Evaluation of peak overlap in migration-time distributions determined by organelle capillary electrophoresis: Type-II error analogy based on statistical-overlap theory

Organelles commonly are separated by capillary electrophoresis (CE) with laser-induced-fluorescence detection. Usually, it is assumed that peaks observed in the CE originate from single organelles, with negligible occurrence of peak overlap. Under this assumption, migration-time and mobility distributions are obtained by partitioning the CE into different regions and counting the number of observed peaks in each region. In this paper, criteria based on statistical-overlap theory (SOT) are developed to test the assumption of negligible peak overlap and to predict conditions for its validity. For regions of the CE having constant peak density, the numbers of peaks (i.e., intensity profiles of single organelles) and observed peaks (i.e., maxima) are modeled by probability distributions. For minor peak overlap, the distributions partially merge, and their mergence is described by an analogy to the Type-II error of hypothesis testing. Criteria are developed for the amount of peak overlap, at which the number of observed peaks has an 85% or 90% probability of lying within the 95% confidence interval of the number of peaks of single organelles. For this or smaller amounts of peak overlap, the number of observed peaks is a good approximation to the number of peaks. A simple procedure is developed for evaluating peak overlap, requiring determination of only the peak standard deviation, the duration of the region occupied by peaks, and the number of observed peaks in the region. The procedure can be applied independently to each region of the partitioned CE. The procedure is applied to a mitochondrial CE.     

29Si nuclear magnetic resonance of poly(methylphenyl)silane

A Cl-terminated poly(methylphenyl)silane is synthesized and analyzed by ²⁹Si-NMR spectroscopy. Assignments are proposed for the different peaks observed. A modification of the chemical shifts of the peaks relative to the main chain atoms is observed upon heating. From these experimental results and computational calculations on model compounds, the assignments of the three main peaks to configurationally different Si atoms are confirmed. © 1996 John Wiley & Sons, Inc.     

A data treatment method for detecting fluorescence anisotropy peaks in capillary electropherograms

A data treatment method is presented to detect fluorescence anisotropy (FA) peaks in capillary electrophoresis electropherograms. The data treatment method converts plots of fluorescence anisotropy vs. time that contain no peaks that are distinguishable from the noise of the anisotropy background into plots that show distinct fluorescence anisotropy peaks. The method was demonstrated using laser-induced fluorescence anisotropy data from individual Aβ (1–42) aggregates separated using capillary electrophoresis. Applying this data treatment method enabled the detection of anisotropy peaks for individual Aβ aggregate fluorescence peaks that were not observed prior to the data treatment method. The data treatment method is not specifically designed for Aβ aggregate analysis or capillary electrophoresis, and it should be applicable to other applications and other separation methods with FA detection.     

GARANT-a general algorithm for resonance assignment of multidimensional nuclear magnetic resonance spectra

A new program for automatic resonance assignment of nuclear magnetic resonance (NMR) spectra of proteins, GARANT (General Algorithm for Resonance AssignmeNT), is introduced. Three principal elements used in this approach are: (a) representation of resonance assignments as an optimal match of two graphs describing, respectively, peaks expected from combined knowledge of the primary structure and the magnetization transfer pathways in the spectra used, and experimentally observed peaks; (b) a scoring scheme able to distinguish between correct and incorrect resonance assignments; and (c) combination of an evolutionary algorithm with a local optimization routine. The score that evaluates the match of expected peaks to observed peaks relies on the agreement of the information available about these peaks, most prominently, but not exclusively, the chemical shifts. Tests show that the combination of an evolutionary algorithm and a local optimization routine yields results that are clearly superior to those obtained when using either of the two techniques separately in the search for the correct assignments. GARANT is laid out for assignment problems involving peaks observed in two- and three-dimensional homonuclear and heteronuclear NMR spectra of proteins. © 1997 by John Wiley & Sons, Inc.     

The relationship between phase transition and luminescence in simple hydrocarbons

A satisfactory correlation between the temperature of radiothermoluminescence (RTL) peaks and phase transition points has been found in simple hydrocarbons. RTL peaks due to a glass transition phenomenon are observed clearly in ethane, propane and other compounds. These compounds were deposited rapidly onto a cooled cooper surface from the vapour (the vapour condensation method). A method for studying phase transitions in several substances by means of RTL is proposed.     

Tuning the lasing wavelength of dye-doped chiral nematic liquid crystal by fluid flow

The influence of hydrodynamic flow on the lasing wavelength and stability of the laser emission average power of a dye-doped chiral liquid crystalline (DDCLC) structure is studied. Multiple lasing peaks at equidistant wavelengths are observed, which are originating from multiple domains that are induced by the hydrodynamic flow of the DDCLC. A simultaneous blue shift over 4 nm for all lasing peaks is observed after increasing the flow velocity to 0.3 mm/s for cells with a thickness of 10 μm. It is observed that the CLC flow dramatically increases the stability of the laser emission compared to conventional cells because dye bleaching and LC molecular reorientation due to pumping beam is reduced.     

Computer simulation and theory of the diffusion- and flow-induced concentration dispersion in microfluidic devices and HPLC systems based on rectangular microchannels

The dynamics of formation of solute peaks in microfluidic systems are investigated by computer simulation. A finite-element numerical procedure is applied to analyze the diffusion- and flow-controlled concentration dispersion in a 40 microm-high rectangular flow-through channel. Two-dimensional concentration profiles are shown for channels with cross sections of large aspect ratio. The final shapes of the peaks are formed during a very short time period, ranging from a few milliseconds to about 1s for low and high flow velocities, respectively. The observed standard half-width sigma of the peaks is found to strictly follow a linear function of t(1/2) over the whole time range. The extrapolated long-term peak characteristics are in perfect agreement with theoretical predictions. For comparison, theoretical results on the concentration dispersion for solute peaks in open-channel liquid-chromatography (HPLC) are re-examined and applied.     

Degenerate two-photon absorption spectra in azoaromatic compounds.

A systematic study of the degenerate two-photon absorption (2PA) spectra of seven azoaromatic compounds in dimethyl sulfoxide solution is reported, which employed the Z-scan technique with femtosecond laser pulses from the bottom of the azo compound absorption bands up to 1100 nm. The 2PA peaks for pseudostilbene-type azo compounds (Disperse Orange (DO) 3, Disperse Red (DR) 13, DR1, DR19, and DR19-Cl) were observed at twice the peak wavelength of the linear absorption. However, such peaks were not observed for other azo compounds (PAMINO and DIAMINO) because of the symmetry of these molecules. A resonance enhancement of the 2PA cross-section was observed for all compounds. The 2PA peak and the nonlinear resonance enhancement behavior could be adjusted with a model based on perturbation theory. Such knowledge can be a guideline to the understanding of the 2PA process in azoaromatic compounds.     

Conformational changes monitored by fluorescence study on reconstituted hemoglobins

Intrinsic steady state fluorescence measurements were performed on a series of reconstituted metal ion and hybrid hemoglobins (Hbs). At 296 nm excitation, the spectrum exhibits a broad and asymmetric feature in the case of copper and nickel reconstituted hemoglobins. Deconvolution of the fluorescence bands clearly reveals the existence of two definite peaks. A similar trend was also observed for hybrid hemoglobins (CuNi, NiCu, CuFe-CO, and NiFe-CO). A guassian fit of the fluorescence bands in these proteins again yields two prominent peaks, which are assigned as due to two different tryptophan (Trp) environments. A relative ratio of the amplitudes of these peaks indicates the percentage of T-character in these molecules. This is in support to our previous findings by other spectroscopic studies on the same molecules. These studies therefore, suggest the presence of two different environments of a tryptophan thereby revealing structural heterogeneity among the subunits.     

Artifacts in four-sector tandem mass spectrometry

Several types of artifacts were shown to be present in 4-sector tandem collision-induced dissociation (CID) mass spectra. In CID spectra of protonated peptides produced by liquid secondary-ion mass spectrometry (LSIMS), peaks corresponding to successive losses of matrix molecules from the precursor ion were observed. In addition, peaks corresponding to MH+ ions of smaller peptides that were also present in the sample/matrix mixture in greater abundance than the selected precursor ion were observed. Both of these types of artifact peaks were shown to originate from the 'peak-at-every-mass' chemical noise at the same nominal mass as that selected by the first 2 sectors (MS1). These noise ions are transmitted through to the collision cell and produce fragments that are analysed and detected in the next 2 sectors (MS2). A second, unrelated, kind of artifact was found to be due to decompositions in the second field-free region of MS2 in an EBEB geometry machine. These artifacts, which are detectable over only a very limited mass range when using a conventional single-point detector, can be present over a much greater mass range when an array detector is used and when the collision cell is floated above ground potential. A clear understanding of the origins of all peaks in a CID spectrum is important in order to have a firm foundation for interpretation, manual or computer-aided, of the spectra of unknown compounds.     

Peak-splitting in reversed-phase,ion-pair high-performance liquid chromatography of sympathomimetic drugs and its probable mechanism

Summary In the determination of ephedrine using reversed-phase, ion-pair liquid chromatography, a chromatographically pure sample was observed to give three peaks under certain mobile phase conditions. The mobile phases which produced maximum peak splitting were determined for ephedrine and a number of other sympathomimetic drugs.A proposal that peak splitting was the result of the composite interplay of two discrete chromatographic mechanisms, was investigated. The results of analysis by GC/MS confirmed that each peak was due to ephedrine, however, only one of the three split peaks was found to contain ion pairs. It is postulated that peak splitting is a physical phenomenon on reversed-phase columns and the separation of these drugs by ion-pair HPLC is based on a mixed rather than a single mechanism.This study has also shown that errors can arise in ion-pair HPLC when multiple peaks are assumed to indicate heterogeneity. Interconvertible species of the same solute can give rise to these peaks.     

Analysis of ice water by the thermally stimulated depolarized current (TSDC) method.

The thermally stimulated depolarized current and temperature profiles (TSDC analysis) on iced water was proved to be an effective tool for the qualitative evaluation of various water samples, each having an independent relaxation process, brought about by a pure dipolar orientation. The method proved to be applicable to different kinds of water samples used in the experiment and tap-water samples from different suppliers. Five main peaks (A to E, in the order of increasing temperature) were observed, of which two peaks (B and D) were found for the first time. The appearance of multiple peaks suggested the existence of multi-states of the hydrogen bond, cleaved by the TSDC process. The TSDC profiles were quite reproducible when the water samples contained practically no cations. A separate addition of each cation at a low concentration level revealed that a cation with a smaller ionic radius shifted peak A to a higher temperature. For ice of tap water, which contained relatively higher amount of cations, the TSDC profiles were quite different in shape compared with the standard ice-water samples (shift of peaks A - D to higher temperature, and a strong increase in the current strength of peaks B - E). However, it was still possible to tell from which districts the water samples were supplied.     

Fractionation of HDPE on quench cooling

In a previous paper we discussed co-crystallization in a LDPE/HDPE blend using TREF and DSC. As part of that study it was observed that pure HDPE showed an unexpected fractionation behavior when quench crystallized in TREF. The overall peak broadened and two peaks appeared instead of the previously observed single peak for slow cooled HDPE.The development of two peaks was observed for all commercial HDPEs investigated, independent of their melting indices and densities. TREF and GPC were used in an attempt to evaluate the origin of the two HDPE components.The authors appreciate support from CAPES-BRAZIL (C.A.F.). Additional thanks go to Dr. G. W. Knight in Dow Chemical Company for kindly providing the polymer samples and performing the GPC analysis.     

Studies on substituted phenols by differential pulse cathodic stripping voltammetry: 1. Pentachlorophenol

A study of chlorophenols using differential pulse cathodic stripping voltammetry (dpcsv) is reported. Of the wide range of chlorophenols investigated, only pentachlorophenol and 2,3,4,6-tetrachlorophenol yield stripping peaks.For pentachlorophenol in water, two peaks are obtained at stripping potentials of –1.2 V and –1.58 V versus SCE. In methanol, pentachlorophenol shows one peak with a stripping potential at –1.6 V versus SCE.In the case of 2,3,4,6-tetrachlorophenol in water, two peaks are observed at –1.06 V and –1.6 V versus SCE, while in methanol only one peak arises at a stripping potential of –1.6 V. A procedure was developed for the determination of pentachlorophenol in natural waters.     

CZE of human alpha-1-acid glycoprotein for qualitative and quantitative comparison of samples from different pathological conditions

Alpha-1-acid glycoprotein (AGP) presents different forms, which may arise from differences in the amino acid sequence and/or in the glycosidic part of the protein. Changes in forms of AGP have been described in literature as a possible tumor marker. While most previous works have approached the study of glycopeptides and/or glycans obtained after fragmentation of the protein, in this work, a CZE method is developed to separate up to eleven peaks of intact forms of AGP. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Electropherograms of AGP samples purified from sera of cancer patients and healthy donors are qualitatively and quantitatively compared. Percentages of correct assignment of AGP peaks close to 100% are achieved by using either the migration time of each peak relative to that of the EOF marker or the effective electrophoretic mobility of the peaks. The computer program permits to select, among different hypotheses for peak allotment, that one providing the highest accuracy of assignment. In this way, some peaks with different charge-to-mass ratio and a different distribution of area percentage of AGP forms are observed when comparing samples from sick and healthy individuals. Thus, a method that permits to compare AGP forms existing in sera of individuals with different pathophysiological situations has been developed. A potential for using AGP forms analyzed by CZE as a disease marker and for using this technique for screening purposes is envisaged.     

Dielectric study of polyacrylonitrile,poly-2-hydroxyethyl methacrylate and their copolymers

A dielectric study on polyacrylonitrile (PAN), poly-2-hydroxyethyl methacrylate (P-HEMA) and copolymers of acrylonitrile and HEMA in the temperature range 30–150° is reported. The loss peaks occur in the frequency region 0.1–100 kHz at temperatures between 110° and 140 for PAN. In copolymers of acrylonitrile with HEMA, these loss peaks were present although their positions on temperature scale and their peak heights depended on the HEMA content. For P-HEMA no relaxation peak was observed in this range of frequency and temperature. The results for copolymers compared with those for the homopolymers suggest that the loss peaks in PAN are due to molecular motion in amorphous regions.     

Electrochemical Processes of Sulfide in NaCl Electrolyte Solutions on Mercury Electrode

The electrochemical processes of sulfide in NaCl electrolyte solutions were studied using stripping cyclic voltammetry, differential capacity and chronoamperometric measurements. Some new effects were observed and are here discussed. At potentials around ?0.1 to ?0.3 V a pair of Faradaic adsorptive peaks, which can only be observed in the presence of chloride, is attributed to the formation of HgCl ion from the deposited layer of HgS. A second pair of peaks of purely capacitive character was observed at ?0.4 V. This is the result of the reorientation and partial desorption or transformation of HgS layers. The formation and dissolution of HgS layers on mercury has been well investigated previously. We observed some new effects after depositing approximately ten layers of HgS and attributed this to the formation of polysulfides at positive potentials.     

Complex cold crystallisation peaks in PET/PS blends

This work describes the investigation of complex peaks observed during the cold crystallisation of polyethylene terephthalate (PET) blended with polystyrene (PS). Blends of PET with 1% to 15% PS content were compounded in a laboratory internal mixer and quenched from the melt to produce substantially amorphous samples. The cold crystallisation behaviour was studied using differential scanning calorimetry (DSC) conducted at 10 °C/min. The complex peaks of the PET/PS blends were resolved as the sum of two simple peaks, and the component peak characteristics were determined. While the total crystallinity was found to be independent of the PS content, the areas under the component peaks depend on the amorphous component content. A simple model based on partial dissolution and micro-segregation of PS is proposed to qualitatively explain the presence of the double cold crystallisation peaks.     

Determination and removal of impurities in 2-D LC-MS of peptides

Problems occurring during operation of a 2-D LC-MS system for separation and identification of neuropeptides, such as contamination of the used salts and column bleed, are described. When using polysulfoethyl aspartamide, which is widely used as a strong cation exchange stationary phase in the first dimension, interfering peaks were observed in the second-dimension reversed-phase chromatograms. The observed peaks, found to be caused by column bleeding, had abundance above the threshold value and influenced the quality of the analyses. The origin of the peaks was verified and appropriate measures are proposed. Additionally, peaks caused by polyethylene glycols (PEGs), covering approximately 5 min of feasible chromatographic time in every fraction, were observed. The commercial ammonium formate salts used to prepare the first-dimension mobile phase were found to contain PEG impurities, and in subsequent work the salt solutions were prepared from formic acid and ammonia to avoid any additional contaminations.