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11. Even in cell position 2, where the absorption interference is reduced by the short radiation paths through the sample, the fluorescence decrease in the presence of 500 r g of Fe(II1) corresponded to a 34% loss of aluminum. The absorbances were 0.65 at 365 nm and 0.42 a t 515 nm for this sample. For the absorption-corrected cell shift results, however, the interference due to iron oxinate was eliminated. The results for up to 500 pg of Fe(II1) are not statistically different at the 95% confidence level from the result when no iron was present. In addition to an increase in accuracy, the use of our instrument in this analysis enabled the sample treatment to be kept to a minimum and the analysis time to be reduced compared to the other corrective procedures mentioned above. The time required for a single measurement cycle and computation of the absorption-corrected result is about 40 s. Performing the aluminum analysis also revealed the disadvantage that our instrument is several orders of magnitude less sensitive than most commercial spectrofluorometers. The detection limit for quinine sulfate, at the 95% confidence level, is about 1 X lo-@M. This is due not only to the excitation source, the low light throughput, and the narrow bandwidth of the monochromators used but also to the significant quantity of radiation that is discarded to ensure that the excitation and emission beams are collimated. Although the sensitivity could probably be increased by an order of magnitude or more with a more intense radiation source and faster monochromators, little improvement could probably be achieved by compromising beam collimation before correction accuracy would be degraded. For this reason it is doubtful that an instrument of this type could ever be as sensitive as a conventional spectrofluorometer. This will obviously limit the usefulness of the instrument for trace analyses for which fluorescence methods have been most useful in the past. At
the same time the usefulness of the instrument will be greater than that of a conventional spectrofluorometer for concentrated samples for which the raw fluorescence intensity is not a linear function of the fluorophore concentration. The poor sensitivity of our instrument is reflected in the moderate measurement precision shown in Table 11. For the absorption-corrected results the precision is even poorer. This effect has been predicted by Novak (3) and will be discussed more completely in a future article.
LITERATURE CITED van Slageren, R.; den Boef, G.; van der Linden, W. E. Talanta 1973, 20.. 501-512. -. ... .. -. Holland, J. F.; Teets, R. E.; Kelly, P. M.; Timnick, A. Anal. Chem. 1977. 49. 706-710. Novak, A: Collect. Czech. Chem. Commun. 1978, 43, 2869-2878. Christmann, D. R.; Crouch, S. R.; Holland, J. F.; Timnick, A. Anal. Chem. 1980, 52, 291-295. Holland, J. F.; Teets, R. E.; Tirnnick, A. Anal. Chem. 1973, 45, 145-153. Britten, A.; Archer-Hall, J.; Lockwood, G. Analyst (London) 1978, 103, 928-936. Christmann, D. R.; Crouch, S. R.; Timnlck, A “Abstracts of Papers”, Second Chemical Congress of the North American Continent, Las Vegas, NV, Aug 24-29, 1980, ANYL 191, American Chemical Society, Washington, DC, 1980. Hoyt, S. D.; Ingle, J. D. Anal. Chem. 1978 48, 232-234. Christmann. D. R. Ph.D. Thesis, Michigan State University, East Lansing, MI, 1980, pp 135-171. Joseph, M. Ph.D. Thesis, Michigan State University, East Lansing, MI, 1979, pp 46-60. Milham, P. J.; Hudson, A. W.; Maguire, M. J.; Haddad, K. S.; Short, C. C. Appl. SpeCtfOSC. 1979, 3 3 , 298-300. Goon, E.; Petley, J. E.; McMullen, W. H.; Wiberly, S. E. Anal. Chem. 1953, 25 608-610. Noll, C. A.; Stefanelll, L. J. Anal. Chem. 1963, 3 5 , 1914-1916.
RECEIVEDfor review July 3, 1980. Accepted November 10, 1980. This work was partially supported by NSF Grants CHE 76-81203 and CHE 79-26490 and by an ACS Analytical Division Fellowship sponsored by Perkin-Elmer Corp.
Microcomputer Fluorometer for Corrected, Derivative, and Differential Spectra and Quantum Yield Determinations A. W. Ritter, P. C. Tway, and L. J. Cline Love* Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
H. A. Ashworth Department of Physics, Seton Hall University, South Orange, New Jersey 07079
The procedures and electronic interface for a microcomputer-controlled spectrofluorometer are described, and its performance is evaluated in various data treatment schemes. It is used to obtain corrected fluorescence spectra which are then integrated for use in calculation of quantum yields. Corrections are made for the light source emission profile, monochromators/optics transmission characteristics, and photomuitipiier wavelength response profile, but possible absorbance by the sample of the excitation and emission radiation is not considered. The instrument is also used to caicuiate and output derivative spectra as well as differential spectra to correct for background interferences.
Fluorescence spectroscopy is useful for the characterization and analyses of many classes of compounds because of the excellent sensitivity and specificity obtainable with relatively simple and inexpensive instrumentation (1). One disadvantage 0003-2700/8 1/0353-0280$01.00/0
of fluorescence spectrometry is that the data represent not the true spectrum of a molecule but rather a composite of the true fluorescence spectrum and various instrumental response functions (2). These instrumental sources of error in a spectrofluorometer have long been recognized and are well documented in the scientific literature (3). Several methods exist for solving these problems, the most common one being the use of hardware or software computer generated correction factors. Many manufacturers of fluorescence equipment offer as an option “corrected spectra” attachments which automatically correct for these instrumental distortions. The circuitry we describe here provides a versatile and low-cost alternative to the commercial instruments. It is easily adaptable to spectrofluorometers for which no corrected spectra attachment is available or to cases in which more complex signal processing is desired. Building our instrument around a personal microcomputer also allows great flexibility in hardware selection (amplifiers and counters, etc.) and software (easy programming 0 1981 American Chemical Society
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and microcomputer. CL1 and CL2 indicate the control lines utilized by the PET microcomputer to advance the monochromators(see text).
for data analysis and manipulation). Corrected spectra were needed in our laboratory to measure the fluorescence quantum yields of compounds by integration of the corrected emission spectrum ( 4 ) and to study theoretical relationships between the physical properties of a molecule and its true fluorescence transition energy maxima (5). This paper describes in detail the interfacing of a laboratory-constructed spectrofluorometer to a PET microcomputer and evaluates its performance in some organic molecular photophysical applications. The interface was made as general as possible so t h a t it can be used with any photomultiplier tube operated in the photon counting mode. The same interface can be used with a fluorescence detector on a liquid chromatograph or with a n ultraviolet-visible spectrometer. Although the system does not provide a dynamic, real-time correction, once data have been acquired by the computer, corrected spectra can be quickly calculated and plotted and the area under the curve calculated for quantum yield measurements. Corrections are made for the light source emission profile, monochromators/optics transmission characteristics and photomultiplier wavelength response profile. Optically dilute solutions are used t o avoid the need t o correct for absorbance by the sample of the excitation and emission radiation. In addition, the same interface can be used with different BASIC software programming t o obtain first and second derivative spectra and differential spectra.
EXPERIMENTAL SECTION Overview of the System. An overall block diagram of the
spectrofluorometer, interface, and microcomputer is given in Figure 1. The appropriate excitation wavelength is selected by the excitation monochromator and focused onto the sample, where absorption and the subsequent fluorescence occurs. The fluorescence emission is observed conventionally a t right angles via an emission monochromator and photomultiplier tube (PMT). The signal from the PMT can be measured in either a digital or analog mode depending on the data treatment and presentation required. Since fluorescence measurements are generally made on optically dilute solutions (absorbance 5 0.05) with low light levels at the PMT, photon counting of the discrete PMT digital signal is clearly the method of choice. This gives better signalto-noise (S/N) ratios at low fluorophor concentration levels and facilitates interfacing of the PMT to the microcomputer. The photon counts from the PMT are amplified to be compatible with standard digital logic levels (pulse amplitudes between 1.5 and 5 V). The scientific literature contains many examples of fast pulse amplifiers (6) which are available and do not merit further discussion here. It is assumed that the output of the PMT is fed into a fast pulse amplifier/discriminator and that the discriminator produces pulses which meet logic level needs. The pulse amplifier should have as high a frequency response as
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possible for it will ultimately limit the photon flux rate that the entire system can handle (7). From the discriminator the PMT signal enters the interface which is shown in more detail in Figure 2. The components of the interface are all “over-the-counter” integrated circuits (IC) and can be obtained a t most electronic supply houses. At this point the pulses from the PMT are shaped to be more uniform in both amplitude and width and then counted by a binary coded decimal (BCD) counter which is controlled by the PET microcomputer. The counter has a maximum reading of 999. The addition of another count sets the counter back to zero and produces an overflow. The normal operation consists of zeroing the counter, unlatching it, and allowing it to count for a preset period of time. During this time the microcomputer monitors the overflow line. If an overflow occurs, a register (previously zeroed) is incremented; this register then corresponds to the 1OOO’s place. After the predetermined count time the BCD counter is latched and the remaining digits are read. These values then, in combination with the number stored in the overflow register of the PET, correspond to the number of counts for the selected time interval. The overflow register itself will overflow if it is incremented 256 times so that the maximum count rate can not exceed 255 999 counts per selected time interval. The counting sequence described above is carried out by using a machine language program both to increase the speed of operation and to allow use of the PETS internal clock. The faster the program can (a) recognize an overflow, (b) increment the overflow register, anc (c) look for another overflow, the higher the photon flux that can be accommodated. The BCD counter, when operated at 5 V, can count a 1.5-MHz signal of regularly spaced pulses accurately, so that the time required to recognize an overflow ideally should be comparable to fully use the range of count rates available. If the pulses occur randomly, the highest accurate count rate is significantly less than 1.5 MHz, probably more likely 25 kHz a t the 2-3% error level. A BASIC program can only access the overflow line 15 times per second which limits the photon flux to approximately 15 kHz. A machine language program can, on the other hand, monitor the overflow line every 20 ps, so that the maximum photon flux is limited by the, BCD counter. The internal clock of the PET microcomputer may be used as a timer with a resolution of 1 ps in machine language programs, compared to a resolution of 16 ms in BASIC programs. The former system minimizes timing errors and is preferred. After the specified period of time, selected by the operator on the PET keyboard, the value of the counter is read into the PARALLEL port of the PET and the computer zeros the counter
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via the IEEE port, advances the emission monochromator by a selected interval, and starts the counter again. In this fashion, a complete fluorescence spectral scan is obtained. When data acquisition is completed, the spectra can be manipulated by a series of BASIC software programs to yield uncorrected, corrected, differential, and/or derivative spectra, as well as integrated spectra for use in the calculation of quantum yields. After appropriate data manipulation, the spectra are fed back out of the computer to an 8-bit D/A converter, the output of which drives a strip chart recorder to obtain hardcopy. Circuitry a n d Interface. We shall discuss the circuitry in three functional sections, referring to Figure 3 throughout. The specific components used and a brief description of their function are given in Table I. The detailed requirements for operations of each IC can be obtained from catalogues or texts (8). BCD Counter. Pulses originating from an operational amplifier are shaped by a monostable (74121) and applied to the input of the BCD (14553) counter. The output of the counter (4 bits of data, 2 bits to indicate which digit) reaches the PARALLEL port of the PET through an octal, tristate buffer (81LS96). This buffer is enabled (Le., presents data to the PET) only when a logical “0” has been latched into output pin no. 10 of the D-Flip Flop (DFF). Gates G1, G2, and G3 encode the three digit-select lines of the counter into two lines that go to the PET. The JK-Flip Flop (4027) is triggered by the overflow signal of the counter. D / A Converter. When a scan has been completed, the data are placed on the PARALLEL port, now used as an output port rather than as an input port. When buffer B2 is enabled the data appear at the inputs to buffers B3 and B4. These buffers translate the 5-V logic signals to the 15-V levels required by the D/A converter. The computer is programmed to present the data at a constant rate so the output of the D/A converter provides a suitable time-varying signal for the strip chart recorder. Control Functions. Outputs from the DFF are used for various control operations. Lines 9 and 10 multiplex the PARALLEL port via buffers B1 and B2. Line 15 is used to reset the BCD
Table I. Components of Interface Circuit integrated circuit function 4011 CMOS Quad 2-input nand gate 4027 CMOS dual JK flip-flop 7401 Quad 2-input nand gate (open-collector outputs) 7416 Hex driver, inverting (open-collector t o 1 5 V) 7475 quad latch (level sensitive) 74121 monostable multivibrator (single, not retriggerable) 81LS96 Tri-State octal buffer (inverting) 14553a CMOS 3-digit BCD counter/driver AD7523b CMOS &bit multiplying D/A converter a Radio Shack, San Antonio, TX. Available as part no. 276-2498. Analog Devices, Norwood, MA.
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counters. The output of line 16 controls the monochromator. When a logical “1”appears at output pin no. 16 of the DFF, the oscillator signal (600 Hz) will reach the monochromator through the inverting buffer G5. The monochromator is simply driven for an amount of time that the oscillator signal passes through G5. There is no mechanism to encode the actual value of the wavelength from the monochromator. Utilization of the DFF is required due to the nature of the IEEE port which presents its data in a transient fashion, in contrast to the PARALLEL port where data remain constant until the program or BCD counter changes the data. The bit parallel, byte-serial mode has been standardized by the IEEE (9) to allow several instruments such as printers and voltmeters to be connected simultaneously to the same port. This handshaking
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
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procedure requires certain control lines to be in particular states at particular times in order for data to be transmitted through the port. The IEEE responder (gates G6, G7, and G8) generates these necessary signals so that operation of the IEEE port continues. The circuitry consisting of DFF, G4, and G5 latches the data and presents it constantly to the rest of the circuit. The latching signals are automatically generated on lines 6 and 11of the IEEE port whenever the proper PRINT command is encountered (10). Reagents. Quantum yields were obtained by the comparative method using reagent grade quinine bisulfate (Aldrich) in 1 N sulfuric acid as the standard (4).Doubly distilled water was used for all aqueous solutions. Hydrochloric acid and sulfuric acid were obtained from Fisher Scientific. The rhodamine B and ethylene glycol were both reagent grade and were obtained also from Fisher Scientific. The anthracene sample was obtained from Matheson Coleman and Bell (Lot 8fl3)and recrystallized three times from ethanol. A Beckman Acta I11 was used to record all absorbance measurements.
RESULTS AND DISCUSSION Two sets of correction factors were needed to calibrate the spectrofluorometer for corrected spectra. Excitation correction factors, which compensate for variations in excitation lamp intensity and excitation monochromator transmission with wavelength, and emission correction factors, which compensate for variations in emission monochromator transmission and the PMT response with wavelength, were calculated by the microcomputer and stored on magnetic tape. An uncorrected fluorescence spectra can then be mathematically corrected with these instrumental profile functions. Thus, the correction factors are determined in a previous experiment and applied to subsequent data, not in real time. This assumes a constant source-emission profile, monochromator transmission characteristics, and P M T response profile. Excitation correction factors were obtained by scanning the excitation monochromator using a narrow slit with the emission monochromator set a t zero order (0 nm) so that it passes all wavelengths of the emitted light. The sample was 8 g/L rhodamine B in ethylene glycol in a triangular cell (Precision Cell Co.) placed in the sample compartment so that the light is directed a t the long side of the sample cell and rear surface detection is used. The position of the sample cell is critical since the sample should appear essentially as a point source and scattered light should be minimized. Rhodamine B, which is a quantum shifter, is used as the sample because it absorbs all wavelengths of light from 300 to 600 nm and emits in a narrow band from 600 to 640 nm. The use of rhodamine B as a sample eliminates the wavelength dependence of the PMT since essentially the P M T sees light from only one narrow wavelength region. T o obtain the combination emission-excitation correction factors, a frosted quartz plate was placed in the sample compartment at a 45' angle to the excitation light source utilizing rear surface detection to minimize scatter, and the two monochromators were scanned synchronously. The combination excitation-emission correction factors thus obtained were divided by the excitation factors to obtain the emission correction factors. The excitation and the emission correction factors were each normalized to values of 0 to 1. Corrected fluorescence emission spectra were obtained by dividing the uncorrected data points by the emission correction factors at each Wavelength. The excitation correction factors were only used if the fluorescence intensity of two compounds which were excited a t different wavelengths were to be compared. Corrected fluorescence spectra of quinine sulfate in 1 N HzS04and anthracene in ethanol were obtained. In all cases the corrected spectra are red shifted from the uncorrected spectra, as shown for quinine sulfate in Figure 4. The corrected peak maximum of the quinine sulfate solution is at 451
360
400
I
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I
440
400
520
Wavelength Flgure 4. The corrected (---) and uncorrected (-) fluorescence spectra of quinine sulfate in 1 N sulfuric acid solution. The compound was excited at 337 nm.
Table 11. Comparison of Measured Quantum Yields witlh Literature Values compound @f anthracene (ethanol) 0.31 i 0.01 atabrine (0.1 N HCl) 0.064 f 0.009 a
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nm which agrees with literature values ( 4 , I I ) . The software program for corrected spectra also integrates the area under the corrected emission spectrum for use in calculating quantum yields. The area under the quinine sulfate fluoresceince curves was reporducible to within &2% on successive runs. The fluorescence quantum yields of some standards determined by the comparative method using corrected spectra from the microcomputer are compared to literature valuer5 in Table 11. The precision of the experimental quantum yield measurements was *5%. The largest source of error in 'the quantum yields was the absorbance measurements. This results because of the poor S/N ratios obtained from optically dilute samples. Simple BASIC programs were written to obtain derivative and differential spectra. Figure 5 shows the normal and first derivative spectra of anthracene in ethanol. The derivative software program allows the operator to choose the order of the derivative spectra and the size of the wavelength interval (Ah). Small wavelength increments give better resolution, but decreased S/Nratio. The best wavelength increment for a specific sample and instrument parameters was generarlly found by experimental trial and error. Higher order derivatives increase the resolution of the signal peaks but markedly decrease the S/N ratio. Since there was no smoothing or damping of the digital output in this work, derivatives higher
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Anal. Chem. 1981, 53, 284-286
determination of phosphorescence quantum yields, for background subtraction, and for background subtraction of micelle stabilized room-temperature phosphorescence spectra. The interface is versatile and can be easily adapted to any photometric instrumentation. Although not as convenient to run as a commercially available corrected spectra unit, this interface allows one to inexpensively upgrade any existing spectrofluorometer to obtain the additional information available from corrected, derivative, or differential spectra and quantum yields.
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ACKNOWLEDGMENT
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The authors thank J. Fenton Williams, Perkin-Elmer Corp., for helpful discussions on corrected spectra procedures.
LITERATURE CITED (1) Guiibault, G. G. “Practical Fluorescence Theory, Methods and Techniques”; Marcel Dekker: New York, 1973. (2) Parker, C. A. “Photoluminescence of Solutlons with Appllcations to Photochemistry and Analytical Chemlstry”; Elsevler:
I I
I
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:r\\
\ \ \
\
350
400
Amsterdam,
1968;pp 246-251. (3) Parker, C. A,; Rees, W. T. Analyst(London) 1980, 85, 587-600. (4) Demas, J. N.; Crosby. G. A. J. Phys. Chem. 1971, 75, 991-1024. (5) Strickler, S.J.; Berg, R. A. J. Chem. Phys. 1982, 37, 814-822. (6) Darland, E. J.; Hornshuh, J. E.; Enke, C. G.; Leroi, G. E. Anal. Chem. 1979, 51,245-250. (7) 2-10. Franklin, M. L.; Horilck, G.; Malmstadt, H. V. Anal. Chem. 1989, 41, (8) Shacklette, L.; Ashworth, H. A. “Using Digital and Analog Integrated Circuits”; Prentice-Hall: Englewood Cliffs, NJ, 1978. (9) ANSIlIEEE Std. 488-1978,Institute of Electrical and Electronic Englneers, Inc.: New York, 1978. (IO) “User Manual”; Commodore Business Machines: Santa Clara, CA. It should be noted that the required command wlll have the form “PRINT #7,A$;” (11) Williams, J. F., Perkin-Elmer Corp., private communicatlon, 1979. (12) Blrks, J. B.; Munro, I. H. In “Progress In Reaction Kinetics”; Porter, G., Ed.; Pergamon Press: Oxford, 1967,vol. 4, p 282. (13) Upton, L. M.; Cllne Love, L. J. Anal. Chem. 1979, 51, 1941-1945.
‘-..
450
500
.
Wavelength ( n m )
~l~~~~5. The (---) and the first derivative fluorescence spectra of anthracene in ethanol solution. The compound was excited at 337 nm and the wavelength increment (Ax) was 2 . 2 nm.
RECEIVED for review August 29, 1980. Accepted November 25, 1980. Research support provided by the State of New Jersey under the Independent Colleges and Universities Utilization Act is gratefully acknowledged. P.C.T. thanks Merck & Co., Inc., for partial financial support.
than second order were too noisy to yield additional useful information. With this interface one can rapidly perform many types of data manipulation on a routine basis. It is being used in our laboratory for the functions described above as well as for
Mixed Ligand Chelate Extraction of Lanthanides in 8-Quino1in01-Tet ra- n -heptylammonium Chloride Systems M. Kawashima’ and Henry Freiser” Department of Chemistry, University of Arizona, Tucson, Arizona 8572 I
A fundamental study of the equilibrium extraction behavior of representative tervalent lanthanide ions, La, Pr, and Yb, from aqueous tartrate solutlons into chloroform solutlons containing 8-qulnollnol (HQ) and tetra-n-heptylammonium chloride (RdNCI) was carrled out. The results showed that, except for La which extracted as a simple ion-palr R,NLaQ,, the Ianthanides extract as the complex R,NLnQ4.HQ. m e separation of lanthanides from each other and the coordinatlon number In the complexes are discussed in comparison with self-adduct which were formed In the absence Of R4NC1u
Separation of individual lanthanides is still a very interesting and formidable problem. This study represents a On study leave from the Faculty of Liberal Arts and Education, Shiga University, Otsu-City, Japan. 0003-2700/81/0353-0284$01 .OO/O
systematic evaluation of the use of reagents such as 8quinolinol and its derivatives as chelating and/or adducting agents. In a previous study (1), the equilibrium extraction behavior of series Of representative tervalent lanthanide ions, La, Pr, Eu, Ho, and Yb, into chloroform solutions containing either 8-quinolinol (HQ) alone or combined with 1 , l O phenanthroline (phen), was studied in detail. Lanthanides were found to extract in the form of the self-adduct complexes LnQ3.2HQ or LnQ3.3HQ, except for La which extracted as a simple chelate, L ~ Q ~ .the presence of phen, mixed ligand chelates of all the lanthanides but La of the fornula LnQ3. 2HQ.phen were formed which further enhanced their separation* Since, in preliminary experiments, both tetraalkylammonium cation (R4N+),and tetraphenylborate anion (BPh4-) were found to enhance Ln3+extractions in almost the same pH ranges, it was decided that an investigation of 0 1981 American Chemlcal Society
