In situ time-resolved fluorescence spectroscopy in the frequency domain in capillary electrochromatography

In situ time-resolved fluorescence spectroscopy for capillary electrochromatography (CEC) is described in the frequency domain. Fluorescence decay of the solute molecules is collected directly in the packed stationary phase of the CEC capillary. The fluorescence lifetime profile of the solute molecules reveals the microenvironments they experience in the C18 chromatographic interface. A quartz flow cell and experimental optimization of the signal-to-noise ratio are described that enable the collection of high-quality decay data and subsequent calculation of fluorescence lifetime profiles of the solute molecules. The distribution of pyrene (PY), 1-pyrenemethanol (PY-MeOH), and 1-pyrenebutanol (PY-BuOH) into the C18 stationary phase and the solute-C18 phase interactions are probed, under separation conditions for CEC. All three molecules display a Gaussian distribution of lifetimes, consistent with an ensemble of heterogeneous microenvironments in the C18 stationary phase. The least polar molecule PY diffuses deeply into and interacts extensively with the C18 phase, experiencing high hydrophobicity and significant heterogeneity of microenvironments. The retention order of PY-MeOH, PY-BuOH, and PY in CEC is determined by their interactions with the stationary phase, revealed by their fluorescence lifetime distributions.     

Imaging solute distribution in capillary electrochromatography with laser scanning confocal microscopy

A method for the direct observation of solute molecules interacting with a C18 stationary phase under real separation conditions in capillary electrochromatography (CEC) is investigated. The experiments were performed in a capillary electrochromatographic mode; however, the method and findings are useful both in CEC and revered-phase liquid chromatography. The distribution of solute molecules in the packed capillary is directly imaged with laser scanning confocal fluorescence microscopy. Conventional imaging techniques produce images where the C18 silica beads cannot be distinctively identified as a result of the deep depth of field. The optical sectioning capability of confocal imaging overcomes this problem to afford clearly defined images of the stationary-phase packing and the surrounding mobile phase. Fluorescein molecules are preferentially distributed in the mobile phase under reversed-phase chromatographic conditions. Nile Red and rhodamine 6G molecules prefer the environments of the porous C18 beads. Intensity distributions over time for areas within the stationary-phase beads differ from distributions of areas outside the beads in the mobile phase. Images taken at different depths into the capillary probe the internal structure of the C18 beads. While the internal structures of most beads are porous, confocal images show a small fraction (2%) of the silica beads have porous shells and nonporous cores. The capability of imaging the stationary phase distinctively from the mobile phase opens the possibilities of studying the quality of stationary phase, the structure of the column packing, and the mechanisms of separation.     

Dynamic windowing algorithm for the fast and accurate determination of luminescence lifetimes

An algorithm for the accurate calculation of luminescence lifetimes in near-real-time is described. The dynamic rapid lifetime determination (DRLD) method uses a window-summing technique and dynamically selects the appropriate window width for each lifetime decay such that a large range of lifetimes can be accurately calculated. The selection of window width is based on an optimal range of window-sum ratios. The algorithm was compared to alternative approaches for rapid lifetime determination as well as nonlinear least-squares (NLLS) fitting in both simulated and real experimental conditions. A palladium porphyrin was used as a model luminophore to quantitatively evaluate the algorithm in a dynamic situation, where oxygen concentration was modulated to induce a change in lifetime. Unlike other window-summing techniques, the new algorithm calculates lifetimes that are not significantly different than the slower, traditional NLLS. In addition, the computation time required to calculate the lifetime is 4 orders of magnitude less than NLLS and 2 orders less than other iterative methods. This advance will improve the accuracy of real-time measurements that must be made on samples that are expected to exhibit widely varying lifetimes, such as sensors and biosensors.     

Practical aspects of the maximum entropy inversion of the laplace transform for the quantitative analysis of multi-exponential data

The number, position, area, and width of the bands in a lifetime distribution give the number of exponentials present in time-resolved data and their time constants, amplitudes, and heterogeneities. The maximum entropy inversion of the Laplace transform (MaxEnt-iLT) provides a lifetime distribution from time-resolved data, which is very helpful in the analysis of the relaxation of complex systems. In some applications both positive and negative values for the lifetime distribution amplitudes are physical, but most studies to date have focused on positive-constrained solutions. In this work, we first discuss optimal conditions to obtain a sign-unrestricted maximum entropy lifetime distribution, i.e., the selection of the entropy function and the regularization value. For the selection of the regularization value we compared four methods: the chi2 criterion and Bayesian inference (already used in sign-restricted MaxEnt-iLT), and the L-curve and the generalized cross-validation methods (not yet used in MaxEnt-iLT to our knowledge). Except for the frequently used chi2 criterion, these methods recommended similar regularization values, providing close to optimum solutions. However, even when an optimal entropy function and regularization value are used, a MaxEnt lifetime distribution will contain noise-induced errors, as well as systematic distortions induced by the entropy maximization (regularization-induced errors). We introduce the concept of the apparent resolution function in MaxEnt, which allows both the noise and regularization-induced errors to be estimated. We show the capability of this newly introduced concept in both synthetic and experimental time-resolved Fourier transform infrared (FT-IR) data from the bacteriorhodopsin photocycle.     

On-the-fly fluorescence lifetime detection of humic substances in capillary electrophoresis

On-the-fly fluorescence lifetime detection was investigated as a tool for studying humic substances in capillary zone electrophoresis (CZE). Humic substances are complex, heterogeneous mixtures of natural products that tend to migrate in a single, broad CZE peak. The intrinsic fluorescence lifetime of five humic substances from the International Humic Substances Society (IHSS) was monitored using excitation at 488 or 364 nm to produce intensity-lifetime electropherograms for each of the substances. Each frequency-domain lifetime measurement, collected at subsecond intervals during the CZE run, contains the equivalent of a complete decay profile. Lifetime analysis of each decay profile was used to construct a lifetime-resolved electropherogram for each lifetime component, from which the variation in relative intensity contributions of each lifetime across the broad CZE peak could be determined. Absorption spectra, fluorescence excitation-emission spectra, and lifetime profiles of batch solutions of the samples were determined as well. It was found that, whereas absorption and fluorescence spectral characteristics tended to discriminate between humic acids and fulvic acids, the batch solution lifetime profiles discriminated instead between samples from different sources, regardless of fraction. On-the-fly lifetime detection provided a more detailed view of the fluorescence decay of the samples, including greater resolution of lifetimes for two of the fulvic acids and greater discrimination among samples based on lifetime profiles across the CZE peaks.     

Phase-sensitive fluorescence lifetime detection in capillary electrophoresis

A simple and highly sensitive fluorescence lifetime detection method for capillary electrophoresis has been introduced. The detection scheme is based on the integrated phase-sensitive fluorescence intensity. The integrative nature of the method results in high sensitivity of lifetime detection. The limit of detection is 7.8 amol of fluorescein injected, representing a 2 orders of magnitude improvement over the detection limits previously reported in the UV-visible region. Rayleigh scattering, Raman scattering, and background fluorescence can be effectively suppressed by setting the detector out of the phase from the background signal. Fluorescence background can be eliminated whether the fluorescence lifetime of the background is longer or shorter than the solute molecules of interest. The signal-to-noise ratio of measurements is optimized by varying the modulation frequency and the detector phase angle.     

On-the-Fly Fluorescence Lifetime Determination with Total Emission Detection in HPLC

The entire fluorescence decay profile during HPLC elution has been directly measured on-the-fly in HPLC at higher sensitivity than in previous literature reports. The fluorescence is excited with the fourth harmonic (266 nm) of a pulsed Nd:YAG laser system and detected broadband with a photomultiplier tube and a digital storage oscilloscope. Detection limits in the range 1-10 ppb are found for several individual polycyclic aromatic hydrocarbons (PAHs) when the total time-integrated fluorescence is analyzed. The chromatograms of PAH mixtures containing 8-10 species were lifetime analyzed with a simple phase plane analysis, in which a single lifetime is determined from the fluorescence decay profile for each point on the chromatogram. The determination of lifetimes under coelution conditions is also illustrated and discussed.     

Comparison of subtractive Kramers-Kronig analysis and maximum entropy model in resolving phase from finite spectral range reflectance data

The maximum entropy model (MEM) and Kramers-Kronig (K-K) analysis were compared with the aim of phase retrieval from reflectance. The object was to test two different phase-retrieval methods when reflectance is known at a finite frequency range and data fitting is not performed beyond the finite frequency band. In addition, it was assumed that the phase is known only at one or two anchor points. As an example, we study the terahertz reflection spectrum related to a semiconductor and an optical spectrum of potassium chloride. It is shown that the MEM resolves the complex refractive index of a medium, in the vicinity of initial and final points of the spectra, better than singly and doubly subtractive K-K relations. Both methods give only satisfactory results in the event of one anchor point, but in the case of two anchor points, the MEM is better than doubly subtractive K-K. It is proposed that the MEM should be used instead of K-K analysis, for a priori information of phase at two anchor points, for the purpose of resolving the complex refractive index of a medium from reflectance with high accuracy.     

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.     

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

Imaging of fluorescent yield and lifetime from multiply scattered light reemitted from random media

The feasibility of employing fluorescent contrast agents to perform optical imaging in tissues and other scattering media has been examined through computational studies. Fluorescence lifetime and yield can give crucial information about local metabolite concentrations or environmental conditions within tissues. This information can be employed toward disease detection, diagnosis, and treatment if noninvasively quantitated from reemitted optical signals. However, the problem of inverse image reconstruction of fluorescence yield and lifetime is complicated because of the highly scattering nature of the tissue. Here a light propagation model employing the diffusion equation is used to account for the scattering of both the excitation and fluorescent light. Simulated measurements of frequency-domain parameters of fluorescent modulated ac amplitude and phase lag are used as inputs to an inverse image-reconstruction algorithm, which employs the diffusion model to predict frequency-domain measurements resulting from a modulated input at the phantom periphery. In the inverse image-reconstruction algorithm, a Newton-Raphson technique combined with a Marquardt algorithm is employed to converge on the fluorescent properties within the medium. The successful reconstruction of both the fluorescence yield and lifetime in the case of a heterogeneous fluorophore distribution within a scattering medium has been demonstrated without a priori information or without the necessity of obtaining absence images.     

Statistical modelling of Poisson/log normal data

In statistical data fitting, self consistency is checked by examining the closeness of the quantity chi(2)/NDF to 1, where chi(2) is the sum of squares of data minus fit divided by standard deviation, and NDF is the number of data minus the number of fit parameters. In order to calculate chi(2) one needs an expression for the standard deviation. In this note several alternative expressions for the standard deviation of data distributed according to a Poisson/log-normal distribution are proposed and evaluated by Monte Carlo simulation. Two preferred alternatives are identified. The use of replicate data to obtain uncertainty is problematic for a small number of replicates. A method to correct this problem is proposed. The log-normal approximation is good for sufficiently positive data. A modification of the log-normal approximation is proposed, which allows it to be used to test the hypothesis that the true value is zero.     

On-column trace enrichment by sequential frontal and elution electrochromatography. 1. Application to carbamate insecticides

An on-column trace enrichment method for CEC of dilute samples is presented. The method involves on-line preconcentration by frontal electrochromatography under conditions of strong solute binding to the stationary phase followed by a step-gradient elution electrochromatography with a mobile phase of high eluting strength. This method is tested with dilute samples of carbamate insecticides using capillary columns of 100-microm i.d. packed with a 5-microm octadecyl silica (ODS) stationary phase. The effectiveness of on-line preconcentration (i.e., zone narrowing) depends on the retention factor, k', of the analyte in the injection solvent as well as in the eluting mobile phase (i.e., the organic solvent content), the applied voltage during sample introduction, and elution and length of the introduced sample plug. Under optimal frontal and elution electrochromatography conditions, a 500-fold sensitivity increase is achieved for carbofuran (a carbamate insecticide) with a UV detector. The method is demonstrated with deionized and tap water samples spiked with carbamate insecticides.     

An integrated fritless column for on-chip capillary electrochromatography with conventional stationary phases

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.     

Maximum entropy method for diffractive imaging

Based on the minimization of the Lagrange formula, which is composed of two kinds of information measure, the maximum entropy method (MEM) is derived for diffractive imaging contaminated by quantum noise. This gives a suitable object corresponding to the maximum entropy principle with an iterative procedure. The MEM-based iterative phase retrieval algorithm with the initial process of the hybrid input-output (HIO-MEM) is presented, and a simple numerical example shows that the algorithm is effective for Poisson noise added to Fourier intensity. The relationship between the newly derived MEM for diffractive imaging and the conventional MEM for structure analysis based on crystallography is revealed.     

Retention mechanisms in reversed-phase liquid chromatography. Stationary-phase bonding density and partitioning

The partitioning model of retention for reversed-phase liquid chromatography, described by mean-field statistical thermodynamic theory, asserts that one principal driving force for solute retention is the creation of a solute-sized cavity in the stationary phase. Beyond a critical stationary phase bonding density, increased grafted chain density should result in enhanced chain ordering, which will increase the energy necessary for solute cavity formation and result in decreased chromatographic partition coefficients. We have evaluated chromatographic partition coefficients over an octadecyl bonding density range of 1.6-4.1 mumol/m2 and have found a maximum in partition coefficient at approximately 3.1 mumol/m2. Retention, however, approximately plateaus due to compensating changes in the partition coefficient and stationary phase volume. This provides unequivocal evidence that partitioning is the dominant form of retention for small nonpolar solutes.     

Bayesian maximum entropy (two-dimensional) lifetime distribution reconstruction from time-resolved spectroscopic data

Time-resolved spectroscopy is often used to monitor the relaxation processes (or reactions) of physical, chemical, and biochemical systems after some fast physical or chemical perturbation. Time-resolved spectra contain information about the relaxation kinetics, in the form of macroscopic time constants of decay and their decay associated spectra. In the present paper we show how the Bayesian maximum entropy inversion of the Laplace transform (MaxEnt-iLT) can provide a lifetime distribution without sign-restrictions (or two-dimensional (2D)-lifetime distribution), representing the most probable inference given the data. From the reconstructed (2D) lifetime distribution it is possible to obtain the number of exponentials decays, macroscopic rate constants, and exponential amplitudes (or their decay associated spectra) present in the data. More importantly, the obtained (2D) lifetime distribution is obtained free from pre-conditioned ideas about the number of exponential decays present in the data. In contrast to the standard regularized maximum entropy method, the Bayesian MaxEnt approach automatically estimates the regularization parameter, providing an unsupervised and more objective analysis. We also show that the regularization parameter can be automatically determined by the L-curve and generalized cross-validation methods, providing (2D) lifetime reconstructions relatively close to the Bayesian best inference. Finally, we propose the use of MaxEnt-iLT for a more objective discrimination between data-supported and data-unsupported quantitative kinetic models, which takes both the data and the analysis limitations into account. All these aspects are illustrated with realistic time-resolved Fourier transform infrared (FT-IR) synthetic spectra of the bacteriorhodopsin photocycle.     

Microscopic origins of band broadening in chromatography. Polarity distribution in C(18) stationary phase probed by confocal ratiometric imaging of Nile red

Band broadening is a major factor that influences the efficiency and resolution of chromatographic separations. Studies of microscopic origins of band broadening, such as the micropolarity distribution of chromatographic stationary phase, can provide a better understanding of many chromatographic phenomena and retention behavior. In this work, we probe the chemical environments of C18 chromatographic stationary phase with quantitative confocal fluorescence microscopy under real reversed-phase liquid chromatography conditions. Ratiometric imaging of C18 interface is achieved by loading the stationary phase with a polarity-sensitive dye, Nile red, and optical sectioning with confocal microscopy. The results reveal that there are uniform micropolarity distributions inside individual chromatographic beads, but the polarity may differ between stationary-phase particles. The homogeneity of micropolarity of individual beads suggests that there are not any spatially large exposed silica sites beyond the optical resolution in C18 stationary phase. The strong adsorption sites are smaller in size than the optical resolution of a few hundred nanometers. The heterogeneity between chromatographic beads indicates that the interactions of Nile red with C18 bonded phase are different between beads. This contributes to the broad overall polarity distribution of the C18 stationary phase and can be one of the factors that cause band broadening in separations. With its high spatial resolution and optical sectioning capabilities, confocal fluorescence imaging is shown to be an ideal method to probe the chromatographic stationary phase. The distribution of micropolarity sheds light on the microscopic heterogeneity in chromatographic processes and its influence on chemical separations.     

Temperature dependence of retention in reversed-phase liquid chromatography. 1. Stationary-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been investigated by examining the temperature dependence of retention, with emphasis on the role of the stationary phase in the retention process. Both chromatographic temperature studies and differential scanning calorimetry were used to examine the role of alkyl chain bonding density on the retention mechanism in RPLC. Phase transitions of reversed-phase stationary phases were observed at bonding densities greater than 2.84 mumol/m2. Thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase (delta H degrees and delta S degrees) were calculated for low bonding density columns, and comparison of these values to previously reported values for the partitioning of a nonpolar solute from the bulk organic liquid to water indicated that the chromatographic retention process is not well-modeled by bulk-phase oil-water partitioning processes. In addition, this data showed that the entropic contribution to retention becomes more significant with respect to the enthalpic contribution as the stationary-phase bonding density is increased, providing additional support that partitioning, rather than adsorption, is the relevant model of retention.