J.W. Hofstraat

SYNCHRONOUS FLUORESCENCE SPECTROMETRY OF FISH BILE - A RAPID SCREENING METHOD FOR THE BIOMONITORING OF PAH EXPOSURE

Abstract The uptake of polycyclic aromatic hydrocarbons (PAHs) by fish can be determined by screening the gall bladder bile for PAH metabolites. Conjugated 1-hydroxy pyrene is a major metabolite in bile of fish exposed to PAH polluted sediment. Synchronous fluorescence spectrometry is presented as a rapid screening technique for the determination of this compound; the complete analysis takes only 2–3 min. HPLC with fluorescence detection was used to validate the assay. Calibration methods, using either free 1-hydroxy pyrene or the pyrene-1-glucuronide conjugate as a reference standard, were investigated. The technique was applied to a mesocosm study in which the uptake of PAHs by flounder ( Platichthys flesus ) from polluted sediment was studied. Direct contact with the sediment proved the most important factor; the uptake through water phase or diet was less significant. The usefulness of 1-hydroxy pyrene analysis in bile as a monitoring tool for PAH exposure is discussed.

Flow cytometric discrimination of phytoplankton classes by fluorescence emission and excitation properties.

The ataxonomic discrimination of phytoplankton species on the basis of fluorescence data obtained by multiwavelength excitation in combination with wavelength selective detection in flow cytometry is demonstrated. The discrimination is based on differences in pigment composition between the species, which are reflected in their spectral characteristics. Classification can be done both by making use of the absolute fluorescence intensities and with fluorescence parameter ratios. The latter approach has the advantage that size-related effects and instrument fluctuations are reduced to a large extent. Photoadaptation does influence the absolute as well as the ratioed parameters that are obtained but does not impede the classification into major ataxonomic groups.

Highly-resolved fluorescence spectrometry of pyrene on a thin-layer chromatographic plate

Abstract Site-selection spectroscopy, by using suitable laser line excitation, gives epectra of molecules on thin-layer chromatographic plates comparable in quality to Shpolskii spectra. Pyrene is investigated as a model compound.

Quantitative Determination of Polycyclic Aromatic Hydrocarbons in Harbour Sediment via High-Resolution Shpol'skii Spectroscopy

Abstract Polycyclic aromatic hydrocarbons (PAHs) in harbour sediment have been determined by means of Shpolskii fluorometry. Fundamental aspects of this cryogenic high resolution technique are discussed. It provides characteristic spectra of aromatic molecules that can be successfully employed for identification purposes. Extra selectivity is obtained by making use of selective excitation, even with a broad-banded light source. Highly specific quantitative determinations can be achieved, due to the quasi-linear character of the spectra. Deuterated PAHs are suitable as internal standards. Calibration curves with linearity from 10−5-10−6 M down to the detection limit are found, yielding a dynamic range from 2 to 4 decades for the compounds investigated. The reproducibility of the quantitative experiment lies between 6 and 20 per cent. The results obtained with Shpolskii spectroscopy are in good agreement with data found by a standard method of analysis employing fluorescence detected HPLC.

Shpol'skii fluorimetric determination of polycyclic aromatic hydrocarbons in complex environmental samples

SummaryHigh-resolution fluorimetry in low-temperature n-alkane Shpolskii matrices is a powerful technique for the analysis of rigid, non-polar compounds like polycyclic aromatic hydrocarbons. Because of the methods sensitivity and selectivity, sample clean-up, preconcentration and even chromatographic separation can often be left out. The Shpolskii analysis of pyrene in crude extracts from marine sediments and from bird meat is demonstrated. Special attention is focussed on the extra possibilities acquired when a laser is used as excitation source (Laser Excited Shpolskii Spectroscopy, LESS).

The applicability of fluorescence line-narrowing spectroscopy in combination with thin-layer chromatography

Abstract Laser excitation at low temperatures of compounds on thin-layer chromatographic plates yields, also after elution, highly resolved spectra. The fluorescence line-narrowed spectra permit identification of spectroscopically alike compounds, as is demonstrated for pyrene and halogenated pyrenes. Detection limits are in the low nanogram region even with relatively simple instrumentation. It is shown that quantitative application is possible if an internal standard is used.

Site-selection polarization measurements on tetracene in stretched polyethylene films

Abstract The polarization of individual vibronic bands in fluorescence spectra can be established with site-selection experiments using stretched polymer films. This is illustrated with an investigation on the 19000–20000 cm −1 region of the fluorescence of tetracene. The merits and possible applications of the method are discussed.

Flow cytometry: fast and quantitative characterization of particles in suspension

Abstract The application of flow cytometers for automated and rapid measurements of particles in suspension is presently widely accepted for biomedical applications. Flow cytometers, however, also offer many advantages for determination of (properties of) particles in other application areas. In this paper the potential of flow cytometry for environmental analysis is demonstrated, using examples taken from the analysis of phytoplankton in sea water. Flow cytometry is applied for automated measurement of the species abundance of phytoplankton in sea water, on the basis of the characteristic optical properties of the various algal species. More specific determination of, for instance, toxic phytoplankton could be achieved by making use of selective stains. Another application area is that of functional analysis of phytoplankton species. Here also specific fluorescent stains are used. Examples are presented of viability analysis and of cell cycle analysis, both of phytoplankton species. Other applications of flow cytometry in analytical sciences are discussed.

Persistent spectral hole-burning effects in fluorescence line-narrowing spectroscopy. Analytical implications

Abstract Persistent spectral hole-burning (PSHB) effects on fluorescence line-narrowing (FLN) spectra of tetracene in glassy and polymer matrices have been studied with emphasis on analytical implications. Attention has been paid to the time-evolution of the FLN spectrum under prolonged laser irradiation as well as to the matrix, laser intensity, temperature and excitation wavelength dependence of hole-burning effects. It is shown that PSHB, that generally is an unwanted phenomenon in analytical FLN spectroscopy, can be reduced or completely removed by using a suitable matrix material, by reducing the laser intensity, by working at somewhat elevated temperatures and by employing excitation to the vibronic region of the analyte.

Temperature effects on fluorescence line narrowing spectra of tetracene in amorphous solid solutions. Analytical implications

Abstract Temperature effects in the highly specific vibrationally resolved fluorescence line-narrowing (FLN) spectra of tetracene as a model compound have been investigated with emphasis on analytical implications. Contrary to what is generally observed in conventional solid state methods, in FLN when excitation in the 0-0 region is employed, the lowest temperatures in most cases do not produce the largest peak heights and intensities. For excitation in the vibronic S1 region the conventional behaviour is observed. The analytically useful temperature interval in FLN spectroscopy generally is more limited than in techniques that use crystalline matrix materials. The spectral bands in FLN have a predominantly Lorentzian shape for excitation in the 0-0 region in our experimental set-up where, at the temperatures used, the excitation source does not contribute significantly to the lineshape. For excitation in the vibronic S1 region a stronger Gaussian contribution to the lineshape is observed. No temperature dependent shift of the narrow lines occurs, contrary to what is generally found in the conventional solid state methods. In addition, the position of the lines in FLN is not solvent dependent. To explain these observations it is crucial to realize that in FLN, high resolution is achieved via selective excitation, so that the spectral emission features are determined by the excitation as well as the emission process.