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ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
(34) Stieg, (35) Seitz, 957.
S.;Nieman, T.
w. R.;
A. Anal. Chem., submitted for publication. Suydam. w. w.; Hercules, D. M. Anal. Chem. 1972, 4 4 ,
RECEIVED for review May 5 , 1979. Accepted July 31, 1979.
This research was supported by the donors of the Petroleum Research Fund, administered by the American Chemical Society; by Research Corporation; and by the National Science Foundation (CHE 78-01614).
Laser Fluorometry of Fluorescein and Riboflavin Nobuhiko Ishibashi, * Teiichiro Ogawa, Totaro Imasaka, and Mikio Kunitake Faculty of Engineering, Kyushu University, Fukuoka 8 72,Japan
The detection limits of fluorescein and riboflavin are determlned to be 0.02 and 0.6 parts-per-trillion, respectively, with the use of a very sensitive fluorometric system ( a nitrogen-iaserpumped dye laser and a pulse-gated photon counter). The dependences of the S I N ratio and the detection limit of the excitation and emission Wavelengths are calculated and expressed in the figures for convenient determination of the optimal condition for ultratrace analysis. The results show that the Stokes shift of the sample molecule as well as the molecule’s absorptivity, fluorescence quantum yield, and fluorescence bandwidth influence the detection limit significantly. The present system incorporates an emission monochromator, and this is especlally useful for the analysis of a molecule with a small Stokes shift.
Ultratrace analysis of fluorescing molecules has made great progress by the use of laser sources for excitation. The unique properties of the nitrogen-laser-pumped dye laser can give rise to substantial gains in improving detection limits ( I , 2). The previously reported detection limits of fluorescein with a laser source and a monochromator (3-5) were in the order of 3&100 ppt (ppt = and are approximately identical to that of a conventional fluorescence spectrophotometer. Recently, however, the fluorescein detection limit, as determined by laser fluorometry, has been reported to be 2 ppt (I). The detection limit of a very sensitive fluorometric system is not always determined by the sensitivity of the instrument. The detection of fluorescein a t the concentration of 1 X M would be possible with an instrument which is capable of detecting the molecular fluorescence a t A+ = 5 x lo-” ( A , absorbance; 4, quantum yield) (6). However, residual fluorescence from the solvent impurities, which cannot be removed by any practical purification methods, interferes with the determination of such weak molecular fluorescence. The signal-to-noise ( S I N ) ratio is defined as the ratio of the fluorescence intensity of the sample molecule to the fluctuation of the background signal. The excitation wavelength has a great influence on the fluorescence intensity and the profile of the background signal. However, the excitation wavelength has no influence on the profile of the fluorescence band. I t is useful to know the effect of the excitation and emission wavelengths on the minimum detectable concentration and to choose the optimal wavelengths for detection with the highest S I N ratio. In this paper an application of the very sensitive laser fluorometric system (6) for the ultratrace analysis of fluorescein and riboflavin is described. The optimal experimental 0003-2700/79/035 1-2096$01.OO/O
condition for the fluorometry is discussed in terms of the analysis of the S I N ratio.
EXPERIMENTAL The spectroscopic apparatus consists of a dye laser and a pulse-gated photon counter, and has been described in detail elsewhere (6). The fluorescence cell is cylindrical, 6 cm in height and 4 cm in diameter. Fluorescence was observed with a double monochromator (JASCO CT-40D) equipped with an HTV R928 photomultiplier. The photoelectron signal was gated and counted. The gatewidth was adjusted to 0-200 ns. The integration time of the photoelectron signal was 50 s for each point in the spectrum, and four runs measured under identical conditions were accumulated. In the measurement of an analytical curve, the intensity of the fluorescence signal of the sample and that of the Raman signal of water were measured 10 times (50 s X IO), respectively, and the ratio of fluorescence signal to the Raman signal was plotted. This ratioing is useful to improve reproducibility when the sample cell is exchanged. The drift of signai is negligibly small during ratioing (6). The fluorescein (Wako, chemically pure grade), and riboflavin (Tokyo Kasei, guaranteed grade) were recrystallized from water. The water was deionized, passed through activated charcoal, and filtered (MF-Millipore, GS). Further distillation did not reduce the background signal in the solution blank. The glassware was thoroughly washed with soap, NaOH, and a chromic acid mixture by using an ultrasonic cleaner and then rinsed with copious amounts of water. The pH of the sample solution of fluorescein was adjusted to 13 with NaOH, and that of riboflavin to 6-7 with acetic acid and sodium acetate. ANALYSIS The proper selection of excitation and emission wavelengths is important for ultrasensitive fluorometry. Calculation and visualization of the dependences of the S I N ratio and of the detection limit on wavelength will be useful for this purpose. In the presence of a background signal, nb ( n ,number of photoelectrons), the actual fluorescence, nf, can be obtained by subtracting nb from the signal of the sample, n,. nf = n, - nb
(1)
The background signal consists of the scattered light of the source radiation, source-induced background, and sourceindependent background. In the present study the sourceindependent backgrounds such as dark counts of the photomultiplier and electrical noise from the nitrogen laser were negligible. Then the S I N ratio is expressed as (7)
SIN = nf/dn,
+ nb
Since the SIN ratio depends on the number of photoelectrons 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Fluorescence spectrum of fluorescein (pH 13, 5 X lo-'' M) (1) and background spectrum of solvent water (2). Excitation source: esculin dye laser (Ae., = 480 nm, LAex= 0.3 nm)
Wavelength( n m)
Figure 1.
coming from the sample fluorescence and the background photoemission, the S I N ratio varies as a function of the excitation and fluorescence wavelengths. The detection limit (DL) of the fluorometry is defined with respect to the concentration a t which S I N = 2. From Equation 2,
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Flgure 2. Fluorescence spectrum of riboflavin (pH 6-7), 1 X lo-' M) (A) and background spectrum of solvent water (B). The Raman band of water is located at 431 nm (not shown). Excitation source: PBD dye laser (Ae., = 377 nm, LAex = 0.4 nm)
I
1
where nf(DL) is the number of photoelectrons of the actual fluorescence a t the detection limit. Thus, the concentration a t the detection limit, c(DL), is expressed as follows; 2(1
c(DL) = k
+ dl + 2 4 )
1st (A,).($
(hem)
(4)
where t is the absorptivity, C$ the quantum yield, I the intensity of the exciting laser, and k a proportionality constant. The S I N ratio and the detection limit can be plotted as a function of the fluorescence wavelength a t a specified excitation wavelength, and this figure is useful for the selection of the optimal experimental condition for fluorometry.
RESULTS Fluorescence Spectrum. The fluorescence spectrum of fluorescein (5 X lo-" M, pH 13) excited by an esculin dye laser is shown in Figure 1,together with the background spectrum from the solution blank; these spectra have been improved in comparison with the previous result (1)by using the double monochromator and the pulse-gated photon counter. The fluorescence band of fluorescein appears at 514 nm; the Raman bands of water appear at 521 and 570 nm. The fluorescence peak height of the 5 x lo-" M sample is nearly equal to that of the Raman band of water. The absorption maximum of fluorescein is located at 491 nm, and its Stokes shift is about 910 cm-' (23 nm), which is considerably smaller than typical organic molecules. The fluorescence bandwidth is 35 nm. The fluorescence spectrum of riboflavin and the background spectrum excited at 377 nm are shown in Figure 2. Riboflavin has maxima at 266,377, and 445 nm, in the absorption and excitation spectra (8)and has a fluorescence maximum at 526 nm. The separations of the absorption and fluorescence
Wavelength (nm) Figure 3. SINratio of fluorescein (pH 13, 5 X lo-'' M). Excitation source: (A) esculin dye laser (480 nm), (B) C M U dye laser (450 nm), (C) Al-Calcein Blue chelate laser (420 nm)
maxima are 19000 cm-' (260 nm), 7500 cm-' (149 nm), and 3500 cm-' (81 nm), respectively, and the last one coincides with the Raman shift of water. The bandwidth of the fluorescence is 85 nm. Wavelength Dependences of S/ NRatio and Detection Limit. The S I N ratios of fluorescein excited at 420,450, and 480 nm were calculated from Equation 2 and are shown in Figure 3. It can be seen that excitation at 480 nm gives the highest S I N ratio. The most preferable analytical condition thus exists when the excitation is a t this wavelength and the observation of the emission is at 510 nm. The decrease of the S I N ratio at 570 nm is due to the presence of the Raman band of water. In the longer wavelength region, the fluorescence is weak, and the S I N ratio decreases. When the excitation wavelength was adjusted to 420 nm, the background signal of the impurity and of the Raman band of water decreased. However, the larger decrease of the fluorescence intensity of fluorescein reduced the S I N ratio. The detection limit of fluorescein calculated from Equation 4 (Figure 4) shows that the most preferable observation wavelength for the ultratrace analysis is in the 510-530 nm
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
h
I
.‘.’.. ,.
’
500
.,..:.....:.....::.:...-;’ 550 6oc
Wavelength ( nm 1 Wavelength (nm) Flgure 4. Detection limit of fluorescein (pH 13). Excitation source: esculin dye laser (480 nm)
Wavelength ( nm 1 Figure 5. S/Nratio of riboflavin (pH 6-7, 1 X lo-’ M). Excitation source: (A) PBD dye laser (377 nm), (B) 4-MU dye laser (450 nm)
region, and the minima of the detection limit appears on both sides of the weak Raman band of water. The steep increase a t 570 nm is due to the strongest Raman band of water, and the increase a t above 620 nm is due to the weak fluorescence. The excitation of riboflavin a t 377 nm gives a higher S I N ratio than that a t 450 nm, as shown in Figure 5. This is due to the smaller background signal when excited a t the shorter wavelength. Furthermore, the overlapping of the fluorescence and the Raman bands reduced the S I N ratio when excitation occurred at 450 nm. The detection limit of riboflavin is shown in Figure 6. The minimum detectable concentration can be measured with emission a t 526 nm. A wide region (500-600 nm) is open for ultratrace analysis, since there is a concomitant decrease of the background signal with the decrease of the fluorescence intensity in this region. Analytical Curve and Detection Limits. The dependence of the fluorescence intensity of fluorescein on concentration is measured. The analytical curve of fluorescein
Flgure 6. Detection limit of ribofhvin (pH 6-7). Excitation source: PBD dye laser (377 nm)
is straight. The observed detection limits of fluorescein and riboflavin are 5 x M (0.02 ppt) and 1.5 X lo-’’ M (0.6 ppt), respectively.
DISCUSSION Factors Affecting the Detection Limit. In ultratrace analysis, spectroscopic parameters such as the absorptivity, the fluorescence quantum yield, and the half-width of the fluorescence band have a great influence on the detection limit. Furthermore, the analysis of the S I N ratio indicates that the Stokes shift of the fluorescing molecule can profoundly influence the detection limit, since it depends on the overlapping between the fluorescence of the sample and the Raman band of the solvent (see Figures 4 and 6). Fluorescent molecules can be categorized into three groups according to the relative magnitude of the Stokes shift (AS) of the molecule and the Raman shift (AR) of the water. ( a ) As < XR (e.g.,Fluorescein). The excitation and emission wavelengths should be carefully adjusted, since the highest S I N ratio, hence, the lowest detection limit, can be obtained in the limited wavelength region (Figures 3 and 4). In this case the double monochromator is especially useful to reduce the scattered light of the laser. ( b ) As N XR ( e . g . ,450-nm Excitation of Riboflauin). The bandwidth of the fluorescence of many organic molecules in the condensed phase is larger than that of the Raman band of water, and there two peaks appear in the S I N ratio as shown in Figure 5 . ( c ) hs > hR (e.g.,377-nm Excitation of Riboflauin). A wide spectral region is available for utlratrace analysis (Figures 5 and 6), since the fluorescence maximum is located a t longer wavelength than the Raman band of water, and since the background signal decreases with increasing separation of the excitation and observing wavelengths. Since it is important to select the optimal condition for ultratrace analysis, the illustrations of the S I N ratio and the detection limit as shown before will be very useful, especially for cases a and b. Comparison of an Emission Monochromator with a Filter. As shown above, the monochromator is very useful for ultratrace analysis in case a, since the emission wavelength can be adjusted to the most preferable position. An optical
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1 9 7 9
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Table I. Detection Limits of Fluorescein exciting source dye laser dye laser
Xe lamp
h e x , nm
Ahex,
480 470 470 480 470 337 469
dc amplifier
boxcar integrator boxcar integrator boxcar integrator charge-to-count data converter
dye laser dye laser N , laser dye laser a
detection apparatus pulse-gated photon counter averager
nm
hem, nm A h e m , nm
0.3 10 10
510
0.5
514
1.6 1.6
514 514 514 514 522
0.4
0.1
