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A study was conducted to synthesize and characterize subphthalocyanine (SubPc)-fused dimers and trimers bearing fluorine, iodine, and thioether peripheral substituents. Absorption spectroscopy and electrochemical studies reveal that the substituents have a strong effect on the electronic properties of the macrocycles and good communication was shown between the subphthalocyaninic moieties within the oligomeric structures.

A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDITOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup. The use of monolithic capillary columns sufficiently resolved intact proteins so that peptide mass fingerprinting analysis by MALDI-TOF MS resulted in the identification of close to 40 unique proteins from 120 ng of sample obtained from a prefractionated MCF10 cell line at pH 6.34, where the identifications of several of these proteins were also confirmed by intact MW and tandem mass spectrometric analysis. The reproducibility of this method has been demonstrated to be sufficient for the purpose of protein identifications. Experimental values of protein intact MW are obtained and compared to that expected for each protein identified.

A demonstration that the 2D-monitoring of the Rh-oxidation state in a catalyst bed could be achieved during the catalytic reaction is presented using a suitable spectroscopic cell with gas supply and on-line mass spectrometry. For this purpose, X-ray images are recorded with the charged coupled device (CCD) camera at each energy around the Rh K-edge with and without the spectroscopic cell.

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a [beta]-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.

Bloodstream infections are an important cause of serious morbidity and mortality. Rapid detection and identification of specific pathogens from blood or other clinical specimens could improve the rational use of antimicrobial therapy in clinical medicine and have a great impact on the outcome of patients with systemic infections. Polymerase chain reaction using generic primers was used to amplify genomic DNA of different bacterial strains. The identification was accomplished by measuring the molecular masses of the PCR products using ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry. DNA from 10 bacterial species was amplified by PCR, and the resulting amplification products were analyzed. In all cases, the measured molecular masses of the PCR products matched the theoretical value for the species-specific DNA sequence. However, three pairs of bacteria could not be distinguished since the theoretical difference in amplicon molecular mass was < 1.0 Da (different sequence, same base composition of amplicon). Determination of intra- and interday mass reproducibility resulted in relative standard deviations of 0.0030 and 0.018%, respectively. The limit of detection of the presented method was shown to be 0.5 genome equivalents/PCR. The specificity of the method in a human background was successfully tested by amplifying and analyzing 1000-10000 genome equivalents of Staphylococcus aureus spiked into human plasma.

Poly(styrene-co-divinylbenzene)-based monolithic capillaries of an inner diameter of 200 [micro]m and a length of 2-5 mm have been used to construct C[a.sup.2+]-, A[g.sup.+]-, and N[a.sup.+]selective electrodes. The membranes consist of a solution of ionophore and ion exchanger in bis(2-ethylhexyl) sebacate or 2-nitrophenyl octyl ether, which are used as plasticizers in conventional PVC-based membranes. With capillaries of low porosity, the potentiometdc responses down to [10.sup.-8]-[10.sup.-9] M solutions do not depend on the composition of the internal solution, which indicates a strong suppression of transmembrane ion fluxes. Thus, no tedious optimization of the inner solution is required with monolith ISEs. The lower detection limits of A[g.sup.+]- and C[a.sup.2]- ISEs are comparable to the best ones obtained earlier with optimized inner solutions. Additionally, a monolithic N[a.sup.+]-selective ISE has been obtained exhibiting a lower detection limit of 3 X [10.sup.-8] M N[a.sup.+]. With monolithic capillaries of higher porosity and fused-silica GC caprilades, the transmembrane flux effects are noticeable but still significantly smaller than with conventional PVC membranes.

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene--divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.

A previously unknown isoform of the type I major antenna protein of photosystem II of spinach was identified, and its amino-terminal sequence was characterized by a novel kinetic digestion approach, in which sequential tryptic digesdon was followed by analysis of both released peptides and truncated proteins by reversed-phase high-performance liquid chromatography--electrospray ionization mass spectrometry. Using nonpolar, monolithic, 200-[micro]m-i.d. separation columns based on poly(styrene/divinylbenzene) copolymer and applying gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid, released peptides and truncated proteins could be separated and mass analyzed in a single chromatographic run. This enabled a straightforward identification of the fragments removed from the amino-terminal ends of the protein, which was essential for the characterization of the antenna isomers showing the most significant sequence variation in the amino-terminal region. The sequences of the amino termini were derived from the differences in molecular mass between intact and truncated proteins and were corroborated by sequencing using tandem mass spectrometry and database searching. The sequence of the 23 amino-terminal residues of the previously unknown isoform differed from that of the other two known isoforms only in one and three amino acids, respectively. Such subtle changes in amino acid sequence are supposed to play an important role in the supramolecular organization of photosynthetic antenna proteins.

Monolithic capillary columns were prepared by transition metal-catalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo, endo-dimethanonaphthalene inside a silanized 200-[micro]m-i.d. fused-silica capillary using a mixture of toluene and 2-propanol as porogen and [Cl.sub.2](P[Cy.sub.3]).sub.2]Ru(=CHPh) as initiator. The synthesized columns allowed the rapid and highly efficient separation of single- and double-stranded nucleic acids by ion-pair reversed-phase high-performance liquid chromatography and of proteins by reversed-phase high-performance liquid chromatography. Compared to 3-mm-i.d. analytical columns synthesized from an identical polymerization mixture, a considerable improvement in the peak widths at haft-height of oligonucleotides in the order of 60-80% was obtained. Significant differences in morphology between the capillary column, where the surface of the monolith was rather soft and rugulose, and the analytical column, where the surface was very sharp and smooth, were observed, most probably due to differences in polymerization kinetics. The synthesized monoliths were successfully applied to the separation of the diastereomers of phosphorothioate oligodeoxynucleotides. To confirm the identity of the eluting compounds on the basis of their intact molecular masses, the chromatographic separation system was on-line hyphenated to electrospray ionization mass spectrometry.

An array of monolithic poly(styrene/divinylbenzene) capillaries with individual column thermostats was constructed to demonstrate its utility for the separation of nucleic acids, proteins, and tryptic digests in combination with UV absorbance detection. Because of polymerization-related variation in surface area of monolithic columns, the concentration of acetonitrile required for elution of DNA fragments in denaturing HPLC may vary sufficiently to affect the degree of denaturation. Modulation of column temperature offers a convenient way to harmonize elution profiles among columns. Individual regulation of column temperature also provides the means to determine rapidly in a single parallel run the optimum temperature for resolution of biomolecules. Given the high reproducibility of separations among columns and the ease with which poly(styrene/divinylbenzene)-based stationary phases can be modified to accommodate different modes of chromatography, such arrays will find broad applicability in proteogenomics.

Research has been conducted on the monochromatic light beam. The use of this beam in differentiating various reaction centers is described.

The authors review the publications on the lowest homologues of phthalocyanines, subphthalocyanines. The topics of interest include synthesis and chemical and physical properties of subphthalocyanines and their relatives subazaporphyrins and subnaphthalocyanines.

An algorithm was developed for the computer-aided interpretation of fragment ion spectra from collision-induced dissociation of multiply charged oligodeoxynucleotide ions generated by electrospray ionization. The method compares the experimental spectrum to the m/z values predicted by employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between measured spectrum and predicted m/z values. To substantiate the identity of investigated sequence and reference sequence, or to identify point mutations or insertions/deletions, the reference sequence is systematically varied by incorporating all four possible nucleotides A, T, G, and C at each position in the sequence followed by identification of the correct sequence by the lowest fitness value. Collision energy was shown to have a major impact on the interpretability of the tandem mass spectra by the comparative sequencing algorithm, and the optimal collision energy depended on the length of the fragmented oligodeoxynucleotide. The analytical system was successfully applied to verify DNA sequences as well as to detect and localize point mutations or insertions/deletions in 5-51-meroligodeoxynucleotides.

Research has been conducted on the terminal alkynes. The rhodium-catalyzed intermolecular hydroamination of these alkynes has been performed and the results demonstrate that the terminal alkynes react with anilines in the presence of the cationic rhodium(I) catalyst under mild reaction conditions.

Supra-molecular architectures containing lanthanide ions because of their intrinsic magnetic, spectroscopic, and optical properties are focused. It is shown that absorption spectra, combined with electrochemical data, demonstrate the dual effect associated with attachment of the sulfonamide group.

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200-(mu)m i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. With gradients of acetonitrile in 100 mM triethylammonium acetate, the synthesized columns allowed the rapid and highly efficient separation of single-stranded oligodeoxynucleotides and double-stranded DNA fragments by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Compared with capillary columns packed with micropellicular, octadecylated poly-(styrene/divinylbenzene) particles, an improvement in column performance of approximately 40% was obtained, enabling the analysis of an 18-mer oligodeoxynucleotide with a column efficiency of more than 190 000 plates per meter. The chromatographic separation system was on-line-coupled to electrospray ionization mass spectrometry (ESI-MS). To improve the mass spectrometric detectabilities, 25 mM triethylammonium bicarbonate was utilized as an ion-pair reagent at the cost of only little reduction in separation performance and acetonitrile was added postcolumn as the sheath liquid through the triaxial electrospray probe. High-quality mass spectra of femtomole amounts of 3-mer to 80-mer oligodeoxynucleotides were recorded showing very little cation adduction. Double-stranded DNA fragments ranging in size from 51 to 587 base pairs were separated and detected by IP-RP-HPLC-ESI-MS. Accurate mass determination by deconvolution of the mass spectra was feasible for DNA fragments up to the 267-mer with a molecular mass of 165 019, whereas the spectra of longer fragments were too complex for deconvolution because of incomplete separation due to overloading of the column. Finally, on-line IP-RP-HPLC tandem MS was applied to the sequencing of short oligodeoxynucleotides.

One major difficulty in the analysis of nucleic acids by electrospray mass spectrometry is represented by the affinity of the polyanionic sugar-phosphate backbone for nonvolatile cations, especially ubiquitous sodium and potassium ions. A simple on-line sample preparation system comprising a microflow pumping system and 45 x 0.8-mm-i.d. microcolumns packed with weak or strong cation-exchange resins is described for the efficient removal of cations from nucleic acid samples. Samples were analyzed by flow injection analysis at a 3-5 [[micro]liter]/min flow of 10 mM triethylamine in 50% water-50% acetonitrile. After on-line desalting, mass spectra of oligonucleotides revealed no significant sodium adduct peaks. Moreover, signal-to-noise ratios were greatly enhanced compared to direct injection of the samples. Electrospray mass spectrometry with on-line sample preparation allowed accurate molecular mass determinations of picomole amounts of crude oligonucleotide preparations ranging in size from 8 to 80 nucleotides within a few minutes. The good linearity of the calibration plot ([R.sup.2] = 0.9988) over at least 2 orders of magnitude and a relative standard deviation in peak areas of less than 9% permitted the sensitive quantitative measurement of oligonucleotides in a concentration range of 0.2-20 [[micro]molar] with selected-ion monitoring. Finally, the on-line sample preparation system was evaluated for the mass spectrometric analysis of complex oligonucleotide mixtures.

An amplified flow immunoassay (AFIA) was developed for cocaine, which combines a noncompetitive immunoenzymometric assay (IEMA) with an on-line detection of the enzyme label alkaline phosphatase (ALP) by a substrate-recycling biosensor. In the IEMA, the analyte cocaine first binds to a labeled polyclonal anti-cocaine antibody. Then, the excess labeled antibody is separated on an affinity column that contains a perfusion chromatography carrier modified by immobilized cocaine. The unbound complexes of the analyte cocaine with the ALP-labeled antibody are detected postcolumn. The detector senses phenol produced by ALP from phenyl phosphate. As detector, an amperometric substrate-recycling biosensor was used, which consists of a Clark-type oxygen electrode covered by tyrosinase and pyrroloquinoline quinone-dependent glucose dehydrogenase. The lower limit of detection is 380 pM (38 fmol) for cocaine. The sampling rate is 26/h. Cocaine could be detected from 'real samples' with an imprecision of [+ or -]10% (n = 3) and with a recovery of 49 [+ or -] 3% for various concentrations. AFIA is generally important as a new approach for the fast detection of picomolar concentrations of haptens.

A capillary electrochromatograph incorporating a gradient-forming system generally employed in HPLC is described, and the use of gradient elution in reversed phase electrochromatography is demonstrated by the separation of PTH-amino acids and steroid hormones. The gradient former employs two reciprocating displacement pumps to control the composition of the eluent in the reservoir at the column inlet with time in a controlled manner. Thus, the composition of the mobile phase flowing through the column and driven by electrosmotic forces can be changed with time in a controlled fashion as customary in HPLC with gradient elution. The design of the system allows also for isocratic elution by pumping the eluent of constant composition through the cavity at the column inlet and thus continuously supplying fresh buffer. The eluent gradient is generated by the two pumps and a 10 [[micro]liter] mixer. From there the liquid passes at a flow rate of 0.1-0.2 mL/min through the 17 [[micro]liter] cavity housing the column inlet and an electrode. The flow of the mobile phase was electrosmotic at an effective overall electric field strength of 500-1500 V/cm through a 50 [[micro]meter] x 20/12 cm capillary column packed with 3.5 [[micro]meter] octadecylated silica particles. Gradient profiles generated in this manner were highly reproducible. The same-day and day-today reproducibilities of the electrosmotic flow were found to be better than 3%. The use of the capillary electro-chromatographic system was demonstrated with isocratic and gradient elution for the separation of complex mixtures of biologically interesting substances. The influence of the column temperature on the electrosmotic flow velocity and retention of PTH-amino acids was also investigated.