Peak modeling approach to accurate assignment of first-dimension retention times in comprehensive two-dimensional chromatography

Modeling of first-dimension retention of peaks based on modulation phase and period allows reliable prediction of the modulated peak distributions generated in the comprehensive two-dimensional chromatography experiment. By application of the inverse process, it is also possible to use the profile of the modulated peaks (their heights or areas) to predict the shape and parameters of the original input chromatographic band (retention time, standard deviation, area) for the primary column dimension. This allows an accurate derivation of the firstdimension retention time (RSD 0.02%) which is equal to that for the non-modulated experiment, rather than relying upon the retention time of the major modulated peak generated by the modulation process (RSD 0.16%). The latter metric can produce a retention time that differs by at least the modulation period employed in the experiment, which displays a discontinuity in the retention time vs modulation phase plot at the point of the 180° out-ofphase modulation. In contrast, the new procedure proposed here gives a result that is essentially independent of modulation phase and period. This permits an accurate value to be assigned to the first-dimension retention. The proposed metric accounts for the time on the seconddimension, the phase of the distribution, and the holdup time that the sampled solute is retained in the modulating interface. The approach may also be based on the largest three modulated peaks, rather than all modulated peaks. This simplifies the task of assigning the retention time with little loss of precision in band standard deviation or retention time, provided that these peaks are not all overloaded in the first or second dimension.

Effects of variation in modulator temperature during cryogenic modulation in comprehensive two-dimensional gas chromatography

Many modulation systems in comprehensive 2D GC (GC×GC) are based on cryogenic methods. High trapping temperatures in these systems can result in ineffective trapping of the more volatile compounds, whilst temperatures that are too low can prevent efficient remobilisation of some compounds. To better understand the trapping and release of compounds over a wide range of volatilities, we have investigated a number of different constant temperature modulator settings, and have also examined a constant temperature differential between the cryo-trap and the chromatographic oven. These investigations have led us to modify the temperature regulation capabilities of the longitudinally modulated cryogenic system (LMCS). In contrast to the current system, where the user sets a constant temperature for the cooling chamber, the user now sets the temperature difference between the cryo-trap and the chromatographic oven. In this configuration, the cooling chamber temperature increases during the chromatographic run, tracking the oven temperature ramp. This produces more efficient, volatility-dependent modulation, and increases the range of volatile compounds that can be analysed under optimal trap-and-release conditions within a single analytical run. This system also reduces cryogenic fluid consumption.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain

A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPARαLBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPARαLBD (aa196–468) was synthesized and ligated into the pDEST17 E. coli expression vector downstream of a MBP solubility fusion tag and an intermittent TEV protease cleavage site. Following auto-induction at 28 °C, PPARαLBD protein was purified to electrophoretic homogeneity by a nickel affinity chromatographic step, on-column TEV protease cleavage followed by Sephacryl S200 size exclusion chromatography. The recombinant protein displayed cross-reactivity with goat anti-(human PPARα) polyclonal antibody and was identified as human PPARα by trypic peptide mass finger-printing. The addition of a PPARα specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPARαLBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPARαLBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPARαLBD construct make it amenable to high through-put screening assays in drug discovery programs.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography

We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector.

Peak picking and the assessment of separation performance in two-dimensional high performance liquid chromatography

An algorithm was developed for 2DHPLC that automated the process of peak recognition, measuring their retention times, and then subsequently plotting the information in a two-dimensional retention plane. Following the recognition of peaks, the software then performed a series of statistical assessments of the separation performance, measuring for example, correlation between dimensions, peak capacity and the percentage of usage of the separation space. Peak recognition was achieved by interpreting the first and second derivatives of each respective one-dimensional chromatogram to determine the 1D retention times of each solute and then compiling these retention times for each respective fraction ‘cut’. Due to the nature of comprehensive 2DHPLC adjacent cut fractions may contain peaks common to more than one cut fraction. The algorithm determined which components were common in adjacent cuts and subsequently calculated the peak maximum profile by interpolating the space between adjacent peaks. This algorithm was applied to the analysis of a two-dimensional separation of an apple flesh extract separated in a first dimension comprising a cyano stationary phase and an aqueous/THF mobile phase as the first dimension and a second dimension comprising C18-Hydro with an aqueous/MeOH mobile phase. A total of 187 peaks were detected.

Phenyl-type and C1 stationary phases for environmentally friendlier chromatography

C1 and phenyl-type stationary phases were assessed in terms of their environmental impact on separations using as test solutes polycyclic aromatic hydrocarbons. Methanol (MeOH) and acetonitrile (ACN) mobile-phase gradients were employed. These stationary phases were examined to determine if different physical and chemical properties possessed by these surfaces decreased the organic solvent consumption, and yet maintained peak capacity. The cumulative energy demand (CED) was used to gauge the environmental impact of the separations. The separation of the polycyclic aromatic hydrocarbon test mixture using current methodologies (i.e. a C18/ACN combination) had a CED of 1.13 MJ-eq, and a peak capacity of 27 peaks (resolving 7 of 12 peak pairs with R_s>1). In comparison, a butyl phenyl stationary phase with a methanol mobile phase had a peak capacity of 26, but with a CED of 0.670 MJ-eq. Monolithic columns containing C18 and C1 phases were also tested. A monolithic C18 column with MeOH had the lowest CED at 0.675 MJ-eq, a peak capacity of 28 peaks and good resolving power (resolving ten peak pairs with R_s>1), suggesting that this is a viable option with respect to reducing environmental impact for these types of analyses.

Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography

Mixed mode stationary phases utilize secondary retention mechanisms to add a dimensionality to the surface of high performance liquid chromatography (HPLC) adsorbents. This approach was used by several authors to improve the separation performance of single dimension separations. We explored the magnitude of these secondary interactions by performing an off-line two-dimensional (2D)-HPLC separation with a Scherzo SM-C18 column of a β-lactoglobulin tryptic digest with a mobile phase pH of 7 in the first dimension and 2 in the second. Mechanism divergence was determined using the peak capacity and a geometric approach to factor analysis, to measure the correlation. This separation was repeated with a C18 stationary phase as a control. It was found that the C18 column had a correlation coefficient of 0.784, smaller than the mixed mode column, 0.884. This indicated that the retention mechanisms of the C18 column were more divergent under these two pH environments than the mixed mode column. However, the SM-C18 still provided alternative selectivity of the peptides to that of the C18 and could be considered as a good alternative for further 2D-HPLC separations.

π-Selective stationary phases: (III) Influence of the propyl phenyl ligand density on the aromatic and methylene selectivity of aromatic compounds in reversed phase liquid chromatography

The retention characteristics of phenyl type stationary phases for reversed phase high performance liquid chromatography are still largely unknown. This paper explores the retention process of these types of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97, 2.50 μmol m⁻²). The aromatic and methylene selectivities increased with increasing ligand density until a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic selectivity than methylene selectivity indicating that these columns are suitable for separations involving aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were observed to be influenced by the ligand density. It is likely that the changing retention characteristics are caused by the different topologies of the stationary phases at different ligand densities. At high ligand densities, the partition coefficient became constant.

π-Selective stationary phases: (I) Influence of the spacer chain length of phenyl type phases on the aromatic and methylene selectivity of aromatic compounds in reversed phase high performance liquid chromatography

Phenyl type stationary phases of increasing spacer chain length (phenyl, methyl phenyl, ethyl phenyl, propyl phenyl and butyl phenyl, with 0–4 carbon atoms in the spacer chain, respectively) were synthesised and packed in house to determine the impact that the spacer chain length has on the retention process. Two trends in the aromatic selectivity, q_aromatic, were observed, depending on whether the number of carbon atoms in the spacer chain is even or odd. Linear log k′ vs ϕ plots were obtained for each stationary phase and the S coefficient was determined from the gradient of these plots. For the phenyl type phases, the S vs n_c plots of the retention factors of linear polycyclic aromatic hydrocarbons vs the number of rings exhibit a distinct discontinuity that between 3 and 4 rings, which increases with increasing spacer chain length for even phases but decreases for odd phases. Accordingly, we suggest that the retention factors depend differently on the number of carbon atoms in the spacer chain depending on whether this number is even or odd and that this effect is caused by different orientations of the aromatic ring relative to the silica surface.