increases with time (29, 30). Thus, the He I1 electron time resolved profile, which shows a loss in intensity during the excitation period, indicates the transition from the high voltage, low current, condition present a t breakdown to a lower voltage, high current condition approaching that steady state situation found in a CW type discharge. (This transition is conceptually similar to the breakdown for a glow discharge (34).) In summary, it has been demonstrated'that the He I1 303.8-A intensity is the greatest a t low helium pressures and high electric fields. Its intensity, relative to atomic emission, was greater in the 4-mm tube than in the 10-mm discharge tube. In the case of pulsed excitation, it was shown that a gated detector further enhanced the observed ionic emission relative to the atomic emission. Consequently, in applications of the He I1 303.8-A line, the severe attenuation by a monochromator may be avoided under the time scheme proposed in this work. The potential success of the application of the pulsed microwave source to PES is indicated in Figures 4 and 5, or in Table I. The maximum He 1584.3-A intensity obtained is only a factor of 20 lower from the pulsed source compared to that from the CW microwave source. The latter has been demonstrated previously (35)to generate sufficient intensities for photoelectron spectroscopy.
ACKNOWLEDGMENT We thank Tom Lloyd of the Chemistry Department machine shop for construting the cylindrical microwave cavity and R. K. Skogerboe for describing the cylindrical cavity to us. John Schrag's electronics advice is appreciated.
LITERATURE CITED (1) W. R. Hunter in "Proc. Xth Colloq. Spectrosc. Intern.", E. R. Lippincott and M. Margoshes Ed., Spartan Books, Washington, D.C., 1963, p 247 ff. (2) M. I. Ai-Joboury and D. W. Turner, J. Chem. SOC.,5141 (1963). (3) S. D. Worley, Ph.D. Thesis, University of Texas, Austin, Texas, 1969. (4) M. E. Levy and R. E.Huffman, Appl. Opt., 9, 41 (1970). (5) F. Paresce, S. Kumar, and C. S. Bowyer, Appl. Opt., I O , 1904 (1971). (6) R . E. Huffman, J. C. Larrabee, and D. Chambers, Appl. Opt., 4, 1145 (1965). (7) L. Minnhagen, B. Peterson, and L. Stigmark, Ark. Fys., 16, 541 (1960).
(8) R . E. Huffman, J. C. Larrabee, and Y. Tanaka, Appl. Opt., 4, 1581 (1965). (9) R. Gorden, Jr., R. E. Rebbert, and P. Ausloos, Nat. Bur. Stand. (U.S.) Tech. Note, 496, 1969. (10) J. A. Kinsinger, W. L. Stebbings, R . A. Valenzi, and J. W. Taylor, Anal. Chem., 44, 773 (1972). (11) J. M. Feidman, J. Appl. Phys., 37, 674 (1966). (12) G. R. Branton, D. C. Frost, T. Makita, C. A. McDowell, and I. A. Stenhouse, J. Chem. Phys., 52, 802 (1970). (13) J. Delwiche, P. Natalis, and J. E. Collin, Int. J. Mass Spectrom. /on Phys., 5, 443 (1970). (14) James A. R. Samson, "Techniques of Vacuum Ultra-Violet Spectroscopy", John Wiley & Sons, Inc., New YorK, N.Y., 1967, Chapter 5. (15) D. W. Turner, "Annual Review of Physical Chemistry", Vol. 21, C. J. Christensen and H. S.Johnson, Ed., Annual Reviews inc., Palo Alto, Calif., 1970, pp 107-128. (16) F. Burgar and J. P. Maier, J. Phys. E, 8, 420 (1975). (17) F. E.Lichte and R. K. Skogerboe, Anal. Chem., 45, 399 (1973). (18) D. Villarejo, R. R. Herm, and M. G. Inghram, J. Opt. SOC.Am., 56, 1574 (1966). (19) Wm. B. Peatman and J. P. Barach, J. Chem. Phys., 58, 2638 (1973). (20) J. A. Hornbeck and J. P. Molner, Phys. Rev., 84, 621 (1951). (21) R . K. Curran, J. Chem. Phys., 38, 2974 (1963). (22) A. i. Maksimov, Sov. Phys.-Tech. Phys., 11, 1316 (1967). (23) T. E. Stewart, G. S.Hurst, D. M. Bartell, and J. E. Parks, Phys. Rev. A, 6, 1991 (1971). (24) D. M. Bartell, G. S. Hurst, and E. B. Wagner, Phys. Rev. A, 7, 1068 (1973). (25) W. L. Weise, M. W. Smith, and B. M. Glennon, "Atomic Transition Probabilities", Nat. Bur. Stand. (US.), National Standard Reference Date Series -4, U.S. GPO, Washington, D.C.. 1966, Voi. I, p 9 ff. (26) T. Holstein, Phys. Rev., 72, 1212 (1947) and 83, 1159 (1951). (27) J. Stevefelt and F. Robben, Phys. Rev. A, 5, 1502 (1972). (28) B. F. Gordiets, L. I. Gudzenko, and L. A. Shelepin, Sov. Phys.-Tech. Phys., 11, 1208 (1967). (29) E. V. Belousov, A. S. Bryukhovetskii, and A. V. Prokopov, Sov. Phys.-Tech. Phys.. 19, 53 (1974). (30) L. Vriens, J. Appl. Phys., 44, 3980 (1973). (31) W. E. Wells, P. Monchicourt, R . Deloche, and J. Berlande, Phys. Rev. A, 8, 381 (1973). (32) F. H. Reder and S. C. Brown, Phys. Rev., 95, 885 (1954). (33) F. LiewellynJones, "ionization and Breakdown in Gases", Meuthen and Co., Ltd., London, 1966, p 28. (34) Ref. 33, p 47. (35) J. A. Kinsinger and J. W. Taylor, lnt. J. Mass. Spectrom. /on Phys., 10,445 (1972/73).
RECEIVEDfor review April 26, 1976. Accepted August 19, 1976. We gratefully acknowledge the support of this work by the National Science Foundation (Grants MPS-23569 and CHE 76-01971) and the Wisconsin Alumni Research Foundation.
Enhanced Analog Detection of Weak Fluorescence by Drift-Free Integration Victor Glushko," Richard Caley, and Carol Karp Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, N. Y. 1002 1
An increase in the signal to nolse ratio of weak fluorescence signals is obtained by drift-free integration at the analog output. By using high resolution voltage to frequency conversion followed by event counting, this flexible and inexpensive procedure overcomes several drawbacks common to analog integrators. The improvement in the measurement precision is used to extend the fluorimeter detection limit at fixed wavelengths by an order of magnitude.
Enhanced detection of weak fluorescence has become a necessary goal for many laboratories involved in analytical or biophysical procedures. The quantitation of inorganic, organic, and biological substances in trace concentrations by fluorimetric analysis, either directly or by a coupled reaction,
has become a popular technique with hundreds of articles published every year. The ability of certain fluorophores to reflect changes in mobility and environment also has spurred interest in using such probes in biological materials ( I , 2). However, in order to reduce perturbation effects upon probe incorporation (31, low concentrations have to be used. In combination with limited sample quantities, the accurate measurement of weak fluorescence intensity can become a difficult task on many fluorimeters. Even a moderate gain in the signal to noise ratio is of direct benefit in improved analysis and interpretation of fluorophcre emission. Several papers (4-6) have dealt with the critical comparison between analog and photon counting detection for the optimum measurement of ultraviolet and visible radiation. At high signal intensities, the measurement of photomultiplier current can tolerate photon fluxes that would saturate a photon dis-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14. DECEMBER 1976
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“Olti
5*r
Figure 1. Block diagram of VFC operation
crimination system. On the other hand, the inherent discrimination and signal averaging features of discrete photon counting offer a significant advantage for the optimum measurement of weak optical signals. Consequently, single photon counting has become the technique of choice (7-9) for those applications that require maximum sensitivity. However, a moderate enhancement is frequently desired in the performance of existing analog instrumentation without conversion to photon counting, which may involve significant modification of the signal processing system. This report describes voltage to frequency conversion (VFC) as an inexpensive approach toward obtaining improved precision by drift-free integration at the analog or recorder output (10). VFC circuits have been used for various functions such as data telemetry, scaling of analog signals, and digital readouts. The relatively recent 10-fold reduction in the cost of VFC models, offering high resolution and a large dynamic range, is bound to increase their use in many measurement procedures. In general, there are two advantages in using a VFC approach. Once pulse or frequency conversion is achieved, pulse definition by a threshold discriminator is a simple procedure that can tolerate a relatively noisy baseline. This provides the basis for data telemetry where the analog signal can be converted to a pulse rate a t the signal source, transmitted and converted to an analog signal without significant degradation of the original measurement precision. Second, the pulse output is readily adaptable to event counting (11). The basic drawback of many analog integrators is the loss or “droop” rate of the stored signal, which begins to represent a significant percentage of the total input after several minutes. The VFC improves this situation by effectively integrating over very short periods of time and using an event counter for nondissipative storage. In general, the errors in VFC operation are limited to the conversion process and are independent of time. However, nonrandom fluctuations and long-term drifts in the analog circuitry will be carried over into the integration measurements. In those cases where a weak signal is obscured by random noise, this procedure can be applied to yield increased signal to noise ratios with longer integration periods.
EXPERIMENTAL Voltage t o Frequency Converter. The VFC unit used for this study was a North Hills Electronics Model DF-12 with a built-in power supply. Analog Devices, Datel, Intech, and other suppliers of integrated circuits offer a variety of models that have different voltage and current ranges including bipolar inputs. Without a power supply, these modules retail for under $100 with some as low as $15. Although there are significant differences in operational capability and response among the available VFC models, their general operation follows the block diagram outlined in Figure 1. From the input VIN. a proportional voltage is accumulated in the storage capacitor until it matches a reference voltage, VREF.A comparison circuit activates the pulser unit, a t positive crossover, producing a single output pulse and discharging the storage capacitor. A common circuit configuration is to continue accumulation while in the discharging phase, thereby preventing loss of signal during pulse output. Alternative circuits that provide voltage controlled oscillator frequencies with precise wave2078
form shapes are available; however, event-counting does not require a precise waveform and the additional expense is wasted. The VFC model used in this report was chosen for its high input impedance, flexibility in operation, and the good linear correlation between voltage input and pulse rate output. The output is a series of 14-V pulses of 5-ps duration with the rate, from 0 to 100 kHz, directly proportional to the input voltage from 0 to 10 V. As shown a t the bottom left of Figure 1, a 2-V input increases the potential on the storage capacitor until it corresponds to the VREFproducing a single pulse every 50 p s for a 20-kHz output; whereas a 10-V input, a t the bottom right of Figure 1,produces a pulse every 10 ps for a 100-kHz output. Conversion from voltage to frequency is rapid, occurring within a cycle of the output frequency. In this model, full scale response from 0 to 100 kHz takes less than 0.1 ms. With an input impedance in excess of 10 MQ,there is negligible circuit loading. In most cases, the VFC unit with the appropriate input range can be connected directly in parallel with the recorder or analog output while maintaining normal instrument operation. Individual VFC modules show some variation in operating parameters over the input range of five orders of magnitude. Trim controls are provided to adjust the output zero level and full scale response. T o determine the stability and conversion characteristics of the specific VFC module used in this study, a Tektronix TM503 System was employed to provide a regulated DC voltage from 3.2 mV to 20 V using the PS-501-1power supply; the input to the VFC was bridged in parallel to a calibrated DM501 digital multimeter. Using this system, it was possible to adjust the desired input voltage to 0.1 mV. A minor fluctuation in the power supply produced a 0.1-mV difference over a period of several hours. Spectrofluorimeter. The instrument (12) used for these measurements is based on a Cary 14 spectrophotometer. The excitation source is an Osram 450-watt high pressure xenon lamp located in a water-cooled housing with a slow nitrogen flush to prevent ozone formation. The excitation monochromator is a dual prism Cary model 50-650-000, similar to the unit used in the Cary model 60 circular dichroism instrument. In addition to the sample and reference cells, the sample compartment contains a separate optical path for illumination of a 20 g/l. solution of Rhodamine B in ethylene glycol, which acts as a quantum counter to monitor fluctuations in the excitation intensity. Optical path selection is accomplished by a dual disk light chopper rotating in phase with the normal light chopper in the Cary 14. The first disk directs illumination toward either the quantum counter or the samples; the second disk alternates illumination between the sample and reference cells. Although other configurations of the optical elements are possible with this instrument, front surface illumination a t 2 3 O to the viewing angle was used exclusively for this study. This fluorimeter was designed for flexibility and multiple user application. However, due to several inherent characteristics, accurate measurement of weak fluorescence intensities can be difficult on this instrument. Both the excitation and emission monochromators, although capable of very high resolution (0.1 nm), are “slow” with an aperture ratio of f/8. The monochromators also contribute to the long optical path of approximately 4 m from the excitation lamp to the photomultiplier compartment, with over 40 optical surfaces along the path. Furthermore, the gating procedure that controls the correct signal routing depends upon rapid relay operation. Although this method has been successfully applied to normal spectrophotometer operation, small shifts in light chopper phasing and slight differences in relay characteristics introduce fluctuations in this fluorimeter that can exceed the true emission intensity of a weak sample. Prior to the application of the VFC procedure, significant instabilities and long-term drifts in the analog circuitry had to be eliminated; otherwise, these factors could have curtailed the potential benefit of drift-free integration. Moreover, access to multiple users required maintaining the original instrument configuration. In this context, a low-cost analog detection system was constructed that overcame the electronic fluctuations arising from relay operation. By using a solid-state gate control and direct integration, similar in concept to VFC operation, a significant improvement in signal measurement was achieved for this instrument while providing dualsample signal processing as in the original design. The electronics block diagram and associated waveforms of the signal handling system are presented in Figures 2 and 3. The circled numbers in Figure 2 correlate with the displayed waveforms in Figure 3 such that the signal treatment can be readily determined. After the amplification stage, as shown in waveform 1,the sample channel reflects the optical chopping, which produces a repeating sequence of signals consisting of channel A dark current, DCA, sample A intensity, A, channel B dark current, DCB, and sample B intensity, B, respec-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
DARK CURRENT SUBTRACT
SAMPLE CHANNEL
GATED
r----1
CURRENT
DARK CURRENT SUBTRACT
----INTEGRATOR
QUANTUM COUNTER CHANNEL Figure 2. Block diagram of fluorimeter electronics tively. The quantum counter emission was measured independently using a self-biasing Hamamatsu S640 photodiode as suggested by Crosby ( 1 3 ) .Due to the light chopper, the quantum counter channel is shifted by a quarter phase as shown in waveform 2, where QA and QB represent the quantum counter intensities for samples A and B respectively. The clock-controlled gate, shown in waveform 3, is used to decode the sample and reference signals; an identifying pulse for channel A allows the electronic system to be phased to the Cary 14 light chopper operation. Although the timing circuitry is not included in Figure 2, the gating control operates a t the points indicated by a Q?. Both the position and duration of the gate can be adjusted to obtain the desired portion of the raw signal. The gate controls a storage capacitor, in effect, producing a brief integration. As shown in waveform 4, the dark current signal is accumulated first; at the completion of the gate, the intensity is inverted and held for the start of the sample accumulation. By this procedure, an automatic background subtract is produced a t the end of each phase since both the dark current and the sample intensity are accumulated for identical times. Losses during integration or during the storage phase in the sample and hold modules are negligible, since the longest interval that a circuit has to handle a signal is 17 ms. T o compensate for minor fluctuations in the excitation source, a ratio between the signal, S, and the quantum counter intensity, Q, is produced at a 30-Hz rate. The output is a step function with each channel “updated” every 33 ms, as illustrated in waveform 5. Procedure. As shown on the right side of Figure 2, the analog output of either channel, or their difference, can be directed to both a recorder and the VFC. The pulse output of the VFC is fed to an Ortec Model 775 counter controlled by a Model 719 timer, with a timing error of less than 0.05 ms for a 1-s interval. Since the VFC provides a t least an 80-fold difference between baseline noise and pulse intensity, a simple threshold discriminator is sufficient for maintaining full count registry. Errors in the total count can arise from inaccurate control of the counter by the timer gate. For a 1-s counting interval, the timer should be accurate to at least 0.1 ms to maintain the inherent precision of the VFC. The total storage capacity for integration is limited by the number of digits in the counter and the pulse rate of the VFC. Operating at the full scale 10-V input, the 100-kHz output of this VFC can be accumulated by a 10-digit counter for over 24 h without overflow. The measurement of a series of weak fluorescent samples consisted of adjusting the amplification of the photomultiplier and quantum counter signals to produce an approximate 1-V output for the most intense sample. Although this instrument is equipped with dual paths for automatic blank or reference subtraction, there are small optical differences between the paths. In order to avoid the introduction of additional variables, the sample and reference blank solutions were alternately measured in the same optical path. Five integrations or total counts were obtained for a fixed time period, a t each change in a variable, whether instrumental or sample related. An alternative approach of measuring the time required to reach a desired number
A
A
A
@ SAMPLE CHANNEL Q OUANTUM COUNTER
CHANNEL
@ GATING CONTROL
@ DARK CURRENT SUBTRACT
@ ANALOG OUTPUT
20
-40 ms
60
80
Figure 3. Associated waveforms at different stages in signal handling
of counts was not used; although this is a common procedure in scintillation counting, our equipment was not as flexible in this configuration. In a more sophisticated approach, an Ortec 6220 multichannel analyzer (MCA) was used in the scaling mode to collect the VFC output as a function of time. The channel dwell time, which acts as the integration interval, controlled the sweep across the 512 or 1024 channels or memory. By scanning the monochromator while sweeping the MCA, a fluorescence spectrum is accumulated in the memory, which could be output on a teletype or X-Y recorder. Reagents. ACS certified cyclohexane and 1.00 N sulfuric acid were purchased from Fisher Scientific and used directly. Pyrene and quinine bisulfate were obtained from Eastman Organic Chemicals. The pyrene was recrystallized twice from ether/ethanol to remove a yellow contaminant.
RESULTS AND DISCUSSION The degree of signal enhancement that can be achieved by the use of the VFC procedure depends upon two characteristics: 1) the correlation between the input voltage and the pulse rate output, and 2) the stability of the output over the period of time desired for signal accumulation. The operational limits of these two characteristics were examined prior to using the VFC for fluorescence measurements. As noted in the Experimental section, individual VFC modules show slight differences in response and stability; a Tektronix test system
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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Table I. Statistical Analysis of Voltage to Frequency Conversion Re1 std dev, %
Pulsesh, Hz
Volts 0.0100 0.0203 0.0405
101.4 f 0.55 205.4 & 0.55 407.2 f 0.45
0.0808 0.1607 0.3208
0.6402 1.oooo 2.000 4.001 8.001 10.000
Error, %
0.54 0.27 0.11
f1.4 $1.2 $0.54
809.2 f 0.45 1608.8 f 0.45 3 2 0 9 . 6 1 0.55
0.056 0.028 0.017
+0.15 so.11 +0.052
6 402.4 f 0.89 10 004.6 f 0.89 20 003.0 f 1.00
0.014 0.009 0.005
+0.006 +0.046 so.015
40 012.2 f 2.6 80 007.2 f 2.3 99 980.4 I: 2.3
0.007 0.003 0.002
+0.005
-
-0.003 -0.020
Table 11. Effect of Prolonged Integration on Intrinsic Noise
,oL_L_-.-L----I-
10-2
lo.’
I
IO
YOlt5
Figure 4. VFC pulse rate as a function of voltage
?.??OjS----------
I
5
IO
x 1000 second5
Figure 5. Variation in VFC output from initial operation
was used to determine if a calibration correction would be required over the anticipated input range. Figure 4 presents the pulse rate as a function of voltage from 3.2 mV to 20 V, with each point representing the mean of five 1-s accumulations. The log-log plot demonstrates the linearity of conversion over several orders of magnitude in input voltage. Although this VFC model is rated for a maximum 10-V input, a linear response in pulse rate is maintained to 14 V with a linear correlation coefficient in excess of 0.999. The inset diagram in Figure 4 presents a linear expansion in the region of saturation, showing a maximum output of 145 kHz. Table I provides a statistical analysis of the conversion precision and accuracy in the input range of 0.01 to 10 V. The error in accuracy is evaluated as the difference between the actual and rated conversion of 10 kHz/V. A minor curvature in the con2080
* ANALYTICAL CHEMISTRY, VOL.
Counting interval, s
Pulsesh, Hz
Signal to noise (dB)
1 10 100 1000 8000
10 003.80 f 0.45 10 004.54 f 0.38 10 004.16 f 0.57 10 003.77 f 0.61 10 003.78 f 0.85
22 231 (87) 26 328 (88) 26 327 (88) 16 400 (84) 11 769 (81)
version is evident from the % error column in Table I. Slightly higher frequencies than expected are produced at low voltages while the reverse occurs near the upper limit of the input range. At mid-scale, the error in both accuracy and precision was less than 1 part in 10 000. Below 0.1 V, several sources contributed to produce an increased error. In addition to small fluctuations in the regulated power supply, a 1.8 f 0.4 Hz offset was found in the zero level setting of the VFC. Since normal operation of the fluorescence detection system produced an amplified signal of 0.1 V or greater, with much greater noise than the error in the VFC, no zero level or linearity correction was made. The stability of an integrator controls the potential improvement in the signal to noise ratio. For effective use of a typical analog integrator, the input rate must be adjusted such that the total storage capacity is not exceeded during the anticipated integration period. At intervals longer than several minutes, the chqrging rate approaches the “droop” rate, and significant losses are encountered (11, 14). An external charging circuit, which matches the decay rate, can be used to compensate for the dissipation; however, this procedure requires adjustment and is not normally practical for longtime integration. The VFC approach, by using analog integration over very short periods of time and a counter for permanent storage, permits continued integration essentially independent of time. The stability of the VFC output over the experimental range of integration was checked using a regulated 1.0000-V input. Operating at the limit of VFC resolution, the intrinsic error or noise in a stable system should be independent of time. Table I1 demonstrates the relatively small increase in noise obtained with prolonged integration. For direct comparison, the values are expressed as frequencies with their associated standard deviations. An evaluation of the intrinsic error in the system can be obtained by considering the signal to noise ratio or the separation between the signal and noise levels expressed in decibels (dB). Although the integration period was increased from 1to 8000 s, less than a threefold increase was noted in the noise level, indicating a negligible deterioration in stability.
48, NO. 14, DECEMBER 1976
Table 111. Increase in S/N with Longer Integration of Counting interval, s 1
4 10 40 100 400 1000 4000
Total counts 1797 f 497 6 843 f 841 16 512 f 1 503 65 559 f 2 796 165 314 f 5 411 668 795 f 11 997 1 695 038 f 14 024 6 816 120 f 32 068
M Quinine Fluorescence
VFC output, Hz Sample Blank 1797 f 497 1711 f 210 1651 f 150 1 639 f 70 -1653 f 54 1672 f 30 1695 f 14 1 704 f 8
In a second approach, a 4.997-V input was used, the output of the VFC was directed to the MCA with the dwell time per channel set a t 20 s. Preliminary experiments indicated a minor decrease in the VFC pulse rate from initial operation. The 4.997-V input was selected to achieve the appropriate scaling upon expansion of the stored sweep. Coincident with the start of the MCA sweep, the VFC power supply was turned on at ambient temperature (cold-start); all other equipment had reached operating stability for several hours. The stored counts in the 512 channels of the MCA are shown in Figure 5 using a 1000-fold expansion, from 999 000 to 1000 000 counts per channel. The initial operation of the VFC reveals a time-dependent 0.1% decrease in the pulse-rate for a stable 4.997-V input. The time dependence can be traced to the “warm-up” or operating temperature of the VFC power supply. Except near the limit of VFC resolution, the thermal effect does not represent a significant factor in signal measurement. However, other VFC modules were found to have a more pronounced thermal sensitivity. Under these conditions where temperature fluctuations are a problem, several manufacturers now offer VFC models with superior thermal characteristics, which should significantly reduce thermal effects. The results in Tables I and I1 demonstrate that the VFC procedure introduces negligible error when compared to the relatively high noise normally encountered in the measurement of weak fluorescence intensities. Upon reaching operating temperature, it is generally unnecessary to apply a conversion correction or to compensate for different counting intervals. If the crucial condition of a steady signal obscured by random noise is met, the VFC procedure can be used to improve the signal to noise ratio by increasing the integration period. The relative detection limit for a fluorimeter can be determined by considering the measurement precision for fluorescent standard solutions. Unfortunately, no one compound possesses all of the desired characteristics ( 1 5 ) to act as a universally accepted standard. Instead, a variety of procedures (13,16) and compounds (15,17) have been proposed that cover the visible and near ultraviolet spectrum. Although quinine has its limitations (17), it is one of the oldest and most widely used compounds with standardized solutions available from several commercial sources. Furthermore, the characteristically broad emission of quinine overlaps the fluorescence of many interesting fluorophore groups (18, 19) including dansyl (20) and fluorescamine (21). By establishing the detection limit for quinine, a relative sensitivity for many other materials is also determined. Fresh quinine solutions from 10-l’ to 10-XM were prepared in 1.0 N HzS04. For optimum detection of fluorescence, broad, 3-mm monochromator slits were used, which correspond to a bandwidth dispersion of 16.2 nm a t 350-nm excitation and 10.2 nm at 450-nm emission. To compensate for the intensity of scattered light and the residual Raman band of water at 450 nm, a 1.0 N HzS04 solution was used as the reference blank. Table I11 presents the measurements obtained for a M
Signal to noise Total Actual
Actual
959 f 430 991 f 237 1031 f 124 985 f 69 1005 f 47 1013 f 23 1011 f 1 2 998 f 6
838 f 657 720 f 317 621 f 195 654 f 98 648 f 72 659 f 38 684 f 18 706 f 10
3.6 8.1 11.0 23.4 30.6 55.7 121.0
213.0
1.3 2.3 3.2 6.7 9.0 17.3 38.0 70.6
quinine solution with the integration period varied from 1to 4000 s. Each total count represents the mean of five sample accumulations with the standard deviation given directly below the value. T o provide a relative comparison, the total count and the standard deviation are divided by the counting interval. This value represents the VFC output frequency or relative sample intensity, I S . In the presence of a background component, I B , it is necessary to subtract this effect from the sample signal to obtain the actual fluorescence, I F .
IF = I s - I B Several reports have dealt with the sources of noise in the analog measurement of optical signals (5, 6,22). Rather than identify and quantitate each term, the effective noise is defined statistically as the standard deviation about the mean for a small sampling population. In this context, all values were assumed to approach a normal distribution such that the standard deviation, U F , for the actual fluorescence, I F ,is given by the following relationship where US and ~ r gare the standard deviations for the respective sample and blank intensities, I s and I B . OF
=
dUS2
+
Ug2
The sample total signal to noise, s / N T , and actual fluorescence signal to noise, S/NA, ratios are therefore:
and
In the absence of large background and non-Gaussian effects,
S/NA and S/NT increase proportionately to the square-root of the counting interval (9).On this basis, a 63-fold improvement would be expected upon increasing the counting interval from 1 to 4000 s. Over this range, the data showed a 59-fold increase in S/NT from 3.6 to 213; and the SINA showed a 54-fold increase from 1.3 t o 70.6. Table IV presents similar data for varying quinine concentrations. Each value, with the associated standard deviation, has been divided by the counting interval and corrected for background intensity as accomplished in Table 111. The values in brackets represent s/NA;nondetectable fluorescence is indicated by a s / N A of less than 0.5. Table IV demonstrates the ability of the VFC procedure to extend the detection limit to M quinine with a 100-s integration period and 10-11 M quinine with a 4000-s integration period. It is important to note that the fluorescence intensities for the M sample reflect a small difference between two much larger numbers of the sample and blank intensities. In comparison with the more concentrated quinine samples, the background components and the inherent error in the VFC conversion process contribute to the reduced improvement in the signal to noise ratio with time. Although further enhancement in detection is possible, the amount of time that must be dedicated to each
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14,DECEMBER 1976
2081
Table IV. Enhanced Measurement of Weak Quinine Fluorescence Counting interval, s 1
4 10
40 100 400 1000 4000
lo-"
M
39 f 566 1
