Precise spectroscopy for fundamental physics

We have applied experimental techniques that were developed for use in atomic frequency standards andclocks to investigations of local Lorentz invariance, the linearity of quantum mechanics, andanomalous long-range spin-dependent forces. These experiments used a hyperfine transition in⁹Be⁺ ions in a Penning trap. Recently, we have studied hyperfine transitions in¹⁹⁹Hg⁺ ions in a linear rf trap. Hg⁺ ions might be used for similar investigations in the future.Work of the National Institute of Standards andTechnology. Not subject to US copyright.     

Automated analysis of terbutaline (BricanylR) in human plasma with liquid chromatography and electrochemical detection using column-switching (multidimensional chromatography)

Summary An automated method based on liquid chromatography has been developed for the determination of terbutaline in human plasma in the range of 5–50 pmole·ml^–1. The necessary sensitivity and selectivity was obtained by using electrochemical detection and a microprocessor-controlled column switching system. A combination of three columns was used: a C₈ type for pre-separation, a C₁₈ type for trapping and, for final separation, a strongly acidic ion exchanger. The accuracy of the method was examined by comparison with a method based on gas chromatography — mass spectrometry. The overall precision was ±3.5% and ±2.2% respectively at 5 and 50 pmole·ml^–1. The total absolute recovery for terbutaline and internal standard at the above concentration levels were in the range 85–106%.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Functionality and stability of heparin immobilized onto poly(dimethylsiloxane)

Poly(dimethylsiloxane) (PDMS) has become an attractive material when working in the field of microfluidics, mainly because of the rapid prototyping process it involves. The increased surface volume ratio in microchannels makes the interaction between sample and material surface highly important, evident when handling complex biological samples such as plasma or blood. This study demonstrates a new grade of non-covalent heparin surface that adds efficient anticoagulant property to the PDMS material. The surface modification is a simple and fast one-step process performed at neutral pH, optimal when working with closed microsystems. The heparin formed a uniform and functional coating on hydrophobic PDMS with comparatively high level of antithrombin-binding capacity. In addition, long-term studies revealed that the immobilized heparin was more or less stable in the microchannels over a time of three weeks. Recalcified plasma in contact with native PDMS showed complete coagulation after 1h, while no fibrin formation was detected in plasma incubated on heparin-coated PDMS within the same time. In conclusion, we see the heparin coating developed and evaluated in this study as a tool that greatly facilitates the use of PDMS in microfluidics dealing with plasma or blood samples.     

Analysis of enzymatically digested proteins and protein mixtures using a 9.4 Tesla Fourier transform ion cyclotron mass spectrometer

A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.     

A comparison of the electrochemical stabilities of metal,polymer and graphite coated nanospray emitters

Chronoamperometry (CA) and cyclic voltammetry (CV) were used to compare the electrochemical behavior of metal, polymer and graphite coated nanospray emitters. It is shown that electrochemical reactions occurring at the emitter surface limit the lifetime of the noble metal coated nanospray emitters while the graphite coated nanospray emitters show good electrochemical stabilities. Although the surface of the graphite coated emitters may be passivated at positive potentials, the conductive coating is not lost as for the noble metal coated nanospray emitters. The graphite coated nanospray emitters still produced a stable nanospray signal despite the presence of a passivated surface. The polymer (i.e. polyaniline) coated nanospray emitters showed very low electrochemical activity and could not be thoroughly tested by CA. The relative short lifetimes seen in the electrochemical tests are qualitatively comparable with those obtained in nanospray experiments, in which only the outmost tip of the emitter is electrochemically active. However, the electrochemical stress during CA far exceeds the stress during ESI, which implies that CA can be used to perform quick and simple estimates of emitter stabilities. To our knowledge, this is the first time the electrochemical behavior of metal, polymer and graphite coated nanospray emitters has been compared.     

Bioactive heparin immobilized onto microfluidic channels in poly(dimethylsiloxane) results in hydrophilic surface properties

A new composition of heparin coating for microfluidic systems made out of poly(dimethylsiloxane) (PDMS) was developed and evaluated. The coating that consists of a conditioning polyamine layer followed by two heparin/glutaraldehyde layers, resulted in channel surfaces with sufficient wettability to obtain flow of human normal plasma by capillary force alone. Hydrophilic channel walls are a desirable characteristic in microfluidic devices, since alternative pumping mechanisms must otherwise be included into the system. The immobilized heparin showed high antithrombin-binding capacity and a low degree of blood–material interaction. Plasma in contact with heparin-coated PDMS formed no detectable fibrin in a spectrophotometric assay by which plasma in contact with non-treated PDMS showed complete coagulation. The quartz crystal microbalance technique with energy dissipation monitoring (QCM-D) was utilized to obtain detailed information regarding adsorption kinetics and structural properties of the different layers composing the heparin coating.     

On-line coupling of a microelectrode array equipped poly(dimethylsiloxane) microchip with an integrated graphite electrospray emitter for electrospray ionisation mass spectrometry

A novel method for the manufacturing of microchips for on-chip combinations of electrochemistry (EC) and sheathless electrospray ionisation mass spectrometry (ESI-MS) is described. The technique, which does not require access to clean-room facilities, is based on the incorporation of an array of gold microcoil electrodes into a poly(dimethylsiloxane)(PDMS) microflow channel equipped with an integrated graphite based sheathless ESI emitter. Electrochemical measurements, which were employed to determine the electroactive area of the electrodes and to test the microchips, show that the manufacturing process was reproducible and that the important interelectrode distance in the electrochemical cell could to be adequately controlled. The EC-ESI-MS device was evaluated based on the ESI-MS detection of the oxidation products of dopamine. The results demonstrate that the present on-chip approach enables full potentiostatic control of the electrochemical cell and the attainment of very short transfer times between the electrochemical cell and the electrospray emitter. The transfer times were 0.6 and 1.2 s for flow rates of 1.0 and 0.5 microL min(-1), respectively, while the electrochemical conversion efficiency of the electrochemical cell was found to be 30% at a flow rate of 0.5 microL min(-1). To decouple the electrochemical cell from the ESI-MS high voltage and to increase the user-friendliness, the on-line electrochemistry-ESI-MS experiments were performed using a wireless Bluetooth battery-powered instrument with the chip floating at the potential induced by the ESI high voltage. The described on-chip EC-ESI-MS device can be used for fundamental electrochemical investigations as well as for applications based on the use of electrochemically controlled sample pretreatment, preconcentration and ionisation steps prior to ESI-MS.     

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.