A Simple, Portable Fluorimeter with a Compact,
Inexpensive Nitrogen Laser Source Bradley 1.Jones, Benjamin W. Smith, Moi B. Leong, Michael A. Mignardi, and J a m e s D. Winefordner University of Florida, Gainesville, FL 3261 1 Although nitrogen lasers (and dye lasers pumped by them) have been &ed as excitation sources for~fluorimetry since a t least 1974 ( l ) ,they have not found widespread use in analvtical molecular fluorimetrv exceot in cases such as lowtemperature Shpol'skii spectrimetr< which requires a narrow excitation spectral bandwidth (2, 3) and in the excitation of eluants in high-performance liquid chromatography, HPLC ( 4 , 5 ) ,where the ability to focus the excitation beam onto a small volume of analyte is highly desirable. Limits of detection for Nz-laser-excited fluorescence detection of HPLC eluants have been quite good, generally in the 1-ng/ mL range (6). T h e applications of Nz-laser systems to what might be called "conventional room temperature fluorimetry" have been few (6-8)undoubtedly because of the relatively high cost and complexity of these laser sources. Nevertheless, the analytical figures of merit that have been ohtained are generally equal to or better than those attainable by nonlaser sources. Condensed phase detection limits have been obtained for a t least 40 compounds and have ranged from single molecule detection (9) by fluorescein tagging of polyethyleneimine to hundreds of ng/mL. Typical values are in the range 1-100 pg/mL. We have designed and evaluated a s i m ~ l NAaser-based e fluorimeter that is compact, relatively inexpensive, and portable. Moreover, i t exhibits analytical figures of merit comparable to much costlier and more com. plex systems.
excitation pulse and was used to provide a trigger for the gated integrator (Model SR 250, Stanford Research Systems, Stanford, CA). The photocurrent pulse from the PMT was stretched slightly by a 1000-0 load resistor and detected with the gated integrator. The resulting voltage pulse had a 500-ns full-width half maximum, FWHM, and was sampled with a 2W-ns gate. Radio frequency noise produced by the firing of the laser was reduced considerably by mounting the laser in a shielded housing provided by the manufacturer. The dimensions of the laser including integral power supply were about 25 X 11X 6 cm. The laser was powered either by a line voltage-operated 12-V dc supply or directly hy a 12-V dc lead-acid motorcycle battery. Battery operation had the advantage of reducing radio frequency noise that is normally transmitted to the detection electronics via the power lines. Solutions of seven compounds tested were prepared by weight in several different solvents. Analytical calibration curves were ob-
Experlmental An inexpensive (-$3000), compact commercial nitrogen laser (Model VSL-337, Laser Science, Inc., Cambridge, MA) with -100pJ pulse energy, 3-11s pulse length, and 20-Hz repetition frequency was mounted vertically in order to illuminate a standard 1-cm quartz fluorescence cuvette through its bottom. The beam was rectangular in shape, 0.3 cm X 0.8 em. Thenitrogenlaser is classified as a IIIb laser, meaning one should not look directly at the beam itself or adirect reflection of it. The resulting fluorescencewas allowed to fill the acceptance cane (f 3.5) of a compact monochromator (Instruments SA, Model H-10, Metuchen, NJ) with 4-nm spectral handpass and was detected with a photomultiplier tube, PMT (R1437, Hamamatsu Corporation, Middlesex, NJ) mounted on a commercial PMT hase. A 350-nm-high pass filter was used to minimize the detection of scattered Laser light. Figure 1indicates schematically the placement of the various components. A photodiode (FND-100, EG G, Salem, MA) was positioned to receive a portion of the
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' Research supported by NIH-GM-11373-23.
Author to whom reprint requests should be sent.
Figure 1. A diagram of the fluorimeter.
Volume 66
Number 4
.
April 1989
357
Analytical Figures of MerH Obtalned by Compacl N,-Laser FluorlrneteP Emissim
LOO
Sensitivily
Literahweb
Compound
Solvem
Maximum (nm)
InslmL)
(mV/ng mL-')
LOO (nglmL)
Flwramhene Quinine Riboflavlne Salicyli~add Proflavine Fiuore~cein Anthranlllc acid
&anal 0.1 N H2S04
430 450 520 415 510 520 405
3 0.3 2 9
0.65 3.9 0.13 0.02 0.20 1.86 1.31
1 2 0.2 10
H20
He0 H20
0.15 M NaOH H20
. L I m r dynamic range in all cases is Wee orders ol magnlMs a &Takenham ref 11.
2 0.2 0.3
-
0.02 1
greatw.
tained by triplicate determinations at each of seven dilutions rangingfrom near the detection limit to about three orders of magnitude hieher. The limit of detection for each analvte was calculated using t.-~. h i -nnalvtieal curve slooe (estimated bv a inear reeression fit)and ~-* the signal equivalent & three times tee standard ;deviation of the limiting blank noise measured with a 1-s time constant (a noise FWHM bandwidth of 4 . 2 5 Hz) ~~~
~
~
Results and Discussion The table summarizes the analytical figures of merit ohtained for the seven compounds. Limits of detection were generally similar to those found in the literature and obtained on conventional fluorimeters. When the present results are worse, i t is usually attributable t o the peak of the absorption spectrum being far from 337 nm. When the absorption maximum is near 337 nm (as is the case for quinine, salicvlic acid. and antbranilic acid). then the limits of detectionior the present study are superior to conventional (nonlaser) fluorimeters. Even in cases where the laser wavelength is far from the peak of the absorption spectrum, the present limits of detection are only about 10X worse than the literature values. The slopes of the analytical curves are given as sensitivities in millivolts of sinnal (referred t o the boxcar input) per unit of concentration (ng mL-'). The double logarithmic analytical curves for the seven compounds are shown in Figure 2. They are linear (log-log slope = 1.00 f 0.03) in all cases for a t least three orders of maenitude. Some curves in Firmre 2 have been arbitrarily shiked along the relative signaiintensity axis to avoid overlao. The relative standard deviation for concentrations well above the detection limit was never worse than lo%, and usually 2-5%. Concluslons We have demonstrated that excellent fluorimetric analytical results can he obtained from a simple, portable, laserexcited fluorimeter. Although excitation a t 337 nm is less than optimal for many compounds, the detection limits that were achieved were still quite remarkable. Acknowledgment The authors would like to thank Laser Science, Inc., Cambridge, MA, for the loan of Nz laser.
358
Journal of Chemical Education
0
1
2
3
4
Log Concentration (ng/rnL)
Figure 2. Calibrallon curvesforlhecompwnds examined. From top to bonom: proflavlne, tluwescein,anlhranilicacid, quinine, fluaanlhene,riboflavine,and sslicycllc add. Literature Cned 1. Smith, B. W.: Plankey. F.W.; Omenctto, N.: Hsrt,L. P.; Winefordner. J. D. Swtraehim. Acto 1974.30A.1459-1M. 2. Maple, J. R; Wehry, E. L.; Mamantw, G. Anal. Chem. 1980.52,9E-924. 3. Wehry. E. L.: Mamantoto, G. And. Chem. 1979.51, MA-6MA. 1. Diebold.G.J.;Kamy,N.;Zarr,R.N.;ki&.L.M. J.Aaaoc. 0ff.Aml.Chem.3579.62. -568. 5. Voig~aan,E:Winefordoer,J. D. Talonto 1985,30,7W. 6. Voiglman, E.; Winefordncr. J. D. J. Liq. Chrom. 1985,5(11), 2113-2122. 7. omenetto. N.:Winefordner. J. D. CRCCnticolRou.Anal. Chem. 1981.13.59-115. s. w e h i E.'L.A ~ them. I . I"o.~~,~~RsoR. 9. winefordaer. J. D. Neu A ~ ~ l i e a t i aofwLasera to Chemistry: Hieftje. G. M. Ed.: Amen- C h e m i d s&&: Washingtongto,DC, 1978. lo. Hirsehfeld. T. Appl. Opt. 1976,15.3135-3139. 11. Helman, D. L. Fluamseema ondPhosphoresceme Doto Comp~ndwm;SLWAminca: Urbana. lL.1975.
