ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
that device. while use of a reference channel monitoring" the dye laser fluctuations would further improve system performance. With these modifications to our apparatus, measurement of induced absorption of 10 or less i q very feasible. Under these conditions, the detection limits of ac-coupled inverse Raman spectrometry should be equal to or better than those obtainable by spontaneous Raman spectrometry.
1799
(7) J. Nestor, T. G. Spiro, and G. Klaumin, F'roc. Natl Atad. Sci , 73, 3328 (1976) (8) D Heiman, R W Hellwarth M D Levenson, and G Martin. Phys Rev L e t t , 36, 189 (1976) (9) M D Levenson and J J Song, J Opt Soc A m , 66, 641 (1976) (10) G L Eesley. M D Levenson, and W M Tolles, IEEE J QuantumEktron , 14 45 (1978) (11) W.J. Jones and 8. P. Stoicheff, Phys. Rev. Lett., 13, 657 (1964). (12) E. S. Yeung, J . Mol. Spectrosc., 53, 379 (1974). (13) A. Owyoung, Opt. Commun., 22, 323 (1977). (14) A. Owyoung and E. D. Jones, Opt. Lett., 1, 152 (1977). (15) A. Owyoung, IEEE J . Quantum Electron., 14, 192 (1978). (16) A. Owyoung and P. S. Peercy, J . Appl. Phys., 48, 674 (1977). (17) D. J. Wallan, G. P. Ritz, and M. D. Morris, Appl. Spectrosc., 31, 475 (1977). (18) A. Langseth and R. C. Lord, J . Chem. Phys., 6, 203 (1938). (19) G. P. Ritz. D. J Wallan, and M. D. Morris, Appl. Spectrosc.. in press. (20) J. H. Whinnery, Acc. Chem. Res., 7, 225 (1974). (21) S. H. Lin, E. S. Reid, and C. J. Tredwell, Chem. Phys. Lett.. 24. 389 (1974). (22) W. Werncke, A. Lau. M. Pfeiffer, H.-J. Weigmann, .:1 Hunsalz, and K. Lenz, Opt. Comrnun., 16, 128 (1976). (23) C.-W. Tsai and M. D. Morris, Anal. Chim. Acta, 76, 193 (1975).
ACKNOWLEDGMENT The authors thank Nestor Clough, Lexel Corporation, for the loan of the argon ion laser used in these experiments.
LITERATURE CITED (1) P. R. Regnier and J. P. E. Taran, Appl. Phys. Len.. 23, 240 (1973). (2) R. F. Begley, A. B. Harvey, and R. L. Byer, Appl. Phys. Lett., 25, 387 (1974). (3) L. A. Caniera and L. P. Goss, 32nd Symposium on Molecubr Specb-oscopy, Ohio State University, Columbus, Ohio, June 1977. paper WE-8. (4) P. K. Dutta, J. R. Nestor, and T. G. Spiro. Proc. Natl. Acad. Sci., 74, 4146 (1977). (5) W. M. Tolles, J. W. Nibler, J. R. McDonald, and A. B. Harvey, Appl. Spectrosc., 31, 253 (1977). (6) M. J. Levenson, Phys. Today, 30 ( 5 ) . 44 (1977).
RECEWED for review May 11, 1978. Amepted August 17, 1978. Work supported in part by National [nstitutes of Health grant GM-22604.
Computer-Controlled Dual Wavelength Spectrophotometer K. L. Ratzlaff' School of Chemical Sciences, University of Illinois, Urbana, Illinois 6 780 1
F. S. Chuang, D. F. S. Natusch, and K. R. O'Keefe" Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523
transmitted light intensities a t one wavelength for separate sample and reference cells, DWS uses light of' two different wavelengths which passes through a single sample cell. Operationally, the advantage of DWS derives from the fact that both beams experience the same sample environment. The implications of this characteristic have been considered in previous papers ( I , 2). The technique was first suggested and subsequently developed by Britton Chance for the determination of reaction rates in biological media (3-9); hobever. it was 1969 before the utility of DWS for equilibrium methods of chemical analysis was realized; a t that time, commercial DWS instruments became available (10-22) and applications were presented for analyses in the presence of spectral interferents (10-22). In the past, DWS instrumentation has been directed toward beam-modulated approaches with high modulation rates. In these techniques, the source illuminates two independent wavelength isolation devices (monochromators or filters) and the phase-separated outputs of these devices illuminate the sample. Another possible approach t o DWS involves wavelength modulation of a single monochromator. This is the method often used in derivative spectrometers. Regardless of the type of wavelength selection used, a dual wavelength spectrophotometer that is to be useful for a range of applications must incorporate a variety of features. These include the following. (1) The spectrophotometer must measure transmitted intensities at two separate wavelengths either simultaneously or alternately. If the latter, the modulation rate should be significantly faster than the half-time of the fastest reaction
The construction and evaluation of a computer-controlled dual wavelength spectrometer for use in making UV-visible molecular absorption measurements is described. An electromechanical modulator is used to step the grating of a Czerny-Turner configuration monochromator to either of two positions at a maximum rate of 2 Hz. A wavelength modulation nm is achieved. The precision of better than f2.5 X spectrophotometer is evaluated in terms of instrumental precision, linearity, and accuracy in typical measurement configurations and several applications of the instrument are described.
T h e measurement of electronic energy transitions in molecules in solution has found widespread application in chemistry with the major limitation of electronic transition (UV-visible) spectrometry being related to sample characteristics. Dual-beam-in-time or dual-beam-in-spaceand time spectrophotometers generally rely for their accuracy on the ability of the experimenter to prepare a reference solution that closely approximates the sample matrix. This ability is often quite limited when dealing with samples that are strongly scattering or that contain appreciable amounts of interferents that absorb in the wavelength region of interest. T h e technique of Dual Wavelength Spectrophotometry (DWS) represents a fundamental departure from conventional ratio spectrophotometry in this regard. Rather than ratioing Present address, Department of Chemistry, Northern Illinois university, DeKalb, Ill. 60115. 0003-2700/78/0350-1799$01 O O / O
C
1978 American Chemical Society
1800
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978 CERAMIC MAGNETS MAGNET MOUNT
SPRINGS
PUSH P I N A \ % s ’ N E
BAR
I INCH L_(
OUNTING PLATE
MICROMETER OUTPUT INTERFACE
TTY
PIN STOP-’ ~~~
, 1
MASS STORPG
Figure 1.
MINICOMPUTER
Block diagram of dual wavelength spectrophotometricsystem
t o be studied. In addition, the modulation frequency should be in a favorable signal-to-noise ratio (S/N) region for the source, detector, and associated electronics. T h a t is, t h e modulation frequency should be higher than the frequency components of instrument drift, but low enough to “average out” high frequency noise. (2) The wavelength step should be precisely adjustable over a wide range to allow a variety of applications and it should be possible t o scan the wavelength in a conventional manner t o allow “derivative” spectroscopy with a variable wavelength step size. (3) I n order t o maximize t h e signal-to-noise ratio, a large proportion of t h e measurement interval should be spent in actual spectral measurement as opposed to mechanically performing the modulation. (4) The instrument should be capable of conventional single wavelength spectrophotometry as well as DWS. ( 5 ) T h e instrument should provide high source intensity since in turbid samples much of t h e incident radiant energy is lost through scattering resulting in a reduced S/N. Wavelength-modulated instruments for molecular absorption have been employed only for derivative spectroscopy where the modulation amplitude need not be large, typically about 1 nm. T h e following methods of direct wavelength modulation have been employed primarily for derivative spectroscopy: (i) rotating t h e entrance mirror (23); (ii) vibrating the exit mirror (24);(iii) vibrating the Littrow mirror of a Littrow monochromator (25); (iv) “wobbling” (vibrating) the exit slit (26); (v) using a dual exit slit (27, 28); (vi) rotationally oscillating a quartz plate in the optical path (2S32); (vii) moving a refractor plate in and out of the beam at a n angle (33);(viii) slewing the grating drive motor between two positions (34);and (ix) rotating a n interference filter (35). With the exception of method (viii) all of these methods suffer from major disadvantages; thus they are limited to small wavelength excursions and/or they exhibit a nonlinear relationship between wavelength change and the amplitude of mechanical movement. The applicability of method (viii) above is limited by the rate a t which the grating can be moved using a drive motor. I n order to reduce or eliminate these problems while achieving the goals stated earlier, a new method of wavelength modulation has been developed and integrated into an automated, computer-controlled dual-wavelength spectrophotometer for use in the UV-visible region of the spectrum. This spectrophotometer has been applied to a variety of analytical problems with excellent results.
INSTRUMENTATION A block diagram of the DWS instrument is shown in Figure 1. The optical system is constructed using modules from the EU
--
~
PUSH ROD/ DRIVE PLATE ASSEMBLY
Figure 2. Modulator assembly for dual wavelength operation
700 spectrometer series (GCA/McPherson Instruments, Acton, Mass.) modified as described. The light source for measurement in the near UV-visible region of the spectrum is a tungsten halogen lamp (type SOOQ, Sylvania Inc., Danvers, Mass.) powered directly from the output of a harmonic neutralized constant voltage transformer (type CVS, Sola Electric Corp., Elk Grove Village, Ill.). The imprecision of photometric measurements due to the approximately 2% ripple in the source output caused by operating the lamp from an ac power supply and using an asynchronous measurement system varies from a maximum of about 0.1% for a 40-ms measurement time to a maximum of about 0.004% for a 1-s measurement time as calculated from the integral of a 60-Hz sine wave over these periods. These values can be reduced so that they make insignificant contributions to overall system imprecision by using measurement times which are exact multiples of the ripple period or by using a dc power supply. The source module also incorporates an externally controlled electromechanical shutter to facilitate computer-controlled dark current measurements. The wavelength modulated monochromator is a modified 0.35-m Czerny-Turner monochromator (EU-700, GCA/ McPherson). The sine bar drive of the commercial monochromator is adapted for wavelength modulation by the addition of an electromagnetic assembly which is mounted on the lead screw follower as shown in Figure 2. In the conventional drive, the sine bar push pin is held firmly against the lead-screw driven push rod by means of a spring. In the modified drive, the retaining spring is removed and the sine bar is free to move between limits set by the push rod and by a second pin stop that is mounted on the disk plate assembly. The position of the pin stop is adjusted by means of a micrometer head mounted on the mounting plate. The sine bar is moved between the two positions by action of magnetic attraction/repulsion. Two ceramic magnets (Edmund Scientific, Barrington, N.J.) are mounted on the sine bar in such a manner that they repel each other. The magnets are within the field of an electromagnet which when activated attracts one ceramic magnet and repels the other, thereby forcing the push pin against either the push rod or pin stop, depending upon the polarity of the dc voltage applied to the winding of the electromagnet. The electromagnet is powered by a computer-controlled bipolar power supply capable of supplying up to h20 V at 1 2 A. The position of the two stops determines the grating angles for the two states of the monochromator and, hence, determines the wavelengths of light that illuminate the sample cell. Because the push rod stop is not changed with respect to the conventional monochromator design, the wavelength selected in this position is as read out from the monochromator wavelength indicator. The wavelength displacement that occurs when the push pin is driven against the adjustable pin stop can be calculated from the design parameters of the monochromator, and is 76 nm wavelength displacement per centimeter of mechanical displacement when used with the standard 1180 line per mm grating. Because of design limitations, a maximum travel of 1.8 cm is available, resulting in a maximum wavelength modulation of 137 nm. Using a micrometer graduated in 0.0001-inch increments, a wavelength setability of 0.02 nm is available. The maximum modulation rate that can be achieved with this design is determined by the time required for the sine bar to move
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978 PtiOTOMULTiPLIER
a dark measurement, CD,is obtained. Measurements subsequently made for the same interval at XI and X2 yield results CAland C,, that can be substituted in Equation 1, viz.,
DS/IOP
gi
1 2 0 BIT COUNTER
DS/IOP’s DS/IOP
FuP FLOP
DS/IOP
1801
Figure 3. Transducer and signal processing circuitry. DS/IOP signals are sequencing signals from the computer. MSW and LSW are the most significant and least significant parts of the 20-bit count
to its new position when the polarity of the power supply changes and the time required for the sine bar to stabilize in the new position. With the design reported here, the travel time varies from 50-100 ms, depending upon the distance moved, and the settling time is about 50 ms as determined from observation of the photomultiplier tube output during modulation. The worst case modulation frequency is thus about 2 Hz assuming equal amounts of time are spent in movement and making a measurement. The addition of the wavelength modulator in no measurable way adversely affects the basic optical or mechanical characteristics of the monochromator. For example, the monochromator can be scanned exactly as with the conventional device if the electromagnet is energized to keep the pin stop against the push rod, or the wavelength can be modulated while scanning to obtain a “derivative” spectrum. Because of the 2-Hz maximum modulation rate, scanning to obtain derivative spectra must be quite slow. The programmable sample chamber used in this work is an EU-721-11 module (GCA/McPherson). This module allows either of two conventional spectrometer cells to be positioned in the optical path under external control, and provides feedback signals to the controller indicating when either cell is in the optical path. The detector is a 1P28A photomultiplier tube housed in an EU-701-30 module (GCA/McPherson) which incorporates a regulated high voltage power supply. The control element in the automated dual wavelength spectrometer is a PDP 8/L minicomputer with 8K words of core memory (Digital Equipment Corp., Maynard, Mass.), a dual drive floppy disk mass storage device (Sykes Datatronics Inc., Rochester, N.Y.), and input/output devices including a CRT display, line printer, Teletype, and plotter. In addition, a custom built interface provides control and data transmission between the spectrometer and the controller. Programming is done either in PAL 111, an assembly level language, or os/8 FORTRAN IV, both of which are supplied by Digital Equipment Corp.
SIGNAL PROCESSING It can be shown ( I ) that the dual wavelength readout, AA, is related to radiant power as
where P, and P2 are transmitted radiant powers and Pol and Po, are incident radiant powers a t XI and h2, respectively. A block diagram of the circuitry used to transduce the radiant powers to proportional electric signals, digitize the resulting signals, and present the resulting numbers to the computer is shown in Figure 3. In the configuration used in this work, amplifier A1 was a FET input operational amplifier (NE536, Signetics, Corp., Sunnyvale, Calif.), I1 was a high precision voltage-to-frequency converter (Model 4705/01, Teledyne Philbrick, Dedham, Mass.), and C1 was made up of five tristate counters (8554, National Semiconductor Corp., Santa Clara, Calif.). The number transferred to the computer by the circuit of Figure 3 is a count accumulated by the counter, C1, and is the sum of a term related to the radiant power illuminating the PM tube and terms that result from offsets in the analog electronics. If a measurement is made with no light illuminating the PM tube,
where CO,X1and Co,X2are the counts obtained with a “blank” solution in the optical path. Equation ‘2 is the basic equation that defines the dual wavelength measurement in the system described in the present work. Operationally, CDis measured with the source shutter closed, Co,X1and Co,X2are measured at X1 and X2 with a blank solution in the cell and the right-hand term of the right-hand side of Equation 2 is calculated. Subsequent measurements of CAIand C,, for any solution placed in the optical path of the spectrometer allow the calculation of AA for that sample.
PERFORMANCE AND RESULTS Modulator Precision. Modulation noise in wavelength modulated instruments has been considered previously ( I ) and it was shown that the uncertainty in the wavelength, q,,,gives rise to a variance in the measured value of AA, (rA2:
(3) In this expression, el and c2 are the absorptivities and M1 and M 2 are t h e slopes ( A nm-’) a t t h e two wavelengths, respectively. For this experiment, a peak of PrCl, in 2 M HC1 a t 444 nm was used. This peak has a width a t half-height of about 4 nm and a peak absorptivity of about 9.25 molar-’ crn-’. If X1 and Xz are located at wavelengths above and below the peak so that el = ez (AA = 0.0) and so that both M1 and M 2 are large, a good case for worst case modulation performance in molecular spectrometry results. T h e slits were opened to 400 ,um so that although photometric accuracy was lost ( 2 ) ,t h e precision was not photon limited. The apparent slopes, determined from a conventional dual cell measurement, were plus and minus 1.15 A nm-’. Since elbc = czbc = 1.05, uA nm. should be 8.0 X lo-, for u, = 5 X The observed standard deviations for three sets of 35 measurements were 0.40 X lo-,, 0.49 X lo-,, and 0.41 X lo-,. T o obtain an indication of whether or not those values were modulation noise limited, the modulator was over-ridden so t h a t t h e transmittance a t a single wavelength was ratioed against itself; this removes the modulation noise contribution t o the variance. A standard deviation of 0.41 x was obtained indicating t h a t U , was less than 2.5 X nm. Photometric Linearity. Photometric linearity of t h e instrument was estimated by measuring AA as a function of concentration for a series of dilutions of an appropriate standard. Basic chromate is a n often used absorbance standard and was selected for this study. A series of K2Cr04 solutions were made u p in 0.05 M KOH with nominal absorbances u p to about 1.1 a t 373 nm. Dual wavelength measurements were made a t 373 and 393 nm and a flow cell was used to reduce sample cell positioning errors. These results were compared with measurements made in a conventional absorbance mode. T h e results of these measurements are shown in Table I. T h e excellent linearity and low intercept for t h e measurements made in a conventional absorbance mode (column labeled A (373 nm)) are consistent with results obtained in other work (36) with similar equipment and indicate the utility of the spectrometer in this mode. Measurements in the dual wavelength mode result in a smaller intercept but slightly worse linearity than the conventional absorbance mode. The lower intercept probably results from reduction in systematic errors such as sample presentation and source drift, while the poorer correlation coefficient is due both to the smaller net absorbance range for the dual wavelength measurements and to poorer measurement precision. This conclusion is sup-
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
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Table I. Potassium Chromate Calibration Curves AA (373, A (373 nm) 393 n m ) relative concn 0 0.0006 0.0008 5 0.0563 0.0277 10 0.1115 0.0565 20 0.2261 0.1148 40 0.4523 0.2302 50 0.5640 0.2870 70 0.7917 0.4023 100 1.1238 0.5697 0.00061 0.00015 intercept slope 0.01258 0.00573 correlation 0.999990 0.999977 coefficient std error of estimate of concn on A or AA 0.15 0.21
L 0
1
2
I
Absorbance 01
Peak Moxlmum
Absorborce
ot Peak Mcximum
Figure 4. Instrurnent precision at various values of photon flux. Relative photon flux: Curve 1, 1; Curve 2, 8; Curve 3, 32; Curve 4 , 73; Curve 5, 120. (A) Base absorptivity 40% of maximum absorptivity. (6)Base
absorptivity 2 2 % of maximum absorptivity ported by comparison of the standard errors of estimate when concentration is regressed against A or 14. The slightly poorer standard error in the dual wavelength mode indicates that, under favorable measurement conditions, dual beam measurements are capable of higher precision than dual wavelength measurements, a condition predicted by earlier theoretical treatments ( I , 2). Photometric Precision. The photometric precision of the dual wavelength spectrometer was evaluated over a wide range of photon flux, both to attempt to determine the major noise sources inherent in dual wavelength measurements and t o define the ultimate precision expected with this instrument in different measurement situations. T h e photometric precision of the system was evaluated by making A d measurements on a series of PrCl, solutions made u p in 2 M HCl and having nominal peak absorbances a t 444 n m of 0-2.0 A. Measurements of AA were made with one wavelength set to the peak absorbance wavelength and the other wavelength selected such that the absorptivity wab 40% or 22% of the peak value. T h e photon flux was varied by changing the slit width of t h e monochromator. Slit widths from 50 to 1000 pm were used t o give a range of photon flux t h a t varied from a relative value of 1 to 120, with the maximum photon flux producing a photocurrent of about 1 PA. T h e results of these experiments are shown in Figure 4. T h e plot labeled a is that for the 40% peak absorptivity case and b is the 22% absorptivity case. T h e percent relative standard deviation (RSD) for 30 measurements on a solution with the peak absorbance shown on the abscissa is plotted in each case. The curves labeled 1-5 in each plot are for relative photon fluxes of 1,8, 32, 73, and 120, respectively. Previous work ( I ) has shown that error will be minimized at about 0.5, 0.9, and infinity for the “constant”, “square root”, and “proportional” error, respectively. In both plots, curve 1
1
-
O0
5
IO
Cu2’ Concentration (~10.~ M)
Figure 5. Spectra and analytical curves for copper-murexide system
experiences a minimum a t about 0.9, apparently due to “square root” error, suggesting photon-limited precision. However, a combination of “constant” and “proportional” errors could also produce that effect. hleasurement at higher photon flux does not support this contention, however, and the precision remains photon limited until the flux-independent “proportional” error due to source flicker is approached a t larger slit widths. Applications. Conventional D WS. One of the classic applications of DWS is the measurement of absorbing species t h a t are in equilibrium with each other in solution. An example of this is the measurement of copper concentration in solution by complexation with murexide (ammonium purpurate). In this case the spectra of the indicator (M) and the complex (CUM)have overlapping spectra as shown in the inset of Figure 5 , so the determination of the CUM is difficult using dual-beam spectrophotometry. The murexide system is especially useful for monitoring the amount of metal ion present in a variety of biochemical systems (37). The spectrometer was evaluated for conventional DWS by measuring Cu(I1) concentrations as the CUM species. Several solutions were prepared with various Cu(I1) concentrations and constant total murexide concentration and dual wavelength measurements were made with various wavelength pairs as shown in the inset of Figure 5 . In all cases the difference between the two wavelengths was 20 nm. The LA values presented are referenced t o a solution containing t h e same total murexide concentration as the samples to give the zero intercept. The slopes of the three analytical curves are different, as expected, and all are linear over the concentration range studied. Correlation coefficients of the three lines are 0.997,0.999, and 0.996 for A, R,and C, respectively, indicating good linearity even with a large, wavelength varying background absorbance. Analysis of Turbid Solutions. .4 second application f o r which the spectrophotometer was evaluated was the determination of a molecular absorbing species in the presence of solution turbidity. For this purpose, a series of K2Cr0, solutions was prepared as before with absorbances a t 373 nm u p to a nominal value of 0.9. Absorbance was measured a t 373 nm and the results are plotted as curve A in Figures 6a and 6b. A second series of solutions contained the same K2Cr0, concentrations, but a scattering material (a calcium caseinate suspension) was added so that the apparent absorbance due to turbidity was about 1.6. These solutions were intentionally prepared so the turbidity varied by f 1 0 % , resulting in the absorbance scatter shown in Figure 6b. The results of these measurements are illustrated in curve B of Figures 6a and 6b. When no turbidity is present (curve A in Figures 6a and 6b), good linearity is demonstrated for either conventional or dual wavelength measurements. However, when turbidity is
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
..
1
I
440
00 Relative Concentration of KICrOI
Figure 6. Analytical curves for K,CrO,
in the presence of turibidty. Curves (A) without and Curves (B) with turbidity. (a) Dual wavelength, (b) Single wavelength
440 460 480 Wavelength (nml
Figure 7. Spectrum and derivative spectrum of PrCI, with LA = 0.3 nm. (A) Spectrum. (B) Derivative Spectrum.
present (curve B), two effects on the analytical curves are noticed. First, an intercept is introduced in the dual wavelength case due to the wavelength dependence of the turbidity, but the intercept is much smaller than for the direct absorbance measurement. Secondly, the sensitivity to variations in turbidity is greatly reduced by use of dual wavelength as shown by the low scatter of the points on curve B of Figure 6a as compared to curve B of Figure 6b. Derivative Spectroscopy. Another application for which dual wavelength spectrometers find wide application is “derivative” spectroscopy, although the output obtained if one modulates the wavelength while simultaneously scanning is not a true derivative spectrum. The DWS spectrometer described herein was evaluated in two modes for derivative spectroscopy: a modulated/scanning mode and a numerical mode. Figure 7 shows the spectrum of PrC1, in 2 M HC1 from 425 to 485 nm (Figure 7A) and also the output of the spectrometer in the dual wavelength mode for a wavelength modulation of 0.3 nm at each wavelength (Figure i B ) . The latter plot is a
1803
.
.
I
1
- 1
460 480 Wavelength (nin)
Figure 8. Effect of baseline on derivative spectrum. Curves A and B are derivative spectra of PrCI, in HCI with and without baseline correction. Curve C is the baseline
I
440
I
I,
I
460 480 Wavelength (nm)
Figure 9. Differentiation by digital computation. First- (B) and second(C) order derivatives of PrCI, spectrum ( A )
dual wavelength “derivative” spectrum (AAIAA vs. A). The method used in this case to correct the spectrum for variations in source intensity is to inove the sample in and out of the optical path a t each wavelength and to determine the intensity ratio for the pair of wavelengths (Equation 2 ) . Previous instruments have employed cams (38)or used a series of potentiometers (39, 40) to adjust the baseline. The alternative in a digital instrument would be to store the baseline if sufficient memory were available and the source sufficiently stable. In Figure 8, the effect of failure to compensate for source variations with respect to wavelength is shown. The corrected (Figure 8A) and uncorrected (Figure 8B) first derivative spectra are shown with the uncorrected baseline (first derivative of the source, Figure 8C). An alternative method of obtaining the derivative spectrum is the use of numerical differentiation, for example, by the method of Savitzky and Golay (41, 4 2 ) . Curve A of Figure 9 is a spectrum of 2 M PrCl, in a solution containing colloidal
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
silica. The effect of the silica is to add turbidity and to cause the baseline to decrease monotonically a t longer wavelengths; however, this monotonic variation has a negligible effect on the derivative. The first derivative, curve B, was produced by digital differentiation as was curve C, the second derivative. Note the similarity of the curve obtained by this technique with that obtained using the modulation/scanning approach. In comparing the dual wavelength and digital methods, the digital method is often more convenient and probably is equally precise and accurate when on-line digital processing is available. I t is certainly faster since, in a dual wavelength system, every value for sample and reference is effectively measured twice as first XI and then X2 past the point. This is true no matter which of the baseline correction methods is used. Second, using digital differentiation, the higher order derivatives are immediately available if required.
LITERATURE CITED (1) K. L. Ratzlaff and D. F. S. Natusch, Anal. Chem., 49, 2170 (1977). (2) K. L. Ratziaff and D. F. S. Natusch, submitted for publication in Anal. Chem , (3) B. Chance, Rev. Sci. Instrum., 13, 158 (1942). (4) B. Chance, Rev. Sci. Instrum., 22, 634 (1951). (5) B. Chance, Science, 120, 767 (1954). (6) B. Chance, D. Mayer, N. Graham, and V. Legallais, Rev. Sci. Instrum., 41, 111 (1970). (7) B. Chance, D. Mayer, and V. Legallais, Anal. Biochem., 42, 494 (1971). (8) B. Chance, V. Legallais. J. Sorge, and N. Graham, Anal. Biochem., 66, 498 (1975). (9) B. Chance, N Graham, J Sorge, and V Legallais, Rev So Instrum , 43. 62 (1972) (10) S.Shiba'ta, M.'Furukawa, and K. Goto, Anal. Chim. Acta, 46, 271 (1969). (11) T. J. Porro, Anal. Chem., 44 (4). 93A (1972).
(12) R. L. Sellers, G. W. Lowry, and R. W. Kane, Am. Lab., March 1973. (13) S. Shibata, M. Furukawa, and K. Goto, Anal. Chim. Acta, 53, 369 (1971). (14) S. Shibata, M. Furukawa, and Y. Ishiguro, Anal. Chim. Acta, 62, 305 (1972). (15) S.Shibata, M. Furukawa, and Y. Ishguro, Anal. Chim. Acta, 65, 49 (1973). (16) S. M. Gerchakov, Spectrosc. Left., 4, 403 (1971). (17) S. Shibata, M. Furukawa, and T. Honkawa, A n d . Chim. Acta, 78, 487 (1975). (18) T. Ohnishi and S. Ebashi, J . Biochern. (Tokyo), 55, 599 (1964). (19) R. Rikmenspoel, Rev. Sci. Instrum., 36, 497 (1965). (20) R. J. De Sa and Q. H. Gibson, Rev. Sci. Instrum., 37, 900 (1966). (21) B. Hess, H. Kleinhans, and H. Schlvtter, Hoppe-Seyler's Z. physiol. Chem., 351, 515 (1970). (22) J. Rapp and G. Hind, Anal. Biochern., 60, 479 (1974). (23) M. Want, University of Illinois, Urbana, Ill., personal communication, 1974. (24) G. Bonfigiioli and P. Brovetio, Appl. Opt., 3 , 1417 (1964). (25) V. Hammond and W. Price, J . Opt. Soc. Am., 43, 924 (1953). (26) R. Hager, Anal. Chem., 45, 1131A (1973). (27) J. Kirkpatrick, P h D Thesis, University of Illinois, Urbana, Ill., 1969. (28) J. Defreese and H. Malmstadt, submitted for publication in Anal. Chem. (29) R. Elser and J. Winefordner. Anal. Chem., 44, 698 (1972). (30) I.McWilliams, Anal. Chem.. 41, 674 (1969). (31) D. Brinkman and R. Sacks, Anal. Chem., 47, 1723 (1975). (32) R. Spiliman and H. Malmstadt, Anal. Chem., 48, 303 (1976). (33) E. C. Stanley, Ph.D. Thesis, University of Illinois, Urbana, IiI., 1972. (34) T. O'Haver, G. Green, and B. Keppler, Chem. Instrum., 4, 197 (1973). (35) R . J. Sydor and G. M. Hieftje, Anal. Chem., 48, 535 (1976). (36) K. R. O'Keefe and H. V. Malmstadt, Anal. Chem., 47, 707 (1975). (37) L. Mila and B. Chance, Biochemistry, 7, 4059 (1968). (38) R. Woodriff and D. Schrader, Appl. Spectrosc., 27, 181 (1973). (39) Aminco Laboratory News, 29 (3), 2 (1973). (40) Perkin-Elmer Corporation, UV/FL Produce Department Technical Memo No. 1 (1970). (41) A. Savitzky and M. Goiay, Anal. Chem., 36, 1627 (1964). (42) J. Steinier, Y. Termonia, and J. DeRour, Anal. Chem., 44, 1906 (1972).
RECEIVED for review December 22, 1977. Accepted August 3, 1978.
Resolution and Instrument Line Shape Effects on Spectral Compensation with Fourier Transform Infrared Spectrometers Robert J. Anderson Department of Chemistry, Ithaca College, Ithaca, New York
14850
Peter R. Griffiths" Department of Chemistry, Ohio University, Athens, Ohio 4570 1
The effect of limited instrument resolution on spectral subtraction experiments performed using Fourier transform infrared spectrometers is examined from a theoretical standpoint and the predicted results are tested experimentally. For weak absorption bands, boxcar truncation is found to yield better compensation than triangular apodiration. For strong bands, whose peak absorbance is greater than one, there are certain conditions under which boxcar truncation leads to better compensation and others where triangular apodization is preferable. The results are discussed in terms of removing strong solvent bands from solution spectra, compensating atmospheric,absorption bands, and measuring the effects of intermolecular interactions. The effect of a triangular slit function on subtraction experiments performed using spectra measured with grating monochromators and the possibility of using other apodization functions to improve the results on Fourier spectrometers are also discussed.
One of the many advantages of having a digital data system as an integral component of an infrared spectrophotometer is the ease with which bands due to a single component can 0003-2700/78/0350-1804$0 1.OO/O
be subtracted from a spectrum having absorption features due to more than one component. A typical procedure for compensating bands due to a component, A, in the spectrum of a mixture involves separately measuring transmittance spectra (usually against an air reference) of the mixture and of a pure sample of A under the same instrumental conditions. The spectra are digitized, converted to a linear absorbance format, and stored. The absorbance spectrum of A is then multiplied by an appropriate factor and the scaled spectrum of A is subtracted from the absorbance spectrum of the mixture. The resultant spectrum should then correspond to that which would be measured if A were not present in the mixture. For N-component mixtures, the spectrum of each pure component can theoretically be obtained provided that N samples are available in which the relative concentrations of the components are varied ( 1 ) . Koenig (2) has given several interesting examples of the application of the scaled absorbance subtraction routine to problems in macromolecular chemistry, and procedures of this type are now routinely applied by most users of mid-infrared Fourier transform spectrometers. The validity of the scaled absorbance subtraction technique depends on two fundamental assumptions. First, the shape C 1978 American Chemical Society
