Chlorophyll and other naturally occurring tracer dyes for laser-induced fluorescence in liquid flow applications
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1 Chlorophyll and other naturally occurring tracer dyes for laser-induced fluorescence in liquid flow applications Katharina Zähringer * Lehrstuhl für Strömungsmechanik und Strömungstechnik, Universität Magdeburg, Germany * correspondent author: katharina.zaehringer@ovgu.de Abstract Tracers commonly used in experimental flow studies are mostly nocuous to the environment and human health. Particularly in large flow installations this can become a problem. Naturally occurring nonharmful substances are an alternative that has been tested in this study. Chlorophyll is examined here for its applicability as a tracer in flow studies. The dependency of its fluorescence intensity on parameters like concentration, laser energy, temperature and ph is determined for two commonly used laser excitation wavelengths (532nm, 355nm). Several vitamins also turned out to be most promising candidates for replacing conventional dyes. Their fluorescence intensity is comparable to those and they could also be used for temperature or ph studies. As an example of application, vitamin B2 has been used for the determination of mixing properties of a static mixer. 1. Introduction Laser-induced fluorescence is a very common tool in experimental flow studies, used for the visualization of flow structures, the determination of concentrations, temperatures or other scalar quantities. Often fluorescent tracer dyes are used for that e.g. (Duwel et al. 2004, Hagemeier et al. 2012, Kling and Mewes 2004, Schulz and Sick 2005) and the question of toxicity of the dye becomes an important issue. This even more, if the volumes needed are important and if recirculation cannot be used. Unfortunately all currently used tracer dyes as e.g. rhodamines, fluorescein, coumarins etc. are more or less toxic, carcinogen or even mutagen. This was the motivation for a systematic research of fluorescent species that are not nocuous for human health and the environment. A rather complete review of fluorescent substances can be found in (Guilbault 1973). Several classes of organic and inorganic compounds are described herein, also naturally occurring, nonharmfull species like vitamins or Chlorophyll. Their fluorescence is also described in (Gitelson et al. 1999, Maxwell and Johnson 2000) for Chlorophyll and in (Aaron and Winefordner 1972, Guilbault 1973, Lenci et al. 2005, Schulman et al. 1999) for vitamins. Absorption and emission spectra for chlorophyll in two different solvents are shown in figure 1. Data is from the database from the Oregon Medical Laser Center. Fig. 1 Absorption (left) and emission (right) spectra of chlorophyll In our study the following species have been considered further : Chlorophyll, vitamin C (ascorbic acid), vitamin B 1 (thiamin), vitamin B 2 (riboflavin), vitamin B 6 (pyridoxine) and vitamin B 9 (folic acid). Their fluorescence has been tested for excitation with doubled (532nm) and tripled (355nm) - 1 -
2 Nd:YAG laser light, since these are commonly used in flow studies. At 355nm the absorption spectra show rather good absorption, at 532nm absorption is rather low, but seemed to be worthwhile to be tested. 2. Experimental technique Chlorophyll has been purchased as Chlorophyll sodium salt from Carl Roth, Germany. The vitamins have been purchased from Acros and Aldrich chemicals as pure substances. All are dissolved in distilled water in different concentrations (given in the corresponding result sections) and filled into small transparent cuvettes (10mmx10mmx45mm). A frequency doubled or tripled Nd:YAG laser is used for excitation and the laser light sheet (thickness 0.5mm) is passed through the center of the cuvette (Fig. 2). Laser energy was always stabilized at 6,7 mj/p for both laser wavelengths, apart from the laser energy dependency studies. The fluorescence light from the cuvette is focalized through a 100mm UV-lens to the entrance slit of an Acton research 300mm spectrometer, with a 600 g/mm grading. The spectra are then recorded by a LaVision Nanostar ICCD-camera. Depending on the fluorescence intensity, one or several bursts are accumulated for one spectrum with a gate time of 20ns each. The mean of 50 frames is corrected for ambient light by a background image, taken with pure distilled water in the cuvette and the spectra are then obtained by summing up the intensity of the pixels in each vertical line of this corrected image. cuvette laser light sheet spectro- meter lens ICCD- camera Fig. 2 Experimental set-up for the parametric study The temperature dependency of fluorescence has been determined by heating up the solutions to about 50 C on a heating plate. Then the solution is filled in the cuvette, in which a K-type thermocouple is introduced and spectra are taken at different temperatures during cooling down of the solution to room temperature. The ph-dependency has been obtained by changing the ph of the original solution by the addition of HCl or NaOH. The ph is measured prior to each spectra recording, with a WTW 330i ph-meter, calibrated before each measurement series. Here, all spectra are represented normalized with the maximum intensity of each measurement set. 3. Results Parameter study for Chlorophyll : Excitation wavelength 355nm The concentration and laser energy dependencies of Chlorophyll fluorescence at excitation wavelength 355nm are represented in figure 3. Growing concentration and laser energy leads, as expected, to increasing fluorescence intensity (fig.3, upper row). The increase of maximum intensity (around 660nm) is, in the considered range, linear with laser energy (fig.3, lower right), but not with concentration (fig.3, lower left). Here, saturation effects could be observed for high concentrations and the curves can be approximated by a polynomial of order 2. The wavelength of the maximum is changing with concentration, as it can be seen on the upper, left figure
3 Fig.3 Fluorescence of Chlorophyll: excitation at 355nm, influence of concentration (left column) and laser energy (right column). The temperature dependency of Chlorophyll fluorescence exists, but is not very strong in the considered range (fig. 4, left). The maximum intensity was attained at 50 C and a linear increase can be observed (center). Also, the wavelength of the fluorescence maximum changes slightly, but linearly with temperature (right). Thus these influences have to be considered when working at constant temperature. For temperature measurements with chlorophyll as an indicator, the effects seem to be too small, at least in the considered range of temperatures. Fig.4 Fluorescence of Chlorophyll: excitation at 355nm, influence of temperature. Fluorescence intensity shows a big increase in the region between 6<pH<8 (Fig. 5, left and center). For smaller and higher values of the ph, the fluorescence intensity is rather constant. A kind of hysteresis has been encountered during the experiments, when starting from the natural ph of about 6.5 by decreasing to acid values and then increasing to the basic (center). This hysteresis - 3 -
4 17th International Symposium on Applications of Laser Techniques to Fluid Mechanics does not exist, when regarding the wavelength change of maximum fluorescence (right). This takes place essentially between 5<pH<7. For higher ph, the wavelength is constant. This means, that chlorophyll could be used for the determination of ph, but only in the range of 5<pH<7. Fig. 5 Fluorescence of Chlorophyll: excitation at 355nm, influence of ph. Parameter study for Chlorophyll : Excitation wavelength 532nm As expected, the fluorescence intensity of chlorophyll increases also with concentration and laser energy at an excitation wavelength of 532nm (Fig. 6). This increase is linear with all considered laser energies (bottom, center), but not with concentration (top, center). The wavelength of the maximum intensity changes with concentration but not with energy (Fig. 6, last column). Fig.6 Fluorescence of Chlorophyll: excitation at 532nm, influence of concentration (top row) and laser energy (bottom row). As for an excitation at 355nm, a slightly linear temperature dependency of fluorescence exists also at 532nm (Fig. 7). This dependency might also be not strong enough to use it for temperature measurements. -4-
5 Fig.7 Fluorescence of Chlorophyll: excitation at 532nm, influence of temperature. The ph dependency of intensity is rather unclear (Fig. 8). As for 355nm there exists a hysteresis, when changing the ph by decreasing from the starting value at about 6.5 and then increasing to basic values. The wavelength dependency of the fluorescence maximum is more suitable for phmeasurements, since it shows a clear increase between 4<pH<7.5 (Fig. 8, right). Fig. 8 Fluorescence of Chlorophyll: excitation at 532nm, influence of ph. Parameter study for Vitamins : Several water soluble vitamins have also been examined for their capability of replacing currently used tracer dyes in flow studies. The results of this study (Zähringer 2014) showed a special suitability of vitamins B 2 and B 6 for the use in fluid mechanical applications (Fig. 9). Their fluorescence intensity is comparable to that of currently used tracer dyes (Fig. 10). First applications of Vitamin B 2 in a static mixer showed the good applicability of this tracer (Fig. 11). Structures can be clearly recognized and e.g. postprocessed frequencies of the scalar field show the same results as obtained before with conventional dyes (Lehwald et al. 2012)
6 17th International Symposium on Applications of Laser Techniques to Fluid Mechanics Fig. 9 Fluorescence spectra of vitamins at excitation wavelength 355nm (left) and 532nm (right) Fig. 10 Comparison of fluorescence intensity at 532nm Fig. 11 Vitamin B (excitation at 532nm) as tracer in a static mixer at Re=562 : inlet to static mixer (left), outlet of mixer element (right) 2 4. Conclusions Naturally occurring dyes like Chlorophyll and vitamins are examined here for their suitability to replace currently used, often harmful tracer dyes for flow studies. The temperature and ph dependency of their fluorescence intensity and maximum wavelength are analyzed, as well as the concentration and laser energy influences. Chlorophyll is thus suitable for the use in isothermal flow analysis. The temperature dependency of fluorescence exists, but is rather weak. The change of wavelength due to ph, could be used for ph determination. First applications show the excellent possibilities of these very cheap and non-harmful dyes. Especially in big flow installations and if tracer is released to the surroundings (spray, windtunnel, etc.) they may show big advantages compared to common tracer dyes. Literature : Aaron J-J, Winefordner JD (1972) Fluorimetric and phosphorimetric characteristics of several vitamins. Talanta 19:21-29 Duwel I, Schorr J, Wolfrum J, Schulz C (2004) Laser-induced fluorescence of tracers dissolved in evaporating droplets. Appl Phys B-Lasers Opt 78: Gitelson AA, Buschmann C, Lichtenthaler HK (1999) The Chlorophyll Fluorescence Ratio F735/F700 as an Accurate Measure of the Chlorophyll Content in Plants. Remote Sensing of Environment 69: DOI Guilbault G (1973) Practical Fluorescence New York: Marcel Dekker, Inc
7 Hagemeier T, Hartmann M, Kühle M, Thévenin D, Zähringer K (2012) Experimental characterization of thin films, droplets and rivulets using LED fluorescence. Exp Fluids 52: DOI /s x Kling K, Mewes D (2004) Two-colour laser induced fluorescence for the quantification of microand macromixing in stirred vessels. Chem Eng Sci 59: Lehwald A, Janiga G, Thévenin D, Zähringer K (2012) Simultaneous investigation of macro- and micro-mixing in a static mixer. Chemical Engineering Science 79:8-18 DOI /j.ces Lenci F, Checcucci G, Sgarbossa A, Martin MM, Plaza P, Angelini N (2005) Fluorescent Biomolecules. In: Encyclopedia of Condensed Matter Physics. eds Bassani, F, Liedl, G and Wyder, P), pp , Oxford: Elsevier Maxwell K, Johnson G (2000) Chlorophyll fluorescence-a practical guide. Journal of Experimental Botany 51: Schulman SG, Di QQ, Juchum J (1999) Organic Chemistry Applications of Fluorescence Spectroscopy. In: Encyclopedia of Spectroscopy and Spectrometry. (ed Lindon, J), pp , Oxford: Elsevier Schulz C, Sick V (2005) Tracer-LIF diagnostics: quantitative measurement of fuel concentration, temperature and fuel/air ratio in practical combustion systems. Progress in Energy and Combustion Science 31:75 Zähringer K (2014) The use of vitamins as tracer dyes for laser-induced fluorescence in liquid flow applications. Exp Fluids 55:1712 DOI /s x - 7 -
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