Search

Hyperfast GC/MS below 60 s of measurement time has been used for the measurement of explosives. The new flow-field thermal gradient GC (FF-TG-GC) utilizes a modified transport process of the explosives at lowered temperatures. In combination with the focusing effect of the gradient, high-resolution chromatograms are obtained even in very short time intervals. The reduction of the elution temperature by applying a thermal gradient along the chromatographic column is demonstrated by the simulation of the migration of analytes through the column. The simulation shows an interesting effect of the difference between maximum temperature and elution temperature of analytes during their separation with the spatial gradient. The results show the benefit of the gradient elution both from a modeling perspective and by measurements of explosives with low limits of detection (LOD) in the range from 0.1 to 20 μg/mL (0.5 to 150 pg of analyte mass on column). Results were compared to state-of-the-art vacuum outlet GC/MS as a reference method. A correlation between the reduction of elution temperatures and lower LODs are found for thermal labile nitrate ester explosives (EGDN, NG, ETN, and PETN), while no significant influence of the reduced elution temperature on LODs of more stable explosives, like DNT and TNT, was found.

LC/ESI/MS is a technique widely used for qualitative and quantitative analysis in various fields. However, quantification is currently possible only for compounds for which the standard substances are available, as the ionization efficiency of different compounds in ESI source differs by orders of magnitude. In this paper we present an approach for quantitative LC/ESI/MS analysis without standard substances. This approach relies on accurately predicting the ionization efficiencies in ESI source based on a model, which uses physicochemical parameters of analytes. Furthermore, the model has been made transferable between different mobile phases and instrument setups by using a suitable set of calibration compounds. This approach has been validated both in flow injection and chromatographic mode with gradient elution.

Gradient elution chromatography is an industrial process used to separate and purify multi-component chemical mixtures. In this article, we consider an optimal control problem in which manipulative...

Perhaps the most consistent trend in the development of high-performance liquid chromatography (HPLC) since its inception in the 1960s has been the continuing reach for ever faster analyses. The pioneering work of Knox, Horvath, Halasz, and Guiochon set forth a theoretical framework that was used early on to improve the speed of HPLC, primarily through the commercialization of smaller and smaller particles. Over the past decade, approaches to improving the speed of HPLC have become more diverse, and now practitioners of HPLC are faced with the difficult task of deciding which of these approaches will lead them to the fastest analysis for their application. Digesting the rich literature on the optimization of HPLC is a difficult task in itself, which is further complicated by contradictory marketing messages from competing commercial outlets for HPLC technology. In this perspectives article we provide an overview of the theoretical and practical aspects of the principal modern approaches to improving the speed of HPLC. We present a straightforward theoretical basis, informed by decades of literature on the problem of optimization, that is useful for comparing different technologies for improving the speed of HPLC. Through mindful optimization of conditions, high-performance separations on the subminute timescale are now possible and becoming increasingly common under both isocratic and gradient elution conditions. Certainly the continued development of ultrafast separations will play an important role in the development of two-dimensional HPLC separations. Despite the relatively long history of HPLC as an analytical technique, there is no sign of a slow-down in the development of novel HPLC technologies.

The theory of the dual-mode gradient elution in liquid chromatography involving any type of simultaneous changes in column temperature and mobile-phase composition is developed following Drake's approach. The theory was tested in the retention prediction of six alkylbenzenes in aqueous eluting systems modified by acetonitrile. Significant delay phenomena, i.e., a lag between the programmed gradient temperature and the temperature in the oven, and a lag between the oven temperature and the effective temperature the analyte feels in the column, were detected. When these phenomena were taken into account, the retention prediction obtained for all solutes under all dual-mode gradient conditions was excellent. The average percentage error between experimental and predicted retention times is below to 2%.

The rigorous derivation of the fundamental equation of the dual-mode gradient elution in liquid chromatography involving any type of simultaneous changes in flow rate and mobile-phase composition is developed following Drake's approach. The equation is a generalization of the already known fundamental equations of single gradient when either the mobile-phase composition or the flow rate is constant. The theory was tested in the retention prediction from isocratic data of 18 o-phthalaldehyde derivatives of amino acids in eluting systems modified by acetonitrile or methanol. The retention prediction obtained for all solutes under all dual-mode gradient conditions was excellent. The average percentage error between experimental and predicted retention times ranged from 0.9 to 2.5%. Two approximations that simplify the calculations considerably without increasing the above error were also proposed.

Theoretical calculations are presented to optimize modulation period for maximum total peak capacity in comprehensive two-dimensional HPLC (2D-HPLC) taking into account the effect of modulation on the apparent peak capacity of the first-dimension (1D) separation. Results indicate that modulation periods are most favorable when they are adjusted to approximately 2.2-4 times the standard deviation of a 1D peak in order to avoid excessively short run times at the second dimension (2D). Data are presented that effective peak capacities of several thousand in 60 min can be expected for practical 2D-HPLC conditions, utilizing 1D gradient elution followed by 2D isocratic elution, that remain at approximately 50-70% of the theoretical maximum peak capacity. This work suggests that lower modulation frequencies and longer 2D separation times than previously proposed are favorable under realistic chromatographic conditions, alleviating some practical problems associated with 2D-HPLC.

The original work carried out by Freiling and Drake in gradient liquid chromatography is rewritten in the current language of reversed-phase liquid chromatography. This allows for the rigorous derivation of the fundamental equation for gradient elution and the development of two alternative expressions of this equation, one of which is free from the constraint that the holdup time must be constant. In addition, the above derivation results in a very simple numerical solution of the various equations of gradient elution under any gradient profile. The theory was tested using eight catechol-related solutes in mobile phases modified with methanol, acetonitrile, or 2-propanol. It was found to be a satisfactory prediction of solute gradient retention behavior even if we used a simple linear description for the isocratic elution of these solutes.

A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.

Gradient scouting is the best way to decide the most suitable elution mode in reversed-phase high-performance liquid chromatography (RP-HPLC). A simple rule for this decision involves the evaluation of the ratio Δt/tG (where Δt is the difference in the retention time between the last and the first peak and tG is the time of the gradient), which is equated to a “magic” reference number. In this article, we demonstrate that the reference number depends on the cosolvent selected. This is illustrated with an example, using methanol as cosolvent. For methanol/water mobile phases, when Δt/tG < 0.15, an isocratic elution can be used, otherwise, gradient elution is advisable. In the example, a gradient elution was needed and a segmented gradient program was used to reduce the time of analysis. This article can serve as a reference and basis for laboratory experimentation in an upper-level analytical chemistry course.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic solvent gradient elution conditions, typically carried out at ambient temperatures. These conditions can however induce conformational effects with proteins as evident from changes in their biological or immunological activities. By monitoring the influence of temperature on the retention and band-broadening characteristics of proteins, the role of conformational processes in these lipophilic environments can be examined. These processes can then be interpreted in terms of a two-state model involving a native (N) and a fully unfolded species (U) or more complex folding/unfolding models. In the present study, the gradient elution RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temperatures between 5 and 85 degrees C with n-octadecyl (C18)- and n-butyl (C4)-silica reversed-phase sorbents. The interaction of these proteins with these reversed-phase sorbents has also been examined in terms of the contributions that the heme prosthetic group of SWMYO and the disulfide bonds in HEWL make to the stabilization of the native conformation of these proteins in these hydrophobic environments. The observed interconversions of multiple peak zones of SWMYO and HEWL in the presence of C18 and C4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conformational intermediates of proteins in these perturbing environments has been examined. Pseudo-first-order rate constants have been derived for these processes from analysis of the dependencies on time of the concentration of the different protein species at specified temperatures. The relationship of these processes to the conformational transitions that these proteins can undergo via molten globule-like intermediates (i.e., compact denatured states with a significant amount of residual secondary structure) in solution has also been examined. This study thus further documents an experimental strategy to assess the folding/unfolding behavior of globular proteins in the presence of hydrophobic surfaces and aquo-organic solvents, whereby the system parameters can potentially affect the preservation of native conformations, and thus the function, of the protein under these conditions.

The peak capacity was measured for an ultrahigh-pressure gradient elution chromatogram of a fluorescently tagged tryptic digest of ovalbumin. The peak widths in the chromatogram were determined by measuring the peak height and the second derivative at the peak maximum. This approach for measuring peak widths was programmed into a computer, and the software accurately determined the general progression of peak widths by measuring 47 peaks throughout the chromatogram in under 10 s. Peak capacity was determined by taking the definite integral of the plot of reciprocal base peak width versus retention time. This calculation of peak capacity is a linear transformation with respect to separation space, so the method is more rigorously accurate than previous methods. The peak capacity for the chromatogram was calculated to be 316.

The chromatographic retention and wave characteristics of β-lactoglobulins A and B were studied experimentally for various nonlinear chromatographic modes of operation, including isocratic and gradient elution and frontal and displacement chromatography. In contrast to typical Langmuirian behavior, β-lactoglobulins A and B show increasing retention times with increasing column loading and diffuse fronts and sharp rears at moderate injection volumes. At higher injection volumes, the retention times increase initially and then drop. Significant tailing fronts are found in frontal chromatography, and the displacement effect is observed at high feed concentrations. The unusual behavior for both modes of overloaded chromatography has been explained consistently with an S-shaped isotherm model. The displacement chromatography of these non-Langmuirian adsorbates has also been analyzed. Serious tailing of displacement trains is observed, and, consequently, resolution is severely affected. The cause of non-Langmuirian behavior is postulated to be aggregation of adsorbed proteins on the stationary phase.

A method based on high-performance liquid chromatography using column-switching is described for the screening of diuretics and probenecid in urine samples. The system uses a 20- x 2.1-mm i.d. precolumn, packed with a Hypersil ODS-C18, 30-microns stationary phase, for the on-line sample cleanup and enrichment. Untreated urine samples are directly injected, and the precolumn is flushed for 1 min with water to eliminate polar matrix components. The retained analytes are then back-flushed by means of a six-port switching valve onto a Hypersil ODS-C18 analytical column (5 microns, 250- x 4-mm i.d.), where they are separated using an acetonitrile/phosphate buffer (pH = 3) gradient elution. Under these conditions, the separation and identification of diuretics and probenecid can be achieved with satisfactory selectivity and sensitivity. The described procedure is very simple and rapid since no off-line manipulation of the sample is required, the total analysis time being ca. 15 min.

A system was developed that generates two gradients simultaneously, 180 degrees out of phase. Essential elements of the system were a pump delivering solvent A at a constant velocity, a pump delivering solvent B at a constant velocity, two mixing chambers, and a ten-port valve. By use of this gradient device and two reversed-phase columns packed with 1000-A pore diameter, 8-microns particle size macroporous poly-(styrene-divinylbenzene) resin, six proteins were separated in 40 s in a single gradient cycle.

An HPLC undergraduate experiment on the analysis of traditional Chinese medicine (TCM) has been developed. Two commonly used herbs (glycyrrhizae radix and cinnamomi ramulus) are studied. Glycyrrhizin, cinnamic acid, and cinnamaldehyde are chosen as markers for the herbs. The dried herbs in their natural state and a TCM preparation in powder form are analyzed. Gradient elution with diode-array detection is used for the separation and identification of the markers. Solid-phase extraction is employed for cleaning the sample solutions.

The amino acid taurine (2-aminoethanesulfonic acid) is present as a nutritional supplement in many sports drinks. An experiment, suitable for a junior-senior level instrumental analysis course, is described to measure the amount of taurine in these sports drinks. A pre-column derivatization with Sanger's reagent, 2,4-dinitrofluorobenzene, is followed by an HPLC separation utilizing a gradient elution, and detection at 360 nm.

Etude de l'influence de la composition de la phase mobile (ethanol-chloroforme) et de la temperature de la colonne, de la relation entre la composition des copolymeres et le volume de retention et entre la masse moleculaire et le volume de retention

Erreurs liees a la fois au concept de «linear solvent strength elution» et a des causes experimentales

Polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF) are present with several hundred organic compounds in raw fly ash extract. A first-step normal-phase semipreparative high-pressure liquid chromatography (HPLC) fractionation with a three-solvent gradient elution program separates PCDD and PCDF into a single fraction with about 100 other components. The PCDD and PCDF in this fraction are further isolated from the accompanying compounds by a second-step HPLC fractionation using a reverse-phase semipreparative column and a two-solvent gradient elution program. The distribution of PCDD and PCDF into five subfractions facilitates isomer identification and their quantitation. The separation of PCDD and PCDF by this procedure shows high recovery and good reproducibility.

High-molecular-weight (> 300 daltons) polycyclic aromatic compounds which were extracted from a carbon black were separated by reversed-phase high-performance liquid chromatography using gradient elution. Fractions were collected and analyzed by mass spectrometry and spectrofluorimetry. While only 15 compounds could be positively identified, 38 others were given tentative structural assignments, among which were several large sulfur heterocycles.

Utilisation de la methode (HPLC-IR en continu) avec des solutions aqueuses, en utilisant un nouveau nebuliseur capable de deposer et d'eliminer rapidement la phase mobile

Complex hydroxyl aromatic fractions isolated from nondistillable coal-derived liquids were separated by a combination of normal-phase and reversed-phase liquid chromatography. The normal-phase chromatographic step effected the separation of hydroxyl aromatics into mono- and dihydroxyl fractions. The mono- and dihydroxyl fractions from the coal-liquid sample were characterized with infrared and field-ionization mass spectrometry (FIMS). A monohydroxyl fraction of oils from a Kentucky coal-liquid sample was separated with an optimized reversed-phase chromatographic system. The monohydroxyl fraction was completely eluted by using a combination of isocratic and gradient elution conditions. The combination of reversed-phase liquid chromatography and FIMS provided a unique method for the characterization of the monohydroxyl fraction of oils from the Kentucky coal-liquid sample.

Generalisation de la theorie de Boehm, Martin, Armstrong et Bui en utilisant la theorie de McMillan-Mayer pour les solutions diluees de polymere

High-performance liquid chromatography (HPLC) is shown to be a powerful tool in the analysis of carotenoid and porphyrin pigments. Columns packed with 5-..mu..m irregular silica gel particles by a high density and high constant pressure method allow efficient separation of mixtures of total nonsaponifiable carotenoids from recent sedimentary situations. Good reproducibility of retention times (within 2%) is achieved in the gradient elution mode. However, attention must be paid to reequilibration of the column after each injection by washing with the less polar solvent for a minimum of 15 min (for carotenoids) or of 30 min (for porphyrins). HPLC appears to be useful in ''fingerprinting'' petroporphyrin distributions in crude oil.

A study was made of the accuracy of quantitative high-performance liquid chromatographic (HPLC) determinations of UV absorbing substances in plant extracts. By use of gradient elution on a reversed-phase column and UV detection, 62 extracts from plant leaves were investigated. The obtained chromatograms provided a distribution function of the relative abundance of observed peak areas. A computer simulation of such plant extract analyses permitted the estimation of the real distribution function of component absorbances. By use of the same simulations, the probability that a given determination could be performed with success was obtained. These data should be helpful to analysts working on the chromatographic analysis of such samples to estimate the accuracy of a particular determination in quantitative terms. Realistic determination limits for phenolic compounds in plant extracts were defined and computed. The same method can be applied to the analysis of other biological samples and to other chromatographic techniques. 7 figures, 1 table.

Publisher Summary This chapter discusses variable gradient device for chromatography. Since the introduction of gradient elution to chromatography several years ago, there have been many applications of the general technique in the fractionation of mixtures of substances having a wide range of elution requirements. In such cases, the use of a gradient greatly facilitates the operational aspects of chromatography, as it automatically encompasses these requirements. Moreover, in the case of substances that have a tendency to be eluted in broad, tailing bands when a single solvent is used, gradient elution provides a means of sharpening the peaks without introducing the artifacts that can result when stepwise changes in eluting agents are made. Several methods for the production of gradients have appeared in the literature. With the exception of those that can utilize differential pumping rates, all require a change in apparatus for each change in the shape of the gradient.

A simple gradient apparatus, consisting of a peristaitic pump in addition to a standard high-pressure pump, is described. The device is used to make a single-run ion chromotographic separation of sulfite, sulfate, and thiosulfate in less than 15 min. This separation required a step gradient with 4.8 mM NaHCO/sub 3//4.7 mM Na/sub 2/CO/sub 3/ as start eluent and 6.9 mM NaHCO/sub 3//8.6 mM Na/sub 2/CO/sub 3/ is final eluent when two (4 x 50) mm Dionex anion precolumns in series were used as separator. The eluent compositions were simplex optimized.