D. E. Smith and W.H. Reinmuth. Anal. Chem., 33 482 (1961). D. E. Smith, Anal. Chem.. 35 1811 (1963). E. R. Brown, T. G. W o r d . D. E. Smith, and D. D. DeFord. AnalChem., 38 1119(1966). D. E. Smith, Crit. Rev. Anal. Chem., 2 247 (1971). D. E. Smith, Appl. Comput.Anal.Chem., 2 369 (1972). D. E. Smith, “Electroanalytical Chemistry”, A. J. Bard., Ed., Marcel Dekker, New York, 1966, Vol. 1, Chap. 1. J. Paynter and W. H. Reinmuth. Anal. Chem.. 34 1335 (1962). G. C. Barker and I. L. Jenkins, Analyst (London), 77 685 (1952). H. Schmldt and M. von Stackelberg, “Modern Polarographic Methods”, Academic Press. New York/London 1963. G. C. Barker and R. L. Faircloth, “Advances in Polarography”, I. S. Longmuir. Ed., Pergamon, Oxford, 1960. p 313. J. H. Sluyters, J. S.M. C. Breukel. and M. Sluytersdehbach,J. Electroanal. Chem.. 31, 201 (1971). G. C. Barker, J. Electroanal. Chem., 41 95 (1973). J. H. Sluyters and M. Sluyters-Rehbach, J. Electroanal. Chem., 34, 542 (1972). S. C. Creason and D. E. Smith, J. Electroanal. Chem., 36, App. 1 (1972). S. C. Creason and D. E. Smith, J. Elecrroanal. Chem., 40, App. 1 (1972). S.C. Creason, J. W. Hayes, and D. E. Smith, J. Electroanal. Chem., 47, 9 (1973). PAR Instruction Manual for Two F’haselVector Lock-in Amplifier Model 129A, 1973. PrincetonApplied Research Corp., Princeton, N.J. H. Blutstein, A. M. Bond, and A. Norris, Anal. Chem., 46 1754 (1974).
clude phase selective f , 2f, and 3f ac polarograms with a triangular wave superimposed onto the dc potential. White noise can be regarded as the summation of sine waves a t all frequencies. The ability of the lock-in amplifier to selectively detect the f , 2f, 3f, and 4f modes of white noise and pseudo-random white noise was demonstrated. However, because white noise consists of frequencies with random phase, data processing to extract meaningful ac polarograms is extremely complex, requiring access to a minicomputer or other facility for performing extensive averaging (21-23) and no work other than demonstrating the feasibility of the lock-in amplifier method of measurement was undertaken.
ACKNOWLEDGMENT Discussions with R. L. Gunther during the course of this work are gratefully acknowledged.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7)
R. De Levie and A. A. Husovsky, J. Electroanal. Chem., 20 181 (1969). R. F. E v i l l and A. J. Diefenderfer, Anal. Chem., 39 1885 (1967). J. B. Flato, Anal. Chem., 44 (11) 75A (1972). D. L. McAllister and G. Dryhurst, Anal. Chim. Acta, 58 273,(1973). H. H. Bauer and D. Britz, Chem. Instrum., 2 361 (1970). R. Neeb, 2.Anal. Chem., 188 401 (1962). H. H. Bauer and P. J. Eking. Anal. Chem., 30 341 (1958).
RECEIVEDfor review November 4,1974. Resubmitted May 12, 1975. Accepted August 8, 1975. Financial assistance from the Australian Research Committee is gratefully acknowledged.
Front-Face Laser Fluorescence Technique with Micro-Absorption Cells M. F. Bryant, K. O’Keefe, and H. V. Malmstadt’ School of Chemical Sciences, University of Illinois, Urbana, Ill. 6 180 I
The 2-mm diameter by 1- or 2-cm pathlength spectrophotometric micro-absorption cells (about 30- to 6O-bl volume) which are frequently incorporated in stopped-flow (1-4) or continuous-flow instruments (5, 6) can also be used for quantitative fluorescence measurements by utilizing a front-surface technique. A new mirror and filter arrangement has been designed and developed f0r.a stoppedflow absorption cell module so that no modification of the microcell is required. Special stopped-flow fluorescence attachments have been built using the right-angle technique (3, 4 ) , but the technique reported here allows the simpler and more standard absorption cell module to be used for fluorescence methods. The general use of front-surface illumination with more conventional cells has been discussed by others (7-IO), and it has been shown that the front-surface technique introduces fewer problems from the inner-filter effect ( 7 ) . With the new design, the tightly focused radiation from a cadmium laser is directed onto the small front window of the 2-mm diameter cell, and the fluorescence radiation back through the front window is isolated and measured. The special design considerations for incorporating frontsurface radiation with the stopped-flow cell arise not only because of the very small front window, but primarily because of the rather thick, high pressure windows which are generally seated deep within pressure seals. Therefore, the angle for directing radiation onto the front surface is very limited and complicates the measurement of fluorescent radiation. The specific design for overcoming these difficulties is presented in detail. Send reprint requests to this author. 2324
The new technique was evaluated with fluorescein solutions and was shown to provide a linear analytical curve over more than a 2000-fold concentration range. The detection limit for the system using fluorescein solutions is 0.8 ppb (2.1 X 10-9M) which is quite similar to detection limits reported by others (11, 12).
EXPERIMENTAL Instrumental Design. The arrangement of the components used in the microcell fluorometric system is shown in Figure 1. A 10-15 mW continuous laser (A = 441.6 nm) is used for the excitation source (Model ML-442 Helium Cadmium Laser, Metrologic Instruments, Inc., Bellmawr, N.J.). Filter 1 is a 3-cavity interference filter with a peak transmittance a t 442 nm, and is used to remove background laser emission (Ditric Optic, Inc., Marlboro, Mass.). A manual shutter isolates the sample from the laser excitation source except during the measurement time. A glass focusing lens with a focal length of 12.5 nm is mounted in an x-y-z positioner and used to focus the laser beam onto the folding mirror. The folding mirror is a quartz optical flat (20-mm diameter, 1 mm thick) with a 1-mm diameter aluminum mirror evaporated onto the center and is used to reflect excitation light into the microcell. The sampling and mixing system is similar to the stopped-flow module recently used in an automated reaction rate instrument (I), and includes two identical mechanically driven syringes, a multiport mixer, a 2-cm pathlength X 2-mm diameter optical cell, a thermistor in the solution stream, and thermostated water jacket around the sryinges and optical cell. Filter 2 is a dielectric-coated filter used to reflect 99.7%of the laser excitation radiation (Valpey Corporation, Hollinston, Mass.). The transmittance is 61% a t the 511-nm analysis wavelength. Wavelength isolation of the fluorescence is accomplished with the GCA/McPherson, Model EU-700 Scanning Monochromator (Acton, Mass.). The RCA 1P28 photomultiplier tube used for measurement of fluorescence radiation is housed in the GCA/McPherson Model EU-701-30 Photomultiplier Modle. A Variable Speed Logbinear Chart Recorder was used for recording the data (EU-201 V, Heath Co., Benton Harbor, Mich.).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
F O L D I N G MIRROR
I l-€6M'CRoCELL 1
MICROCELL
- - ?
SHUTTER
3
FII
MONOCHROMATOR
Figure 1.
'W
Block diagram of fluorescence system
I
I QUARTZ
Reagents. Disodium fluorescein (Eastman Kodak) was used to prepare standard solutions in 0.05M disodium phosphate (AR Grade). Procedure. All solutions were automatically introduced into the cell by the stopped-flow system. After delivery, the samples were irradiated for approximately 2 minutes while the fluorescence was measured. The temperature for the measurements was 23.5 f 0.5
Figure
RESULTS AND DISCUSSION The linearity and detection limits of the new front-surface design for microcells were determined using the wellcharacterized fluorescence from sodium fluorescein standard solutions (12). The excitation maximum for sodium fluorescein solutions is at 490 nm but, since the excitation spectrum is broad, the cadmium laser wavelength of 441.6 nm is suitable. The fluorescence maximum was observed a t 511 nm with Filter 2 and the monochromator in the optical path as compared to 520 nm maximum reported by other workers (12),because of the band structure of Filter 2. The analytical curve for sodium fluorescein shown in
2. Front-surface optical design
/
r
Li 0 0 h
f +
-
f
l
z 3
"C. New Optical Design. The new optical design is shown in more detail in Figure 2. An adjustable mount is attached to the cell block with a front plate supporting an optical bar, and thus requires no modification of the microcell. The front plate is designed for z-axis (vertical) adjustment of the optical bar, and the optical bar design allows x-y adjustment of the focusing lens and the folding mirror. The system can be interchanged very easily between the fluorescence and absorbance modes by removing the front plate assembly. The 1-mm laser beam enters the stopped-flow cell compartment, passes through the focusing lens, is focused on the 1-mm diameter folding mirror and diverges t o approximately 1.8 mm a t the cell window surface. The 20-mm diameter quartz flat is mounted t o give a window size of 18 mm and, a t the 45' angle, provides a sufficient window size for the diverging fluorescence to enter the 2-mm by 12-mm monochromator entrance slit. The mirror used to reflect the beam into the cell blocks a certain percentage of the fluorescence which is emitted toward the slit; however, this percentage, based on the 45' angle of the mirror, is less than 20%. Figure 2 also illustrates the problem of recessed windows when a high pressure cell is used. The pressure seal is made with a metal screw with a 2-mm hole in the center for the light path. This construction prevents the use of front-surface angles other than near zero degree. Using this optical arrangement can cause a problem with scattered and reflected light from the cell window. The zerodegree observation causes a stray light problem in isolation of the fluorescenc with the monochromator, but to reduce this problem Filter 2 in Figure 1 is used to remove 99.7% of the excitation radiation that would otherwise enter the monochromator slit. The background signal from the scattered and reflected laser radiation is then negligible compared to the blank fluorescence. An alternative to the use of Filter 2 to reduce this radiation, is to modify the metal screw used for the pressure seal to allow front-surface illumination a t an angle of 10-20'. This would reduce the stray light reaching the monochromator entrance slit and Filter 2 would not be necessary.
PLATE
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3 0 0 t
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3. Analytical curve (plot of log fluorescence signal vs. log concentration)of fluorescein in 0.05M disodium phosphate
Figure
Figure 3 is linear over a 2000-fold concentration range (1.9 ppb to 3.8 ppm). A linear least squares fit to the data collected by averaging five successive analytical curves resulted in a slope of 1.01 and a correlation coefficient of 0.9994. No apparent deviation is noted in the linearity at the highest concentration used for this work, thus indicating possible extension of the analytical curve. A detection limit of 0.8 ppb was calculated using the method described by Gabriels ( 1 3 ) .This is quite similar to the detection limits reported by other workers (11, 12). The system has an absolute detection limit of 52 pg for the determination of fluorescein in the 60-pl cell; however, this does not account for solution to flush the cell between samples (I). The precision for the fluorescence measurements was 0.7 to 6% RSD: 6% for the 1.9 ppb solution but less than 2% for solutions greater than 19 ppb. A major component of the system noise was the 2% peak-to-peak fluctuation in the source intensity, causing a variation of the observed background fluorescence from the optical components. This background could be reduced with higher quality optical components since the background fluctuation presently limits the precision and sensitivity of the system; however, use of a dual-beam-in-space measurement system could correct for this fluctuation and improve both the sensitivity and precision of the fluorescence system ( 1 ) .
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
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CONCLUSION The work presented here demonstrates that use of a stopped-flow absorption microcell system for fluorometric analysis can be readily accomplished without modification of the microcell itself. The front-surface illumination-observation technique can be used by attachment of a reflecting mirror, focusing lens, and a filter. Application of this instrument for reaction-rate analysis of clinical and biological samples is under way, and incorporation of a dualbeam-in-space measurement system will be used to improve the sensitivity and photometric precision of the system.
(3) J. E. Stewart, "Durrum Application Notes No. 7". Durrum Instrument Co., Palo Alto, Calif., 1970. (4) Bulletin B-2437A-J, American Instrument Company, Silver Spring, Md.. 1973. (5) Bulletin 3195-2-3/Rj-4-5M, "The SMAC System", Technicon Instrument Corporation, Tarrytown, N.Y., p 9. (6) J. G. Atwood, Bulletin BL 12/73 1, "The Perkin-Elmer Kinetic Analyzer Model KA-150", Perkin-Elmer Corporation, Norwalk, Conn., 1973. (7) L. Brand and B. Witholt, Fluorescence Measurements, in "Methods in Enzymology", XI, C.H.W. Hirs, Ed., Academic Press, New York, N.Y.. 1967, p 776. (8)G. Winkelman and J. Grossman, Anal. Chem., 39, 1007 (1967). (9) J. McHard and J. D. Winefordner, Anal. Chem., 44, 1922 (1972). (10) S. Ainsworth, Anal. Chem., 37, 537 (1965). (11) T. 0. Tiffany, C. A. Burtis, J. C. Mailen, and L. H. Thacker, Anal. Chem.. 45, 1716 (1973). (12) D. C. Harrington and H. V. Malmstadt, Anal. Chem.. 47, 271 (1975). (13) R. Gabriels. Anal. Chem., 42, 1439 (1970).
LITERATURE CITED (1) K. R. O'Keefe and H. V. Malmstadt, Anal. Chem., 47, 707 (1975). (2) A. C. Javier, S. R. Crouch, and H. V. Malmstadt, Anal. Chem., 41, 239 (1969).
RECEIVEDfor review May 22, 1975. Accepted August 1, 1975.
Geometry Factors and Flux Corrections in Neutron Activation Analysis Stephen R. Piotrowicz,' James L. Fasching,2Dianne D. Zdankiewicz, and Rudolph W. Karin Department of Chemistry, University of Rhode Island, Kingston, R.I. 02887
Two possible sources of error in neutron activation analysis are variations in the neutron flux over the length, width, and height of a sample and geometry factors during counting of the activated sample. Flux monitors are the main method ( I ) used to evaluate flux variations to make the appropriate activity corrections to samples and standards. Geometry problems in counting have been more severe for beta counting than for gamma counting. Scattering and self-absorption of electrons can introduce significant uncertainties in beta counting. The attainment of a suitable, reproducible counting geometry is therefore necessary in beta counting ( 2 ) .Chemical separations and subsequent precipitation to an identical physical form are the usual methods used to minimize geometry problems in beta counting. The use of a NaI(T1) well detector has helped to minimize geometry problems in gamma spectrometry, but for counting with NaI(T1) and Ge(Li) detectors, geometry problems still exist. The authors have developed a sample preparation, handling, and analysis system designed to minimize these two problems as well as reducing contamination problems. The procedure basically involves four steps: 1)spotting a known volume of sample solution or standard solution on a filter disc and allowing it to dry in a laminar-flow clean bench; 2) sealing the disc between two layers of plastic; 3) irradiation; and 4) counting. The filter discs are punched from Whatman 41 filter paper using a sharpened 2.22-cm arch punch (C. S.Osborne Co., Harrison, N.J., No. 149), a mallet or hammer and a thick piece of leather as a cutting block. A leather cutting block is desirable as it does not dull the cutting edge of the arch punch as rapidly as a wooden cutting block. T o minimize contamination, the filter paper is sealed in a plastic bag and punched while in the bag. All work is done in a laminar-flow clean bench. Four'thicknesses of filter paper can easily be punched in this manner. The punched discs are removed from the center of the punch using Teflon-coated tweezers, the plastic is discarded, and Graduate School of Oceanography, U n i v e r s i t y of Rhode Island, Kingston, R.I. 02881. A u t h o r t o whom r e p r i n t requests should be addressed.
the discs are placed in a clean plastic bag for storage. Using a sheet of filter paper 46 cm X 57 cm folded in quarters and allowing for waste on the edges where handling has occurred (even though all handling is done wearing untalced plastic gloves), over 400 discs can be punched from a single sheet of paper giving a relatively constant blank for a large number of samples. The plastic discs are punched in a similar manner only using a slightly larger arch punch, 3.02 cm, from plastic 4 mils thick. We have begun using 6-mil thick plastic instead of the more commonly found 4-mil thick plastic as it is less sensitive to heat and therefore easier to seal than 4-mil plastic. These dimensions were chosen so as to fit the "rabbits" used in the State of Rhode Island Nuclear Science Center's pneumatic tube sample delivery system; however, any other dimensions for different systems could be used. A solid Teflon sealing plate (20.32 cm X 30.48 cm X 1.27 cm thick) which has been machined out to accept 15 separate discs a t a time is used to prepare the samples for irradiation (Figure 1A). Each disc holder will accept the 3.02cm plastic disc and has a raised island in the center for the 2.22-cm filter disc. A rounded groove has been cut around the raised center to the outside edge of each disc holder. A plastic disc is placed in the disc holder and a 2.22-cm filter disc is then placed on top of the plastic disc. The sample solution or standard solution is then pipetted on this filter disc and allowed to dry. We commonly use volumes of 20 and 50 yl for aqueous solutions and standards. Solutions up to 200 yl have been used; however, this volume takes over 1 hour to dry completely. The pipets used are calibrated gravimetrically. After the solution has dried (usually 10 to 20 minutes), a second plastic disc is placed on top of the filter disc and the two plastic discs are then heat sealed using an ordinary soldering iron fitted with a special, Teflon-coated, aluminum tip (Figure 1B). The method could also be adapted to ion exchange papers or filters from ring ovens where large volumes have been processed. The aluminum tip was manufactured with dimensions such that it would f i t the disc holder and with a rounded edge to fit the groove a t the bottom of the disc holder (Figure IC). The tip is recessed in the center so as not to con-
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