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RECEIVEDfor review May 7,1990. Accepted September 13, 1990. This research was supported by the ICP Information Newsletter, Dow Chemical Michigan Division, and Department of Energy Contract DE-AC02-77EV04320.
Use of Solid Carbon Dioxide To Alleviate Fluorescence Quenching by Oxygen Sadao Matsuzawa,* Akihiro Wakisaka, and Mitsuhisa Tamura National Research Institute for Pollution and Resources, 16-3 Onogawa, Tsukuba, Ibaraki 305, J a p a n INTRODUCTION Fluorescence quenching by molecular oxygen is a serious problem for fluorometric analysis and the lifetime measurement of fluorescent compounds in many fields. Compound detectability is reduced and the lifetime shortened by quenching. Deoxygenation is the most effective means for eliminating quenching. Various methods, suitable for liquid samples, have been used for the deoxygenation and include nitrogen ( I , 2 ) or argon (3) purging, evacuation (4), freezepump-thaw cycles ( 5 ) , ultrasonification (6), chemical scavenging (7,8), and the use of permeable membranes (9-11). However, they require much time and are rather tedious. Here, we report a simple and efficient method for alleviating fluorescence quenching by oxygen, through the addition of small amounts of solid carbon dioxide (COz) to solutions of fluorescent compounds and by sealing off the solution vessels.
EXPERIMENTAL SECTION Instrumentation. Fluorescence intensity was measured on a Hitachi fluorescence spectrometer 650-60. The excitation source was a 150-W xenon arc lamp. Fluorescence lifetimes were measured by the time-correlated single-photon-counting method, wing a Horiba nanosecond time-resolved spectrometer NAES-1100. 10 The flash lamp was a Horiba NFL-111 (25 kV, 1MPa of Hz, kHz at maximum, full width at half-maximum flash profile was below 2 ns). The photomultipliers for start and stop pulses were IP28 and R212 (Hamamatsu), respectively. In both measure-
ments, a sample solution was taken into a cuvette cell (1X 1 X 4.5 mm) with a stopcock.
Reagents. Aromatic hydrocarbons were from various sources and used as received. n-Hexane, cyclohexane, and ethanol as solvents were all of liquid chromatographicgrade. Commercially available solid carbon dioxide (COJ was used to eliminate the quenching. Procedure. Small pieces (0.02-0.03 g each) of solid C02were successively added to a solution (3 mL) in a cuvette cell through a hole in the stopcock, which, after the solid C02had completely dissolved, was turned off to prevent redissolving of oxygen from the atmosphere. The slight dew that formed on the surface of the cuvette cell was removed by blowing with nitrogen. "he extent of fluorescence enhancement by adding solid COz to the solution was determined for naphthalene (1 X lo4 M) and pyrene (2 X 10+ M) in n-hexane and ethanol and compared with that by purging gaseous COz, nitrogen, and argon. There was greater enhancement with I x / I 0 , (10, 12), where Ix and Io2 are the fluorescence intensity with and without deoxygenation treatment, respectively. X stands for the material used for deoxygenation. This method was used to measure the fluorescence lifetimes of aromatic hydrocarbons, and the values obtained were compared with literature values (13).
RESULTS AND DISCUSSION Figure 1 shows enhancement of fluorescence intensity for naphthalene, 1 X lo4 M in n-hexane and ethanol, due to the addition of solid COPto 3 mL of solution at room temperature.
0003-2700/90/0362-2654$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO.
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Flgure 1. Enhancement of fluorescence intensity for naphthalene (1 X lo4 M) by adding solid CO, (abscissa) to 3 mL of solution. Solvent: (a) n-hexane; (b) ethanol. Accurate measurement was not possible due to sublimation. Fluorescence intensity was measured at
336 nm.
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Time
(min)
Table I. Fluorescence Lifetimes of Aromatic Hydrocarbons
Flgure 2. Fluorescence enhancement for naphthalene (1 X lo-' M) in n-hexane by adding solid CO, compared with that by gas purging. Gases used for purging: (1)carbon dioxide (CO,); (2) nitrogen; (3) argon. Flow rate of the gas was 20 mL/min. Enhancement by adding solid CO, is indicated by a horizontal line ( I x / I o 2 = 17.5).
This parameter gradually increased with the amount of solid COP,becoming constant above 0.2 g. The btal amount of solid COz added did not dissolve in the solvent; most of it was sublimed. On addition of 0.2 g of solid COz to 3 mL of nhexane, as much as 0.009 g dissolved, as determined by the infrared absorption method, while the value calculated by using the solubility (14) at 20 "C was about 0.011 g. In nhexane, as shown in Figure la, I ~ o , / I o ,(enhancement factor) a t an excess amount of COPattained a constant value of about 17.5, this being the same as that of the sample degassed by the most reliable freeze-pump-thaw cycles method. Thus, the present rapid method, in which about 0.1 g/mL of solid COPis added, can be used efficiently for deoxygenation. The effect of decreasing the temperature by the addition of solid COz on fluorescence yield is negligible since the temperature of the solution never decreases below 10 "C and the fluorescence intensity for naphthalene at 10 "C is virtually the same as that at room temperature. In ethanol, Ico,/Io, showed a constant value of about 5.5 (Figure l b ) , possibly due to the lower solubility (14) of oxygen in ethanol (one-tenth that in n-hexane). l ~ ~ , /for I ~pyrene , in n-hexane and ethanol were about 68.0 and 20.0, respectively. Fluorescence enhancement for naphthalene in n-hexane by adding solid COPis compared, in Figure 2, with that by purging with gaseous COP,nitrogen, and argon. Although purging with gaseous COP(curve 1 of Figure 2) enhanced fluorescence to the same extent as these inert gases, the enhancement is less than that (17.5, as indicated by a horizontal line) by adding solid COP. Differences in dissolution rates for solid and gaseous COz may be the reason for this; the dissolution rate of solid COPwas higher. That is, in the case of gaseous COP purging, the solution was still not saturated with COz. No
393 nm.
compd benzene naphthalene anthracene phenanthrene pyrene
in ethanol, ns in cyclohexane, ns lit. COz method lit. value" C02 method 26.5 107 5.3 55.3 425
29 96 4.9 57 450
14.9 93.8 5.3 52.7 370
31 105 5.3 61 475
"These values are cited from ref 13. bThe lifetimes measured in polar solvent such as alcohol and EPA.
change in the spectrum of naphthalene could be found by dissolving COz. It should be pointed out that the redissolving of oxygen for a solution containing COz is much slower than that containing argon or nitrogen. Figure 3 shows variation in the value of Ix/Io, for pyrene after opening the stopcock of the cuvette cell. The rate of decrease in the enhancement factor for a COz-treated sample was less than those for argonand nitrogen-treated samples, regardless of the solvent used [(a) n-hexane; (b) ethanol]. The effect of COzwas particularly remarkable in ethanol. These differences may arise from intermolecular force between the substrate and gaseous molecules and by interaction between the solvent and gaseous molecules. Long-lived species such as pyrene are generally susceptible to quenching by oxygen, causing a decrease in lifetime. The elimination of quenching is thus indispensable for measuring fluorescence lifetime. Table I shows this parameter for aromatic hydrocarbons in cyclohexane and ethanol, as measured by the present method (the COz method) for deoxygenation. This was done just after dissolution of the solid COP Lifetime values by the solid COz method generally agreed with literature values (13). The values for benzene and pyrene in ethanol were much less than the literature values. Difficulty in the replacement of small amounts of oxygen, trapped among the hydrogen-bonding network of ethanol, with COz may be the reason for this.
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
CONCLUSION The method proposed here is applicable to most cases of fluorometric analysis for greater detection sensitivity and accurate lifetime measurement for organic compounds soluble in nonpolar solvents. It should also find rapid and economical applications to improving the efficiency of photosensitizing reactions or laser oscillations whose underlying principle is fluorescence emission.
LITERATURE CITED (1) (2) (3) (4)
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RECEIVED for review June 18, 1990. Accepted September, 4, 1990.
