C. Gooijer

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.

Wavelength-resolved laser-induced fluorescence detection in capillary electrophoresis: naphthalenesulphonates in river water

Abstract Capillary electrophoresis (CE) is combined with wavelength-resolved laser-induced fluorescence detection for the environmental analysis of naphtalenesulphonates (NS) in river water samples. The method enables the detection and the identification of non- as well as hydroxy- and amino-substituted NS. An approximate 30-fold sample concentration is performed by off-line solid-phase extraction prior to the CE separation. The native-fluorescent NS are excited at 280 nm or 325 nm by an excimer dye laser combination providing tunable UV radiation. Wavelength-resolved fluorescence-emission spectra are recorded on-line by an intensified diode-array detector mounted on a spectrograph. Since the emission spectra are strongly affected by the type and pattern of substitution, they provide much structural information. In combination with migration times identification is possible, if standards are available. The low to sub-μg 1−1 detection limits achieved enabled the identification and quantitation of two trisulphonate and an amino-disulphonate NS in River Elbe samples. The data obtained satisfactorily agree with those obtained by ion-pair HPLC.

Coupling of column liquid chromatography and surface-enhanced resonance Raman spectroscopy via a thin-layer chromatographic plate.

Abstract Surface-enhanced resonance Raman (SERR) spectroscopy was used to characterize compounds separated by column liquid chromatography (LC). Three percent of the effluent from a conventional-size LC column were immobilized on a moving thin-layer chromatography (TLC) plate using a spray-jet solvent-elimination interface. Next, colloidal silver was applied to the analyte spots and in situ SERR spectra were recorded with a multichannel micro-Raman spectrometer. Storage of the LC effluent avoids the need to use a continuous flow of colloidal silver and, in principle allows compounds to be detected independently of LC conditions like eluent composition and flow rate. Using dyes as test compounds, the method was optimized and aspects were studied such as type of TLC plate, LC separation of the dyes, preservation of LC integrity during immobilization, and SERR analysis of the deposited compounds. With a silica TLC plate as deposition substrate, good-quality and characteristic SERR spectra were obtained for the dyes which were separated on a cyanoproyl-modified silica LC column with methanol-water containing the volatile additives ammonium acetate and triethylamine as eluent. The minimum identifiable concentration of the dyes was about 250 ng ml −1 (750 pg applied on the plate). For some strong Raman scatterers such as nile blue and pararosaniline, the major spectral peaks could be observed down to concentrations of 25 ng ml −1 .

Identification of herbicides in river water using on-line trace enrichment combined with column liquid chromatography-Fourier-transform infrared spectrometry

Trace enrichment of herbicides on a 10×2.0 mm I.D. precolumn packed with Polygosil C18 material, was combined on-line with column liquid chromatography-Fourier-transform infrared spectrometry (LC-FT-IR). The isocratic LC separation was carried out on a 200×2.1 mm I.D Rosil C18 column using acetonitrile-phosphate buffer as eluent. The LC-FT-IR coupling was based on post-column on-line liquid-liquid extraction and solvent elimination, followed by FT-IR microscopy. The feasibility of the complete system was demonstrated by analysing river water spiked with triazines and phenylureas at the low-μg/l level. Identifiable spectra were obtained for all analytes, which included several isomers. Using 50–100 ml water samples, the identification limit for the herbicides typically was 1 μg/l. The usefulness of six spectral library search algorithms to correctly identify the recorded spectra was tested. A Peak-Search routine which is based on the matching of peak frequencies alone was found to be the most suitable to identify the analytes at the trace level.

On-line coupling of liquid chromatography to thin-layer chromatography for the identification of polycyclic aromatic hydrocarbons in marine sediment by fluorescence excitation and emission spectroscopy

Abstract Storage of the effluent from a reversed-phase column liquid chromatography separation on a thin-layer chromatography plate offers two advantages: (1) detection principles that are not applicable on-line with flow systems can be used; (2) the thin-layer chromatography plate itself allows a second separation to be carried out. Both advantages are demonstrated for the separation and identification of polycyclic aromatic hydrocarbons in a marine sediment sample. The effluent from a microbore liquid chromatography column was deposited on a linearly moving thin-layer chromatography plate through a spray jet assembly interface. As with isocratic liquid chromatography, not all polycyclic aromatic hydrocarbons present in the sample could be separated; a second separation of the immobilized chromatogram was applied by means of thin-layer chromatography. The overlapping peaks were then resolved completely. Eleven polycyclic aromatic hydrocarbons were identified by measuring their fluorescence excitation and emission spectra on the thin-layer chromatography plate with a conventional spectrometer.

Coupling of narrow-bore liquid chromatography to thin-layer chromatography

Abstract The coupling of liquid chromatography (l.c.) on narrow-bore columns to thin-layer chromatography (t.l.c.) is described. The effluent from a l.c. column can be deposited on a t.l.c. plate after a normal-phase or reversed-phase separation without serious loss of chromatographic information. Both silica and alkyl-modified silica plates can be used for storage. The interface is a fused silica capillary which connects the column outlet to the spray jet assembly of a Linomat applicator for t.l.c. The stored chromatogram can serve as starting point for a new separation, but also allows the use of detection principles which are normally not compatible with l.c. The chromatography of some polynuclear aromatic hydrocarbons is used to illustrate the possibilities of the combinations.

Coupling of reversed-phase liquid column chromatography and Fourier transform infrared spectrometry using postcolumn on-line extraction and solvent elimination.

An on-line postcolumn extraction module was used in conjunction with a solvent elimination interface for the semi-on-line coupling of reversed-phase liquid chromatography (RP-LC) and Fourier transform infrared spectrometry (FT-IR). The extraction module consisted of a phase segmentor, an extraction coil, and a phase separator. Dichloromethane was used as extraction solvent. The organic phase delivered by the separator was evaporated by a spray-jet assembly that simultaneously deposited the extracted analytes onto a zinc selenide window, which was subsequently analyzed by FT-IR microscopy. The method is evaluated by studying parameters such as postcolumn band broadening, phase separation efficiency, evaporation efficiency, extraction yield, eluent composition, and use of nonvolatile buffer salts. Good-quality spectra were obtained for test compounds (phenylureas and quinones), which were separated by RP-LC using a buffered eluent with high water content. Large-volume injections allowed FT-IR detection at the submicrogram per milliliter level.

Spray jet assembly interface for the coupling of reversed-phase narrow-bore liquid chromatography and Fourier transform infrared spectrometry

Abstract Liquid chromatography was coupled to Fourier transform infrared spectrometry via solvent elimination prior to infrared detection. The method allows the immobilization of analytes separated by reversed-phase liquid chromatography. Using a spray jet assembly, the effluent from a narrow-bore reversed-phase liquid chromatography column was continuously sprayed onto a linearly moving substrate suitable for infrared detection. The deposited compounds were analyzed Fourier transform infrared microscopy. Transmission measurement using zinc selenide as substrate appears to be preferable to measurement in the reflection mode on an aluminum surface. With polycyclic hydrocarbons and quinones as model compounds, it was shown that the chromatographic separation is hardly effected during the immobilization process. The identification limits for these compounds were 10–20 ng. Aqueous methanol liquid chromatography eluents containing up to about 20% water cn be handled.

Coupling of narrow-bore liquid chromatography to thin-layer chromatography : Part 2. Application of fluorescence-based spectroscopic techniques for off-line detection

Abstract The effluent from a narrow-bore liquid chromatographic (l.c.) separation can be immobilized on thin-layer chromatographic (t.l.c.) plates with little loss of resolution. The deposited compounds are then available for further inspection. For off-line detection, direct fluorescence emission, fluorescence excitation emission spectra, and fluorescence line-narrowing spectroscopy are investigated with tetracene and benz[k] fluoranthene as model compounds. Detection based on direct emission measurements and on measurements for which complete spectra are obtained for the separated compounds, is suitable for identification and determination. Detection limits are of the same order of magnitude as those for on-line detection in narrow-bore l.c. The fluorescence spectra of immobilized compounds can be obtained with a conventional fluorescence spectrometer equipped with a solid-sample accessory. No other special apparatus is needed. The immobilized chromatogram is also suitable for techniques incompatible with flow systems, e.g., fluorescence line-narrowing spectroscopy, which yields fluorescence spectra via laser excitation of low-temperature solid samples. Very selective narrow-line fluorescence spectra were obtained for tetracene deposited on t.l.c. plates in amounts down to the low picogram level.

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.