A laser-based fluorometry system for investigations of seawater and porewater fluorescence

Marine Chemistry, 31 (1990) 219-230

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Elsevier Science Publishers B.V., Amsterdam

A laser-based fluorometry system for investigations of seawater and porewater fluorescence Robert F. Chen and Jeffrey L. Bada Scripps Institution of Oceanography, University of California, San Diego, A-OI 2-B, La Jolla, CA 92093 (U.S.A.) (Received November 15, 1989; revision accepted June 1, 1990)

ABSTRACT Chen, R.F. and Bada, J.L., 1990. A laser-based fluorometry system for investigations of seawater and porewater fluorescence. Mar. Chem., 31: 219-230. A highly sensitive laser-induced fluorescence (LIF) system has been developed to study the fluorescence of dissolved organic carbon (DOC) in the marine environment. The LIF detector has a detection limit of ~ 10 attomoles ( 10 X 10- ~s moles) of pterin and eliminates internal quenching in highly fluorescent samples such as anoxic porewaters encountered when using conventional fluorometry. LIF analysis is rapid, reproducible, and uses only 100/zl of a sample. This small size requirement permits fluorescence analyses of samples often available only in limited amounts, such as porewaters, hydrothermal vent waters, and rainwaters. In addition, the LIF detection system may greatly simplify extraction and separation procedures required to characterize the fluorescent components of DOC.

INTRODUCTION

Whereas the fluorescence (2ex = 320-365 nm, '~-em = 420-470 n m ) of natural waters such as rivers and estuaries is intense, and thus easily measured, because of the presence of terrestrial humic acids (Zimmerman and Rommets, 1974; Smart et al., 1976; Dorsch and Bidleman, 1982), open ocean seawater fluorescence (2ex= 300--350 nm, ~,em=400-500 nm) is much lower and more difficult to evaluate (Traganza, 1969; Duursma, 1974; Zimmerman and Rommets, 1974; Hayase et al., 1987, 1988; Donard et al., 1989). Although conventional fluorometry using commercial instrumentation can be used to measure seawater fluorescence (Kramer, 1979; Hayase et al., 1987, 1988), several problems limit the routine study of this important property of seawater. For example, the 3-4 ml required by conventional fluorometry has so far limited studies to filtered ocean waters. A highly sensitive fluorometer would permit investigators to study small fluorescence variations of open ocean 0304-4203/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.

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R.F. CHEN AND J.L. BADA

waters, as well as to investigate the fluorescence of limited samples such as porewaters, rainwaters, hydrothermal vent waters, and dissolved organic carbon (DOC) fractions isolated by small-scale resin extractions and high-performance liquid chromatography (HPLC); all of these are not readily measured using conventional fluorometry. The sensitivity of fluorescence measurements is controlled by Rayleigh scattering from sample cell walls, Raman scattering of the solvent, noise in the excitation source, solvent and sample cell contamination, and stray light (Parker, 1968; Matthews and Lytle, 1979; Cheng and Dovichi, 1988 ). These considerations have not been minimized by conventional fluorometry. Internal quenching and broad-band excitation resulting in low selectivity in the emission spectrum are also problems inherent in conventional fluorometry. Additional complications associated with these static measurements include temperature changes, photochemical degradation, and sample manipulation during the measurements. Although a flow-through system is possible with conventional fluorometry, this results in either a lower sensitivity or a large sample volume requirement. The recent emphasis on laser excitation and miniaturization to reduce sample and solvent quantities (Yeung and Sepaniak, 1980; Dovichi et al., 1983; Zare, 1984; Shelly and Edkins, 1988) has led to the development of laserinduced fluorescence (LIF) detection which, because of its high sensitivity, potentially has a wide spectrum of applications in the study of seawater fluorescence. For example, a highly sensitive pulsed nitrogen laser-based spectrophotometer has been used to differentiate emission spectra of coastal and marine waters (Donard et al., 1989 ). In addition, we have recently used an LIF system to measure the bulk fluorescence of small samples of filtered seawater and porewaters (Chen and Bada, 1989). LIF detection is also ideal for monitoring HPLC effluents (Hershberger et al., 1979; Folestad et al., 1982; Gluckman et al., 1984; Berthod et al., 1987), such as those encountered in the characterization of the fluorescent components of DOC. The LIF-based methodology is rapid, sensitive and requires only small amounts of sample. In this paper, we describe our LIF system and demonstrate its application to fluorescence studies of marine samples. METHODS

Reagents and conventional fluorometry instrumentation Sodium chloride, lauryl sulfate, dimethyldichlorosilane, pterin, and xanthopterin were obtained from Sigma Chemical Company. Quinine sulfate monohydrate was obtained from Aldrich Chemical Company. The NaC1 was baked for 24 h at 500 °C before use. Millipore Milli-Q water was used to prepare standards and the 0.5 NaC1 solution.

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A Farrand model MK 2 scanning spectrofluorometer equipped with a xenon lamp, excitation and emission monochromators, and a photomultiplier was used for conventional fluorometry. The Farrand excitation and emission bandwidths were set at 10 nm. A 1 cm × 1 cm X 4 cm quartz fluorescence cell was used for the measurements.

General description of LIF system The LIF system we have developed uses as an excitation source the 325-nm beam of an HeCd laser (model 4240 NB; Liconix, Sunnyvale, CA ). This laser beam is focused with a spherical lens (Esco Products, Inc, Oak Ridge, N J) with a 1-in focusing length into a 100-/~m core diameter fused silica optic fiber (Polymicro Technology, Phoenix, AZ). The optic fiber is used to transport the excitation light to a 200-/tm i.d. deactivated fused silica capillary column (Science Glass Engineering, Austin, T X ) . The flow-through cell was made by carefully removing about 0.5 cm of the polyimide coating on the fused silica column with a razor blade. The actual placement and orientation of the excitation optic fiber with respect to the scraped region of the fused silica capillary column is discussed below. A 0.5 N NaCI solution was used for column elution and was delivered to the capillary column by a Model L6200 p u m p (EM Science, Cherry Hill, NJ) at a flow rate of 0.5 ml min -t. Samples were introduced with a Valco Model C6W injection valve (Valco Instruments, Houston, T X ) fitted with a 100-/A sample loop. The emission radiation was collected using 600-/tm fused silica optic fibers (Polymicro Tech. ) positioned at right angles to the excitation beam. The collected emission radiation was first passed through a 400-nm high-pass cutoff filter (Model FSR GG400, Newport Research Corp., Fountain Valley, CA) and then into a grating m o n o c h r o m a t o r (Model 77250, Oriel Corp., Stratford, CT) set at 450 n m with a 10-nm exit slit. The exiting light was collected with a Centronic Model 4249B photomultiplier tube (Newbury Park, CA), and the resulting current was converted to voltage with a Model AI-101 preamplifier (Thorn EMI, Fairfield, N J). A Datamark Servocorder SR6253 (Cole Scientific, Calabasas, CA) was used to collect data. The optic fibers and capillary column were positioned with X Y Z positioners and fiber chucks (Newport Research Corp. ). An aluminium box (2-in on a side ), which was used for the detector housing, was h o m e m a d e as were the interfaces into and out of the monochromator. The fused silica capillary was connected to the injector by inserting it into a l-in length of 0.010-in (254#m ) Teflon tubing (Alltech Assoc., Deerfield, IL) and using a standard Valco 1/ 16-in nut and ferrule. A general diagram of our LIF system is shown in Fig. 1.

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LIF cell design The heart of the LIF detector is the flow-through cell. Several different designs have been previously evaluated (van Vliet and Poppe, 1985; McGuffin and Zare, 1986; Cheng and Dovichi, 1988; Shelly and Edkins, 1988). In our system, fiber optics are used to deliver the excitation light to the cell and to collect the emission radiation. We tested several cell geometries and configurations (Fig. 2 ) to ascertain the optimal arrangement for the measurement of seawater fluorescence. In the first arrangement (Fig. 2a), the excitation beam is positioned outside the flow-through cell at right angles to a single emission fiber. A substantial amount of the excitation power is lost by scattering off the cell wall, as seen by the small peaks produced with this arrangement. In addition, there is significant noise, probably derived from fluorescence impurities in the fused silica capillary tubing. This noise is greatly reduced, and the excitation power is conserved, when the excitation fiber is placed directly inside the capillary column flow-through cell (Fig. 2b). The remaining noise is probably related to column-solvent-solute surface interactions, as the column was used as supplied by the manufacturer in this arrangement. In the optimum configuration, which is shown in Fig. 2c, the noise associated with the arrangement shown in Fig. 2b was greatly reduced by deactivating the column as described below. The signal strength was also increased by the ad-

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dition of a second optic fiber to collect more of the emission light. With two emission fibers, the cell box maintains a simple geometry, while still allowing the user to see into the cell box through a hole on one side fitted with a removable nut to aid in positioning and examining the flow-through cell for contamination and bubbles. The configuration in Fig. 2c was thus used for measuring the fluorescence of seawater and porewaters.

Column chemistry Surface chemical interactions between free silanol groups on the walls of the fused silica capillary column and solute molecules can cause peak broadening and irreproducible peak heights and peak areas (Lee and Wright, 1980; Tarbet et al., 1988 ). Although the fused silica column we initially used was stated by the manufacturer to be deactivated, surface interactions were still a problem. Therefore, further column deactivation was carried out using dimethyldichlorosilane to inactivate the remaining free silanol groups with hydrophobic methyl groups. The resulting peaks were reasonably reproducible, but the peak shapes were still found to be unsatisfactory. Because ionic strength and pH affect fluorescence (Laane, 1982; Lakowicz, 1983; Willey, 1984), a solution with an ionic strength and pH similar to that of the sample must be used to insure unaltered fluorescence signals in a flowthrough system. For our seawater investigations, we used a 0.5 N NaCl (pH ~ 7) solution for elution. In addition, we found that adding a non-fluorescent surfactant such as lauryl sulfate to the 0.5 N NaC1 solution helped maintain a hydrophobic surface and thus undistorted peak shapes and reproducible peak heights. Still, mixing at the head or the tail of the injection volume makes peak areas less reproducible, whereas peak heights are consistently less affected. Therefore, peak heights were a better measure of fluorescence intensity than peak areas. Fluorescence measurements were found to be unaffected by the amount of sample injected; the same fluorescence intensities were obtained when 100 and 500/tl of a seawater sample were injected.

Standards Because our excitation and emission wavelengths are different from those used by Kalle (1963), Dorsch and Bidleman (1982), and Hayase et al. ( 1987 ), 2.8/tg 1-~ quinine sulfate monohydrate at pH 2 was arbitrarily set to 45 fluorescent units (flu) on the Farrand spectrofluorometer. Quinine sulfate was not suitable on the LIF detector, however, because NaC1 was found to quench the fluorescence intensity by about an order of magnitude. Pterin and xanthopterin in 0.5 N NaC1 were evaluated, but these compounds are so light sensitive that, even when stored in amber glass bottles, it was difficult to ob-

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tain reproducible peak heights over the period of a day. Phenanthrene and pyrene are almost insoluble in water and so it is difficult to prepare reproducible standards. Instead, it was found that deep seawater maintained a very constant signal over several months when stored at room temperature in an amber bottle if care was taken to avoid contamination. Therefore, a 4-1 bottle of filtered seawater from 1500 and 1900 m in the San Clemente Basin (SCB), located about 75 km offshore from San Diego, was used as a standard on the LIF detector. This SCB deep water was measured vs. quinine sulfate on the Farrand spectrofluorometer each day that LIF measurements were made. If an aliquot ( ~ 100 ml) of this seawater standard was replaced every week or so from the 4-1 reference bottle, the fluorescence signal was stable ( ___< 3%) over many months. It is our belief that any seawater sample can be used as a standard on the flow-through system as long as it is calibrated against quinine sulfate on another instrument the same day.

Seawater and porewater samples Seawater samples were collected in Niskin bottles and were immediately filtered through 0.7-am precombusted (5 h at 500°C) glass fiber filters (Whatman GF-F) and then frozen in precombusted (5 h at 500°C) amber glass bottles fitted with clean Teflon-lined caps. Santa Barbara porewater samples were obtained from box and Kasten cores, filtered through 0.45-gm membrane filters, and frozen in small amber vials with Teflon-lined caps (Chen and Bada, 1989). RESULTS AND DISCUSSION

System performance The LIF detector has a dynamic range of at least three order of magnitudes. Peak heights were found to increase linearly (r2= 0.9996 ) from 1.5 nM to 10 #M pterin in 0.5 N NaC1. Reproducibility of peak height measurements was good, with standard deviations of about + 2% as determined by replicate (n = 3-10) injections of either seawater or standard. Signal-to-noise ratios were generally around 100: I for deep waters and 30:1 for surface waters, so even small variations in fluorescence are resolvable. With a signal-to-noise ratio of two, the lowest pterin concentration which can be accurately measured is thus ~ 75 pM. With this injected concentration and an illuminated volume of the flow-through cell of ~ 150 nl, this gives an absolute detection limit of only ~ 10 attomoles ( 10 × 1O- 18 moles ) of pterin. The main source of noise appears to be fluorescent impurities in the glass and the solvent as well as a small amount of scattered excitation light that passes through the filters. The background noise is dependent on laser power,

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so the sensitivity remains constant over a wide range of laser power. Light leaks and Raman scattering seem to be minimal. If higher sensitivity is required, noise could be greatly reduced by adding a chopper and frequency modulated detection (Doherty, 1983).

Examples of the LIF analysis of seawater and porewaters, and a comparison with results obtained using conventional fluorometry Figure 3 shows seawater fluorescence profiles obtained using both the LIF system and the Farrand conventional fluorometer for the San Clemente Basin (Fig. 3a), the Straits of Florida (Fig. 3b), and the Sargasso Sea (Fig. 3c). As can be seen, the agreement between the two methods is excellent. We have previously reported similar comparative results for the Santa Barbara Basin (Chen and Bada, 1989). All of the oceanic fluorescence profiles we have obtained so far show similar characteristics, namely a surface minimum and a constant, high deep value. The low fluorescence in near-surface waters seems to be homogeneous in the mixed layer, and is apparently caused by photodegradation (Kramer, 1979; Hayase et al., 1987; Chen and Bada, 1989). The sources of deep-water fluorescence components could be remineralization of organic matter and release of humic substances from particles in correlation with nutrients (Hayase et al., 1987, 1988), solubilization and diffusion from the sediments (Chen and Bada, 1989 ), or in situ production (Kalle, 1949; Duursma, 1974). The fluorescence profiles for Santa Barbara porewaters obtained by LIF detection and conventional fluorometry are shown in Fig. 4a. The porewater fluorescence is much more intense than that of the overlying seawater, and increases with depth within the sediment. In the deeper part of the core, the porewater fluorescence determined by LIF is significantly greater than that Fluorescence 0

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measured by the Farrand fluorometer. We have previously suggested (Chen and Bada, 1989 ) that in highly fluorescent samples this difference may be the result of internal quenching of the Farrand signal because of the large sample volume required in the conventional fluorometer cell. To test this, we sequentially diluted one of the Santa Barbara porewaters with 0.5 N NaC1 and measured the corresponding fluorescence using both techniques (Fig. 4b ). As can be seen, with increasing dilution, the porewater fluorescence intensity measured by the two methods became similar. This demonstrates that with highly fluorescent samples, LIF detection minimizes internal quenching problems associated with large volume cells, and thus provides a more accurate measure of the actual fluorescence than does conventional fluorometry. It should be noted that corrections to quenched conventional measurements can be made to determine 'absolute fluorescence' (Duursma, 1974), but this requires emission and excitation light absorption coefficients for each sample. This necessitates the use of a spectrometer and a computer to carry out the calculations and therefore increases the time and effort needed for each sample. In the Santa Barbara Basin, fluorescence correlates with porewater alkalinity and ammonium ion concentration, which suggests that the release of fluorescent material is associated with remineralization of organic matter (Chen and Bada, 1989 ). Further analyses are necessary to determine the sources and sinks of porewater fluorescence in diverse sedimentary environments.

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Advantages of LIF detectionfor studies offluorescence in marine samples A comparison with the conventional Farrand fluorometer is useful to characterize the advantages and disadvantages of LIF detection. The mass sensitivity ( 10 attomoles for the LIF detector vs. 400 femtomoles for the Farrand) is greatly enhanced (by a factor of 40 000), whereas the concentration sensitivity (75 vs. 400 pM) is only 5-6 times greater. With the LIF system, the sampling time is faster, and the sampling process could be automated with an autoinjector. Only ~ 1-2 min per sample is needed with LIF, whereas to rinse carefully and change samples in the quartz cuvette of a conventional fluorometer takes ~ 4-5 min per sample. LIF detection requires only 100/tl of a sample compared with ~ 1 ml to rinse the cell and 2-3 ml to make a measurement with the Farrand fluorometer. Thus, LIF detection allows routine measurement in limited samples such as porewaters, hydrothermal vent waters, and rainwater. Also, when separating and isolating fractions of fluorescent organic matter by techniques such as XAD resin extraction, Sep-Pak C-18 cartridges, activated charcoal, etc., LIF detection greatly reduces solvent and sample sizes, and thus minimizes contamination problems encountered in large-scale extractions and analyses (Momzikoff, 1969; Dunlap and Susic, 1985; Harvey and Boran, 1985). Conventional fluorometry exposes the sample and the cell to operator contamination. The sample can photodegrade before and during measurement and the temperature can drift. These problems are greatly reduced using the rapid flow-through system in LIF detection. Full excitation and emission scans are possible with the Farrand fluorometer, however. With the LIF detector, a loss in sensitivity would arise ifa diode array detector were used for monitoring the emission radiation, and the wavelength of the excitation laser beam is fixed. The LIF emission monochromator could be scanned employing the correct filters if necessary, but this would require a slow flow rate or a large injection volume. The excitation light, however, is better defined, and thus more selective, when using a coherent laser beam, whereas a xenon lamp is less selective and excites a broader range of electronic states of fluorescence. Raman and Rayleigh scattering are also broadband phenomena when using a xenon lamp, whereas laser-induced Raman and Rayleigh scattering is very narrowband and can therefore be easily filtered out. CONCLUSIONS

In light of the recent advances and problems in the study of dissolved organic carbon (e.g. Williams and Druffel, 1987; Sugimura and Suzuki, 1988), it is clear that new techniques and app'roaches in this field are becoming increasingly valuable. Although the fluorescence of dissolved organic matter in

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seawater has been investigated for over 40 years, the increase in sensitivity, reliability, and speed of the LIF detector described in this paper allows detailed studies of the sources, sinks, and pathways of the fluorescent fraction of DOC in the oceans. The LIF detector is not limited to bulk analyses, but is also currently being evaluated as an on-column detector in HPLC, microbore HPLC, and open tubular liquid chromatography. The great mass sensitivity of LIF should permit the detection and thus characterization of the components of the highly complex mixture of fluorescent compounds in the marine environment. ACKNOWLEDGEMENTS

We thank Drs. Richard Zare and Mark Roach for sharing their knowledge about LIF detection. This work was supported by ONR contract N00014-87K0005/JB and grant N00014-89-J- 1422/JB. REFERENCES Berthod, A., Li, K.P., Yu, T. and Winefordner, J.D., 1987. Low-volume fluorescence detector for high-performance liquid chromatography. Anal. Chem., 59: 1485-1488. Chen, R.F. and Bada, J.L., 1989. Seawater and porewater fluorescence in the Santa Barbara Basin. Geophys. Res. Lett., 16: 687-690. Cheng, Y.F. and Dovichi, N.J., 1988. Laser-induced fluorescence detection using the sheathflow cuvette for capillary zone electrophoresis. In: E.R. Menzel (Editor), Fluorescence Detection II. The Society of Photo-Optical Instrumentation Engineers, 910:112-115. Doherty, H., 1983. Techniques of low level light measurement. Lasers and Appl., July 1983:4145. Donard, O.F.X., Lamotte, M., Belin, C. and Ewald, M., 1989. High-sensitivity fluorescence spectroscopy of Mediterranean waters using a conventional or a pulsed laser excitation source. Mar. Chem., 27:117-136. Dorsch, J.E. and Bidleman, T.F., 1982. Natural organics as fluorescent tracers of river-sea mixing. Estuarine Coastal Shelf Sci., 15:701-707. Dovichi, N.J., Martin, J.C., Jett, J.H. and Keller, R.A., 1983. Attogram detection limit for aqueous dye samples by laser-induced fluorescence. Science, 219: 845-847. Dunlap, W.C. and Susic, M., 1985. Determination of pteridines and flavins in seawater by reverse-phase, high performance liquid chromatography with fluorometric detection. Mar. Chem., 17: 185-198. Duursma, E.K., 1974. The fluorescence of dissolved organic matter in the sea. In: N.G. Jerlov and E. Steeman Nielsen (Editors), Optical Aspects of Oceanography. Academic Press, London, pp. 237-256. Folestad, S., Johnson, L., Josefsson, B. and Galle, B., 1982. Laser induced fluorescence detection for conventional and microcolumn liquid chromatography. Anal. Chem., 54: 925-929. Gluckman, J., Shelly, D. and Novotny, M., 1984. Laser fluorimetry for capillary column liquid chromatography: high sensitivity detection of derivatized biological compounds. J. Chromatogr., 317: 443-453. Harvey, G.R. and Boran, D.A., 1985.Geochemistry of humic substances in seawater. In: G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Editors), Humic Substances in Soil, Sediment, and Water. Wiley, New York, pp. 233-247. Hayase, K., Yamamoto, M., Nakazawa, I. and Tsubota, H., 1987. Behavior of natural fluores-

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cence in Sagami Bay, and Tokyo Bay, Japan - - vertical and lateral distributions. Mar. Chem., 20: 265-276. Hayase, K., Tsubota, H., Sunada, I., Goda, S. and Yamazaki, H., 1988. Vertical distribution of fluorescent organic matter in the North Pacific. Mar. Chem., 25: 373-381. Hershberger, L.W., Callis, J.B. and Christian, G.D., 1979. Sub-microliter flow-through cuvette for fluorescence monitoring of high performance liquid chromatographic effluents. Anal. Chem., 51: 1444-1446. Kalle, K., 1949. Fluoreszenz und Gelbstoff im Bottnischem und Finnischen Meerbusen. Dtsch. Hydrogr. Z., 2: 9-124. Kalle, K., 1963. Ober das Verhalten und die Herkunft der in den Gewassern und in der Atmosphare vorhandenen himmelblauen Fluoreszenz. Dtsch. Hydrogr. Z., 16:153-166. Kramer, C.J.M., 1979. Degradation by sunlight of dissolved fluorescing substances in the upper layers of the eastern Atlantic Ocean. Neth. J. Sea Res., 13: 325-329. Laane, R.W.P.M., 1982. Influence of pH on the fluorescence of dissolved organic matter. Mar. Chem., 11: 395-401. Lakowitz, J.R., 1983. Principles of Fluorescence Spectroscopy. Plenum, New York, 496 pp. Lee, M.L. and Wright, B.W., 1980. Preparation of glass capillary columns for gas chromatography. J. Chromatogr., 184:235-312. Matthews, T.G. and Lytle, F.E., 1979. Blank limitations in laser excited solution luminescence. Anal. Chem., 51: 583-585. McGuffin, V.L. and Zare, R.N., 1986. In: Satinda Ahuja (Editor), Applications of Laser Fluorimetry to Microcolumn Liquid Chromatography. Am. Chem. Soc. Symp. Ser. 297:120-136. Momzikoff, A., 1969. Recherches sur les compos6s fluorescents de l'eau de mer. Identification de l'isoxanthopterine, riboflavine et du lumichrome. Cah. Biol. Mar., 10:221-230. Parker, C.A., 1968. Photoluminescence of Solutions. Elsevier, New York, 544 pp. Shelly, D.C. and Edkins, T.J., 1988. Miniature laser fluorescence detector for capillary liquid chromatography. In: E.R. Menzel, Fluorescence Detection II. The Society of Photo-Optical Instrumentation Engineers, 910:116-122. Smart, P.L., Finlayson, B.L., Rylands, W.D. and Ball, C.M., 1976. The relation of fluorescence to dissolved organic carbon in surface waters. Water Res., 10:805-811. Sugimura, Y. and Suzuki, Y., 1988. A high-temperature catalytic oxidation method for the determination of non-volatile dissolved organic carbon in seawater by direct injection of a liquid sample. Mar. Chem., 24: 105-131. Tarbet, B.J., Bradshaw, J.S., Markides, K.E., Lee, M.L. and Jones, B.A., 1988. The chemistry of capillary column technology. LC-GC, 6: 232-248. Traganza, D., 1969. Fluorescence excitation and emission spectra of dissolved organic matter in sea water. Bull. Mar. Sci., 19: 897-904. Van Viler, H.P.M. and Poppe, H., 1985. The performance of some cell designs for laser-induced fluorescence detection in open-tubular liquid chromatography. J. Chromatogr., 346: 149160. Willey, J.D., 1984. The effect of seawater magnesium on natural fluorescence during estuarine mixing, and implications for tracer applications. Mar. Chem., 15: 19-45. Williams, P.M. and Druffel, E.M., 1987. Radiocarbon in dissolved organic matter in the central North Pacific Ocean. Nature (Lond.), 330: 246-248. Yeung, E.S. and Sepaniak, M.J., 1980. Laser fluorometric detection in liquid chromatography. Anal. Chem., 52: 1465A-1481A. Zare, R.N., 1984. Laser chemical analysis. Science, 226: 298-303. Zimmerman, J.T.F. and Rommets, J.W., 1974. Natural fluorescence as a tracer in the Dutch Wadden Sea and the adjacent North Sea. Neth. J. Sea. Res., 8:117-125.