Detection Limit of Fluorescein as Determined by Fluorometry with an Esculin Laser Source The fluorescein was recrystallizedfrom water. The water was distilled, deionized, and redistilled. The sodium hydroxide (guaranteed grade) was used for adjusting the pH of the solution without further purification.
Sir: Fluorometric analysis provides a useful method for the determination of the ppb level of the fluorescing molecule. In principle, the sensitivity can be raised by using a more intense exciting source such as a laser, since the fluorescence intensity is proportional to the brightness of the source. The detection limits of the fluorometric analyses with a laser source and a monochromator reported previously (1-4), however, are in the order of 30-100 ppt (ppt = and are approximately identical to that of the conventional fluorescence spectrophotometer; thus, the merit of the laser has not been fully appreciated. The nitrogen laser pumped dye laser is especially suitable for an excitation source (5),since its rapid repetition rate allows it to be regarded as a quasi-continuous source, and since the wavelength is tunable. In this study, the detection limit of fluorescein, excited by a nitrogen laser pumped esculin laser, was investigated a t concentrations much below those of previous work.
RESULTS AND DISCUSSION The fluorescence spectra of fluorescein (10-lo M, 5 X M, pH 13) excited by an esculin laser are shown in Figure 1. The fluorescence maximum was observed a t 514 nm. The band at 554 nm is assigned to the Raman band of the solvent water. The wavelength of the broadband laser emission of the esculin laser is preferable for the fluorescence measurement of fluorescein, since the Raman band does not superimpose on the fluorescence band maximum. The band undulates owing to the fluctuation of the intensity of the exciting laser and to that of the dark current of the photomultiplier. The detection limit, which is defined as a 2:l ratio of the fluorescence signal-to-background noise level with a solution blank, is determined to be 5 X M (2 ppt) from Figure 1. The precision of this experiment was investigated at the concentration of 6 X M. Three successive traces of the spectra are shown in Figure 2, and the relative standard deviation was determined to be 2%. A linear analytical curve for fluorescein was observed from to 5 X M for the esculin laser excitation. An upper limit to the linearity occurs a t 1.8 X M ( A = 0.02, E = 1.1 X lo4 at 470 nm) where self-quenching and reabsorption of the luminescence causes nonlinearities. The fluorescence intensity a t the concentration of 8 X M is nearly equal to that of the Raman band of water, and this relation can be used as an internal standard of the analytical curve for the trace fluorometric analysis.
EXPERIMENTAL The experimental apparatus consists of a dye laser of esculin pumped by a nitrogen laser, and a fluorescence detector. The esculin laser is used in the broadband oscillation; the wavelength and the bandwidth of the laser emission are 470 nm and 10 nm. The fluorescence was measured with a Jasco CT-100 monochromator and an EM1 9558QB photomultiplier. The signal was amplified by a Teledyne Philbrick 1029 OP amplifier with a time constant of 4.7 s. The detection limit of the present apparatus was compared with that of a conventional Hitachi MPF-4 fluorescence spectrophotometer (a xenon lamp was used for the excitation).
I
550
500
nm
600 a
Flgure 1. Fluorescence spectra of fluorescein
n m 600
550
500
Figure 2. Three successive traces of the fluorescence spectra of fluorescein at the concentration of 6 X M
M solution of fluorescein. (2) 5 X IO-’* M solution of fluorescein. (3)
(1) Solution blank
lo-’’
Table I. Detection Limits of Fluorescein Excited by the Tunable Laser, He-Cd Laser, and Xenon Lamp Detection limita Exciting source Esculin laser Xenon lampc Dye laser Dye laser N2 laser Dye laser He-Cd laser
Xem
Ahem
Xem
Axe,,
nm
nm 10
nm
nm
470 470 480 470 337 469 442
10 0.4 0.1
... 1 ...
514 514 514 514 514 522 511
1.6 1.6
... ... ... 4
...
Estimated, PPt
0bserved, PPt
2 70
2 70
b b (1)
lOOd
40d 30 70 700
Ref.
700 1700
(2) (2) (3) (4)
a The concentraton of 2 X 10-1O M fluorescein solution corresponds to 70 ppt, since the molecular weight of the fluorescein ion (fluorescingspecies) is used for the conversion. Present study. Hitachi MPF-4. Whether this is the estimated value or the observed value is not indicated.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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The detection limits of fluorescein determined by the various exciting sources so far reported are shown in Table I. The sensitivity of the fluorometric analysis excited by the esculin laser in this work exceeds those in the previous works and that by a conventional spectrophotometer by more than an order of magnitude. Thus the dye laser was proved to be useful as an exciting source for the trace fluorometric analysis. At the limit of detection, the major source of noise of the conventional spectrophotometer is the dark current of the photomultiplier and that of the previous apparatus with the laser source was concluded to be noise on the background signal, caused by fluorescence of impurities in the solvent (2). In the present investigation, the discharge circuit and the electronic devices for the photodetection were shielded carefully by metal enclosures; the background fluorescence of solvent impurities was reduced by repeated purifications of the solvents; the optical system was strictly adjusted to reduce the scattering light of the excitation laser. Thus, the background signal was reduced to allow the observation of such weak fluorescence as 2 ppt of fluorescein. Either a stray light of the monochromator, or the fluorescence of the residual impurities in the distilled water may contribute to the background signal from the solvent water. The detection limit of fluorometry can be improved by using the interference filter instead of the emission mono-
chromator. For example, the sub-part per trillion detection of Rhodamine 6G was reported (6).However, the Raman band cannot be used as the internal standard, and the fluorescence from the unwanted species cannot be resolved on the fluorescence spectrum. Thus, the use of a monochromator, as reported here, has definite advantages over such a simple method. Totaro Imasaka Hidekazu Kadone Teiichiro Ogawa Nobuhiko Ishibashi* Faculty of Engineering 36 Kyushu University Fukuoka 812, Japan LITERATURE CITED (1) B. W. Smith, F. W. Plankey, N. Omenetto, L. P.Hart, and J. D.Winefordner, Spectrochim. Acta, Part A, 30, 1459 (1974). (2) T. F. V. Gel1 and J. D. Winefordner, Anal. Cbem., 48, 335 (1976). (3) D. C. Harrington and H. V. Malmstadt, Anal. Cbem., 47, 271 (1975). (4) M. F. Bryant, K. O'Keefe, and H. V. Malmstadt, Anal. Chem., 47, 2324 (1975). (5) T. Imasaka, T. Ogawa, and N. Ishibashi, Bunseki Kagaku, 26, 96 (1977). (6) A. E. Bradley and R. N. Zare, J. Am. Cbem. SOC.,98, 620 (1976).
RECEIVEDfor review November 10,1976. Accepted January 3. 1977.
Composition Differences in Commercial Polyethylene Bottles and Their Relation to the Stability of Stored Part-per-Billion Mercury(l1) Solutions Sir: During a study of the suitability of linear polyethylene bottles for storage of trace Hg(I1) solutions, we have found several lots of commercial bottles which are a mixture of 45% polypropylene and 55%linear polyethylene, and which differ significantly from 100% linear polyethylene bottles with respect to the types and levels of additives. The losses of Hg(I1) incurred when 1ppb Hg(I1) solutions are stored, and the interaction of the container with the preservative solution are quite different in 100%linear polyethylene bottles and in 45% polypropylene bottles. Such differences in the stability of trace level Hg(I1) samples stored in the two types of bottles constitute a potentially serious source of variance in Hg(I1) analysis of samples stored for an appreciable time as is often necessary in environmental studies that involve large numbers of samples. Because polyethylene bottles of the two compositions may well exert differential effects on other samples, we wish to alert others to the possibility of unexpected variations in losses when trace samples are stored in commercial linear polyethylene bottles. Differences in the efficiency of preservatives in stabilizing 1 ppb Hg(I1) solutions stored under apparently identical conditions in bottles from different manufacturers' lots first suggested that, although the bottles within each lot are relatively uniform, the various lots represent two distinct types of bottles, which we have denoted LPE I and LPE 11. In their behavior toward 1ppb Hg(II), solutions stabilized with 5% v/v HN03-0.05% w/v K2Cr207, as recommended by Feldman (I), the two types of bottles differ in two important respects. First, losses of Hg(I1) after 10 days storage, as monitored by a cold vapor atomic absorption technique similar to that of Kopp et al. ( 2 )were 43%in the LPE I bottles but only 6% in the LPE I1 bottles. Second, the dichromate preservative is often reduced in the LPE I1 bottles. The degree of reduction varies substantially from one bottle to another, and occasionallythe 668
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
dichromate suffers complete reduction after several days. The bottles have a capacity of 500 mL, so that complete reduction represents the loss of 0.85 mmol of dichromate. In contrast, little if any of the dichromate is reduced in LPE I bottles, even after storage for a month or longer. The infrared spectra of thin films of the LPE I plastic and the LPE I1 plastic in Figures 1 and 2, respectively, clearly demonstrate that the LPE I plastic is linear polyethylene and that the LPE I1 plastic contains substantial quantities of polypropylene. The spectrum of LPE I shows, in addition to the C-H bands of aliphatic hydrocarbons, bands at 907 and 989 cm-l characteristic of terminal unsaturation, and an olefinic C=C stretching band at 1638 cm-l is detectable if much thicker films are used. The 907 and 989 cm-l bands indicate that LPE I is a low pressure, linear polyethylene, and the spectrum corresponds closely to representative spectra of linear polyethylenes given by Hummel and Scholl(3). The spectrum of LPE I1 exhibits a number of bands characteristic of crystalline isotactic polypropylene ( 4 ) , the more prominent of which are the six moderately intense bands at 809,840,898, 970,998, and 1165 cm-l, and an intense methyl deformation band at 1375 cm-l. A strong doublet with maxima at 718 and 728 cm-l, characteristic of crystalline polyethylene ( 5 ) appears in the spectra of both LPE I and LPE 11. The polypropylene content of the LPE I1 polymer as estimated using four methods based on the intensities of appropriate infrared bands, is approximately 45% polypropylene by weight. The first method, based on the relative intensities of the 718 cm-l polyethylene band and the 970 cm-l polypropylene band (6),yields a composition of 45% polypropylene. The second method, using the relative intensities of the 718 cm-1 polyethylene band and the 1165 cm-l polypropylene band (7) also indicates that the polypropylenecontent of LPE I1 is 45%. The third method utilizes the film thickness and the
