Generation of ultrahigh peak capacity LC separations via elevated temperatures and high linear mobile-phase velocities

The use of a combination of ultraperformance liquid chromatography at approximately 11,000 psi on sub 2-microm particles combined with reversed-phase gradient chromatography at a temperature of 90 degrees C is described as applied to the analysis of endogenous and drug metabolites in human and animal urine. By using elevated temperatures, back pressures can be reduced while maintaining high flow rates and chromatographic efficiency, with peaks 1-3 s wide at the base. Application to urine samples provided a peak capacity of approximately 700 for a 10-min analysis and greater than approximately 1000 in 1 h. Despite the narrow nature of the peaks, good quality mass spectra were also obtained, allowing the identification of typical drug and endogenous metabolites. These ultra-high-resolution chromatograms should be ideal for the analysis of complex samples in, for example, metabolite identification, impurity identification, and metabonomic/metabolomic studies. Applications in natural product analysis and proteomics can also be envisaged.     

Comprehensive two-dimensional gas chromatographic separations with a microfabricated thermal modulator

Rapid, comprehensive two-dimensional gas chromatographic (GC × GC) separations by use of a microfabricated midpoint thermal modulator (μTM) are demonstrated, and the effects of various μTM design and operating parameters on performance are characterized. The two-stage μTM chip consists of two interconnected spiral etched-Si microchannels (4.2 and 2.8 cm long) with a cross section of 250 × 140 μm(2), an anodically bonded Pyrex cap, and a cross-linked wall coating of poly(dimethylsiloxane) (PDMS). Integrated heaters provide rapid, sequential heating of each μTM stage, while a proximate, underlying thermoelectric cooler provides continual cooling. The first-dimension column used for GC × GC separations was a 6 m long, 250 μm i.d. capillary with a PDMS stationary phase, and the second-dimension column was a 0.5 m long, 100 μm i.d. capillary with a poly(ethylene glycol) phase. Using sets of five to seven volatile test compounds (boiling point ≤174 °C), the effects of the minimum (T(min)) and maximum (T(max)) modulation temperature, stage heating lag/offset (O(s)), modulation period (P(M)), and volumetric flow rate (F) on the quality of the separations were evaluated with respect to several performance metrics. Best results were obtained with a T(min) = -20 °C, T(max) = 210 °C, O(s) = 600 ms, P(M) = 6 s, and F = 0.9 mL/min. Replicate modulated peak areas and retention times were reproducible to <5%. A structured nine-component GC × GC chromatogram was produced, and a 21 component separation was achieved in <3 min. The potential for creating portable μGC × μGC systems is discussed.     

Liquid chromatographic arsenic speciation with gas-phase chemiluminescence detection

We present a newly developed gas-phase chemiluminescence (CL) detection method for the separation and quantification of inorganic and organic arsenic species. Arsenite, arsenate, dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA) were separated by anion exchange using carbonate-bicarbonate and NaOH eluents with step-gradient elution. The separated species were passed through a UV photooxidation reactor which decomposed the organic species and converted them to inorganic As(V). Subsequent on-line hydride generation with acid and sodium borohydride produces AsH3 and H2, which are separated from the liquid in a gas-liquid separator. The produced AsH3, driven by H2, reacts with ozone in a small reflective cell located atop a photomultiplier tube, resulting in intense CL. In the present form, the limits of detection (LODs, signal-to-noise = 3), based on peak height, for arsenite, arsenate, MMA, and DMA are 0.4, 0.2, 0.5, and 0.3 microg/L, respectively, for a 100 microL injected sample. This analyzer demonstrates the robustness of the CL detection system for arsenic and provides an affordable alternative to atomic spectrometry for use as a detector after chromatographic speciation. We found no significant practical interferences.     

Liquid core waveguide for full imaging of electrophoretic separations

We employed a liquid core waveguide to image both DNA electrophoresis separations and isoelectric focusing of proteins. The utility of the system is demonstrated for DNA fragment sizing and protein separations. The system utilizes the liquid-core waveguide as an efficient window for both the excitation of separated samples and the collection of light through total internal reflectance, with an ability to detect target molecules in the zeptomolar range. Scanning the excitation laser along the length of the electrophoresis capillary excites individually separated analyte bands, while the fluorescence is collected end-on by an optical fiber coupled to a photomultiplier, thus, creating an image of the separation along the length of the capillary.     

Speciation of metallothionein-like proteins of the mussel Mytilus edulis at basal levels by chromatographic separations coupled to quadrupole and double-focusing magnetic sector ICPMS

Characterization and partial purification of metallothionein-like proteins (MLPs) of the mussel Mytilus edulis from natural populations of three coastal regions in Spain were performed. Size exclusion chromatography (SEC) with quadrupole (Q-ICPMS) or double-focusing inductively coupled plasma mass spectrometry (DF-ICPMS) detection was used first for speciation of cadmium in such natural samples and those of mussels exposed to 500 mg x L(-1) Cd in an aquarium tank. SEC results showed always a single Cd-MLP peak (MLP fraction). The contents in Cd, Cu, and Zn of this MLP fraction, of the high molecular weight protein pool (HMW), and of the whole cytosol were then measured by DF-ICPMS. Then, a given aliquot (50 microL) of MLPs with the highest values for UV molecular absorption at 254 nm (also the maximum sulfur and Cd, Cu, or Zn contents) was used to further fractionation. Fast protein liquid chromatography "on line" with Q-ICPMS was used for the purpose. Two Cd-MLP isoforms (MLP-1, MLP-2), with retention times (tR) of 15.7 and 16.0 min, were then detected in cytosols of the mussel samples of aquarium tank and also of the industrial area and Galicia coast. Conversely, wild coast mussels did not show any Cd-MLP signals at all. Analysis of essential elements copper and zinc in such cytosols by FPLC-Q-ICPMS revealed that these two metals were associated just to MLP-1. These results tend to indicate a different role for the two MLP isoforms detected in mussels (i.e., essential metals' homeostasis role seems to be tied to the MLP-1 isoform only). They illustrate the fact that trace metal speciation of unknown species in biological materials is becoming a challenge and points to the use of several complementary analytical techniques to obtain the required speciation information.     

添加剂对沥青燃烧性能的影响

温拌改性剂可以有效地降低摊铺或碾压温度,有利于隧道等特殊环境的路面施工,但关于温拌改性剂是否会影响沥青燃烧性能的研究却很少.分别选取3种不同添加剂ADZ、ADW、ADS加入到不同沥青中,利用数显氧指数仪测试其氧指数.探讨添加剂的加入在实现降低摊铺或碾压温度的情况下是否会影响沥青的燃烧性能,是否会对阻燃剂的阻燃效果产生影...     

Theory and use of the pseudophase model in gas-liquid chromatographic enantiomeric separations

The theory and use of the "three-phase" model in enantioselective gas-liquid chromatography utilizing a methylated cyclodextrin/polysiloxane stationary phase is presented for the first time. Equations are derived that account for all three partition equilibria in the system, including partitioning between the gas mobile phase and both stationary-phase components and the analyte equilibrium between the polysiloxane and cyclodextrin pseudophase. The separation of the retention contributions from the achiral and chiral parts of the stationary phase can be easily accomplished. Also, it allows the direct examination of the two contributions to enantioselctivity, i.e., that which occurs completely in the liquid stationary phase versus the direct transfer of the chiral analyte in the gas phase to the dissolved chiral selector. Six compounds were studied to verify the model: 1-phenylethanol, alpha-ionone, 3-methyl-1-indanone, o-(chloromethyl)phenyl sulfoxide, o-(bromomethyl)phenyl sulfoxide, and ethyl p-tolylsulfonate. Generally, the cyclodextrin component of the stationary phase contributes to retention more than the bulk liquid polysiloxane. This may be an important requirement for effective GC chiral stationary phases. In addition, the roles of enthalpy and entropy toward enantiorecognition by this stationary phase were examined. While enantiomeric differences in both enthalpy and entropy provide chiral discrimination, the contribution of entropy appears to be more significant in this regard. The three-phase model may be applied to any gas-liquid chromatography stationary phase involving a pseudophase.     

Two-dimensional gas-phase separations coupled to mass spectrometry for analysis of complex mixtures

Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-flight (TOF) MS. Implementation of FAIMS/IMS and IMS/MS interfaces using electrodynamic ion funnels greatly improves sensitivity. Evaluation of FAIMS/IMS/TOF performance for a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions and, hence, the benefit of FAIMS filtering prior to IMS/MS. The effective peak capacities in analyses of tryptic peptides are approximately 500 for FAIMS/IMS separations and approximately 10(6) for 3-D FAIMS/IMS/MS, providing a potential platform for ultrahigh-throughput analyses of complex mixtures.     

Intergranular pressure solution in internally wetted polycrystals: Effect of additives

Wetting of grain boundaries in polycrystalline materials leads to considerable changes in their physicochemical and mechanical properties. Under a constant compressive load, internally wetted materials display an enhanced deformability; creep rate increases sometimes by several orders of magnitude. The dominant creep mechanism is known as dissolution–precipitation or pressure solution; a stress-induced excessive chemical potential provides a driving force for dissolution of material within grain contacts, diffusion through the grain boundary solution film and re-precipitation elsewhere. Sensitivity of pressure solution rate to the chemical composition of the intergranular liquid was reported earlier, but the underlying mechanisms were poorly understood. In the present work, the creep experiments were carried out on poly- or monocrystalline sodium chloride in the presence of NaCl aqueous solution (pure or containing additives such as copper, magnesium and lead chlorides, K₄Fe(CN)₆ and urea). In all cases, pressure solution has been shown to be the main deformation mechanism. Creep rate decreases in the presence of additives which are known to affect the dissolution and growth processes of sodium chloride or its concentration in the brine. Rate-limiting stage (dissolution or diffusion) in various environments has been identified.     

Comparison of electrokinetics-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of human salivary proteins

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of high-abundance proteins such as amylases, mucins, proline-rich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITP/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1% false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.     

Effects of mobile-phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents

The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion models. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]- ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations.     

Retention of ionizable compounds on HPLC. 8. Influence of mobile-phase pH change on the chromatographic retention of acids and bases during gradient elution.

The relationships between retention and mobile-phase pH in gradient elution are studied for acids and bases. The apparent pH shift caused by the increasing amount of acetonitrile and methanol has been determined starting from a wide range of pH values. It is shown that good relationships between the retention of ionizable compounds and the pH of the aqueous buffer can be established if the same type of buffer (ammonium acetate in this work) is used for all pH points. Equations are proposed to fit the gradient retention data to the pH of the aqueous buffer. The proposed equation gives an account of the relative variation of the pKa of the compound in the reference to the variation of the pH of the buffer as both parameters change during gradient elution.