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ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
Time-Resolved Phosphorescence Spectrometry with a Silicon Intensified Target Vidicon and Regression Analysis Methods Douglas E. Goeringer and Harry L. Pardue" Department of Chemistry, Purdue University, West Lafayette, Indiana 4 7907
This paper descrlbes the development of a silicon intensified target vidicon camera system for phosphorescence studies and describes applications to room-temperature phosphorescence of salts of organic acids deposited on filter paper. The instrument permits time resolved spectra to be recorded with a minimum scan time of 8 ms/scan. Spectral decay data are processed by a variety of regression methods to obtain rate constants, lifetimes, and initial intensity. The methods include provision for multiple data rates, multiwavelength data for each component, and for an internal standard procedure that reduces effects of experimental variables. The internal-standard method reduces overall imprecision by factors of 2 to 5 depending upon the component. Results are reported for one-, two-, and three-component mixtures of 1-naphthoic acid, 2-naphthoic acid, 4-biphenylcarboxylic acid, 2naphthol-6,8-disulfonlc acid, and p-aminobenzoic acid.
Advances in instrumentation and methodology for phosphorimetry prior to 1975 have been reviewed ( I ) and the potential advantages of time-resolved spectra were mentioned. One instrument system designed for time-resolved phosphorimetry uses a computer to control stepwise changes in an emission monochromator with 100 decay curves being averaged a t each wavelength position ( 2 ) . The process requires about 1h to record a time-resolved spectrum of a component with a lifetime of 30 ms. While there have been numerous applications of imaging detectors t o other types of analytical spectrometry, e.g., spectrofluorometry (31, there have been no reports of applications of imaging detectors for phosphorimetry. This paper describes the design and performance characteristics of a system t h a t utilizes a silicon intensified target (SIT) vidicon detector with a pulsed source, and illustrates applications for room temperature phosphorescence of organic acids deposited on filter paper ( 4 - 7 ) . The system generates time-resolved spectra with the fastest scan repetition rate being 8 ms/scan and the longest time required to obtain a time-resolved spectrum being limited by the decay time of the phosphorescence process. T h e paper also describes some d a t a processing options t h a t are unique in comparison with procedures t h a t have been applied previously t o phosphorescence data. Unique features of the work include regression programs t h a t permit fits of many data points a t several wavelengths t o first-order decay models for one-, two-, and three-component mixtures, the use of a kinetic internal standard method t h a t compensates for variations in experimental variables such as sample preparation, sample matrices, sample placement, excitation source intensity and duration, etc., and a n iterative data processing program t h a t requires only initial estimates of decay rate constants and computes peak intensities t h a t are consistent with whatever rate constants are applicable for the conditions under which each sample is run. T h e paper includes results for one-, two-, and three-component samples of organic acids. EXPERIMENTAL Instrumentation. A block diagram of the instrument system is presented in Figure 1, and the system is discussed under 0003-2700/79/035 1 - 1054$0 1.OO/O
headings relating to major units which are the optical system, the SIT vidicon and associated circuitry, and the computer control system. Optical S y s t e m . The excitation source is a 300-W xenon flash lamp (N725, Xenon Corporation, Wilmington, Mass. 01887) in an air cooled housing (Model FH1298, Xenon Corporation) containing a mirror, M1, that refocuses radiation back onto the source, and a fused quartz lens, L1 (fil.5, 6-cm focal length), that collimates the excitation energy. Another fused quartz lens, L2 ( f / 2 , 10-cm focal length), is used to focus excitation radiation back onto the sample. The flssh lamp is driven by a pulser (Model 457 Micropulser, Xenon Corporation) operated at about 9 kV. In these experiments, the sample was irradiated with undispersed radiation from the flash lamp. The sample holder is similar to that described earlier ( 5 ) . The sample is positioned at the intersection of the excitation and emission optical axis and at a 45' angle to each axis. Dry argon is passed through stainless steel tubing and into the quartz cuvette in which the sample is held. Emission energy is collected by lens L3 cf/2,25-cm focal length) and focused on the slit, SL, of the spectrograph by lens L4 cf/3, 6.5-cm focal length). A high speed shutter, SH(Unib1itz Model 225L, Vincent Associates, Rochester, N.Y. 14607), with a total opening time of less than 6 ms is located between L4 and the spectrograph. This shutter is closed during the time the sample is being irradiated. The emission polychromator is a prototype version of an f / 3 spectrograph (UFS-200, Instruments, S.A,, Inc., Metuchen, N.J. 08840) and is designed to provide a flat field focal plane approximately 10 mm outside of the housing to facilitate applications with imaging detectors. The spectrograph is adapted to the SIT vidicon detector housing with a fitting that permits necessary wavelength and focusing adjustments. The spectrograph has a reciprocal linear dispersion of 24 nm/mm that displays a spectral range of about 300 nm of the 12.5-mm wavelength axis scanning format used with the SIT vidicon. Energy throughput was increased at the expense of resolution by replacing the standard 50-pm slit with a 5OO-bm slit that increased the bandpass t o 12 nm. The spectral range used in most of these studies was 360 to 660 nm. SIT V i d i c o n and Circuitry. This discussion is limited to the most important features of the SIT vidicon camera system; circuit schematics can be provided upon request. The intensifier section of the SIT vidicon (4804/PS, Radio Corporation of America, Lancaster, Pa. 17604) is operated at -8 kV and the focus/gating electrode is operated at 98% of the photocathode voltage by a resistive voltage divider. The silicon vidicon circuitry is similar to that used in other studies ( 8 , 9 ) and the scan format consists of a 31.25-kHz vertical scan format and horizontal scan times adjustable between 8.192 and 65.54 ms in steps of 8.192 ms. The horizontal (wavelength) axis is scanned in 256 steps with data recorded at 125 wavelength positions per scan. These and other functions are controlled by a digital computer as discussed in the next section. C o m p u t e r and Control Circuitry. A Digital Equipment Corporation PDP-B/M computer configured as described earlier (9) was used for system control and data acquisition control functions and data acquisition cycles are best discussed with the aid of the timing diagram in Figure 2. Prior t o each run, the target is scanned repetitively with the flag FF, fire FF, and ADC F F all cleared and the shutter closed. The horizontal scan counter produces an end-of-scan pulse (EOS PLS) at the end of each horizontal scan and these EOS pulses are used to synchronize other functions. An experiment is initiated by a manual switch that triggers the FLAG OS which signals the 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979 LP
L
MI
!
POWER SUPPLY
I
1055
,SA
,
POWER EMISSION
where I , is the intensity a t time t at the selected wavelength, and IO,A is the intensity a t t = 0. To facilitate the inclusion of data from different wavelengths, we define a term, xx = IA/IA(max), which is the time independent ratio of the intensity a t any wavelength to the peak intensity. Using this term, Equation 1 then becomes
SUPPLIES
This expression has the advantage that intensity data a t any wavelength can be referenced to the peak wavelength. For a two-component mixture, Equation 2 can be written as COMMAND
DlFF AMP
REAL -TIME
DECODER
INTERFACE COMPUTER TAPE
POP 8 / M
Figure 1. Block diagram of SIT based time-resolved phosphorimeter PLS
ADC F-F
n-7
-
Figure 2. Timing diagram for data acquisition sequence
computer that the experiment is initiated. The computer then sets the FLAG FF, and when the next EOS flag is sensed, it sets the flip-flop that controls firing of the lamp (FIRE FF) so that the FIRE OS will be triggered and the lamp will be fired when the computer senses the next EOS flag. This first flash is used to prime the lamp and no data are collected. After the priming flash, the computer counts 256 EOS flags and then causes the lamp to fire for a run on the next EOS flag. The FIRE FF is cleared and after the next EOS flag, the computer clears the shutter flip-flop (SHTR FF) to open the shutter. Following the next EOS flag, the computer sets the analog-todigital converter flip-flop and data are acquired a t a software controlled rate. The frequency with which data are collected is controlled by counting EOS flags. In the specific example illustrated in Figure 2, data are collected on each alternate scan; however, any number of scans can be skipped between conversions so that data acquisition rates can be adjusted to be appropriate for different decay lifetimes. In experiments with different species with different lifetimes, it was convenient to change data acquisition rates during experiments. Data rates were adjusted so that equal numbers of data points are collected during four half-lives of each phosphorescent decay process (10). It is important to note that different data rates are selected by skipping wavelength scans and not by changing the scan frequency. D a t a Processing. A major goal of this work was to use both temporal and spectral data to analyze data for single-, two-, and three-component mixtures in a manner analogous to that reported by Ridder and Margerum (IO). Because many of the curve fitting procedures used in this work have been discussed in detail earlier (10-12), these procedures are discussed briefly here. The decay curve at a given wavelength, A, for any given species is represented by
and an analogous equation can be written for three components. For single component runs, Equation 2 is converted to the logarithmic form and In (IO,h(max)) and k are evaluated from a linear-least-squares fit in In ( I t , h / ~ h ) vs. t . For two components in which both k , and k 2 have been previously evaluated, the linearization procedure described earlier ( I I) is used and lIo,A(max) and 210,Aimax) are evaluated from a least-squares fit of I,, vs. lXxe-kl' and 2X,e-k2r. For two component samples in which the initial and rate constant, k Z ,for only one component intensity, 210,himax), are known, Equation 3 can be rearranged to give
which can be solved explicitly for In lZo,Aim) and k l by the method of least squares using It,hvs. t data. For two or more components in which neither initial intensities nor rate constants are known for any species, we used Taylor's Series expansions of the exponential terms in Equation 3 and iterative procedures that include Marquardt's compromise discussed earlier (12) to evaluate best estimates of rate constants and intensities. When this procedure was used, first estimates for initial intensities were obtained by application of one of the previously described methods using estimates of rate constants obtained from single component experiments. This method was used primarily with internal standard procedures described later so that effects of experimental variables that could affect rate constants would be compensated for in the final results. Sample Preparation. The organic acids, 1-naphthoic acid (1-NA), 2-naphthoic acid (2-NA),4-biphenylcarboxylic acid (4-BI) (Aldrich Chemical Co., Inc. Milwaukee, Wis.), 2-naphthol-6,8disulfonic acid, dipotassium salt (2-DL) (Eastman Organic Chemicals, Rochester, N.Y.), and p-aminobenzoic acid, sodium salt (PABA) (Sigma Chemical Co., St. Louis, Mo.) were all used as received. Desired amounts of each acid were dissolved in 0.2 mol/L sodium hydroxide (Matheson, Coleman, and Bell Manufacturing Chemists, Norwood, Ohio) and individual samples were prepared by spotting 5 pL of the solution on 5 mm X 10 mm strips of filter paper (Eaton Dikeman 613). Each sample was air dried for 10 min and then placed in a desiccator over Drierite for 10 to 12 h. Procedure. Each sample was transferred from the desiccator to the sample holder and remained under a stream of dry argon (passed over Drierite) for 1 min before phosphorescent spectra were recorded. Sixty spectra consisting of 125 wavelength points per spectrum were recorded for each sample during a period that depended upon the decay rate constant as discussed earlier. Three such experiments were averaged for each sample except for the three-component mixtures where results for five samples were averaged. AU spectra were corrected for dark current and amplifier offset; the blank signal from the filter paper was negligibly small. All data reported below are based upon a single wavelength for each component except that the normalization factors, x,, in Equations 2-4 are used to compute deconvoluted spectra for individual components in mixtures.
RESULTS A N D DISCUSSION P e r f o r m a n c e Characteristics. Because there are relatively few data published on characteristics of SIT vidicons for analytical spectroscopy, it was necessary to evaluate t h e
1056
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
L 2-lLLU-4 5..
1 I
-4-Ad+L.L
&*J444L
IC
s : t v . r,wt\-
9
I-.
m?
L+
Flgure 3. Effects of slgnal amplltude and slgnal averaglng on SIN for SIT detector. Slngle run: (A)Slope = 0,98 f 0.09;S, = 0.04;r = 0,995; ( 0 ) Slope = 0,46f 0.08;S, = 0,07; r = 0.96. Average of 100 runs: (0)Slope = 0.98 f 0.15; S, = 0.07; r = 0.99; (0)Slope = 0.50 0,08;S, = 0,07; r = OS7
*
performance of the camera system prior to applying it to phosphorescence studies. Results of that evaluation are reported here, Wavelength Calibration, Resolution. A mercury pen lamp was used to calibrate positions along the horizontal axis in wavelength units and to evaluate the resolution of the system. A linear least-squares fit of wavelength VI. position for mercury lines at 366, 405, 436, 546, and 578 nm gave an intercept of 358 f 3 nm and a slope of 1.20 f 0.03 nm/step where there are 256 steps along the horizontal axis. The correlation coefficient and standard error of estimate (s,) for the fit were 0.99993 and 1.3 nm, respectively. With a 50-pm wide by 8-mm high slit, the full width a t half maximum (fwhm) for typical peaks was about 3 steps corresponding to fwhm of 3 steps X 1.2 nm/step or about 3.6 nm. The expected value based on the manufacturer's specifications is about 4 nm. When the slit height was reduced to 1.5 mm, the resulting fwhm decreased to 2.4 nm compared to an expected value near 1.2 nm suggesting that the S I T detector is imposing limitation on the ultimate resolution that can be expected with these small slits. Phosphorescence studies were conducted with a 500-pm slit such that the fwhm is limited to about 1 2 nm by the reciprocal linear dispersion of 24 nm/mm. Linearity. T h e linearity of the system response was evaluated with a precision optical attenuator ( L T / S Model 2001, Technometrics, Inc., Lafayette, Ind. 47907). Results reported are the average of 32 scans at each intensity, and the gain settings were changed for each decade change in intensity. Regression equations of measured absorbance (y) VI. expected absorbance ( x ) were y = (1,009 f 0 . 0 1 ) ~- 0.003 f 0.025 with r = 0.9998 and s, = 0.025 for 19 equally spaced points between 0.3 and 4 absorbance units at 450 nm and y = (0.993 f 0.009)~ 0.02 f 0.02 with r = 0.9998 and s, = 0.023 for similar experiments at 500 nm. When the absorbance was changed over a range of 0.3 to 3.5 at 450 nm and measurements were made with fixed gain, the regression equation was y = (1.014 f 0.009)~- 0.016 f 0.017 with r = 0.9999 and s, = 0.012. For this fixed gain experiment, the measured absorbances began to deviate significantly from expected values above 3 absorbance units. While t h e detector is linear over about four orders of magnitude of intensity, the 12-bit ADC limits the linear dynamic range to about three orders of magnitude at fixed amplifier gain. This was judged to be adequate for phosphorescence studies. Noise Characteristics. T o evaluate the signal-to-noise ratio (S/N) characteristics of the system, repetitive runs were made at 450 nm with different intensities set by an optical attenuator. In one set of experiments, standard deviations were computed from ten replicate runs at each signal level, and the average standard deviation and average signal were computed from five such runs. In a second set of experiments, a similar
+
Flgurr 4. Effect of slgnal amplltude on response tlme of the SIT vldlcon. = (-0.48 f 0.03)~ 2.8 f 0,05;S, = 0,07; r = 0,96
y
+
procedure was followed, except that the ten replicate runs from which standard deviations were computed were each based upon averages of 100 runs. Results, including regression statistics, are presented in Figure 3. For single-scan and 100-scan ensemble average experiments, the 95% confidence level current detection limits are about 0.4 nA and 0.025 nA, respectively; S / N increases linearly with signal up to about 10 and 2 nA, respectively; and S/N increases linearly with S1/* for currents above 10 and 2 nA, respectively. The S1I2 dependence extends to lower currents when 100 ensemble averages are used because a random component of the amplifier noise that predominates a t lower currents is reduced by signal averaging. Other experiments were carried out to evaluate the effects on S / N of summing signals from different wavelengths. Results fm the low current range where the system is amplifier noise limited (see Figure 3) are similar to those reported earlier (13) for a silicon vidicon (Figure 2, Curve D (13))while results for higher currents are similar to those reported for an image dissector with 128 ensemble averages (Figure 2, Curve B (14)), and are not presented here. Dynamic Response. In order to evaluate the effect of signal level upon the time required to obtain a signal that accurately represents source intensity, the electronic shutter was used to apply step functions of increasing and decreasing light intensity at different intensity levels. Figure 4 shows the effect of signal level on the time required for the target to be charged to 95% of the maximum value for different scan times when the tube goes from dark to illuminated conditions quickly. Virtually identical data obtained for the decrease of current to 5 % of the maximum value (light to dark) gave a slope of 4 5 0 f 0.05 and an intercept of 2.86 f 0.06 in good agreement with the data in Figure 4, These data lead to an expression of the form
tB6= 6.3
X
102/S1'2
for the time required to recharge the vidicon surface to 95% of its final value, where S is the signal current expressed in nA and t is expressed in ms. The response curve approaches first-order behavior so that the more conventional risetime (90% response) would be approximated by teo N 4.8 X 102/S'J2, What these observations mean is that for a fixed intensity and fixed electron beam flux, the time required to recharge the active surface will be fixed, and the faster each individual scan, the more scans will be required to read an accurate signal. They also mean that during the time scale of phosphorescence experiments to be conducted in this study, or stopped-flow experiments carried out in other studies, the silicon vidicon detectors d o not reach what might be considered dynamic equilibrium during the transient observations. Rather, they operate in some sort of steady-state charge/discharge condition away from equilibrium, and the reliability of kinetic
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7 , JUNE 1979
Table I. Effect of Sample Positioning and Ensemble Averaging on Relative Error ensemble rate constant peak current sample avk, RSD, RSD, position erages 8-l %O I , nA %" fixed fixed repositioned
1 6 6
1.01 0.94 0.94
3.7 1.9 2,O
17.39 17,91 14.09
I
1057
A
11.3 8,3 10.6
Based on five runs, observations will depend upon the rate of processes being monitored. Ridder and Margerum (10) have examined this phenomenon and noted that first-order processes with rate constants of 54 smlor smaller can be monitored reliably but t h a t vidicon detectors will introduce distortion into systems with larger rate constants. Phosphorescence processes included in this study have rate constants well below this upper limit and detector lag is not expected to be a significant factor. Analytical Results. Five organic acids, 2-naphthoic acid (2-NA), 1-naphthoic acid (1-NA), 4-biphenylcarboxylic acid (4-BI), 2-naphthol-6,8-disulfonic acid (2-DL), and p a m i n o benzoic acid sodium salt (PABA), were used in a variety of experiments to evaluate the S I T based system for phosphorescence studies. Pure Components. The camera system and measurement approach were evaluated initially with single component studies on each of the five acids. Figures 5-A and 5-B represent selected time resolved spectra and a decay plot for 2-NA t h a t has one of the more complex spectra and slower decay rates, and Figures 6-A and 6-B represent similar data for PABA t h a t has simpler spectra and a faster decay rate. Spectra and decay curves for the other acids are similar to those shown for 2-NA and PABA. Rate constants, lifetimes, and ,peak intensities were obtained by fitting these data to a first-order model. A 0.5-pg sample of 2-NA was used to evaluate effects of ensemble averaging on within-run and between-run errors. Results presented in Table I show that ensemble averaging can improve the reliability of rate constants, but has a relatively small effect on the between-run imprecision of intensity measurements even when the sample is not repositioned between runs. Similar results obtained without ensemble averaging or repositioning of the samples are presented in Table I1 for 2.5 pg of each of the five acids. Within-run errors that reflect the smoothness of individual response curves (Figures 5-B and 6-B) are observed to be in the range of 0.5 to 270, However, between-run errors that reflect variations in the excitation source are observed to be in the range of 1 to 5% for rate constants and 6 to 18% for peak intensities. These data show that the detector performance is significantly better than the supporting methodologies for these acids.
\ $1 Bt
9
gb
---
2, 1%
-.--
~
~L----&---...-_ i -
- =
&
mo Flgure 5. Tlme resolved spectra and decay curve for 2-naphtholc acid. (A) Top to bottom: Spectra at 286.7, 491 5, 696.3, . ., 2539.5 rns @ 40.96 ms/scan '!E 1*6FZ'
0
3. A \
o-L...--.-.---___
*
.13-P Figure 8. Tlme resolved spectra and decay curve for p-amlnobenrolc acld. (A) Top to bottom: Spectra at 114.69, 196.61, 278.53, , . , 1015.81 ms @ 16.384 ms/scan -
':Y r%k
T h e linearity of signal current with amount of acid was evaluated for 2-NA, 1-NA, and 4-BI in the range from 100 to 1000 ng with two data processing options. In one option, a
--Table 11. WithinmRun and Between-Run Uncertainties for 2.5 pg of Each of the Five Acids within-run 92 ~m,, lifetime, acid 11 m k * SD,a s-l I T , ms I , ?r SD,O nA k 61.9 i 11.4 0.56 1-NA 526 1.88 i 0.06 633 * 14 0.61 618 1.10i 0.05 909 3 4 2 68.6 ?: 6,s 2-NA 0.24 4-BI 485 1.16 i 0.03 864 i 22 194 * 1 2 2,2 97 i 1 3 PABA 430 3.04 k 0.15 330 i 1 7 0.8 226 i 3 16.7 3 0.4 641 4.43 i 0.05 2-DL a
Standard deviation €or four runs.
RSD,
between-run RSD, 5%"
I,,
k
0.62 0.87 0.23
2.7 4.6
2.11 0,86
6.2
18.4 11.6 6.2 13.2
1.2
6.1
2.6
I"
1058
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
Table 111. Regression Results for Intensity vs. hlass Data' correlation compd
slope
SD, nA/ng
i
intercept
i
SD, nA
std error, nA ordinate range, nA
coeff
Single point, fixed time 1-NA 2-NA 4-BI
0.028 i 0.0017 0.023 i 0.0008 0.089 + 0.0043
2.2 1.4 3.1
i
*
t
0.52 0.25 1.33
0.49 0.23 1.26
3-14 2-1 2 10-45
0.997 0.999 0.998
Multipoint regression 1-NA 2-NA 4-BI
0.044 t 0.0027 0.033 t 0.0012 0.108 i 0.0052
2.8 t 0.84 1.7 3 0.37 3.7 i 1.63
Internal standard (intensity ratio vs. ng-' unitless 1-NA 2-NA 4-BI 4-BI 4-BIC
0.0022 0.0013 0.0043 0.0018 0.0025
i
t
0.00005 0.00004
i
0.00008
2
0.00016 0.00012
i
-0,079 0.026 -0.042 0.107 0.079
i i
*
f
?:
0.80 4-22 0.35 3-1 7 1.55 11-54 mass (ng))-two component unitless unitless
0.015 0.011 0.026 0.049 0.039
0.014 0.010
0.024 0.046 0.037
Internal standard (intensity ratio vs. mass (ng))-three 1-NA 4-BI a
0.0055 0.0084
100-500 ng of each acid.
ng- ' i 0.00026 i 0.00014
uni tless -0.019 t 0.057 0.034 i- 0.015
Peak wavelengths for 4-BI and 1-NA.
Flgure 7. Peak intensity (computed from a fit of I vs. t data to a first-order model) vs. analyte concentration. (A)2-NA; y = (0.033 f
0.001)~ -t 1.7 f 0.4; S, = 0.34; r = 0.999; (0) 1-NA; y = (0.044 f 0 . 0 0 3 ) ~-I-2.8 f 0.8; S, = 0.8; r = 0.996; (0) 4-BI; y = (0.108 f 0 . 0 0 5 ) ~ 3.7 f 1.6; S, = 1.6; r = 0.998
+
fixed time approach was used in which the intensity after 327.68 ms for 2-NA, after 245.76 ms for 1-NA, and after 229.38 ms for 4-BI was measured. In the other approach, intensity vs. time data a t A,, were fit by a regression method to a first-order model so that projected values of peak intensity were obtained. Figure 7 shows data obtained with the regression method for the three acids. The projected peak intensity increases linearly with concentration up to about 500 ng for each species after which the plot curves toward the mass axis. Similar behavior was observed for the other data processing option. The detector is linear to much higher intensities and the nonlinear behavior above 500 ng is almost certainly related to other aspects of the system or phosphorescence process (6). T h e solid lines represent least-squares fits of the data between 100 and 500 ng, and statistical parameters for both data processing options are included in Table 111. Sensitivities for 1-NA and 2-NA are comparable near 0.03 to 0.04 nA/ng while that for 4-BI is 2 to 3 times larger. Using the standard error as a measure of the scatter about the line, detection limits (a95% confidence level) for the three acids are estimated
unitless 0.03 7 0.010
0.22-1.1 0.13-0.7 0.43-2.1 0.18-0.9 0.25-1.2
0.996 0.999 0.999
0.9995 0.9992 0.9996 0.9 93 0.998
component uni tless 0.55-2.7 0.84-4.2
0.999 0.9999
Short wavelength shoulder for 4-BI.
Figure 8. Normalized spectra for PABA, 4BI, and 1-NA mixture. PABA: 2.5 pg; Ix(mx) = 97 nA. 4-BI: 2.5 pg; Ix(mex,= 194 nA. 1-NA: 2.5 19; IA(mx, = 62 nA
to be 36,21, and 29 ng for 1-NA, 2-NA, and 4-BI, respectively, and these values are generally consistent with values of 28, 28, and 19 ng, respectively, based on standard deviations of 100 ng samples of each component. While these data show t h a t it is feasible to perform phosphorescence analyses with the system described to this point, they also show that there are serious problems resulting from experimental variables other than the detection system. T h e principal sources of difficulty are fluctuations in the excitation source, preparation of samples, and positioning of samples. A traditional method for reducing the effects of such problems is the use of an internal standard. Although we are not aware of applications of the internal standard approach with either phosphorescence methods or solution kinetic methods in general, multiwavelength data available with the SIT system, and the multicomponent kinetic data processing methods developed in this laboratory (10-12) appear to make this a feasible approach. Internal-Standard M e t h o d , T w o Components. The internal-standard method was evaluated for four two-component combinations; three combinations contained 500 ng of PABA as the internal standard and variable amounts (100-1000 ng) of 1-NA, 2-NA, and 4-BI as analytes, and a fourth combination contained 750 ng of 1-NA as the internal standard and
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7 , JUNE 1979
1059
Table IV. Uncertainties for Phosphorescence Measurements with and without Internal Standard wavelength, nm rate constant, s - ' relative std. deviation internal analyte standard analyte std analyte std within-run between-run 2-NA 2-NA 2-NA 2-NA 1-NA 1-NA 4-BI 4-BI 4-BIC 4-BId
PABA PABA None PABA None PABA None PABA 1-NA 1-NA
518 51 8 51 8 518 526 5 26 485 485 485 469
430 430
... 430
...
430
...
430
1.10 1.10 1.10 1.10 1.88 1.88 1.16
3.04 3.04
... 3.04
...
3.04
...
3.04
5 26
1.16 1.16
1.88
526
1.16
1.88
2. ga 2.6b 0. 4' 2. 7' 0. Ba 3. 7' 0. 5a
4.Za 3.6' 4.2O
2. oa
2.lb 10.oa
2. oa 13.4a 3. 3a 8.'iQ 3.Ba A. 1Q 3. 2a
Average a Average % RSD for three runs each on three different samples at each of six mass amounts (100-1000 ng). Peak wavelength for 4-BI. Short wavelength shoulder % RSD for three runs on each six samples (i00-1000 ng). for 4-BI.
Figure 10. Unresolved and deconvoluted spectra for mixture of PABA, 4-61, and 1-NA at 196.61 ms. (1) Mixture: 250 ng PABA; 100 ng 4-BI; 200 ng 1-NA. (2) PABA. (3) 4-BI. (4) 1-NA Figure 9. Time resolved spectra for 2-NA/PABA mixture. Data rates of 16.384 ms/scan and 81.920 rnslscan 100-1000 ng of 4-BI as the analyte. Figure 8 illustrates the spectral properties of the combinations examined. It is noted that there is relatively little spectral overlap near the peak intensity wavelengths for 1-NA and PABA and this is typical for 2-NA and 4-BI with PABA. However, there is significant spectral overlap for 4-BI and 1-NA. Analyses for all combinations were based on data at peak wavelengths for analyte and internal standard and analyses for 4-BI with 1-NA were also based on data at the short-wavelength shoulder on the spectrum for 4-BI. Although most wavelength combinations give reasonably good spectral resolution, there is some overlap in all cases and all combinations will depend somewhat upon kinetic differences for component resolution. The peak wavelength combinations for 4-BI and 1-NA will depend almost exclusively upon kinetic differences for component resolution. Figure 9 represents a typical intensity vs. time vs. wavelength response surface for 2-NA with PABA. This plot shows the change in data rate used for different rates of decay. In the analytical procedure, a multiple linear regression program is used to project the intensity values at selected wavelengths back to zero time, and analyte concentration is computed from ratios of initial intensity for the analyte to the initial intensity for the internal standard. Plots of intensity ratio vs. analyte mass are similar to the plots in Figure 'i and regression equations for the linear regions of the plots (100-500 ng) are included in Table 111. T h e internal-standard method does not improve the range of linearity; its principal advantage is in the improvement of the between-run precision of results as illustrated by data summarized in Table IV. T h e most important observations to be made from the data in Table IV are that the within-run and between-run errors
are very similar when internal standards are used and that errors for results with internal standards are 2 to 5 times smaller than are the errors for results without an internal standard. The first two entries for 2-NA show that sample-to-sample variations have little effect on the imprecision of results. The last two entries for 4-BI depend much more upon kinetic resolution and much less on spectral resolution than do the other combinations. Uncertanities are not judged to be significantly different for 4-BI with the different internal standards suggesting that kinetic differentiation is effective. Comparison of the within-run errors for single component samples with the within-run errors for two-component samples (analyte with internal standard) provides a direct measure of the loss in precision resulting from the spectral/kinetic differentiation among species. The degradation of within-run precision is more than compensated by the improvement in between-run precision given by the internal-standard method. Internal-Standard M e t h o d , Three Components. The internal-standard method was evaluated for one combination of three components in which 250 ng of PABA was used as the internal standard and 50-250 ng of 4-BI and 100-500 ng of 1-NA were used as analytes. Figure 8 shows the normalized spectra of pure components. While the peak intensity wavelength for PABA is reasonably well isolated from the other components, there is significant overlap between 4-BI and 1-NA, and differentiation between these species depends heavily upon kinetic differences as noted earlier. T h e rate constant ratios, kpABA:kl.XA:kQ.BI were 2.6:1.6:1.0. T h e upper plot in Figure 10 is a typical spectrum of a three-component mixture and the three lower plots represent deconvoluted spectra for the individual components based upon kinetic determination of peak intensities and computations of intensities a t other wavelengths from these peak intensities and ratios computed from pure component spectra.
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1 9 7 9
The deconvoluted spectra are similar to spectra shown for pure components. Plots of intensity ratios (ana1yte:internal standard) showed the same nonlinear behavior observed for one- and twocomponent samples. Regression data for the linear regions of the three-component samples are included in Table 111. Some comparisons can be made among the three data sets in Table 111. First, because the amount of PABA in the two-component samples was twice that in the three-component samples (500 ng vs. 250 ng), the slopes for 1-NA and 4-BI are expected to differ by ratios of 1:2. Slopes for 4-BI (0.0043 vs. 0.0084) are very close to the expected ratio; however, values for 1-NA (0.0022 vs. 0.0055) differ a bit more than expected and we are unable to explain this difference. T h e relative uncertainties in slopes are significantly less for the twocomponent mixtures than they are for either the singlecomponent or three-component samples. The ratios of standard errors to the averages of the ordinate values are about twice &S small for the two- and three-component samples as they are for the one-component samples (-2.5% vs. 3.5 to 6 % ) suggesting that the internal-standard method does improve the scatter in the calibration plots by a significant amount. It is clear from these data that the internal-standard method offers significant advantages for the phosphorescence studies, and the method is made possible by the multiwavelength SIT detector and the simultaneous multicomponent kinetic methods developed in this laboratory. In this work, we used only one wavelength for each component. Earlier work with absorbance data has shown that multiwavelength data obtained with imaging detectors can
be used to resolve multicomponent mixtures (15). It has also been shown that multiwavelength absorbance and kinetic data can be combined effectively to resolve mixtures (10). It may be that the combination of these techniques to the phosphorescent data would improve the within-run and between-run imprecision of mixtures toward the limiting value of the within-run imprecision for single components. We are presently investigating this possibility.
LITERATURE CITED (1) C. M. O'Donnell and J. D. Winefordner, Clin. Chem. (Winston-Salem, N . C . ) , 21, 285 (1975). (2) R. M. Wilson and T. L. Miller, Anal. Chem., 47, 256 (1975). (3) Y. Talmi. D. C. Baker, J. R. Jadamec, and W. A. Saner, Anal. Chem., 5 0 , 937A (1978). (4) E. M. Schulman and C. Walling, J. Phys. Chem.. 77, 902 (1973). (5) R. A. Paynter, S. L. Wellons, and J. D. Winefordner. Anal. Chem., 46, 736 (1974). (6) S. L. Wellons, R. A. Paynter. and J. D. Winefordner, Specfrochirn. Acta. Part A. 30, 2133 (1974). ( 7 ) E. M. Schulman and R. T. Parker, J. Phys. Chem., 81. 1932 (1977). (8) M. J. Milano. H. L. Pardue. T. E. Cook. R. E. Santini. D. W. Maraerum. . and J. M. T.'Raycheba, Anal. Chem.,' 46, 374 (1974). (9) H. L. Felkel, Jr.. and H. L. Pardue. Anal. Chern.. 49, 1112 (1977). IO) G. M. Ridder and D. W. Margerum, Anal. Chem., 49, 2098 (1977). 11) B. G. Willis. W. H. Woodruff, J. R. Frysinger, D. W. Margerum. and H. L. Pardue, Anal. Chem., 42, 1350 (1970). 12) G. E. Mieling and H. L. Pardue, Anal. Chem., 50, 1611 (1978). 13) H. L. Felkel. Jr.. and H. L. Pardue. Ciin. Chem. ( Winston-Saiem. N.C.h I
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Received for review January 11, 1979. Accepted March 2, 1979. This work was supported by Grant No. CHE 75-1550 AD1 from the National Science Foundation.
Chemical Analysis and Structural Characterization of Transition Metal Disulfide Intercalates Steven L. Suib, Larry R. Faulkner, and Galen D. Stucky" School of Chemical Sciences and Materials Research Laboratory, University of Illinois, Urbana, Illinois 6 180 1
Richard J. Blattner Materials Research Laboratory, University of Illinois, Urbana, Illinois 6 180 1
Controlled potential electrolysis has been used to synthesize intercalates of single crystal transition metal disuifldes. Electrochemical experiments using cyclic voltammetry were undertaken to study the process of intercalation. Cyclic voltammetric studies of aqueous solutions of CuS04 and RhCI, have revealed two quasi-reversible couples for single crystal 2H-MoS2 working electrodes and irreversible behavior with 2H-TaS2 single crystal electrodes. Auger electron spectroscopy (AES), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and X-ray powder dlffractometry have been used to determine the analytical and structural composition of these intercalates. Our results indlcate that thlck and laterally nonuniform eiectropiatlng occurs with ~H-MOS,, whereas intercalation predominates with 2H-TaS2.
Over the past several years, chemists, physicists and materials scientists have studied intercalation of layered compounds. Recently, intercalates of transition metal di0003-2700/79/035 1-1060$01.OO/O
chalcogenides have been of interest for a number of theoretical and practical reasons, including superconductivity and battery studies ( 1 , 2 ) . Whittingham ( 3 ) has shown that metal dichalcogenide intercalates of group 4B and 5B can be produced electrochemically by controlled potential electrolysis of aqueous salt solutions using metal chalcogenide cathodes. Other workers (4-6) have reported similar findings. Little work has been reported concerning the chemistry of these compounds and virtually no spectroscopy has been applied to compounds of this type. Little concentrated effort has been made to clearly identify both the chemical composition and structural characteristics of the metal dichalcogenide intercalation compounds. In the present paper, Auger electron spectroscopy has been employed to analyze the surface composition of the intercalates, to check for changes of in-depth composition, and to obtain information about impurities introduced during the intercalation process. At the same time, scanning electron microscopy coupled with energy dispersive X-ray analysis experiments has been carried out to monitor the bulk analytical composition and structure of these materials. Our (C 1979 American Chemical Society
