UV Laser-induced Fluorescence Research Articles

Recent advancements in amino acid analysis using capillary electrophoresis

Recent advances in the analysis of amino acids using capillary electrophoresis are addressed. This area of research continues to receive increased attention as is evident from the 62 references reviewed. This review discusses current detection strategies including UV absorbance, laser-induced fluorescence, electrochemical, and others. Separation methodologies for both derivatized and underivatized amino acids are reviewed. Both direct and indirect enantiomeric resolution of amino acids are addressed. Applications utilizing capillary electrophoresis for the analysis of amino acids are discussed. This review covers literature published in 1997 and 1998.

Radiative properties of neutral germanium obtained from excited-state lifetime and branching-ratio measurements and comparison with theoretical calculations

Using time-resolved UV laser-induced fluorescence from a laser-produced plasma, natural radiative lifetimes have been measured for 21 levels of Ge I belonging to the odd-parity $4p4d,4p5d,$ and $4p6s$ configurations. Stimulated Brillouin scattering in water has allowed us to compress Nd:YAG laser pulses pumping a dye laser (where YAG denotes yttrium aluminum garnet), thus yielding 1-ns tunable laser pulses to enable accurate measurements of short lifetimes. Branching ratios of Ge I have been measured by inductively coupled plasma emission spectrometry, the intensity calibration being performed by means of Ar lines emitted by a hollow-cathode lamp. The experimental lifetimes and branching ratios have been combined in order to provide a set of accurate transition probabilities for the $4p\ensuremath{-}5s$ and $4p\ensuremath{-}4d$ transitions. A relativistic Hartree-Fock calculation, taking configuration-interaction and polarization effects into account, has been combined with a least-squares optimization procedure of the Slater and spin-orbit integrals in order to test the ability of this approach to correctly predict radiative properties of the group-IV elements. Good agreement between experimental and theoretical transition probability values has been achieved for most of the transitions considered.

Highly strained turbulent rich methane flames stabilized by hot combustion products

The structure of rich turbulent methane flames, with an equivalence ratio of 2.0, at high strain rates has been investigated based on simultaneous and instantaneous line profile measurements of the species mass fractions of CH 4, O 2, N 2, H 2O, CO 2, CO, H 2 and OH radical, together with gas temperature. The flames are stabilized by the hot combustion products from a stoichiometric large pilot flame surrounding a Bunsen burner. Two flames have been investigated at overall stretch rates of 2500 s −1 and 4167 s −1. One-dimensional combined UV Raman, Rayleigh, and laser-induced predissociation fluorescence (LIPF) technique has been applied. Measurements at different axial positions enable the flame structure to be studied at different turbulence levels. The instantaneous species mass fractions and temperature profiles are conditioned on both mixture fraction and a reaction progress variable, namely the H 2O mass fraction, allowing a proper description of the partially premixed flame structure. The components of the scalar dissipation rate of both mixture fraction and progress variable in the radial direction are also measured. The flames are stabilized at rich conditions by radicals and heat transferred from the stoichiometric pilot flame. Stretch effects are found to be dominant in these flames, leading to local flame structures that correspond to flames in the distributed-reaction-zones regime. The pdf of the progress variable is found to be monomodal in all cases. The conditional mean scalar dissipation rate profile in mixture fraction space exhibits a local minimum at the reaction zone due to differential polynary diffusion effects. The scalar dissipation rates of the progress variable are of the same order of magnitude as those of the mixture fraction, when these are appropriately normalized. This indicates that molecular mixing in both variables is equally important. This also suggests that a two-variable formulation is necessary to model these flames.

Characterization of micelles by capillary electrophoresis.

A practical approach for the characterization of pure micelles, and binary or ternary mixed micelles by capillary electrophoretic methods is presented. Interest is focused on the determination of mobility and composition of the micelles. The investigations are performed with bile salts, phosphatidylcholines and fatty acids. Pure bile salt micelles are characterized with the help of marking and displacement methods. Binary bile salt/phospholipid and ternary bile salt/phospholipid/fatty acid micelles are analyzed using capillary electrophoresis (CE) techniques with standard and improved UV detection, laser-induced fluorescence and electrospray ionization-mass spectrometry (ESI-MS). For both the binary and the ternary systems, only one stable mixed micellar phase is found with a high negative mobility.

Dynamics of the H+HCl gas-phase reaction: absolute reactive cross-section for [formula omitted] atom formation

The reaction of atomic hydrogen with HCl was studied in the gas phase using the laser photolysis/vacuum–UV laser-induced fluorescence (LP/VUV–LIF) `pump-and-probe' technique. Translationally energetic H atoms with an average energy of E cm=1.0 eV in the (H–HCl)–center-of-mass system were generated by laser photolysis of H 2S at 248 nm. Doppler profiles of nascent Cl( 2 P 3/2) atoms produced in the reactive collision of the H atoms with room temperature HCl molecules were measured under single collision conditions by VUV–LIF. For the H+ HCl→ H 2+ Cl( 2 P 3/2) reaction an absolute reaction cross-section σ R(1.0 eV)=(0.34±0.05) Å 2 was determined by means of a photolytic calibration method using the 248 nm laser photolysis of PCl 3 as reference for well-defined concentrations of Cl atoms. Small amounts of Cl in the ( 2 P 1/2) excited state were observed in the H+HCl experiments. An upper limit of (0.02±0.01) Å 2 was measured for the Cl ∗( 2 P 1/2) formation cross-section in the H+HCl reaction at 1.0 eV.

Lifetime measurements in odd-parity Rydberg series of neutral lead by time-resolved laser spectroscopy

Natural radiative lifetimes have been measured in the odd-parity Rydberg series 6pns (n=7-13) and 6pnd (n=6-13) of neutral lead using time-resolved UV laser-induced fluorescence from a laser-produced plasma. A relativistic Hartree-Fock calculation taking configuration interaction into account in a detailed way has also been performed for odd-as well as even-parity states for testing the ability of this approach to correctly predict radiative properties of heavy atoms. A generally good overall agreement between experimental and theoretical lifetimes has been achieved except for a few levels.

Determination of methamphetamine and related compounds by capillary electrophoresis with UV and laser-induced fluorescence detection

Methods for the simultaneous determination of methamphetamine (MP) and its related compounds (MPs) using capillary electrophoresis (CE) with UV absorbance and laser-induced fluorescence (LIF) detection are described. In UV detection, MPs were applied to CE without any derivatization procedure and detected at 210 nm for a rapid and simple analysis. Capillary zone electrophoresis (CZE) and electrokinetic capillary electrophoresis (MEKC) were used. MP, amphetamine (AP), 1-phenylethylamine (1-PA as an I.S.), 2-phenylethylamine (2-PA), 4-hydroxymethamphetamine (4-HMP) and 4-hydroxyamphetamine (4-HAP) were separated within 15 min by both CZE and MEKC. Detection limits of MPs were in the range 48–72 fmol/injection for CZE and 85–191 fmol/injection for MEKC. MEKC was successfully applied to the determination of MPs in urine. For a highly sensitive analysis, LIF detection was also examined using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. By the method, in which MPs derivatives were separated within 45 min by MEKC, 22–40 amol/injection of primary amines (AP, 4-HAP and 2-PA) and 690 amol/injection of MP and 300 amol/injection of 4-HMP were detected. The concentration of MP and AP in 50 μl urine from MP addicts were successfully determined. A comparison of the characteristics for both UV and LIF detections was also discussed.

Determination of metal cations in microchip electrophoresis using on-chip complexation and sample stacking

A method for the determination of metal cations in aqueous solution using a monolithically integrated microfabricated (microchip) device which allows sample stacking, electrophoretic separation, and postcolumn derivatization is described. The metal cations are detected by UV laser-induced fluorescence following complexation with 8-hydroxyquinoline-5-sulfonic acid. To make use of electrokinetic stacking phenomena, the sample is added to a reservoir containing a low-conductivity buffer and is then introduced into a main separation channel using a gated injection scheme. The complexing agent is added downstream from the injection cross. For two model compounds, magnesium and calcium, detection limits were 0.5 and 18 ppb, respectively. The reproducibility for multiple injections was 1.5% relative standard deviation. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 313–319, 1998

Short chain fatty acids analysis by capillary electrophoresis and indirect UV detection or laser-induced fluorescence

Short chain fatty acids are difficult to study using conventional techniques such as gas chromatography, because of their high volatility. Capillary electrophoresis (CE) and UV detection or conductimetry was used to study fatty acids of 6 carbon lengths or less. Difficulties in dissolving longer acids in aqueous buffer prevented analysis by CE. Recently indirect laser-induced fluorescence was used to study C 6 to C 18 fatty acids and the sensitivity of the detection was in the sub-micromolar range. In this paper we studied C 5 to C 18 branched, hydroxy or linear fatty acids using CE and UV indirect detection and we succeeded in obtaining good separations but with very poor sensitivity (limited to 10 −5 M). In a second attempt we studied fatty acids after 5-bromomethylfluorescein derivation and analysis by CE and laser-induced fluorescence (LIF). The sensitivity was in the sub-nanomolar (10 −10 M) range but we could only study C 8 to C 11 fatty acids. Using CE-LIF, we quantitated these acids in normal and pathological serae.

Line Raman, Rayleigh, and laser-induced predissociation fluorescence technique for combustion with a tunable KrF excimer laser

We have applied a line UV Raman, Rayleigh, and laser-induced predissociation fluorescence technique for measurement of turbulent hydrocarbon flames. The species concentration of CO(2), O(2), CO, N(2), CH(4), H(2)O, OH, and H(2) and the temperature are measured instantaneously and simultaneously along a line of 11.4 mm, from which the gradients with respect to mixture fraction and spatial direction are obtained. The technique has been successfully tested in a laminar premixed stoichiometric methane flame and a laminar hydrogen diffusion flame. In addition the technique has been tested in a highly turbulent rich premixed methane flame. The data show that the technique can be used to provide instantaneous measurements of local profiles that describe the local flame structure in highly turbulent flames.

Insertion vs. Abstraction in the Reactions of NH(a1Δ, v = 0, 1) with H2 and D2

Abstract Gas-phase reactions of NH(a1Δ, v = 0,1) + H2 (1) and NH(a1Δ, v = 0,1) + D2 (2) were studied in the photolysis of HNCO/H2 (or D2) mixtures. NH(a1Δ) and product H (D) atom were detected by UV and vacuum-UV laser-induced fluorescence. The observed rate constants at room temperature (295 ± 2K) and activation energies in the range of 295 — 407 K were; in units of 10−12 cm3 molecule−1 s−1 and kJ mol−1. The product branching ratio in reaction 2 was determined to be [D]/[H] = 3.2 ± 0.76, in which no temperature dependence was indicated. These results are consistent with insertion mechanism having a loose entrance transition state structure, which is suggested by ab initio CI calculations. In contrast, the contribution of direct abstraction channel is supposed to be negligibly small.

Analysis of monoclonal antibody and immunoconjugate digests by capillary electrophoresis and capillary liquid chromatography

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.

Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.

Direct determination of zolpidem and its main metabolites in urine using capillary electrophoresis with laser-induced fluorescence detection

Zolpidem is a new sleep inducer belonging to the imidazopyridine class. We wish to report a method for the determination of zolpidem and its main metabolites in urine without extraction using capillary electrophoresis with UV laser-induced fluorescence detection with a He-Cd laser. A 10-nl sample of urine can be directly applied to the capillary. The separation is carried out within 10 min, and the limit of detection is 2 ng/ml. This procedure is very simple and fast. No organic solvents are necessary.

植物葉からのUVレーザ誘起蛍光を利用した生体情報の測定

植物葉からのUVレーザ誘起蛍光を利用した生体情報の測定

Indirect detection in capillary electrophoresis comparison between indirect UV and indirect laser-induced fluorescence detection for the determination of isoprenyl pyrophosphates

Three different detection modes were compared for the determination of isoprenyl pyrophosphates by capillary electrophoresis, viz., direct UV, indirect UV and indirect laser-induced fluorescence (ILIF). The composition of the background electrolyte was varied accordingly. On the basis of separation efficiency ( N = 600 000) and limit of detection (ca. 0.5 μM ), the ILIF method gave the best results. Separation was complete in less than 6 min.

Analysis of trace level peptides using capillary electrophoresis with UV laser-induced fluorescence

In order to measure trace level peptides (< 1 μM) in exceedingly small sample sizes (< 100 pl) from neuronal or subneuronal samples, sophisticated separation and detection methods are required. Novel instrumentation is described that employs UV laser excitation of fluorescamine labeled peptides for fluorescence detection in capillary electrophoresis. Small injections of standard analyte mixtures or biological samples are efficiently separated on the basis of their electrophoretic mobilities in an electric field applied across the capillary. For sensitive detection, we use the 354 nm line from a helium-cadmium laser for excitation, a reflecting microscope objective for fluorescence collection, and a photomultiplier tube for photon counting. A large excess of fluorescamine derivatizes amino acids and peptides down to concentrations of 10 nM with a previously unreported but manageable fluorescence background resulting from reagent hydrolysis and impurities. This detection method gives a linear working range of four orders of magnitude and a limit of detection for labeled Aplysia californica egg-laying hormone of 2.3 × 10 −9 M. Finally, assays of individual Aplysia c. neurons produce electropherograms unique to the identified neuron type and peaks corresponding to peptide standards.

Vibrational relaxation of NO(X 2Π, ν=3) by NO, O 2 and CH 4

A two-laser (IR overtone pump and UV laser-induced fluorescence probe) technique has been used to measure vibrational relaxation for NO(X 2Π, ν=3) colliding with NO, O 2, and CH 4 at 295 K. The total removal rate coefficients are: (3.26±0.24) × 10 −12 cm 3 s −1 for NO, (1.46±0.15) × 10 −13 cm 3 s −1 for O 2, and (1.20±0.12) × 10 −13 cm 3 s −1 for CH 4.

Micellar electrokinetic chromatography of the anti-human immunodeficiency virus (HIV) agent michellamine B with absorption and laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) was applied to the separation of the anti-HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium deoxycholate and 10-20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high-performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser-induced fluorescence detection (LIF). Using the sodium dodecyl sulfate-containing buffer and LIF, the limit of detection for michellamine B was approximately 2 ng/mL. The sensitivity was degraded approximately 100-fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.

Capillary zone electrophoresis of malto-oligosaccharides derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid

Malto-oligosaccharides were derivatized via their reducing end with 8-aminonaphthalene-1,3,6-trisulfonic acid by reductive amination, and the separation and electrophoretic migration behavior of the labelled sugars were investigated by capillary zone electrophoresis. Series of linear malto-oligosaccharides were found particularly suitable for both the study of the effect of the operating conditions on the separation and the investigation of the relationship between the electrophoretic mobility and the molecular size of the homologues. The electrophoretic mobility of the malto-oligosaccharide conjugates was found to be a linear function of the molecular mass to the negative two-thirds power. The sugar derivatives employed here carry three negative charges due to the presence of the dissociated sulfonic acid groups even at strongly acidic pH. Therefore, the analytes can migrate in the electric field without interference by electroendosmotic flow and/or wall adsorption in uncoated silica capillaries at low pH. As a result, the separation of these carbohydrate conjugates can be carried out under such conditions with high speed and efficiency in free solution, i.e., without an anticonvective medium such as a gel or a viscous polymer solution. Appropriate use of triethylammonium phosphate buffer, pH 2.5, as the background electrolyte improves not only the reproducibility, but also the efficiency and speed of the separation. The labelled sugars allow monitoring of the separation by UV detector or laser-induced fluorescence detector with concomitant enhancement of analytical sensitivity.