvent carrier. One of the major advantages of capillary SFC is that both liquid chromatography and gas chromatography detectors are readily adapted for use as SFC detection systems.

Supercritical fluid chromatography (SFC) serves as a high resolution chromatographic technique for the analysis of high molecular mass solutes that are beyond the volatility range of gas chromatography (GC). Multidimensional SFC is a relatively new technique which is growing at a rapid pace. SFC also serves as a high resolution chromatographic technique for the analysis of high molecular mass solutes that are beyond the volatility range of GC. SFC instrumentation has some similarities to both LC and GC. A syringe or reciprocating pump delivers the SFC mobile phase as a liquid to the injection valve. SFC is often applied to the analysis of high molecular mass samples such as fossil fuels, but such samples generally become more complex with increasing molecular mass because the number of possible isomers increases. In SFC, more than one of the operating parameters can be varied simultaneously; for example, the temperature may be continuously increased during a density/pressure program.

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Abnormal accumulation and aggregation of amyloid-β-peptide (Aβ) eventually lead to the formation and cerebral deposition of amyloid plaques, the major pathological hallmark in Alzheimer's disease (AD). Oleuropein (OE), an Olea europaea L. derived polyphenol, exhibits a broad range of pharmacological properties, such as antioxidant, anti-inflammatory, and antiatherogenic, which could serve as combative mechanisms against several reported pathways involved in the pathophysiology of AD. The reported noncovalent interaction between Aβ and OE could imply a potential antiamyloidogenic role of the latter on the former via stabilization of its structure and prevention of the adaptation of a toxic β-sheet conformation. The established β-sheet conformation of the Aβ hydrophobic carboxy-terminal region and the dependence of its toxicity and aggregational propensity on its secondary structure make the determination of the binding site between Aβ and OE highly important for assessing the role of the interaction. In this study, two different proteolytic digestion protocols, in conjunction with high-sensitivity electrospray ionization mass spectrometric analysis of the resulting peptide fragments, were used to determine the noncovalent binding site of OE on Aβ and revealed the critical regions for the interaction.

Amyloid beta peptide (Abeta) aggregation leads to the senile plaque formation, a process that is strongly influenced by oxidative stress and is considered as the molecular basis of various neurodegenerative diseases, such as Alzheimer's disease (AD). Endogenous antioxidants or dietary derived compounds may down-regulate this process. In this study, the interaction of two antioxidants, oleuropein (OE) and melatonin (M), with Abeta is monitored through nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The concerted application of these two analytical techniques provides new experimental evidence and residue-specific insights into the interacting Abeta peptide amino acids that are implicated in this process. Both antioxidant compounds interact in a similar way with the peptide and cause chemical shift variations. The most pronounced resonance changes have been observed for the 1H-15N signals of N-terminal region and Leu17-Phe20 residues, as monitored by NMR titration studies.

Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 microm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step.

Ions that are observed in a mass spectrum obtained with electrospray mass spectrometry can be assumed to originate preferentially from ions that have a high distribution to the surface of the charged droplets. In this study, a relation between chromatographic retention and electrophoretic mobility to the ion distribution (derived from measured signal intensities in mass spectra and electrospray current) within electrosprayed droplets for a series of tetraalkylammonium ions, ranging from tetramethyl to tetrapentyl, is presented. Chromatographic retention in a reversed-phase system was taken as a measure of the analyte’s surface activity, which was found to have a large influence on the ion distribution within electrosprayed droplets. In addition, different transport mechanisms such as electrophoretic migration and diffusion can influence the surface partitioning coefficient. The viscosity of the solvent system is affected by the methanol content and will influence both diffusion and ion mobility. However, as diffusion and ion mobility are proportional to each other, we have, in this study, chosen to focus on the ion mobility parameter. It was found that the influence of ion mobility relative to surface activity on the droplet surface partitioning of analyte ions decreases with increasing methanol content. This effect is most probably coupled to the decrease in droplet size caused by the decreased surface tension at increasing methanol content. The same observation was made upon increasing the ionic strength of the solvent system, which is also known to give rise to a decreased initial droplet size. The observed effect of ionic strength on the droplet surface partitioning of analyte ions could also be explained by the fact that at higher ionic strength, a larger number of ions are initially closer to the droplet surface and, thus, the contribution of ionic transport from the bulk liquid to the liquid/air surface interface (jet and droplet surface), attributable to migration or diffusion will decrease.

Beta amyloid peptide (Aβ) is the major proteinaceous component of senile plaques formed in Alzheimer’s disease (AD) brain. The aggregation of Aβ is associated with neurodegeneration, loss of cognitive ability, and premature death. It has been suggested that oxidative stress and generation of free radical species have implications in the fibrillation of Aβ and its subsequent neurotoxicity. For this reason, it is proposed that antioxidants may offer a protective or therapeutic alternative against amyloidosis. This study is the first report of the formation of the noncovalent complex between Aβ or its oxidized form and the natural derived antioxidant oleuropein (OE) by electrospray ionization mass spectrometry (ESI MS). ESI MS allowed the real time monitoring of the complex formation between Aβ, OE, and variants thereof. Several experimental conditions, such as elevated orifice potential, low pH values, presence of organic modifier, and ligand concentration were examined, to assess the specificity and the stability of the formed noncovalent complexes.

Miniaturized biochemical devices in glass, silicon and polymer materials are starting to find their way from the academic laboratories to real-life applications. However, most attention has been given to miniaturize the downstream functions of various microfluidic systems, leaving the sample introduction and preparation steps to more conventional, bulkier solutions. For point-of-care diagnostics in particular, it becomes crucial to be able to handle complex human samples in a miniaturized format.In this work, we report on a microsystem for on-chip sample preparation that is able to remove blood cells from whole blood. The hybrid system consists of a commercially available membrane filter incorporated into a poly(dimethylsiloxane) (PDMS) casted device. Membrane materials were evaluated on the bases of low nonspecific adsorption of free and protein-bound testosterone as analyte substance. The hybrid system including a hydrophilic polypropylene filter successfully removed blood cells from diluted human whole blood. Surface oxidation was sufficient to make the plasma filtrate flow through the membrane filter and the channel system by capillary force alone and thus no external pumping source was needed.

Pressurized fluid extraction has been used to obtain environmentally benign and fast extraction of squalene and a tocopherol from a low-value biomass in the olive oil production; olive oil pomace. The extraction conditions, i.e., temperature (80-190 degrees C), extracting solvent (acetonitrile, acetone, ethanol, 1-propanol, 2-propanol, 2-butanol, ethyl acetate, toluene) and extraction time (2-18 min.), were optimized through an experimental design to obtain the highest yield. Temperature was found to be the most important parameter for effective extraction of squalene from the olive biomass. The best results were obtained when 2-propanol or ethanol was used as a solvent at a temperature of 190 degrees C, an extraction time of 10 minutes times three cycles. The content of squalene was found to be in the range of 0.2-0.5 mg/g, while the recovery of a-tocopherol was in the range of 0.01 mg/g using the same extraction parameters. The developed method can be used both in an analytical context and as a starting point for up-scaled "green" processes.

Onion waste is a renewable raw material, rich in different molecular species of the antioxidant quercetin. To utilize this resource, an environmentally sustainable procedure has been developed, using pressurized hot water to extract the quercetin species, followed by biocatalytic conversion of the quercetin glycosides to quercetin and carbohydrates. Two different recombinantly expressed thermostable β-glucosidases, Thermotoga neapolitana β-glucosidase A and B, were utilized as catalysts. These enzymes maintain activity at temperatures around 90 °C, and are therefore ideal to use in combination with hot water extraction. Our results, based on experimental design, showed that they converted quercetin glycosides to active quercetin in less than 10 min reaction time in water at 90 °C, pH 5.0. Experimental design showed that the optimal extraction conditions included three 5 min extraction cycles with water at 120 °C and 50 bars, giving a total extraction time of 15 min. Several different types of quercetin and isorhamnetin glycosides as well as kaempferol were detected in onion waste using LC-MS/MS analysis. After converting the different glycosidic compounds to their respective aglycones, the quercetin content was 10 to 50 mg g−1 dry weight of onion waste (RSD 8%). In summary, our research demonstrates that subcritical water extraction followed by β-glucosidase-catalyzed hydrolysis is a rapid method to determine the content of quercetin and isorhamnetin in onion samples, and is environmentally sustainable as it only uses water as solvent and enzymes as catalysts.

Using a slight overpressure, a urine sample is loaded onto a monolithic photopolymerized sol–gel column that has been derivatized with hydrophobic carbon chains and then the complex urine matrix is washed with aqueous solution. A buffer containing organic solvent is used to elute the adsorbed peptides by an applied voltage and the sample is then introduced into a mass spectrometer by sheath flow electrospray. The importance of desalting this type of sample is demonstrated by an experiment that shows that the signal intensity of a test solution with neurotensin, sprayed directly into the mass spectrometer, decreased from 4.5×104cps to no detectible signal when just 10% urine is added to the sample solution. We suggest that this procedure may find general application for desalting biological samples prior to mass spectrometric analysis.

Information on protein expression, disease biomarkers or surrogate markers and genetic disorders can nowadays be achieved from analysis of complex biological samples by liquid separation coupled to mass spectrometric (MS) detection. This paper describes fast multidimensional separation by on-line liquid chromatography (LC) and capillary electrophoresis (CE), followed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) MS detection. This detector provides ultrahigh resolution of the detected ions, mass accuracy at the ppm-level and high sensitivity. Most of the challenge of this system lies in the development of a new interface for the on-line coupling of LC to CE. The interface developed in poly(dimethylsiloxane) provides a RSD for injection repeatability of <3.5% and surface control for unspecific binding by deactivation with a cationic polymer, PolyE-323. We have evaluated the interface, as well as the overall system, with respect to robustness and deconvolution ability. Sequence coverage for bovine serum albumin (BSA) of 93% showed a high recovery of sample in the different transfer steps through the system. The detection limit for identification is 277 ng mL(-1) (or 280 nM) on average for peptides. In the future, we expect LC-CE-MS to be a novel strategy for elucidating the chemistry of biological matrices.

Pressurized Hot Water Extraction and Enzyme-Catalyzed Conversion of Polyphenolic Glycosides in Onion Waste