Design and Evaluation of a Vidicon Based Derivative Spectrometer Thomas E. Cook,' Robert E. Santlnl, and Harry L. Pardue" Department of Chemistry, Purdue Universw, West Lafayette, Indiana 47907
A vklkon based rapM scan spectrometer which sknuttaneously generates flrst derlvatlve and Intensity spectra Is descrlbed. The wavelength modulation used to produce the derlvatlve slgnal Is created by the electron beam scan pattern. The system operates in the vlslble reglon over a selectable range of 40 or 400 nm and Is Interfaced to a rnlnlcomputer. Scan tlme Is flxed at 100 ms. The performance characterlstlcr of the system are evaluated via atomlc emlsslon and molecular absorption In the gas and liquid phase. It was found to be h e a r over at least two orders of magnltude with good SIN characterlstlcs. The analytlcal utllky of the system is Illustrated with the determinatlon of blllrubln In the presence of albumln. A crltlcal comparison ot electrostatlc vs. magnetlc deflectlon methods In the derlvative experiment is provlded. The proper optlrnlzatlon of the derivative spectrum as a function of wavelength modulatlon amplitude Is dlscussed from an emplrlcal polnt of vlew.
Several authors have discussed advantages of derivative spectroscopy in separating spectral information from background, discriminating overlapping spectra in the analysis of mixtures, and improving the sensitivity of ultraviolet methods for gaseous species. Applications have included atomic emission ( I ) and atomic absorption (2) spectra, molecular absorption (3-7) and molecular fluorescence (8,9).Methods used to generate derivative spectra have included direct computation by digital (7,10,11) and analog (8) computers and by mechanical modulation of the wavelength dispersion optics ( I - 4 , 9 ) . The basic difference between the mechanical modulation and computational methods is that the former produces a representation of the derivative of intensity or absorbance with respect to wavelength while the latter produces a derivative of intensity or absorbance with respect to time which is assumed to be directly related to wavelength (3). An early report from this laboratory described derivative spectra computed from the output of a silicon target vidicon based spectrometer (7). A subsequent report gave a brief description of a vidicon based derivative spectrometer in which first derivative spectra were generated directly by the spectrometer used in conjunction with a phase sensitive lock-in amplifier (12). In this latter system, wavelength modulation is accomplished by incorporating the modulation into the electronic deflection of the interrogating electron beam in the vidicon along the wavelength axis. The principal advantages of this system are that it eliminates the need for mechanical modulation, and in the process permits scan rates to a t least ten scans per second. This paper represents a more complete description of the design features and characteristics of the system than were presented earlier. Two derivative systems are discussed: one is based upon a vidicon spectrometer developed in this laboratory (13)and one is based upon modifications of a commercially available system. The primary difference between these systems is that
the former utilizes a vidicon tube with magnetic deflection of the interrogating beam while the latter employs electrmtatic deflection. Accordingly our data will reflect the relative merits of these deflection modes for derivative spectroscopy. Although these systems are referred to in the text as the custom designed and modified commercial systems, these fundamental differences should be kept in mind. General Considerations. Hager (3) and Green and 0'Haver (8)have given lucid descriptions of the manner in which wavelength modulation methods coupled with tuned amplifiers generate fiist and second derivatives. The wavelength axis is moddated at a frequency significantly greater than the scan frequency. If the detector signal is constant over the modulation range, then the ac component of the signal will be zero. However, if the detector signal is not constant over the modulation range, then the ac component of the signal will have a finite value related to the rate of change of the detector signal with wavelength. It is easily shown (3) that the ac component of the signal corresponding to the first harmonic of the modulation frequency is related to the first derivative of the signal vs. wavelength profile and the ac component corresponding to the second harmonic is related to the second derivative. In this work, we take advantage of the two-dimensional character of the vidicon detector in which the vertical axis is parallel to the slit axis and the horizontal axis corresponds to the wavelength axis. To produce an effect equivalent to wavelength modulation with the vidicon, a high frequency, low amplitude signal is superimposed on the low frequency horizontal deflection ramp signal (see Figure 1B). As the electron beam moves along the vertical (slit) axis, it is deflected horizontally by a small amount so that it interrogates different wavelength resolution elements as it moves from top to bottom along the slit axis. If the horizontal modulation frequency is equal to the vertical scan frequency, then one modulation cycle will be completed for each vertical sweep cycle so that the effect is very similar to that experienced when a narrow band of wavelengths is swept back and forth (mechanically modulated) across the exit slit of a monochromator. Thus, the operational discussions for mechanical systems (3,8) apply also to this vidicon based system. An amplifier tuned to the first or second harmonic of the modulation frequency will generate a signal proportional to the fiist or second derivative of the intensity-wavelength profile. This paper is concerned with fiist derivative spectra for atomic emission and molecular absorption processes. For emission and luminescenceprocesses, the first derivative of the intensity-wavelength profile (intensity derivative, dI/dX) generated by either the mechanical modulation or vidicon based derivative spectrometer is proportional to analyte concentration. However, for absorption processes, the intensity derivative must be further processed to obtain a signal proportional to concentration. Starting with absorbance expressed in terms of unattenuated and attenuated intensities (Io and I),it is easily shown that the absorbance derivative (dA/dX) is given by
1Present address, The Procter and Gamble Company, Ivorydale Technical Center, Cincinnati, Ohio 45217 ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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Simplified interrogation pattern providing wavelength
Flgure 1.
modulation and using Beers law, it is easily shown that the expression explicit in concentration is
where axand b are the absorptivity and cell path length. Thus, the intensity derivatives must be combined with intensity values to obtain absorbance derivatives. The intensity data can be obtained either from a separate conventional scan or as the dc output from the modulation experiment. The first approach has the advantage that the spectral resolution of the intensity data is not degraded by the modulation process while the latter approach has the advantages of being most efficient and of minimizing effects of time dependent variables such as source and detector drift. Although both approaches were used in different parts of this work, most results reported below were obtained with the latter approach. For those examples in which intensity data were obtained from modulation experiments,data are included which permit an evaluation of the effects of modulation amplitude on the absorbance spectra and which illustrate unique problems which can develop when the modulation amplitude is large compared to the natural width of lines in source spectra. For an ideal source and detector which would yield a perfectly flat signal vs. wavelength response, the dIo/dX term would be zero and a simple proportionality would apply. However, with real systems, this dIo/dX term will usually be finite, even for small modulation amplitudes, and a background correction may be necessary. This is especially true with arc lamps (xenon,hydrogen, mercury) which have narrow line spectra. Some additional details of the interrogation pattern and some of its consequences are represented in Figure 1. Figure 1B depicts the horizontal deflection ramp signal which is broad because a high frequency ac component is superimposed upon
the ramp. The figure includes an expanded segment of the ramp to emphasize the modulation signal. Figure 1C represents the vertical deflection signal on a time scale comparable to the expanded segment in Figure lB, and Figure 1A represents the net effect (wavelength axis expanded) on the interrogation pattern of the interaction of the horizontal and vertical signals. The solid lines in each of the expanded scale figures represent one complete cycle for the horizontal modulation and vertical deflection signals and the effect on the deflection pattern. It is important in this application that the active surface of the detector be illuminated uniformly along the vertical axis and that the area illuminated be at least as large as the area interrogated by the electron beam. Best results were obtained when the same frequency was used for the vertical deflection and the wavelength modulation signals. Even when no intentional modulation signal is superimposed upon the horizontal deflection pattern, the output is modulated by the vertical sweep frequency both through “crosstalk” between the vertical and horizontal deflection systems and uncharacterized effects within the target. If the vertical deflection and wavelength modulation frequencies are not identical, many beat frequencies appear and the intensity of the derivative signal is significantly reduced. Also, it is desirable that the modulation frequency be much larger than the horizontal scan frequency. This is the consideration which influenced our choice of a maximum scan repetition rate of 10 Hz.
INSTRUMENTATION Two instrument systems were used and evaluated in this work. One was a modified version of a custom designed instrument built in this laboratory (13),and one was a modified version of a
commercially available instrument (Model 520, Tektronix, Inc., Beaverton, Ore. 97005). Details of the modifications required on the commercial instrumentwill be supplied to interested persons. Deflection and Measurement Circuits. Custom Designed Systems. A simplified diagram of this system is presented in Figure 2. This system utilized a silicon target vidicon tube (No. 4532A, Radio Corp. of America, Harrison, N.J. 07029) with magnetic deflection coils (No. CY100-3031-1,Penn Tran Corp., Bellefonte, Pa. 16823) mounted external to the tube. The vertical deflection coils were driven by a triangular wave generated by integrating the square wave from a crystal clock and associated divider. Vertical sweep frequencies of 100,62.5,and 50 KHz were used in this study. The horizontal deflection signal is a 1000-step staircase ramp generated by a digital-to-analog converter (No. VlOBBD, Date1 Systems, Inc., Canton, Mass. 02021) driven by a crystal clock operating through a binary counter to yield a 100-ms scan time. The horizontal modulation signal was superimposed on the horizontal deflection signal by summing a suitably scaled portion of the vertical deflection triangular waveform with the staircase signal at the input of a summing amplifier. The amplitude of the modulating signal wm adjusted by a variable resistor
dI/dX
HORIZONTAL DRIVER
I INPUT
3
I
VIDICON
AMPLIFIER
VERTICAL
DRIVER
Figure 2.
872
Block diagram of vidicon based derivative spectrometer
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
at the input of the summing amplifier. The vertical deflection waveform was also used as the reference frequency for the lock-in amplifier. The output from a preamplifier used to sense detector charging current is sampled both by a scaling amplifier (for intensity measurements) and by a lock-in amplifier (No. 126, Princeton Applied Research Corp., Princeton, N.J. 08540) scaling amplifier combination (for derivative measurements). Both outputs were normalized for full scale values of f1.00 V to be consistent with the analog-to-digital converter (ADC) used with the computer. Commercial Instrument. Because of the lag in the vidicon detector and frequency band width of the lock-in-amplifier(LIA), the 1-MHz vertical deflection of the Tektronix 520 System was replaced by a 20-KHz (-45V peak-to-peak) signal generated by passing the reference signal from the LIA through an audiofrequency step-up transformer (BIT-250-56, TRW, Inc., Los Angeles, Calif. 90024) with a center tap biased at +265V. The 20-ms scan time of the instrument was increased to 100 ms by increasing the integrating capacitor in the analog ramp generator and reducing the magnitude of the input voltage to the integrator. A fraction of the 20-KHz reference signal from the LIA was added to the ramp signal via a summing amplifier inserted between the ramp generator and horizontal deflection amplifiers. The output from the detector preamplifier was passed via a voltage follower to the lock-in amplifier and a scaling amplifier as discussed earlier in the custom designed system. Optics. Custom Designed System. Holographic grating dispersion optics (H-20 Monochromator, J-Y Optical Systems, Metuchen, N.J. 08840) with f14.2 optics and a reciprocal linear dispersion of 4 nm/mm was used with this system. An entrance slit of 125pm was used and the exit slit was removed so the active surface of the vidicon could be placed in the output focal plane. A tungsten-halogen lamp (No. 2331, General Electric Company, Nela Park, Cleveland, Ohio 44112) powered by a stabilized supply (PAR-l2C, Kepco, Inc., Flushing, N.Y. 11352) was used for absorption experiments. A conventional 10-mmcuvette was used for solution absorption experiments and a 15-cm glass tube with quartz windows and input and exit ports was used for gas absorption experiments. Quartz lenses focused light through the absorption cell onto the entrance slit of the dispersion optics. An air-acetylene burner (No. 02-100380-00,Varian Instrument Div., Palo Alto,Calif. 94303) was used for flame emission experiments. The neon emission spectrum from a hollow cathode lamp (No. WL 23059A, Westinghouse Electric Corp., Elmira, N.Y. 44902) was used with this and the modified instrument as a source of narrow lines. Commercial System. This system has f/6 dispersion optics mounted in the same housing with the vidicon detector. Interchangeable gratings provide optical windows of 40 and 400 nm. Using a tungsten-halogen lamp, the useful spectral range was from about 360 to about 800 nm. A concave mirror was used to collect energy from the lamp and focus it through a 1-cm quartz cell mounted on the entrance to the dispersion optics and onto a 100-gm entrance slit. When the wider of the two wavelength ranges was used, quartz filters were used to produce a response throughout the spectral range that is more uniform than that for the tungsten-halogen source alone. Data Acquisition and Processing. A digital computer (PDP-12-30, Digital Equipment Corporation, Maynard, Mass. 01754) with 8K of core memory, two magnetic tape drive units, and a built-in 10-bit, 30-KHz, ADC ( f l V) with eight buffered and multiplexed inputs was used for data acquisition and processing. A total of 512 data points were recorded at a data rate of 5.12 KHz during each scan and up to 16000 scans could be averaged in double precision (24 bits) before memory word overflow occurred. Only the 12 most significant bits were saved for subsequent processing. When intensity and derivative data were recorded simultaneously,256 data points for each signal were collected using the multiplexed input of the ADC. All intensity measurements were corrected for dark current. When signal averaging was to be performed, the dark current signal was inverted and averaged for a period of time equal to the averaging time to be used for the signal; then points in each spectral scan were added algebraically to corresponding points in the dark current “spectrum” so that the time required for dark current correction was minimized.
Table I. Comparison of Precision Data for Absorbance and Derivative Signals for Iodine Vapor Wavelength Absorbance
(See Fig. 4)
RSD, %
0.131 0.103 0.124
1.9 +0.357 1.6 0.848 0.7 2.6 -0.450 1.4 1.154 0.6 3 1.9 +0.562 0.8 1.156 0.6 Average of eleven values. Average of seven values. 1
2
a
Signala
Derivative Peak Peak-t o-Peak RSD, SigRSD, Signalb % naP %
RESULTS AND DISCUSSION An earlier report included preliminary data to show that the vidicon based system is a viable approach to derivative spectroscopy and illustrated that derivative signals are linear with intensity over a t least two orders of magnitude (12). In our continuing work with this system, we concluded that some problems we encountered were related to the fact that our custom designed system utilized a magnetic deflection tube, and our study was expanded to include an electrostatic deflection tube. Results for a variety of chemical systems and light sources are presented here to demonstrate more fully the characteristics of the derivative concept when implemented with magnetic and electrostatic deflection tubes. In the following discussion all uncertainties in slopes, intercepts, average values, etc. are reported at the 95% confidence interval. Custom Designed System-Magnetic Deflection Tube. Two chemical systems, the flame emission of sodium and the absorption of iodine vapor, were used to evaluate this system. Sodium Flame Emission. The first derivative spectra for aqueous solutions of sodium excited in an air-acetylene flame were used as an early test of the viability of the concept. These spectra were generated with a 200-ms integration period followed by a 100-ms scan time without dark current correction, and were similar to those reported earlier (12). Regression analysis of sodium concentrations computed from peak-to-peak derivative signals vs. added amounts in the range from 0.01 to 1.0 mg/L yielded a slope of 1.02 f 0.04, an intercept of -0.026 f 0.018 and a correlation coefficient of 0.999. The small intercept resulted from contributions to the derivative signal by the dark current. These data confirm our earlier conclusions that response is linear with intensity over a t least two orders of magnitude. Absorption by Iodine Vapor. One of the promising applications of derivative spectroscopy is for the determination of trace components in gaseous samples (3), and we have included a gaseous sample as an example in our studies. Figures 3A and 3B represent the absorption and derivative spectra obtained when a few crystals of iodine are permitted to equilibrate at 20 “C in a 15-cm absorption tube described earlier. The iodine concentration was estimated from vapor pressure of tables (14) to be about 330 mg/L. Each spectrum displayed is the average of 100 scans and the derivative spectrum was corrected for dark current but not for the background derivative [(dIo/dA)/Zo]. Derivative data were quantitated both as the peak values (peak amplitude above zero crossing) and the peak-to-peak values (difference between one peak or valley and next adjacent valley or peak). Precision data for absorbance (obtained without modulation) and derivative data a t the three labeled wavelengths (Figure 3) are presented in Table I. The relative imprecision of the absorbance and peak derivative values are comparable, but the imprecision of the peak-to-peak derivative values is only about 50% of that of the absorbance values. Although these linearity and precision data were encouraging, there were serious limitations in this system. UnANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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644 5
6027 WAVELENGTH ( n m )
Flgure 4. Integrals of the derivative spectra measured for neon emission from a hollow cathode lamp. A. Magnetically deflected vidicon. B. Electrostatically deflected vidicon. C. Electrostatically deflected vidicon with dark current correction
Flgure 3. Absorption (A) and derivative (B) spectra of Iodine vapor. The derivative ordinate is in arbitrary units distorted derivative data could be obtained only from a small segment of the wavelength axis at a time. Although any segment of the active surface could be selected by careful adjustment of the lock-in amplifier reference phase, the useful range for any particular setting was judged to be too narrow for general use. The distortion is believed to have resulted from three sources: namely, astigmatism in the concave grating dispersion optics on either side of the normal slit position, distortions in the electron optics which resulted in beam landing errors, and interactions between vertical and horizontal deflection coils. The astigmatism problem was confirmed by modifying the input optics to the monochromator and observing a predictable decrease in astigmatism, and the deflection coil interaction was confirmed by using small “pick-up” coils to observe noise and “ringing” superimposed on the modulation signal. These observations led us to consider an alternate experimental approach. Because the Tektronix 520 Rapid-Scanning Spectrometer incorporates a specially designed tube which incorporates electrostatic deflection and reportedly has improved focusing and reduced beam landing errors relative to more conventional tubes (15),and because we had one of these systems available, we elected to modify this instrument for derivative spectroscopy and evaluate its characteristics. The problems observed with the custom designed instrument were reduced with this latter system; the remainder of our more detailed evaluations were carried out on the modified commercial instrument. Modified Commercial Instrument-Electrostatic Deflection Tube. Systems used to evaluate this derivative 874
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-11 595
I
I
1
1
’
1
WAVELENGTH (nm)
1
’
I
1 I
637
Flgure 5. Derivative spectrum of neon emission from a hollow cathode lamp without dark current correction spectrometer included the neon emission from a hollow cathode lamp, bilirubin absorbance in the presence of a variety of interferences including albumin and hemoglobin, and absorption spectra of praseodymium. Comparison of Instruments. Our first goal was a comparison of the two instrument systems. Both instruments were used to measure the derivative spectrum of neon in a hollow cathode lamp; and then the emission spectrum was reconstructed by integration of the derivative spectra. The integrated spectra proved to be very sensitive diagnostics of distortion in derivative spectra. Figures 4A and 4B represent the emission spectra reconstructed by numerically integrating the derivatives obtained with the magnetic and electrostatic deflection systems, respectively. Dark current correction does little to improve the larger background signal apparent in the magnetic deflection system. However, it is clear from Figure 4C that the smaller background signal inherent in the modified commercial system is reduced still further by dark current correction. These and other experiments led us to focus our attention on the modified commercial system. All of the data reported below were obtained with this system. Linearity. Figure 5 represents the derivative spectrum of 11 neon emission lines in the range from 600 to 640 nm. Averages of four scans of these 11lines, attenuated by factors of 1.0 to 100 with neutral density filters, were used to evaluate the linearity of the system. The peak-to-peak derivative value for each line at each attenuation level was normalized to the
01
10.0
0
Flgure 6. Absorption and derbative spectra of bilirubin at two modulation amplitudes. a. Absorption, modulation amplitude = 6 . 7 % , b. Absorption, modulation amplitude = 53%. c. Derivative, modulation amplitude = 6 . 7 % . d. Derivative, modulation amplitude = 53%.
maximum value, and the results were compared to the transmittance values of these filters measured at the same wavelengths on a Cary 14 recording spectrophotometer. Linear regression of these data which ranged from 1.6% to 100% T yielded a slope of 1.OOO f 0.003, an intercept of 4.001 f 0.002, and a correlation coefficient of 0.9998, Standard deviations averaged 0.6% T for both instruments and the maximum deviation between results on the two instruments was 0.9% T a t 31% T. Considering all sources of bias in these experiments, these results represent excellent agreement between the two instruments. Modulation Amplitude. Because the wavelength modulation amplitude can effect an increase in the sensitivity of a derivative spectrum at the expense of resolution, we chose to examine this variable in detail. We have chosen to represent the modulation amplitude as a percentage of the apparent width at half height of the spectral bands being examined. For example, the apparent half width of the bilirubin spectrum (Curve a in Figure 6) is about 72 nm. Thus, 0.72 nm would represent 1% modulation for this band. The qualitative effect of the modulation amplitude on the absorbance spectrum of bilirubin is apparent from curves a and b in Figure 6 for which the modulation amplitudes were 6.7% and 53%, respectively. Curves c and d illustrate the effects of these same degrees of modulation on the amplitude of the derivative signal and emphasize the dramatic increase in derivative signal amplitude with modulation amplitude. Figure 7 demonstrates quantitatively the effect of modulation amplitude on the absorbance (Curve a) and derivative (Curve b) signals measured at the maximum for each signal. The derivative signal is observed to increase linearly with modulation amplitude until the modulation bandwidth becomes wide enough to cause the apparent absorbance to decrease from its narrow band width value. The decreased dependence of the derivative signal on the modulation amplitude results from both the decrease in the dI/dX term and the increase in the intensity term in Equation 1. The break in the derivative curve occurs at a bandwidth at or near 40% of the width at half height for the bilirubin band. A similar conclusion was reached in a recent theoretical study (16). The absorbance curve exhibits little if any dependence on the modulation amplitude up to about 40% modulation. This observation suggests that it is feasible to record both absorbance and derivative data simultaneously. It is reemphasized that the absorbance data included in Figure 7
60
MODULATION AMPLITUDE I:)
Figure 7. Effect of modulation amplitude on the absorbance (a)and derivative (b) values measured for bilirubin. For curve b, y = (0.261 f 0.008)~ 0.03 f 0.04,r = 0.998
+
i: x-
A
x-
x-
Figure 8. Effect of modulation amplitude on line spectra. A. Mercury lines at three levels of modulation amplitude. B. Interrogation pattern illustrating sampling of line spectra at different times by electron beam
represent values at the maximum in the absorbance spectrum and that values on either side of the maximum will show a greater dependence on modulation amplitude. Figure 8 illustrates another effect of bandwidth modulation, which we shall call peak splitting. The first frame in Figure SA represents emission signals for two mercury lines (404.7 nm and 407.8 nm) with about 0.24 nm modulation. The second and third frames show what happens as the modulation amplitude is increased to 0.72 nm and 1.2 nm. At 0.72 nm modulation the peak is broadened significantly while at 1.2 nm modulation, peak splitting has begun to occur. The three frames in Figure 8B are used to illustrate how this splitting occurs. The interrogation pattern is shown during three separate short time intervals. Initially (At.1)the leading edge of the scan pattern interrogates the wavelength channel (shaded area). During a later time interval (At3), the trailing edge of the scan pattern interrogates the same wavelength channel. At an intermediate time interval ( A t z ) ,only a small section of this wavelength channel is scanned. If the wavelength channel corresponds to an intense narrow line in a spectrum, the measured intensity will be reduced during the intermediate time interval. This behavior can be troublesome when a source containing line spectra is used for absorbance derivatives because the intensity terms in Equations 1 and 2 will be badly distorted. It is difficult to remove these effects completely with background correction and we used tungsten-halogen lamps instead of arc lamps to avoid this problem. Signal-to-Noise Ratio. It has been suggested that the signal-to-noiseratio (S/N) will usually be better for absorbance measurements than for derivative measurements (49).Three ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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1.5
0.
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I
I
I
I
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I
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b
Y
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z
s
K
2rn a
1.0 360
Figure 9.
WAVELENGTH (nm)
Y
U
z
m q
d
YI m
U
0.c
640
Absorption and derivative spectra of an aqueous praseo-
dymium solution
chemical systems were used to compare the S/N relationships for absorbance and derivative signals measured with the vidicon based spectrometer. The species chosen were praseodymium oxide (dissolved in hot dilute perchloric acid and diluted with water), bilirubin, and albumin which had relatively narrow, moderately broad, and very broad absorption spectra respectively. Preliminary work with the bilirubin spectra showed that the S/N values for absorbance data were essentially independent of the modulation amplitude up to about 40% modulation, and then increased slightly with increasing modulation amplitude. Accordingly, in order to simplify experimental procedures and to minimize effects of extraneous experimental variables on the comparisons, precision data were computed from simultaneously recorded absorbance and derivative data. Absorption and derivative spectra for praseodymium are included in Figure 9 and absorption and derivative spectra for bilirubin and albumin are included in Figures 10A and 1OB. The modulation amplitudes were 9.6 nm for praseodymium and 16.8 nm for the bilirubin and albumin data. All data reported here are based upon averages of 256 scans. Signal-to-noise ratios reported for praseodymium are averages of data collected a t the three wavelengths for each spectrum represented by arrows in Figure 9. Those for bilirubin absorption and derivative spectra were measured at 453 and 483 nm, respectively. Absorption and derivative data for albumin were collected a t the same wavelength between 370 and 500 nm because there is no maximum in either spectrum. Signal-to-noise ratios were determined at several values of absorbance and the results for absorbance and derivative values, were grouped into absorbance ranges and averaged using a weighting factor of (n- 1)'''. Results for the different solutions are presented in Figure 11. The S/N values for absorbances fall on a similar curve for all three solutions. However, there are significant differences among the S / N values for derivative signals, with S/N decreasing as the slope (da/dX) of the spectral band decreases. The precision of derivative data for bilirubin and praseodymium is observed to be equal to or better than that for absorbance data for these species. Ideally, the signal-to-noise ratio of the derivative signal would be expected to be about the same for all species if the ratio of the modulation amplitude to the slope of the spectra were kept constant. However, this is not achievable in practice for very broad band spectra such as that for albumin because the response features of the source and detector are sharper than those for albumin and increases in modulation amplitude 876
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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-1
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Figure 10. Absorption and derivative spectra for solutions of bilirubin and albumin. A. Absorption spectra; a. Bilirubin 0.855 pM, albumin 0.08mM. b. Bilirubin 0.855 pM, albumin 1.54 mM. c. Albumin 1.54 mM. E. Derivative spectra; a. Bilirubin 0.855 pM, albumin 0.08 mM. b. Bilirubin 0.855 WM,albumin 1.54 mM. c. Albumin 1.54 mM
5
Figure 11. Comparison of measured SIN of derivative and absorption Praseodymium, absorbance measurements. (0)Praseodata. (0) dymium, derivative measurements. ( 0 ) Bilirubin, absorbance measurements. (V)Bilirubin, derivative measurements. (A)Albumin, absorbance measurements. (A)Albumin, derivative measurements
required to achieve significant improvement in S/N would result in distorted derivative spectra.
0
ALBUMIN (mM1
1.6
Figure 12. Comparison of effect of albumin on absorbance and derivation signals for bilirubin. Bilirubin concentration = 0.5 mg/L. a, Absorbance signal (456 nm). b, Derivative signal (483 nm). I, The ordinate is the ratio of the signal at a given albumin concentration to that extrapolated to zero albumin concentration
Bilirubin. Bilirubin is used as an example to illustrate potential advantages of the derivative method compared to more conventional absorbance measurements. Figures 10A and 10B include absorption and derivative spectra of bilirubin and albumin as well as similar spectra for a solution containing both species. It is observed that albumin contributes a significant absorbance at all wavelengths, but that the derivative spectra of the bilirubin and bilirubin-albuminmixtures (curves a and b in Figure 10B) are virtually superimposed on the long-wavelength side of the derivative spectrum. This suggests greater selectivity of the derivative signal for bilirubin over albumin than is possible with absorbance measurements. To evaluate this effect quantitatively, measurements were made on solutions containing the same bilirubin concentration and variable amounts of albumin. Results presented in Figure 12 indicate that the derivative signal is affected much less by the presence of albumin than is the absorbance signal.
Similar studies on the effects of hemoglobin and turbidity on bilirubin derivative spectra were carried out with the goal of determining bilirubin in biological fluids. Preliminary data for synthetic solutions were very promising. However, attempts to determine bilirubin in sera were unsuccessful because of significant variations in spectral properties of bilirubin in sera and in available standards. Because of the sensitivity of the derivative spectrometer to peak position, this instrument could be very useful in studies aimed at understanding these spectral changes. Projections. Although a digital computer was used to compute absorbance derivatives from intensity and intensity derivative data, the operations in Equation 1 could be implemented with either analog or digital hardware to produce a “stand-alone” system. Also, derivative spectra can be computed from intensity data recorded in the conventional scanning mode (7). We are investigating these different approaches at this time and will report on the relative merits of each at a later date. LITERATURE CITED (1) W. Sneliman, T. C. Rains, K. W. Yee, H. D. Cook, and 0. Menis, Anal. Chem., 42, 394 (1970). (2) R. C. Elser and J. D. Winefordner, Anal. Chem., 44, 698 (1972). (3) R. N. Hager, Anal. Chem., 45, 1131A (1973). (4) J. W. Strojek, D. Yates, and T. Kuwana, Anal. Chem., 47, 1050 (1975). (5) G. Bonfiglioli and P. Brovetto, Appl. Opt., 3, 1417 (1964). (6) D. T. Williams and R . N. Hager, Appl. Opt., 9, 1597 (1970). (7) H. L. Pardue, A. E. McDowell, D.M. Fast, and M. J. Milano, Clin. Chem. (Winston-Salem, N.C.),21, 1195 (1975). (8) G. L. Green and T. C. O’Haver, Anal. Chem.. 48,2191 (1974). (9) T. C. O’Haver and G. L. Green, Anal. Chem., 48, 312 (1976). (IO) A. Savitzky and M. J. E. Golay, Anal. Chem., 38, 1627 (1964). (11) F. Grum, D. Paine, and L. Zoeller, Appl. Opt., 11, 93 (1972). (12) T. E. Cook, H. L. Pardue, and R. E. Santini, Anal. Chem., 48, 451 (1976). (13) M. J. Milano, H. L. Pardue, T. E. Cook, R. E. Santini, D. W. Margerum, and J. M. T. Raycheba, Anal. Chem., 48, 374 (1974). (14) A. N. Nesmeyanov, “Vapor Pressure of the Chemical Elements”, Eisevier Publishing Company, New York, N.Y., 1963. (15) J. Marrs, “Real Time Analytical Spectrometry”, FACSS Meeting, Indianapolis, Ind., Oct. 1975. (16) R. J. Hanisch and G. P. Hughes, Rev. Sci. Insfrum., 48, 1262 (1975).
RECEIVED for review October 8,1976. Accepted February 23, 1977. This work was supported in part by Research Grant No. GM13326-10 from the NIH, USPHS and by Grant No. CHE75-15500 A01 from the National Science Foundation.
Effect of Chloride Ion and Ionic Strength on the Response of a Copper(l1) lon-Selective Electrode G. B. Oglesby, W. C. Duer,” and F. J. Miller0 Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida 33 149
The potentiometric response of the copper(I1) ion-selective electrode (Ag2S/CuSmembrane)was investigated in aqueous solutions containing background electrolyte. For background electrolyte containing chloride, the electrode response, aithough linear with the logarithm of copper(I1) activity, was not Nernstlan. For background electrolyte which contained no chloride, the electrode response was Nernstian irrespective of background ionic strength.
The potentiometric response of the Ag,S/CuS copper(I1) ion-selective electrode has been the subject of some consideration in the literature (1-3). It has been shown that above
a certain copper(I1) ion concentration, the electrode exhibits Nernstian response in binary aqueous solution (1,3). It is also known that chloride ion in solution reduces the upper concentration limit of utility for the electrode because of a compositional change in the electrode’s solid phase (1). Further, the electrode has been used to determine copper(I1) in seawater although the response slope was larger than Nernstian (2). However, whether the increase in response slope is due solely to chloride ion or the accompanying increase in ionic strength has not been demonstrated. It is the purpose of the present communicationto document the response of the electrode in solutions which contain a single background electrolyte. To fulfill this purpose, the electrode responses were monitored as a function of ionic ANALYTICAL CHEMISTRY, VOL. 49, NO. 6,MAY 1977
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