High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.     

Total synthesis of the monoterpenoid alkaloid (±)-tangutorine

A novel approach to a formal total synthesis of the monoterpenoid indole alkaloid (±)-tangutorine has been developed starting from an α,β-unsaturated cyclic dehydroamino ester. Synthesis of the rather unusual trans-substituted 2,3-indoloquinolizidine substructure was accomplished via Cu(II)-mediated conjugate addition and organozinc/copper coupling as the key steps, thereby setting the stage for ring-closing metathesis to produce the quinolone substructure. Finally, Bischler-Napieralski cyclization gave rise to the pentacyclic system of (±)-tangutorine thereby realizing a formal synthesis in an overall yield of 5.2% in eight consecutive steps.     

Catalytic chain transfer copolymerization of methyl methacrylate and methyl acrylate

Bis(aqua)bis((difluoroboryl)dimethylglyoximate)cobalt(II) (COBF) has proven to be a very effective catalytic chain transfer agent in the copolymerization of MA and MMA. The chain transfer activity depends on the fraction of MMA in the monomer feed and the total radical concentration. The polymerization can be described by a model that combines features of catalytic chain transfer for MMA homopolymerization and cobalt mediated controlled radical polymerization of MA. According to the model part of the COBF is covalently bonded to MA‐ended polymeric radicals and cannot take part in the chain transfer step. The model can also account for the observed inhibition time that occurs at high chain transfer agent concentration and low fraction of MMA in the monomer feed.     

Potential of poly(styrene-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) monolithic columns for the LC-MS analysis of protein digests

Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample. Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak capacity, and also increases protein identification, as observed from higher identification scores and a larger number of identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm column is higher.     

Kinetic optimisation of open-tubular liquid-chromatography capillaries coated with thick porous layers for increased loadability

The kinetic optimisation of open-tubular liquid chromatography (OTLC) columns has been revisited by taking the thick-film effects for porous coatings on retention, column resistance, band broadening and mass loadability into account. Considering the most advantageous case (i.e. where the retentive layer allows for the same high internal diffusion coefficient as conventional porous particles), calculations show the need for the development of coating procedures leading to porous films filling up approximately 50-70% of the total column diameter. Furthermore, to achieve optimum kinetic performance for separations of small molecules with total analysis times of less than 8h (k'=9), total column diameters should be less than 6 μm with lengths typically greater than 0.8m for N values of 125,000-500,000 at a pressure of 400 bar. The use of elevated temperature LC (90°C) is also shown to increase the allowable total column diameter to up to 9 μm for a larger range of N values (100,000-880,000).     

Novel Selectivity-Based Forensic Toxicological Validation of a Paper Spray Mass Spectrometry Method for the Quantitative Determination of Eight Amphetamines in Whole Blood

Paper spray tandem mass spectrometry is used to identify and quantify eight individual amphetamines in whole blood in 1.3 min. The method has been optimized and fully validated according to forensic toxicology guidelines, for the quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), para-methoxyamphetamine (PMA), para-methoxymethamphetamine (PMMA), and 4-fluoroamphetamine (4-FA). Additionally, a new concept of intrinsic and application-based selectivity is discussed, featuring increased confidence in the power to discriminate the amphetamines from other chemically similar compounds when applying an ambient mass spectrometric method without chromatographic separation. Accuracy was within ±15% and average precision was better than 15%, and better than 20% at the LLOQ. Detection limits between 15 and 50 ng/mL were obtained using only 12 μL of whole blood.

Open image in new window

Graphical abstract ?

     

Determination of major carotenoids in vegetables by capillary electrochromatography

A simple and rapid method for the isocratic separation and determination of carotenoids (carotenes and xanthophylls) in vegetables by CEC is described. The capillary column (100 microm ID, 25 cm effective length) was packed with 3 microm Hypersil ODS particles. The optimized mobile phase contained 60% ACN, 35% THF and 5% of a 5 mM Tris aqueous buffer of pH 8. beta-Carotene, lycopene and lutein were separated with efficiencies of 66 000-128 000 plates/m within a short time (less than 12 min for the last peak eluted, 13/13'-cis-beta-carotene). An excellent resolution of the three carotenoids, as well as partial resolution of their geometrical isomers, was achieved. Application to the determination of the analytes in carrot, tomato, spinach and corn was demonstrated.     

Bound and free water distribution in wood during water uptake and drying as measured by 1D magnetic resonance imaging

Knowledge on moisture transport in wood is important for understanding its utilization, durability and product quality. Moisture transport processes in wood can be studied by Nuclear Magnetic Resonance (NMR) imaging. By combining NMR imaging with relaxometry, the state of water within wood can be identified, i.e. water bound to the cell wall, and free water in the cell lumen/vessel. This paper presents how the transport of water can be monitored and quantified in terms of bound and free water during water uptake and drying. Three types of wood from softwood to hardwood were selected covering a range of low to high density wood; pine sapwood and oak and teak. A calibration is performed to determine the different water states in each different wood type and to convert the NMR signal into moisture content. For all wood types, water transport appeared to be internally limited during both uptake and drying. In case of water uptake, free water was observed only after the cell walls were saturated with bound water. In case of drying, the loss of bound water starts only after vanishing of free water, irrespective of the position. Obviously, there is always a local thermodynamic equilibrium of bound and free water for both uptake and drying. Finally, we determined the effective diffusion coefficient (D _eff ). Experimentally determined diffusion constants were compared with those derived by the diffusion models for conceptual understanding of transport mechanism. We found that diffusion in the cell wall fibers plays a critical role in the transport process.     

Deceleration and electrostatic trapping of OH radicals

A pulsed beam of ground state OH radicals is slowed down using a Stark decelerator and is subsequently loaded into an electrostatic trap. Characterization of the molecular beam production, deceleration, and trap loading process is performed via laser induced fluorescence detection inside the quadrupole trap. Depending on the details of the trap loading sequence, typically 10(5) OH (X2Pi(3/2),J=3/2) radicals are trapped at a density of around 10(7) cm(-3) and at temperatures in the 50-500 mK range. The 1/e trap lifetime is around 1.0 s.     

Multiple packets of neutral molecules revolving for over a mile

The level of control that one has over neutral molecules in beams dictates their possible applications. Here we experimentally demonstrate that state-selected, neutral molecules can be kept together in a few mm long packet for a distance of over one mile. This is accomplished in a circular arrangement of 40 straight electrostatic hexapoles through which the molecules propagate over 1000 times. Up to 19 packets of molecules have simultaneously been stored in this ring structure. This brings the realization of a molecular low-energy collider within reach.