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In this preliminary study, we evaluated four different types of substrate for the at-line coupling of capillary electrophoresis and surface-enhanced (resonance) Raman spectroscopy, CE-SER(R)S, with emphasis on spectral repeatability. We tested Sub1: etched silver foil, Sub2: a vapour-deposited silver film, Sub3: a silver oxalate-precoated silica TLC plate and Sub4: a silica TLC plate on which colloid and poly(L-lysine) were manually added to the analyte spots, used earlier in at-line CE-SE(R)RS. All substrates were first tested by manual spotting using trans-1,2-bis(4-pyridyl)ethylene (BPE) as a model compound for SERS and crystal violet (CV) as a model compound for SE(R)RS. The spectral features of the SE(R)RS spectra of BPE and CV showed a most satisfactory repeatability on all substrates. As expected, the signal intensities varied considerably between runs; this implies that quantification in at-line CE-SE(R)RS should rather be done by means of an on-line absorbance detector. In addition, the suitability of Sub1, Sub2 and Sub4 as deposition substrates after CE was explored using two cationic dyes: CV and basic fuchsin (BF). Good-quality SERRS spectra could be recorded on all three substrates. Although Sub1 and Sub2 have a poor water-sorptivity, they were found to be good substrates for at-line CE-SERRS. They do not require any post-deposition addition of silver colloid and could therefore become attractive alternatives for Sub4. © 2003 Elsevier B.V. All rights reserved.

A wide-ranging overview of room temperature phosphorescence in the liquid state (RTPL1) is presented, with a focus on recent developments. RTPL techniques like micelle-stabilized (MS)-RTP, cyclodextrin-induced (CD)-RTP, and heavy atom-induced (HAI)-RTP are discussed. These techniques are mainly applied in the stand-alone format, but coupling with some separation techniques appears to be feasible. Applications of direct, sensitized and quenched phosphorescence are also discussed. As regards sensitized and quenched RTP, emphasis is on the coupling with liquid chromatography (LC) and capillary electrophoresis (CE), but stand-alone applications are also reported. Further, the application of RTPL in immunoassays and in RTP optosensing—the optical sensing of analytes based on RTP—is reviewed. Next to the application of RTPL in quantitative analysis, its use for the structural probing of protein conformations and for time-resolved microscopy of labelled biomolecules is discussed. Finally, an overview is presented of the various analytical techniques which are based on the closely related phenomenon of long-lived lanthanide luminescence. The paper closes with a short evaluation of the state-of-the-art in RTP and a discussion on future perspectives.

The fluorescence behaviour of 19 flavonoids was studied. Three isoflavones (formononetin (F), ononin (FG) and daidzein (D)) were found to exhibit large Stokes’ shifts, possibly due to a change of the structure of the molecule from non-planar in the S0 state to planar in the S1 state. Lifetime measurements were carried out using time-correlated single photon counting spectroscopy to further characterise the mechanism. These large shifts provide a high selectivity, so that fluorescent isoflavones can be readily detected in plant samples by means of reversed-phase LC with fluorescence detection. Attention has to be paid to possibly fluorescent impurities in flavonoid standards, as was observed for daidzin (DG), which is not fluorescent itself, but has a fluorescent isomer. To distinguish between the two compounds, LC with fluorescence and MS detection was used to separate and identify the impurity.

The question is addressed, whether in capillary electrophoresis (CE) the laser-based forward degenerate four-wave mixing (F-D4WM) detection method can become a realistic alternative to conventional ultraviolet (UV) absorption detection. In order to answer this question F-D4WM experiments at 351 nm have been performed. As test analytes the herbicides dinitro-ortho-cresol and dinitro-(tert-butyl)-ortho-phenol (trivial names DNOC and dinoterb, respectively) were used without derivatisation. These compounds show moderate absorption (molar absorptivity 4000 M−1 cm−1) at 351 nm under the conditions (buffer pH 8.2) used for CE separation. The detection limits obtained (1 μM) were 25-fold better than with on-column absorption detection at 351 nm. This result is shown to be typical for CE, where commonly buffer solutions with a high water content are used. For micro-separation systems that are compatible with liquid solutions with a low water content F-D4WM detection has a much wider potential.

The feasibility of a direct coupling of the low-temperature, high-resolution, fluorescence line-narrowing spectroscopy (FLNS) technique and the autoradiographic 32 P -post-labelling technique for DNA adducts is explored. FLNS has been used to identify analytes on various types of thin-layer chromatography (TLC) plates, but not on poly(ethylene)imine-cellulose (PEI-cellulose) TLC sheets involved in the 32 P -post-labelling technique which includes a two-dimensional separation. PEI-cellulose was found to exhibit a high fluorescence background. Its fluorescence excitation and emission characteristics were studied in detail and the signal-to-noise ratios of the FLNS spectra were optimized using background subtraction, time-resolved detection and a new synchronous scanning procedure. Benzo[a]pyrene (B[a]P) tetrols and a standard B[a]P–DNA adduct were used as model compounds. Limits of detection were in the low-picomole range. The technique was applied for the identification of spots extracted from PEI-cellulose TLC sheets obtained after a 32 P -post-labelling procedure of DNA containing B[a]P adducts at a level of two adducts per hundred bases.

Analytical procedures are developed for the identification and quantification of pesticides in the marine environment. Concentrations in the marine environment are expected to be at or below the low ng/l level, which requires preconcentration of analytes and improved detection. Off-line solid phase extraction (SPE) was combined with large-volume injection gas chromatography (GC) by means of a programmable temperature vaporiser (PTV). The selectivity of the detection procedure was considerably increased by using tandem mass spectrometry (MS/MS) instead of single-MS operation. Two ionisation modes, i.e. electron impact (EI) and positive chemical ionisation (PCI) with acetonitrile as reagent gas, were studied for the determination of five test analytes, i.e. atrazine, desethylatrazine, simazine, alachlor and metolachlor. The detection limits for both ionisation modes were essentially the same, and ranged between 0.2 and 5 ng l−1 when using a sample volume of 200 ml. With this modest sample volume, simultaneous filtration and extraction of even low-salinity samples could be performed without the filter being blocked by the large amount of suspended matter present in such samples.

The possibility of on-line diode laser-based intracavity absorption detection (LIAD) in liquid chromatography is examined. Diode laser-based LIAD should, in principle, be very sensitive because of the inherent stability of the light source compared to that of other lasers. Because many diode lasers are sold in packages that also contain a photodiode for optical feedback stabilization, the detection set-up is very simple. However, contrary to earlier reports, little signal gain is observed for LIAD compared to direct absorption detection. This is mainly due to the inefficient coupling of the diode laser and the external cavity. Because LIAD is more sensitive to mechanical instabilities, while rationing with a reference signal as well as feedback stabilization is not possible, the actual detection limit achieved for the model compound mitoxantrone is 7 × 10−7 M or 1 × 10−4 absorption units. These values are about two orders of magnitude less favourable than those observed with double-beam absorption detection using the same diode lasers.

Liquid chromatography (LC) was coupled semi on-line to Fourier transform infrared spectrometry (FT-IR) using a spray-jet assembly interface to eliminate the LC eluent prior to IR detection. The usefulness of the LC-FT-IR system in the identification of closely related compounds in complex mixtures is demonstrated by the analysis of a chlorinated pyrene sample which contains a number of chloropyrene isomers and congeners. Characteristic FT-IR transmission spectra of all constituents could be recorded. Since most of these compounds have not been analysed by IR before, spectral assignment was mainly based on the empirical Hansen—Berg rules for substituted pyrenes. The identification limits for the chloropyrenes typically were 5–10 ng injected.

A combination of conventional-size liquid chromatography (LC) and visible diode-laser induced fluorescence (DIO-LIF) detection has been applied for the detection of photosensitizers used in photodynamic therapy. Of these photosensitizers, a mixture of disulfonated aluminum phthalocyanines (AlPcS 2 ) has been chosen as a model. First, the influence of pH and solvent on the spectroscopic properties of di- and tetrasulfonated aluminum phthalocyanine has been examined, indicating the presence of multi-step ionization equilibria and the formation of aggregates. Direct fluorescence detection of AlPcS 2 is found to be unsatisfactory. For biological samples, the introduction of LC showed clear advantages, especially for faeces extracts. A detection limit of 7 × 10 −12 M was found for AlPcS 2 ( S/N = 3 for the major component) in both rat urine and faeces extracts using the DIO-LIF system, which is 20-fold more sensitive than a conventional fluorescence detection system, in combination with chromatography.

Organotin compounds (OTs) were analyzed after derivatization using a methyl Grignard reagent with gas chromatography with ion trap detection (GC-ITD) for detection. GC-ITD allows full scan mass spectrometry at trace levels and thus allows good verification of the presence of the OTs. With the described methods all currently used OT biocides can be determined in water, sediment and suspended matter: tributyltin (TBT), triphenyltin (TPT), tricyclohexyltin (TCT) and fenbutatin oxide (hexakis-(2-methyl-2-phenylpropyl)-distannoxane, FBTO). Additionally in water also some degradation products of the above mentioned biocides can be determined: dibutyltin (DBT), monobutyltin (MBT), diphenyltin (DPT), monophenyltin (MPT) and dicyclohexyltin (DCT). Detection limits are 1–40 ng/l (as tin) for water samples and 2–10 ng/g (as tin) for suspended matter and sediment samples. Transport routes of triphenyltin acetate (TPT) used in potato culture in the Flevoland polder in the Netherlands were studied. TPT levels were detected in freshwater and suspended matter and the sorption coefficient (Kd) was calculated. On the basis of concentrations in water and suspended matter it was calculated that only a negligible portion of TPT used is pumped out of the polder by the pumping-engines. Evaporation of TPT is probably a much more important transport route. Therefore a first attempt was made to investigate concentration of OTs in rainwater. The results show that at distances of over 20 km from potato fields, TPT in rainwater is around the proposed general quality objective for freshwater of 10 ng/l (as tin). These observations support the modelling study by Baart and Diederen, indicating that evaporation of TPT is one of the transport routes of TPT.

The simultaneous separation of the N -dimethylcarbamate pirimicarb and its metabolites V–VII was carried out by liquid chromatography (LC) using diode array detection (DAD). Two columns, a 250 × 4.6 mm i.d. column packed with 5-μm particles with a cyanopropyl modified silica stationary phase and a 150 × 4.6 mm i.d. column packed with 3-μm particles with a base deactivated C 8 modified silica stationary phase, were compared. The addition of a buffer in the eluent, for both columns, was needed to improve the peak shape of metabolite V and the resolution of the metabolites V–VII and consequently the pH dependence of the retention time was tested. Additionally chromatograms of two soil samples of different origin, spiked at a concentration level of 1 μg g −1 for pirimicarb, analysed by LC-DAD, gas chromatography (GC) with nitrogen phosphorus detection and GC-mass spectrometry are shown. Detection limits and reproducibility data of primicarb in a candidate reference sample using LC-DAD and a cyanopropyl column are reported.

The characteristics of laser-induced fluorescence (LIF) detection and state-of-the-art conventional fluorescence (CF) detection in the UV region below 300 nm in conventional-size column liquid chromatography are compared. An argon-ion laser system is used to provide four laser lines, i.e. 257 and 293 nm obtained by frequency-doubling of visible light and the UV lines at 334 and 352 nm. Light intensities inside the detector cell were measured by means of actinometry. Flicker noise, especially important for LIF detection was suppressed by simultaneously monitoring the laser light and ratioing sample and reference signals. On-line removal of fluorescent impurities in the eluent by means of a precolumn filled with carbon material resulted in a three-fold reduction of the background signal for LIF detection with 257-nm excitation. It is shown that LIF detection at 257 nm is more suitable than LIF at 293, 334 and 352 nm, not only for some pharmaceutical compounds that can only be excited below 30 nm, but also for polyaromatic hydrocarbons that absorbe at wavelengths longer than 300 nm. Compared with CF detection at optimum excitation and emission settings, the detection limits obtained with the LIF system were improved typically by 5 to 10-fold.

The efficient control of fermentation processes requires reliable monitoring systems. This paper describes a fully automated monitoring system based on the on-line combination of ultrafiltration and column liquid chromatography. An evaluation of the performance of a hollow-fibre ultrafiltration module is provided and a method for the determination of sugars, alcohols and organic acids during a gluconic acid and a beer fermentation is presented. The system can be used for at least 250 h with complex fermentation broths under both aerobic and anaerobic conditions without any detrimental effect on either the fermentation or the chromatographic part. Depending on the application 3–5 analyses can be performed every hour, using only 3.0 μl of sample per analysis. Using refractometric or direct UV detection the linear dynamic range is from 0.2 to 150 g l −1 for most of the compounds, while with a simple post-column derivatization procedure sugars can be determined in the low mg l −1 range.

The potential of coupled column liquid gas chromatography (LC) for the trace determination of a single polar compound by direct large-volume injection of aqueous samples was studied and a general approach to method development for this class of compound is discussed. As a new application, the determination of methyl isothiocyanate (MITC) by means of reversed-phase (RP) LC column switching and UV detection at 237 nm is reported. With direct injection of 770-μl samples, the detection limit is 1 μg l−1 and the time of analysis is about 10 min. For a further increase in selectivity and detectability (down to a level of about 0.1 gmg l−1), a rapid liquid-liquid extraction followed by an organic-aqueous phase switch on a silica cartridge is required prior to RPLC. No residues of MITC were found in over 200 surface water samples investigated.

A fully automated column liquid chromatographic separation system using on-line trace enrichment, gradient elution and diode-array detection for the trace-level determination of polar pollutants is described. Automation of the system was achieved by means of an automated cartridge-exchange system (PROSPEKT). Relevant parameters such as pH, volume and ionic strength of the sample, flow-rate during the enrichment step and wavelengths and band widths during detection were optimized for eighteen pollutants in various types of water at concentration levels below 5 μg l−1. The determination limit for all test compounds in liquid chromatographic grade water was 0.1 μg l−1, and identification, via diode-array spectra, could be performed at the same level. The mean relative standard deviations of the peak areas and the retention times for all the test compounds were 10% and 0.3%, respectively, at the 5 μg l−1 level for river Rhine water.

The influence of metal ions on the peroxyoxalate chemiluminescence reaction was investigated using batch experiments. A number of metal ions showed an improvement of the chemiluminescence reaction of bis(2-nitrophenyl) oxalate with hydrogen peroxide in the presence of 3-aminofluoranthene as the fluorophore and imidazole as the catalyst. Copper(II) gave the highest enhancement, of about one order of magnitude. The reason for the increase in the chemiluminescence intensity by metal ions is probably a change in the mechanism of the peroxyoxalate chemiluminescence reaction due to stabilization of the oxalate ester-imidazole complex by the metal ion, leading to a higher efficiency of the chemiluminescence reaction.

In recent years, much progress has been made in the development of liquid chromatographic (LC) detection systems based on chemiluminescence (CL) reactions. Peroxyoxalate CL (PO-CL) is now one of the most sensitive detection methods in LC. For many compounds detection limits are in the low picogram range. In the present review attention is devoted to the optimization of PO-CL detection with emphasis on background chemiluminescence and kinetic aspects. Further, derivatization reactions for various types of analytes with labels possessing good CL characteristics are described in some detail. An overview of, often biomedically oriented, applications is given and the potential of PO-CL for the detection of trace amounts of hydrogen peroxide generated by post-column enzyme reactions is illustrated.

An automated method is described for the routine determination of 3′-azido-2′,3′-dideoxythymidine (AZT), the best known drug against acquired immunodeficiency syndrome (AIDS). The method is based on on-line dialysis to remove matrix macromolecules, followed by selective preconcentration and clean-up with a silver(I)-thiol stationary phase. After desorption of the solute with a small plug of perchloric acid, chromatography is applied using an octadecyl-modified silica column. Using UV absorbance detection at 269 nm, the minimum detectable concentration in plasma is 20 ng ml−1 (600-μl sample). The within-day reproducibility at the 20 ng ml−1 level is 4.4% and at least 128 samples can be analysed unattendedly without exchanging the dialysis membrane, the precolumn or the analytical column.

A method for flow-through liquid scintillation counting in reversed-phase liquid chromatography (RP-LC) based on the continuous extraction of aqueous column eluates with a water-immiscible liquid scintillator is evaluated in terms of sensitivity, reproducibility and extra-column band broadening. 3H- and 14C-labelled phenylthiohydantoinamino acid derivatives of widely different polarity serve as model compounds. For extractable derivatives, counting efficiencies of over 30 and 80% can be obtained for 3H and 14C, respectively. The reproducibility and extra-column band broadening depend on the mixing ratio of scintillator and LC eluent; relative standard deviations (peak areas) of less than 3% can be obtained. The sensitivity of flow-through counting can be increased at least 150-fold by storing the segmented scintillator/eluate stream in a capillary storage loop. After the separation is complete, the stream is re-introduced into the radioactivity detector at reduced flow-rates to increase the mean residence time, i.e., the counting time, in the detector.

A flow injection analysis (FIA) system with peroxyoxalate chemiluminescence (CL) detection was optimized by factorial design analysis in the absence and presence of Cu(II) ions. The carrier stream was a mixture of acetonitrile and an aqueous imidazole solution of pH 7.O. The effect of the metal ions was determined by adding Cu(II) nitrate to the imidazole solution. A mixture of 5 mM bis(2-nitrophenyl)oxalate solution in acetonitrile and hydrogen peroxide was used as reagent solution. The fluorophore used was 3-aminofluoranthene dissolved in acetonitrile to a concentration of 12.5 nM. The parameters of the factorial design experiment in the absence of Cu(II), were the concentration of hydrogen peroxide and imidazole, the percentage of acetonitrile and the length of the mixing coil; those of experiments in the presence of the metal ions, the concentration of imidazole and Cu(II) and the length of the mixing coil. The enhancement of the chemiluminescence intensity by Cu(II) in the FIA system was found to be 70%.

The mono-ortho chlorobiphenyls (CBs) 60, 74, 114, 123, 157, 167 and 189 were determined in Aroclor mixtures and aquatic organisms by multidimensional gas chromatography with electron capture detection (MDGC/ECD), using a combination of an Ultra 2 and an FFAP column. MDGC/ECD is recommended as the most suitable technique for direct determination of these CBs, without a liquid chromatographic (LC) pre-separation of mono-ortho CBs from the other CBs. Dependent, to some extent, on the stationary phase used, single-column determinations of these CBs easily yield too high results due to the presence of interferences. The contribution of the mono-ortho CBs studied to the total 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs) from CBs in fish is highly dependent on the toxic equivalency factors (TEFs) used and varies between 0.02 and 22%. This contribution is mainly due to CBs 74, 114, 157 and 167 which may easily be determined by taking three heart-cuts and combining them into one MDGC/ECD run. The analytical error is negligible compared with the huge uncertainty of the TEFs. A need for more precise TEFs is emphasized. © 1995.

The applicability of two-photon excitation (TPE) for fluorescence detection in flow dynamic systems was explored. Emphasis was on conventional-size liquid chromatography (LC) and a direct comparison was made with one-photon excitation (OPE) by the use of standard laser- and lamp excitation. Simultaneous two-photon excitation (STPE) with visible laser light was used for fluorescence detection of UV-absorbing analytes; in consecutive two-photon excitation (CTPE) fluorescence from higher excited states was detected. In both TPE modes fluorescence is measured at the short-wavelength side of the laser light, a spectral region with relatively low background signals. STPE fluorescence will only have potential if the background measured in real samples is sufficiently low; this was investigated for the LC analysis of urine spiked with indole-3-acetic acid. The relative intensities of signals from the urine matrix and indole-3-acetic acid were not improved; that is, no extra selectivity was gained by applying STPE. In CTPE, one photon is sufficient to bring the analyte in an excited electronic state so that in this mode, in principle, both normal and short-wavelength fluorescence can be measured. CTPE fluorescence has a distinct potential; it was observed for aminoanthraquinones, one of them has a very low fluorescence quantum yield in protic eluents. Detection limits were at the nM level. Mitoxantrone, a potent anti-tumour drug, could be detected in urine at a similar level by applying excitation at 625 nm and detecting higher excited state emission in a 290–400 nm window; sample treatment prior to LC was very simple.

A new type of interface has been developed for the direct coupling of packed-capillary reversed-phase liquid chromatography with electron-capture detection. This interface allows the detection of relatively non-volatile and/or polar compounds, and simply consists of a fused-silica capillary (50 μm I.D.) inside of a stainless-steel capillary (0.32 mm I.D.) connecting the analytical column with the detector through the oven wall. The make-up gas is used for cooling of the fused-silica capillary. With flow-injection analysis the influence of several parameters (e.g. flow-rate of the make-up gas and mobile phase, oven temperature) on the performance of the total system (i.e. band broadening, sensitivity) has been studied. Using optimal conditions (flow-rate 2 μl min−1, make-up gas 40 ml N2 min−1, oven temperature 300 °C, injection volume 60 nl) separation and detection of mononitroanilines, chlorophenols, pesticides and benzodiazepines have been performed. Detection limits are in the range of 10–450 pg (0.2–7 mg 1−1) under reversed-phase conditions. Large-volume injections up to 15 μl, using a non-eluting solvent (water) to focus the analytes on top of the column, have been performed to increase the sensitivity of the system. This technique increased the concentration sensitivity with 2–3 orders of magnitude allowing the detection of ca. 150 ng 1−1 of chlorophenols in river Meuse water.

The influence of the analytical error in the multidimensional gas chromatographic determination of mono-ortho substituted chlorobiphenyls in biota on the calculation of their TCDD equivalents is negligible compared with the influence of the high uncertainty of toxic equivalency factors reported in the literature.