Unusual properties of the aspartate receptor cytoplasmic fragment: Flexible structure and slow dissociation

The transmembrane receptors which provide the information input for bacterial chemotaxis consist of a periplasmic ligand-binding domain, two transmembrane helices, and a cytoplasmic signaling domain. The cytoplasmic domain is central to the generation of intracellular signals via a phosphorylation cascade, as well as sensory adaptation through covalent modification. We are seeking insights into the mechanisms of signaling by investigating the changes which occur in a cloned 31 kDa cytoplasmic domain fragment (c-fragment) of the aspartate receptor. Point mutations in the cytoplasmic domain have been found which lead to either of two behavioral extremes of constant tumbling or constant smooth-swimming, mimicking the two signaling states of the receptor. Recent studies have revealed a monomer-oligomer equilibrium and correlated the smooth-swimming behavioral phenotype with the tendency to form oligomers. We have characterized the association/dissociation process by studying the dissociation kinetics for two oligomer-forming c-fragments using gel filtration chromatography and circular dichroism. We have also used CD to study the secondary structure of isolated monomer and oligomer forms. These studies indicate that the slow dissociation process proceeds through a highly unfolded intermediate and that the monomeric c-fragments are partially unfolded. A model is proposed to describe these results where intermolecular coiled-coil interactions in the oligomer are substituted by intramolecular coiled-coil interactions in the monomer. This change in subunit contacts may mimic the structural change involved in transmembrane signaling. We have also probed the tertiary structure of the c-fragments and found that they are globally dynamic proteins under physiological solvent conditions. Comparison of proteolysis of the c-fragment with that of the cytoplasmic domain of the intact receptor suggests that the intact receptor possesses similar flexibility. Thus the unusual flexibility of the c-fragment may persist in the intact receptor, and ligand-modulation of this flexibility may play a role in the mechanism of transmembrane signaling

Model for predicting comprehensive two-dimensional gas chromatography retention times

A model for approximating the relative retention of solutes in comprehensive two-dimensional gas chromatography (GCxGC) is presented. The model uses retention data from standard single-column temperature-programmed separations. The one-dimensional retention times are first converted into retention indices and then these indices are combined in a simple manner to generate a retention diagram. A retention diagram is an approximation of the two-dimensional chromatogram that has retention order and spacing in both dimensions similar to that found in the experimental chromatogram. If required, the retention diagram can be scaled to more closely resemble the two-dimensional chromatogram. The model has been tested by using retention time data from single-column gas chromatography-mass spectrometry and valve-based GCxGC. A total of 139 volatile organic compounds (VOCs) were examined. Approximately half of the VOCs had a single functional group and a linear alkyl chain (i.e., compounds with the structure Z-(CH(2))(n)-H). The retention diagrams had primary retention orders that were in excellent agreement with the GCxGC chromatograms. The relative secondary retention order for compounds with similar structures was also accurately predicted by the retention diagram. However, the relative secondary retention for compounds with dissimilar structures, such as acyclic alcohols and multi-substituted alkylbenzenes, were less accurately modeled. This study demonstrates how readily available single-column retention time data can be used to provide an a priori estimate of the relative retention of solutes in a GCxGC chromatogram. Such a capability is useful for screening possible combinations of stationary phases

Comprehensive Two-Dimensional Gas Chromatography with Pattern Modulation

Comprehensive two-dimensional gas chromatography (GC×GC) modulators normally transfer primary column effluent to the head of the secondary column as a series of sharp pulses. Such pulses are produced with time-varying temperature gradients in thermal modulation or with time-varying flow patterns in flow modulation. Thermal modulators produce narrow peaks at optimal flow rates, but require large amounts of consumables or a highly engineered heating/cooling system. Flow modulators involve simpler hardware and no additional consumables. However, flow modulators require a large increase in secondary column flow or transfer only a small portion of the primary effluent to the secondary column. This study examines a new method of producing GC×GC separations with a flow modulator. Instead of injecting narrow pulses, the modulator transfers primary effluent to the secondary column in the form of an intricate injection pattern. The detector signal is deconvoluted and converted to a two-dimensional chromatogram. The high duty cycle of the technique (\u3e50%) leads to deconvoluted peaks with twenty times greater intensity than those produced by conventional modulation with a Deans switch modulator. Pattern modulation can be produced without requiring elevated carrier flows. This study evaluates the efficacy of pattern modulation GC×GC by analyzing a standard mixture of 43 oxygenated organic compounds and an E85 fuel sample

Multidimensional Gas Chromatography: Fundamental Advances and New Applications

Multidimensional gas chromatography (MDGC) is a technique for isolating and identifying volatile and semi-volatile organic compounds present in complex mixtures. MDGC separations employ two or more gas chromatographic separations in a sequential fashion. The separation produced by each stage is maintained, at least in part, so that the resolving power of the composite separation exceeds that of the individual stages. Although MDGC has been in existence for more than 50 years, each year brings improvements and insights that allow a wider range of samples to be analyzed with more informative, higher-resolution separations. This review summarizes the advances and applications of MDGC that have been described in nearly 200 articles published between January 2011 and November 2012. The vast majority of MDGC separations use two columns and so they are classified as two-dimensional gas chromatography (2-D GC). These separations frequently fall into one of two categories: heart-cutting 2-D GC or comprehensive two-dimensional gas chromatography (GC x GC). While these two techniques use similar hardware, they are implemented in very different ways. Heart-cutting 2-D GC is a more mature technology than GC x GC and as a result there have been far fewer research articles in the past two years devoted to heart cutting 2-D GC. This review examines studies aimed at improving the materials and methods used for conducting MDGC separations. Recent applications of MDGC in a wide range of fields are also considered

Iterative Trapping of Gaseous Volatile Organic Compounds in a Capillary Column

The iterative trapping method has been developed for concentrating gaseous volatile organic compounds (VOCs) prior to gas chromatographic analysis. VOCs are trapped in a 50 cm x 0.53 mm metal capillary column coated with a 7 µm thick film of polydimethylsiloxane (PDMS). Iterative trapping does not employ the two-step thermal desorption approach used by most VOC concentrating techniques. Instead, a four-step cycle involving synchronized changes in flow direction and temperature is repeated throughout the sampling process. This iterative process causes VOCs to accumulate within the capillary well past the level where a standard two-step method reaches its saturation limit. Iterative trapping is capable of sampling and desorbing C5 through C11 n-alkanes with uniform efficiency. This new technique, in its current form, is most appropriate for focusing VOCs from gas volumes on the order of 10 mL. Iterative trapping increases the focusing power of a weak sorbent like PDMS and allows narrow chromatographic peaks to be generated without the use of high desorption temperatures or a secondary focusing stage

Method for reducing the ambiguity of comprehensive two-dimensional chromatography retention times

Comprehensive two-dimensional chromatography generates a two-dimensional chromatogram from a one-dimensional signal array. This process can only be done unambiguously when the range of secondary retention times is less than the modulation period. However, complex samples often produce wider ranges of secondary retention times. Peaks with retention times that exceed the modulation period are said to be wrapped-around . A simple algorithm has been developed that determines absolute retention times when wrap-around occurs. A sample is first analyzed under standard modulation conditions and then re-analyzed with a modulation period that is increased by an integer fraction of the original modulation period. Retention shifts along the secondary axis are used to determine absolute retention times. A theoretical analysis has been performed to optimize the implementation conditions and characterize the technique limitations. The efficacy of this algorithm has been tested through a series of isothermal GC x GC separations. This method has been found to be particularly useful during the initial stages of method development

The Multi-Mode Modulator: A Versatile Fluidic Device for Two-Dimensional Gas Chromatography

A fluidic device called the multi-mode modulator (MMM) has been developed for use as a comprehensive two-dimensional gas chromatography (GC x GC) modulator. The MMM can be employed in a wide range of capacities including as a traditional heart-cutting device, a low duty cycle GC x GC modulator, and a full transfer GC x GC modulator. The MMM is capable of producing narrow component pulses (widths \u3c50 ms) while operating at flows compatible with high resolution chromatography. The sample path of modulated components is confined to the interior of a joining capillary. The joining capillary dimensions and the position of the columns within the joining capillary can be optimized for the selected modulation mode. Furthermore, the joining capillary can be replaced easily and inexpensively if it becomes fouled due to sample matrix components or column bleed. The principles of operation of the MMM are described and its efficacy is demonstrated as a heart-cutting device and as a GC x GC modulator

Analysis of Biodiesel/Petroleum Diesel Blends with Comprehensive Two-Dimensional Gas Chromatography

Comprehensive two-dimensional gas chromatography (GCxGC) is used to analyze petroleum diesel, biodiesel, and biodiesel/petroleum diesel blends. The GCxGC instrument is assembled from a conventional gas chromatograph fitted with a simple, in-line fluidic modulator. A 5% phenyl polydimethylsiloxane primary column is coupled to a polyethylene glycol secondary column. This column combination generates chromatograms where the fatty acid methyl esters (FAMEs) found in biodiesel occupy a region that is also populated by numerous cyclic alkanes and monoaromatics found in petroleum. Fortunately, the intensities of the petroleum hydrocarbon peaks are far lower than the intensities of the FAME peaks, even for blends with low biodiesel content. This allows the FAMEs to be accurately quantitated by direct integration. The method is calibrated by analyzing standard mixtures of soybean biodiesel in petroleum diesel with concentrations ranging from 1 to 20% v/v. The resulting calibration curve displays excellent linearity. This curve is used to determine the concentration of a B20 biodiesel/petroleum diesel blend obtained from a local retailer. Excellent precision and accuracy are obtained

Analysis of siloxanes in hydrocarbon mixtures using comprehensive two-dimensional gas chromatography

A comprehensive two-dimensional gas chromatography (GC × GC) method for separating siloxanes from hydrocarbons has been developed using a systematic process. First, the retention indices of a set of siloxanes and a set of hydrocarbons were determined on 6 different stationary phases. The retention indices were then used to model GC × GC separation on 15 different stationary phase pairs. The SPB-Octyl × DB-1 pair was predicted to provide the best separation of the siloxanes from the hydrocarbons. The efficacy of this stationary phase pair was experimentally tested by performing a GC × GC analysis of gasoline spiked with siloxanes and by analyzing biogas obtained from a local wastewater treatment facility. The model predictions agreed well with the experimental results. The SPB-Octyl × DB-1 stationary phase pair constrained the hydrocarbons to a narrow range of secondary retention times and fully isolated the siloxanes from the hydrocarbon band. The resulting GC × GC method allows siloxanes to be resolved from complex mixtures of hydrocarbons without requiring the use of a selective detector

High speed Deans switch for low duty cycle comprehensive two-dimensional gas chromatography

A new high-speed valve-based modulator has been designed and tested for use in comprehensive two-dimensional gas chromatography (GC × GC). The modulator is a Deans switch constructed from two micro-volume fittings and a solenoid valve. Modulator performance was characterized over a wide range of device settings including the magnitude of the switching flow, the gap between the tips of the primary and secondary column, the primary column flow rate, and the carrier gas identity. Under optimized conditions, the modulator was found to be capable of generating narrow pulses (\u3c50 ms) of primary effluent with a 2 mL/min secondary column flow. This capability will ultimately allow the modulator to be used with GC × GC separations employing a wide range of detectors and secondary column geometries. The main disadvantage of this modulator is that it employs a low sampling duty cycle, and thus it produces separations with sensitivities that are lower than those produced with thermal modulators or differential flow modulators. The efficacy of the new high-speed Deans switch modulator was demonstrated through the GC × GC separation of a hydrocarbon standard and gasoline. Precise quantitation of individual components was possible provided the modulation ratio was kept greater than 2.0