Novel comprehensive multidimensional liquid chromatography approach for elucidation of the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 strain.

Shikimic acid is a intermediate of aromatic amino acid biosynthesis and the preferred starting material for production of the most commonly prescribed anti-influenza drug, Tamiflu. Its six-membered carbocyclic ring is adorned with several chiral centers and various functionalities, making shikimic acid a valuable chiral synthon. When microbially-produced, in addition to shikimic acid, numerous other metabolites are exported out of the cytoplasm and accumulate in the culture medium. This extracellular matrix of metabolites is referred to as the microbosphere. Due to the high sample complexity, in this study, the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 was analyzed by liquid chromatography and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection. GC analysis of the trimethylsilyl derivatives was also carried out in order to support the elucidation of the selected metabolites in the microbosphere. The elucidation of the metabolic fraction of this bacterial strain might be of valid aid for improving, through genetic changes, the concentration and yield of shikimic acid synthesized from glucose. Graphical abstract.

Longitudinal On-Column Thermal Modulation for Comprehensive Two-Dimensional Liquid Chromatography.

Longitudinal on-column thermal modulation for comprehensive two-dimensional liquid chromatography is introduced. Modulation optimization involved a systematic investigation of heat transfer, analyte retention, and migration velocity at a range of temperatures. Longitudinal on-column thermal modulation was realized using a set of alkylphenones and compared to a conventional valve-modulator employing sample loops. The thermal modulator showed a reduced modulation-induced pressure impact than valve modulation, resulting in reduced baseline perturbation by a factor of 6; yielding a 6-14-fold improvement in signal-to-noise. A red wine sample was analyzed to demonstrate the potential of the longitudinal on-column thermal modulator for separation of a complex sample. Discrete peaks in the second dimension using the thermal modulator were 30-55% narrower than with the valve modulator. The results shown herein demonstrate the benefits of an active focusing modulator, such as reduced detection limits and increased total peak capacity.

Multidimensional GC using planar microfluidic devices for the characterization of phenolic antioxidants in fuels.

A multidimensional gas chromatographic approach using planar microfluidic devices for Deans switching has been developed and implemented for the characterization of sterically hindered phenolic compounds used as antioxidants in fuels. Detection and quantitation was conducted with MS in selected ion monitoring mode. A complete analysis is conducted in less than 15 min with precision greater than 5.5% at 1 and 25 ppm w/w (ppm(w)). LODs of 50 ppb w/w (ppb(w)) or better in selected ion monitoring mode and a linear range of 100 ppb(w) to 100 ppm(w) with a correlation coefficient greater than 0.998 were attained for all analytes. Unique to this analytical configuration is the use of a mass spectrometer capable of monitoring the column effluent from either dimension by incorporating a high-temperature rotary valve and a three-port planar microfluidic device. High-molecular-weight (C25-C40) fuel contaminants eluting from the first column can be selectively sent to the mass spectrometer for profile characterization in scan mode. These compounds would otherwise be retained substantially by the low-phase-ratio analytical column employed in the second dimension.

Selectivity tuning via temperature pulsing using low thermal mass liquid chromatography and monolithic columns.

Low thermal mass LC was applied to the capillary LC separation of a complex insecticide mixture by increasing temperature and decreasing gradients, as well as fast selected temperature pulses to increase resolution of overlapped components. The technology was applied using a new generation of capillary monolithic stationary phases. Considerable peak shifts and selectivity changes were observed for given temperature conditions. The concept of temperature pulsing during an elution profile shows promise for increasing resolution in difficult separations and can provide a relatively simple means to solve coelution problems.

Applications of planar microfluidic devices and gas chromatography for complex problem solving.

The application of planar microfluidic devices in GC for the separation of components of interest otherwise difficult to separate in a single analysis is presented. A variety of configurations were used for parallel chromatography, column effluent splitting, back flushing, selectivity tuning, valve less switching and column isolation, heart cutting, and comprehensive multidimensional chromatography. The synergies of recently commercialized planar microfluidic devices combined with the resolving power of fused-silica capillary columns are demonstrated. Difficult separations were accomplished in one single analysis, such as light hydrocarbons in air with high-moisture content, fixed gases in hydrocarbons, trace sulfur containing compounds in natural gas, and oxygenated compounds in hydrocarbons, among others.

Direct measurement of part-per-billion levels of dimethyl sulfoxide in water by gas chromatography with stacked injection and chemiluminescence detection.

Dimethyl sulfoxide (DMSO) is a chemical of industrial significance with many important applications. DMSO is used as an industrial solvent, in drug delivery and healthcare applications, among others. Analysis of DMSO in water typically involves extensive sample preparation, enrichment, and derivatization to improve solute detectability. A novel gas chromatographic procedure has been developed for the direct measurement of trace levels of DMSO in an aqueous matrix, such as potable water. The technology utilizes stacked injection techniques for in-column solute enrichment, a precolumn to enhance solute focusing effects, and sulfur chemiluminescence detection for matrix suppression and sensitivity. A detection limit of 2 parts per billion (ppb) (v/v) of DMSO in water was attained. Relative precision of less than 7% at the concentration of 10 ppb (v/v) of DMSO was demonstrated. A correlation coefficient of 0.9988 was obtained over a range of 2 ppb (v/v) to 100 ppb (v/v). No detectable carry-over was found at the 5 ppb (v/v) level whereas less than 4% carry-over was observed at the 100 ppb (v/v) level. Various sample storage media including glass, polyethylene, and polycarbonate were also studied to minimize solute loss. Recoveries greater than 84% were achieved with all storage media tested. The method was found to be reliable and simple to implement.

Temperature pulsing for controlling chromatographic resolution in capillary liquid chromatography.

In this study we introduce the implementation of rapid temperature pulses for selectivity tuning in capillary liquid chromatography. Short temperature pulses improved resolution in discrete sections of chromatograms, demonstrated for ion-exchange chromatography (IC) and hydrophilic interaction chromatography (HILIC) modes. Using a resistively heated column module capable of accurate and rapid temperature changes, this concept is first illustrated with separations of small anions by IC using a packed capillary column as well as a series of nucleobases and nucleosides by HILIC using a silica monolithic column with zwitterionic functionality (ZIC-HILIC). Both positive (increasing temperature) and negative temperature pulses are demonstrated to produce significant changes in selectivity and are useful approaches for improving resolution between coeluted compounds. The approach was shown to be reproducible over a large number of replicates. Finally, the use of temperature gradients as well as other complex temperature profiles was also examined for both IC and HILIC separations.

Comprehensive two dimensional gas chromatography review.

Advances, developments and applications in 2-D comprehensive GC (GC x GC) from 2007 through October 2008 are reviewed, with emphasis on modulation, and data handling, and applications of current relevance. Industrial perspectives as well as suggestions where further developments would be beneficial are summarized in the concluding remarks.

Determination of short-chain branching content in polyethylene by pyrolysis comprehensive multidimensional gas chromatography using low thermal mass column technology.

A research effort was undertaken to utilize the pyrolysis process to create fragments of polyethylene that could be indicative of branching, and allow quantitiation of said short-chain branches by pyrolysis comprehensive 2-D GC (Py-GC x GC). Several strategies for sample introduction and pyrolysis such as the in-column pyrolysis device and the programmed temperature vaporizer (PTV) were studied. The chromatographic separations were executed using low-thermal mass (LTM) comprehensive 2-D GC (GC x GC). A series of polyethylene-co-hexene samples were analyzed and a linear correlation of 1-hexene content with branching peak ratio was found. Correlation coefficients were determined as 0.97 for the measurements performed.

Instability of hexane-acetonitrile mobile phases used for the chromatographic analysis of triacylglycerides.

Comprehensive 2-D LC is an emerging separation technique that has seen a rapid increase in applications in the last decade. The technique has been applied for the separation of numerous complex mixtures including triacylglycerides (TAG). Determination of TAG in food products such as rice, palm, and canola oils have been previously described and the technique of choice utilizes a silver-modified silica column with hexane-ACN as the mobile phase. Repeated retention time inconsistencies were experienced in our studies when this mobile phase was applied to the separation of natural and synthetic mixtures containing TAG. The present report summarizes a study performed to determine the relative stability of ACN, propionitrile (PCN), and butyronitrile (BCN) at concentrations ranging from 0.43 to 2.8% in hexane and heptane. The data obtained suggest that unless evaporative loss of the mobile phase is prevented, TAG retention time irreproducibility can be significant when using mobile-phase mixtures prepared with ACN or PCN. BCN should be used as the solvent modifier in cases where evaporation cannot be prevented.