(20) R. J. Widlar, "Fairchild Application Bulletin App-116," Fairchild Semiconductor, Mountain View, CA. Feb. 1966. (21) M. K. Vander Kooi, "L144 Programmable Micro-Power Triple Op Amp, Siliconix Application Note." Siliconix Inc., Santa Clara, CA, Dec. 1973. (22) "Fairchild kA735 1 Data Sheet," Fairchild Semiconductor, Mountain View, CA, May 1973. (23) J. A. Dean and J. P. Rupley, "RCA ApDlication Note ICAN-6267," RCA, Harrison, NJ, March 1971. (24) "COS/MOS Digital Integrated Circuits," RCA Solid State Databook Series, Somerville, NJ, 1973. (25) S. 0. Farwell and R. D. Geer, "A Voltammetry Waveform Generator Controlled by Digital Logic," presented at the 26th Northwest Regional Meeting of the American Chemical Society, Bozeman, MT, June 1971. (26) K. H.Pool, C. J. Johnson, G. I. Connor, and G. H.Boehme, Anal. Cbem., 45, 437 (1973). (27) F. G. Cottrell. 2.Pbys, Cbem., 42, 385 (1902). (28) R. N. Adams. "Electrochemistry of Solid Electrodes", Marcel Dekker, Inc., New York, NY. (29) R. S.Nicholson and I. Shain, Anal. Cbem., 36,706 (1964). (30) L. Eberson and H. Schafer, "Organic Electrochemistry", Springer-Verlag, New York. NY, 1969, pp 256-271. (31) P. T. Allen in "Analytical Metods for Pesticides, Plant Growth Regulators, and Food Additives", Vol. V, G. Zweig, Ed., Academic Press, New York, NY, 1967, Chapter 3.
(32) G. J. Hoijtink and J. Van Schooten. Rec. Trav. Cbem., fays-Bas, 71, 1089 (1952). (33) J. D. Stuart, R. R. Kennan, R. J. Fenn, R. G. Jensen, and W. J. Pudelkievicz, "Degradation of Polychlorinated Biphenyls as Studied by Oxidative Electrochemistry", presented at the PCB Symposium of the American Chemical Society, Division of Water, Air and Waste Chemistry, Aug. 1972. (34) S. 0. Farwell, F. A. Beland and R. D. Geer, "Reduction Pathways of Organohalogen Compounds, Parts I and II", submitted for publication in J. Nectroanal. Chem.
RECEIVEDfor review February 4, 1974. Accepted January 20, 1975. Contribution from Agricultural Experiment Station, Montana State University, Bozeman, and published as Journal Series No. 490. Work supported in part by regional research funds, Project W-45. Taken from the Ph.D. thesis of S. 0. Farwell, Montana State University, August 1973.
Dye-Sensitized Continuous Photochemical Analysis: Identification and Relative Importance of Key Experimental Parameters R. White'
V.
and J. M. Fitzgerald2
Department of Chemistry, University of Houston, Houston, TX 77004
Important operating conditions for a new analytical technique, based on dye-sensitized reactions carried out in flowing streams, are evaluated. The photoredox reaction used is a form of triplet energy transfer wherein a colored organic dye absorbs light to generate the excited state triplet, which then abstracts electrons from a suitable substrate. The products of the reaction are colorless reduced dye and substrate oxidation products; spectrophotometric measurement of dye photobleaching is employed. The rate and stoichiometry of photoredox reactions are dramatically affected by the structures of dye and substrate used, dissolved oxygen, and reaction pH. Other important parameters, largely associated with the apparatus used, are dyecarrier flow rate, photolysis source power, and reaction temperature. Data for DCPA measurements, using Methylene Blue and Rose Bengal, are reported for the model substrates ascorbic acid, epinephrine, nicotine, and caffeine. Appropriate operating conditions yield low micromolar limits-of-detection and 1 YO precision.
Continuous flow analysis has been extensively applied in clinical chemistry with great success; a recent review details state-of-the-art developments ( I ). New continuous flow methods are often developed by adapting well-established manual procedures to machine execution. A second approach to continuous flow analysis involves development of new methods designed expressly for automation. Analytical methods based on photochemical reactions have been recently reviewed ( 2 ) . The theories of photochemical and Present address, M e d i c a l Laboratory Associates, 1025 S. 1 8 t h St., B i r m i n g h a m , A L 35205. Address r e p r i n t requests t o t h i s author.
*
related processes which an excited state molecule can undergo have been developed in detail (3-8). Intensely colored dyes are used as the photoactive reagent for the present work. Thus, the extent of the photochemical reaction can be measured spectrophotometrically, and the modest energies (40-50 kcal/mole) of visible light are well suited for use with biochemical substrates. We have previously reported the continuous determination of ascorbic acid (AA) by sensitized photooxidation with Methylene Blue (MB) (9). There exists an extensive literature detailing similar dye-sensitized reactions (6, 1015); most experiments were carried out batchwise in Warburg apparatus. Nevertheless, this literature provides a base for development of new analytical methods employing dye-sensitized photochemical reactions carried out in flowing streams. This technique will be referred to as "DyeSensitized Continuous Photochemical Analysis", and abbreviated hereafter as "DCPA". Although a study of the MBIAA system showed DCPA to have analytical potential ( 9 ) , it became clear that better understanding of the interactions between the photochemistry and operating conditions for DCPA was badly needed. The following objectives were selected for the present work: first, identify and evaluate those experimental parameters which have a significant effect on rate and extent of dye photobleaching reactions; second, determine whether experimental parameters for one given dye-sensitizer vary with the substrate photooxidized; third, develop guidelines for future study of dye-sensitized reactions to be tested for DCPA. Numerous dyes are potentially applicable as photosensitizers for DCPA; the exceptional paper by Gomyo et al. ( 1 1 ) served as a screening study. Based on their data, MB (a phenothiazine dye), and Rose Bengal, RB (a xantheine dye), were chosen because they have different wavelengths ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, M A Y 1975
9
903
of maximum absorption, molar absorptivities, pKa’s, and reduction intermediates. Therefore, differences in photosensitizing ability could be expected. Selection of substrates posed a more formidable problem. Photooxidations with nitrogen heterocycles ( 1 6 ) , amino acids (11, 13, 14), polypeptides (12), nucleic acids ( 1 7 ) , EDTA (18), and amines (10, 19-21) have been reported. The best understood reactions are those of amines; a screening study is described in the experimental section. The chemical reaction which is the basis for DCPA involves reaction of photoexcited dye with substrate. Although other photochemical reactions are known to proceed via formation of singlet oxygen (22), there are a number of kinetic ( 6 ) ,electron spin resonance (23),gas saturation (9, 10-17, 21), and flash photolysis experiments (21, 24, 25) which demonstrate that the reactive photoredox species is the metastable excited state triplet of the dye molecule. DCPA is based on excited state oxidation-reduction and a generalized reaction can be written: Dye-Sensitizer (excited triplet)
+
Substrate (ground state)
electrm
trans fer
Reduced Dye-Sensitizer + Substrate Oxidation Products
(1)
The spectroscopic requirements for high quantum efficiency of the photoredox reaction have been summarized (4, 5, 7, 8 ) ; the dyelsubstrate combinations selected for this study meet these requirements. In reality, the photoredox reaction is a sequence of stepwise reactions which sum up to the generalized version in Equation 1. A number of papers describe the stepwise nature of the actual reduction of MB (6, 7, 21). First, one electron is abstracted from the substrate to produce “SemiMethylene Blue”.
The destruction of extended conjugation results in a colorless product. This intermediate then adds another electron and a proton t o produce the leuco dye.
ent is then subject to possible free radical attack, which may affect the desired photobleaching reaction. For example, amine-oxidation product distributions have been reported to depend strongly on the presence or absence of dissolved oxygen (21). The electron releasing ability of the substrate will also dramatically affect the stepwise dye reduction. The possible kinetic complications have been discussed in detail (21). For DCPA, one can simplify the kinetic problems to variations of two distinct cases. First, reduction can proceed slowly dictating slow flow rates. Second, a rapid back reaction can result in transient stability of the reduced dye; requiring fast flow rate to the detector. Slow flow rates also cause diffusion broadening as the dyelsubstrate “plug” moves through the apparatus, which reduces sensitivity. Flow rate also has a critical effect on the number of photons absorbed by the system:
No. of hv = (Source Intensity)(Exposure T i m e ) = f ( L a m p Wattage) x f(F1ow Rate)
(4)
(5)
For complete photolysis, the number of photons must be greater than that required by the reaction stoichiometry. Therefore, reactions with high quantum efficiency can be run a t fast flow rates; the opposite is true for reactions with low quantum efficiency. The intensity and spectral distribution of the tungsten-filament source used here are described by the black-body equations (29). For this reason, lamp wattage studies must be performed. Dissolved oxygen is an important parameter because it affects the products and extent of photobleaching (21 1. The problem reduces to analytical sensitivity for DCPA. In our study of the MB/AA system, removal of dissolved oxygen did not increase sensitivity sufficient to justify nitrogen purging for routine work (9). However, this fact must be checked for each new dyehubstrate combination. Temperature dependence of rate constants for triplet energy transfer reactions have been reported (30). Therefore, DCPA apparatus must include temperature control in the photolysis zone because of heat given off by the source. Spectrophotometric detection introduces additional constraints on the experimental parameters. Both the sensitivity (slope of the calibration line) and limit of detection (first change in Abs in excess of that due to simple dilution of dye) will depend on the change in dye concentration (Beer’s law): AAbs = EdyebACdye
The actual reduction product of RB has not been characterized a t this time. Since the potentials of both the dye and the substrate are pH dependent (26-28), it is possible to make a particular dyelsubstrate reaction spontaneous or non-spontaneous by adjustment of reaction pH; previously reported dye-sensitized reactions occur within well-defined pH corridors (10-21). However, one must consider the stability of the dye and substrate a t a given pH. A further restriction would be any change in dye molar absorptivity with change in pH. The pK,’s of MB (11.6) and RB (4.3) are such that these two dyes allow one to carry out studies over the entire p H range. That is, MB can be used in acidic solution while RB is stable in base. Even if the redox behavior of dye triplet and substrate is favorable for the photoredox process, the stability and further reactivity of the substrate oxidation product may complicate the overall stoichiometry. Furthermore, since the primary process in the stepwise reduction of the dye is a one-electron transfer from the substrate to the dye, then free radical intermediates must exist. Any substance pres904
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
Note the actual measurement is the decrease in dye concentration ( h c d y , is negative). The convention used here for AAbs is defined as follows: the minimum numerical absorbance read from the spectrophotometer is subtracted from the “base-line” absorbance due to unbleached dye. This yields a positive number which increases with increasing substrate concentration injected. For two different dyesensitizers with different molar absorptivities, the dye with the larger molar absorptivity would give better sensitivity and limit of detection, provided that the reaction stoichiometry for Equation 1 is the same for both dyes. However, stoichiometric ratios are reported to vary depending on the dye/substrate combination in question ( I 1 ). MB has been reported to form dimers a t dye concentrations exceeding 10d4M (31, 32). Spectrophotometric consequences of dimer formation have been considered elsewhere (33); dimer formation could also cause problems in the photochemical processes. Therefore, DCPA investigations should be carried out a t dye concentrations lower than where dimerization occurs.
Table I. Effect of Carrier Stream Flow Rate on Substrate Dilution and Sample Repetition Rate
-w
1 DRAIN
Figure 1. Block diagram of DCPA apparatus' R1, Air-saturated MB reservoir; R2, air-saturated RB reservoir: RJ, nitrogensaturated MB reservoir: R,, narogen-saturated RB reservoir: V1, V p , VI, dye carrier solution selection valves; I, sample injector (motor driven syringe); P.R.. Photochemical reactor (water jacketed spiral): D, detector (digital readout spectrophotometer): pH, flow-through pH electrode and associated electronics; V,, flow rate control valve and associated flow meter. Arrows indicate direction of flow
EXPERIMENTAL Apparatus. A conceptual block diagram of the apparatus used is shown in Figure 1. The actual apparatus is a modification of that previously described (9); only changes and additions will be presented here. The entire flow system is constructed of 6-mm i d . glass tubing. Based on previous experience (9),the method of sample injection was modified to provide better reproducibility and capability of operation a t high back pressures due to fast dye-carrier flow. A motor-driven syringe injector (Sage Instruments, Model No. 197) was used; the reproducibility was determined to be better than 0.1% gravimetrically. The charge-discharge cycle is complete in one second; a 0.8-ml sample is injected from a 1-ml syringe (Becton-Dickinson, Model 2005). With addition of the syringe injector, accurate determination of sample dilution when injected a t various dye-carrier flow rates was possible. A Beer's law calibration for MB was prepared by passing solutions of known concentration through the spectrophotometer flow cell. A plot of Abs vs. MB concentration was linear up to 1.650 (MB = 3 X 10-5M). Then 0.8 ml of a known concentration MB was injected into the system with only water flowing through the apparatus. Measurement of the maximum absorbance was converted to maximum MB concentration (Beer's law) and the dilution factors were calculated. Data are summarized in Table I. I t can be seen that fast flow rates favor both minimum sample dilution and rapid sample-injection repetition rate. Four dye reservoirs (20 1. each) were used for study of two dyes under both air and nitrogen-saturated conditions. A combination of stopcocks allowed rapid change of dye-carrier solution used. Reservoirs were pressurized to 5 psi over atmospheric with air or nitrogen. This served two purposes: first, efficient removal of oxygen from the nitrogen-saturated dyes was possible and, second, the pressurized system allowed a wider range of flow rates than did the previously used siphon (9). The sensitivity of spectrophotometric detection depends on the ability to distinguish small changes in dye concentration ( i t . , ACdye, Equation 6); a digital readout spectrophotometer (Bausch & Lomb, Spectronic 100) was added. Changes in solution absorbance as small as 10.001 (hereafter f l mAbs) were read directly. Digital readout and a 10-mm path, micro flow-cell (Savant Cells, Type 40) improved sensitivity over the previous detection system (9).
Since photolysis temperature could be an important experimental parameter ( 3 0 ) , a new photochemical reactor was constructed with a water jacket for thermostating. The reactor spiral was twice the length of the original ( 9 ) , doubling exposure time. The water jacket had a 5-cm dia. inner opening down the central axis of the reactor for placement of the lamp; the jacket was 15-cm 0.d. Original cooling air blower (100 cfm) and lamp power supply were retained (9).A 1-kW projection lamp (General Electric, Model DGS) served as the photolysis source for the present study. An in-line, flow-through, combination pH electrode (Benchmark, Model 737) was installed downstream from the spectrophotometer. The pH of the dye/substrate mixture corresponding to a maximum photobleaching could thus be measured (BeckmanZeromatic pH Meter).
Substrate
Time t o
Flow rate,
dilution
peak max,
Clearance
mllmin
factor a
secb
time, secC
210 42 6 24 170 50 8 45 115 57 10 60 90 65 13 78 70 80 19 138 a Ratio of known MB concentration injected (0.8 ml) into water carrier to a maximum MB concentration measured in detector cell. Reciprocal of this number X concn substrate injected gives maximum substrate concn in dye stream. * Elapsed time between injector drive and maximum AAbs read a t detector. Elapsed time between injector drive and return to base-line Abs. New sample may be injected after ca. half of this time. Reagents. Commercial MB (Color Index No. 52015) and RB (Color Index No. 45440) were used (Eastman Organic Chemicals). However, even the best available grades of both dyes were impure. Manufacturer's assay reported MB to be 91% by weight, while R B was 87% by weight. Previous workers have found multiple recrystallizations improve dye purity only a few percent ( 2 4 ) .Dye solution is discarded after use and a t such a rate (50 to 300 ml/min) that purification of the dye used was deemed impractical. Carrier solution was prepared by adding solid dye to distilled water in the reservoir; mixing and gas-saturation were accomplished by passing compressed nitrogen or air through the solutions for 1 hr prior to use. Solutions were stored under 5 psi pressure of gas. The concentration of both MB and RB was found to be not critical (range 2.5 to 3 X 10-5M). The observed maximum AAbs for bleaching with a given substrate concentration injected did not change with small changes in dye carrier concentration. The dye concentration was near the upper limit of linearity (Abs ca. 1.5) for the spectrophoMB = 665 nm; , , ,A RB = 550 nm). High abtometer used,,A,( sorbances should be used for best photochemical efficiency (2). Adjustment of dye pH was accomplished with HC1, "3, or NaOH. Solutions a t all pH values were stable for a t least two days; stability increased the greater the difference between solution pH and dye pKa. The best commercially available grades of the following amines were used for a preliminary screening study: caffeine, diethylamine, dopamine, epinephrine, morphine, nicotine, norepinephrine, and proline. DCPA was tested for all substrates with both air and nitrogen-saturated solutions of both dyes. Based on this survey, four substrates were selected for detailed investigation. AA was retained as a reference compound to which other substrates were compared. Epinephrine (E) exhibited the best photobleaching of any catechol amine screened. Caffeine (C) showed kinetic complications which caused a dramatic dependence of photobleaching on flow rate. Nicotine (N) was found to have a large temperature coefficient. Stock 10-2M solutions of the four substrates were prepared fresh daily by weight from reagent grade chemicals. Lower concentrations for DCPA runs were prepared by dilution and gas saturated just prior to injection. All dye and substrate solutions were prepared in distilled water. Deionized water was found to be unacceptable for DCPA; the presence of trace aromatic plasticizers from resin columns resulted in erratic photobleaching. Procedure. The general sequence of events for making DCPA ,,A measurements is as follows. The spectrophotometer is set for of the dye to be used, water only is passed through the flow cell and zero Abs set. The reservoir of appropriate dye, adjusted to the desired pH and under air or nitrogen pressure, is selected. The flow rate is set with the Teflon (DuPont) needle valve and measured with the in-line flowmeter (9).The water circulating through the reactor jacket is adjusted to the desired temperature f ( 2 "C). The lamp blower and power supply are activated; generally the applied power is 1 kW. A water blank is connected to the injector syringe valve. The syringe is rinsed by carrying out 10 charge-discharge cycles. When the photometer stabilizes to il mAbs, individual blanks are injected. A second injection is made after the AAbs maximum from the preceding sample has passed through the detection system (Table I). The change in absorbance, AAbs, between base line and the (negative) peak due to simple dilution of the carrier stream by the blank are read digitally to the nearest ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
905
Table 11. Values of Experimental Parameters Used and Statistical Treatment of Results for Measurements of Model Substrates with DCPA Flow rate, Substrate
Dye-gap
PH
ml/minc
Reactor
power t o larnp,ivd
temp., ' C
Concn range,.\ie
slopef
Comments
MB-Air 2 73 ( 2 1 ' ) 850-1000(p) NCg 10-5-10-3 458 (A) MB-N, 2 73 ( 2 1 , ) 800--1000(p) NCg 10-6-1 0 ' 3 857(*1) k RB-Air 9 11O(2l') >lOOO(1) NCP 10-4-1 0-3 256(*2) > lOOO(1) NCg 10-6-1 0-3 480(+1) RE N , 9 110(7(~) Epinephrine MB-Air 11 91(11') >1000(1) 20 10-4-1 0-3 80 i, k >1000(1) 20 10-5-1 0-3 216(*1) k 11 91 (11') (E ) MB-N, R B-Ai r 10 9 1( 2 1 , ) >lOOO(1) 20 10-4-1 0-3 110 j 20 1 o-5-10-3 251 (i.1) h >lOOO(1) RE-N, 10 91 ( I C ) Nicotine MB-Air 9 9 10 1 ) 900-1000(p) 50 10-2 3 j , ~2 (N ) MB-N, 9 91M 900-1000(p) 50 10-5-1 0-3 176(+1) 700-1000(p) RB-Air 11 91(d 50 10-4-1 0-3 88 j REN, 11 91 ( n ) 700-1000(p) 50 I 0-6-1 0-3 472 (i.1) k Caffeine MB-Air 10 260(r) >lOOO(1) 35 10-2 2 j, > 1OOO(1) MB-N, 10 260(r) 35 1 0-~-1 o-~ 50 j RB-Air 10 91 (w) INDM 50 10-~-1 o-~ 85 i 50 10-5-1 0-3 247(i.2) k >lOOO(?ij REN, 10 91 ( 2 1 . ) a MB = Methylene Blue, RB = Rose Bengal. Both dyes approximately 3 X 10-5M. This concn reflects upper limit of spectrophotometric linearity. Air: solutions saturated with compressed air. Nz: solutions saturated with nitrogen. * Maximum photobleaching observed over range of f 0 . 3 pH unit from value listed. Range of flow rates over which same photobleaching observed indicated by: (u)= &20 ml/min of listed value. (n)= +10 ml/min of listed value. ( r ) Maximum flow rate possible. Dye consumption rate too high for routine use of this system. Watts of power applied to 1000-W tungsten filament photolysis source. ( p ) = plateau of wattage exists over which no change in photobleaching observed. ( 1 ) = linear increase in photobleaching with increasing applied wattage. (s) = reaction independent (IND) of lamp wattage from 0 to 1000 W applied. ( u ) = nonlinear increase in photobleaching; approaching plateau a t 1000 W applied. e Range of linearity of AAbs vs. concn substrate injected for listed conditions. Lower concn is the limit of detection, taken as A of 5 mAbs greater than solvent blank. Slope of calibration line in units of AmAbslmM substrate injected, from linear-regression analysis. Standard error of estimate for slope, in mAbs units, listed in parentheses. g Ascorbic acid exhibits no temperature dependence with either dye, temperature not critical (NC). Conditions resulting in highest sensitivity for substrate in question. J Slope signficantly less than with other conditions for the same substrate. Statistical parameters not evaluated. pH near pK, for MB; E not stable at this pH. Undesirable condition. Photobleaching tw small for accurate measurement, estimated slope reported; not analytically useful. Ascorbic Acid (AA)
(c)
mAbs unit. Typically, the blank is 20 to 23 mAbs. Next, a test substrate solution is connected to the injector, rinsed for 10 cycles, individual injections are made, and the AAbs is measured; generally five replicates of each concentration are run. A calibration line is then plotted as AmAbs vs. concentration of substrate injected (in mM); alternatively, data can be subjected to least-squares linear regression analysis (9). The slope (sensitivity) and limit of detection (AAbs = 5 mAbs greater than water blank) for the dye/substrate combination under investigation are obtained from leastsquares data treatment. The range of substrate concentrations which correlate linearly with AAbs is also evaluated. The effects of the key experimental parameters on the extent of photobleaching observed are evaluated in the following way: variables are studied in the order pH, flow rate, lamp wattage, and reactor temperature for each dye, saturated with the appropriate gas. For determination of the best pH range, substrate solutions of varying p H are injected into the dye carrier a t natural p H (MB = 8, RB = 5). Flow rate, lamp wattage, and reactor temperature are set at arbitrary values. The reaction p H of the dye/ substrate mixtures are read using the in-stream p H electrode and the AAbs for this pH is measured. A plot of AAbs us. reaction pH clearly shows the p H range providing maximum photobleaching. The pH of both dye and substrate solutions are adjusted to this range. Dye flow rate is then varied in 10 ml/min increments and substrate injected a t each value. For most dye/substrate combinations, a range of suitable flow rates, representing a trade-off between diffusion broadening (slow flow) and insufficient exposure time (fast flow), can be located from a plot of AAbs vs. flow rate. Of the four substrates, only C/MB gave unusual results (discussed later). The flow rate is then set in the range giving best photobleaching and lamp wattage decreased in 100-W steps with injection of substrate a t each value. Plots of lamp wattage vs. AAbs for the four substrates studied exhibited three classes of behavior to be discussed later. The lamp power is then returned to 1 kW and the reactor temperature varied. If a dependence of photobleaching with temperature is found, the effect should be investigated in detail; AA is the only substrate studied to date which has no temperature coefficient. Temperature range available for this study was 20 to 55 oc. All experimental parameters are finally set to the optimum values for a given dye/gas/substrate combination and a series of substrate concentrations are run using the general procedure given 906
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
above. Sensitivity and limit of detection are determined from the calibration line (9). Data for the four substrates studied are summarized in Table 11.
RESULTS AND DISCUSSION The i m p r o v e m e n t s in t h e a p p a r a t u s design r e s u l t e d in significant i m p r o v e m e n t s i n t h e DCPA m e a s u r e m e n t s . The i m p r o v e d s a m p l e injector r e s u l t e d in f a s t e r s a m p l e r e p e t i t i o n r a t e s ( T a b l e I), and b e t t e r precision ( T a b l e 11).Digital r e a d o u t significantly i m p r o v e d precision over the earlier analog method (9). If the slope of the calibration line is g r e a t e r than 100 m A b s / m M s u b s t r a t e injected, r e a d i n g e r rors of f l mAbs r e s u l t in precision b e t t e r t h a n 1%.T h e r e fore, least-squares analyses were carried out only for d y e / s u b s t r a t e c o m b i n a t i o n s w i t h sensitivities g r e a t e r t h a n 100 m A b s / m M ( C o l u m n 8 of Table 11).Obviously, b e s t limits of d e t e c t i o n ( C o l u m n 7 of Table 11) a r e o b t a i n e d with highest sensitivities. T w o b r o a d generalizations can b e d r a w n f r o m t h e inform a t i o n in T a b l e 11. Sensitivity for D C P A with n i t r o g e n - s a t u r a t e d d y e is always s u p e r i o r t o that o b t a i n e d w i t h a i r - s a t urated dye. T h e role of oxygen in dye-sensitized p h o t o r e d o x reactions is c u r r e n t l y a m a t t e r of d i s a g r e e m e n t b e t w e e n investigators (6, 7, 21, 22). Nevertheless, good analytical data c a n s o m e t i m e s be o b t a i n e d e v e n in the p r e s e n c e of oxygen, e.g., A A / R B E/RB, AA/MB. W i t h the exc e p t i o n of AA, s u b s t r a t e s yield better sensitivity w i t h RB than w i t h MB ( E q u a t i o n 6). The f a c t that MB is s u p e r i o r for AA indicates that t h e p h o t o r e d o x reaction stoichiomet r y a n d r a t e of a given s u b s t r a t e c a n be d i f f e r e n t for e a c h of t h e t w o dyes; t h i s has b e e n observed b y o t h e r s ( 1 1 ) . F o r e a c h d y e h u b s t r a t e c o m b i n a t i o n listed i n T a b l e 11, a definite p H corridor exists over which the p h o t o r e d o x react i o n is s p o n t a n e o u s . Also, t h e b e s t reaction pH for a specific d y e / s u b s t r a t e c o m b i n a t i o n is t h e same for a i r o r n i t r o g e n - s a t u r a t e d solutions. T h e r e a r e t w o pH e x t r e m e s in
Table 11. Neither dye nor substrate are stable a t the p H ( 1 1 ) required for the E/MB system; however, adequate data could be obtained even under these adverse conditions. The dye of choice for E determination would obviously be RB. On the other hand, both dye and substrate are quite stable a t p H 2 for the AA/MB system. Other systems tabulated here lie between the two extremes. A definite range of flow rates within which maximum photobleaching occurs is found for all dyehubstrate combinations with the exception of C/MB. This latter system shows a continuous increase in photobleaching with increasing flow rate from 30 to 260 ml/min, which is the upper limit of the present apparatus. This behavior can be explained on the basis of a rapid, postphotochemical, back reaction (21). In such a case, the maximum AAbs is observed when the bleached dye is swept rapidly from the photochemical reactor to the detector. Although mechanistically intriguing, C/MB is not the DCPA system of choice because of low sensitivity and impractically high flow rates. T h e usual flow rate maximum is observed for C/RB; sensitivity with RB was five times that obtained with MB, and so the C/MB reaction was not studied further. Ideally, DCPA should be run under conditions where an excess of photons is available to drive the photoredox reaction to completion. This situation prevails for substrates listed in Table I1 where a range of applied wattages results in the same amount of photobleaching, e.g. AA/MB, N/ MB, N/RB. At the other extreme, some photoredox systems are so inefficient that the number of photons is, in fact, the limiting reagent, e.g. AA/RB, E/MB, E/RB, C/ MB. For these systems, a linear dependence of photobleaching with lamp wattage, up to the maximum available, is noted in Table 11. The intensity dependence of the C/RB system is unusual. In the presence of oxygen, the reaction is spontaneous, and the same 4Abs is measured with or without irradiation. This is the only case observed to date, but suggests interesting analytical possibilities; however, this particular DCPA system has poor sensitivity. The C/RB-N:! system is well behaved and is the best DCPA system for C determination. AA provides a good example of the interdependence of flow rate, lamp wattage, and dye structure for DCPA reactions. The data in Table I1 indicate a trade-off between source intensity and residence time for the AA/RB system. AA/RB is photon-limited (i.e., no lamp wattage plateau exists). On the other hand, the AA/MB system shows constant photobleaching with source power between 800 and 1000 W. These observations can be explained as follows: a dye, such as RB, with a short wavelength of maximum absorption has more available energy for the photoredox reactions, but the absorption band of the dye does not match the spectral emission from the black-body source (29) as well as dye with a longer wavelength maximum. Hence, MB is a more efficient absorber than is RB with the source used in this study. Figure 2 summarizes data for the temperature dependence of each nitrogen-saturated dye/substrate combination. In this figure, bleaching (normalized to 20 OC) is plotted vs. reactor temperature. As can be seen, there is no general behavior pattern. AA has no temperature dependence with either dye, and serves as a reference to which other substrates can be compared. E exhibits a negative temperature dependence with both dyes; this is not a photochemical effect but rather is due to decomposition of E into a colored product in basic solution. The observed temperature effect for E is due to competition between thermal and photoredox reaction pathways. The temperature dependence of N shown in Figure 2 is exponential; this would be
re 0 t I
8
*I 6 +I 4
A Abs
t I
2
tl
0
T to e AAbs20 + O 6
to
4
to
2
00 -0 2 -0 4
REACTOR TEMPERATURE
'C
Flgure 2. Temperature dependence for model substrates measured by DCPA (1AbsdAAbs20). Normalized photobleaching: AAbs at a given reactor temperature, T, divided by AAbs for same substrate concentration at 20 ' C . 1Abs defined in text. A, data points for nitrogen-saturated RE: 0, data points for nitrogen-saturated ME; A, ascorbic acid; C, caffeine: E, epinephrine: N, nicotine; all other Darameters at values listed in Table II
predicted by Arrhenius theory for a rate-limiting, postphotochemical reaction (3, 5, 30). C exhibits bizarre temperature dependence with both dyes which cannot be explained a t present, but unusual DCPA behavior of C has been noted above. Examination of the DCPA conditions summarized in Table I1 would indicate that selective photobleaching by one substrate in the presence of a mixture of the model substrates might result from judicious selection of experimental parameters. This point was not a major objective of the present study; however, a preliminary check of some more obvious cases was carried out. Adjustment of reaction p H is an obvious method of achieving selectivity. This was previously found true for the AA/MB system (9). This finding was again verified by injecting an equimolar (10-4M) sample of all four substrates used for this study into MB-N2 adjusted to p H 2. No interference in AA photo-bleaching, either positive or negative, was found. A second method of achieving selectivity is suggested by the high flow rate required by the C/MB system. Equimolar (10-3M) solutions of C and N, injected into MB-N2 a t p H 10, gave the same photo-bleaching as did C alone, if a flow rate of 260 ml/min was used. Although not photochemical in nature, the C/RB/air system was also found to be selective when equimolar (10-3M) C and N was injected a t p H 10 with the photolysis source off. These preliminary findings are encouraging; further studies invoking selective operating conditions are presently under way.
CONCLUSIONS Several critical parameters which must be considered during the development of any new DCPA system have now been identified. Clearly, nitrogen purging always yields superior sensitivity. The pH used is extremely important, but cannot be predicted for a new dye/substrate combination a t present. Furthermore, both the photoredox (kinetics and stoichiometry) and spectrophotometric (c,Amax) properties of any dye will be key factors in determining suitability for DCPA. Parameters of lesser importance include flow rate, lamp power, and reactor temperature. These three parameters are closely associated with the particular equipment used; any new DCPA apparatus should include provision for their control and variation. Photochemical methods are currently a novelty in both clinical and analytical chemistry; it is hoped that the research described here will hasten their wider acceptance. ANALYTICAL CHEMISTRY, VOL. 47,
NO. 6,
MAY 1975
907
LITERATURE CITED (1) M. K. Schwartz, Anal. Chem., 45, 739A (1973). (2) J. M. Fitzgerald, Ed., "Analytical Photochemistry and Photochemical Analysis: Solids, Solutions, and Polymers", Marcel Dekker Inc., New York, NY, 1971, Chapters 4 and 5. (3) J. G. Calvert and J. N. Pitts, Jr., "Photochemistry", John Wiley and Sons, New York, NY, 1967, Chapters 3-6. (4) R. S. Becker, "Theory and interpretation of Fluorescence and Phosphorescence", W. A. Benjamin & Co., New York, NY, 1969, Chapters 3, 7, 9. 15, 17. (5) N. J. Turro, "Molecular Photochemistry". W. A. Benjamin & Co., New York, NY, 1965, Chapters 5 and 6. (6) J. D. Margerum, A. M. Lackner, M. J. Little, and C. T. Petrusis, J. Phys. Chem., 75, 3066 (1971). (7) W. G. Herkstroeter, A. A. Lamola, and G. S.Hammond, J. Am. Chem. Soc., 88, 4537 (1964). (8) R. P. Wayne, "Photochemistry", American Elsevier. New York. NY, 1970, Chapters 4-6. (9) V. R. White and J. M. Fitzgeraid, Anal. Chem., 44, 1267 (1972). (10) L. Weil, Science, 107, 426 (1948): (11) T. Gomyo, Y. Yang, and M. Fujimaki, Agric. Biol. Chem., 32 1061 (1968). (12) L. Weil, T. S. Seibies, and T. T. Herskovits, Arch. Biochem. Biophys., 111, 308 (1965). (13) L. Weil, Arch. Biochem. Biophys., 110, 57 (1965). (14) J. S.Bellin and C. A. Yankus, Arch. Biochem. Biophys., 123, 18 (1968). (15) J. S. Bond, S. H. Francis, and J. H. Park, J. Bid. Chem., 245, 1041 (1970). (16) M. I. Simon and H. VanVonakis, Arch. Biochem. Biophys., 105, 197 (1964).
(17) F. W. Morthland, P. P. H. DeBruyn, and N. H. Smith, Exp. CellRes., 7, 201 (1954). (18) G. Oster and N. Wotherspoon, J. Am. Chem. Soc., 79, 4836 (1957). (19) L. Weil and J. Maher, Arch. Biochem., 29, 241 (1950). (20) R. F. Bartholomew and R. S.Davidson. Chem. Commun., 1970, 1174. (21) R. F. Bartholomew and R. S.Davidson. J. Chem. Soc., C, 1971, 2347. (22) C.S.Foote and R. W. Denny, J. Am. Chem. SOC.,93,5168 (1971). (23) L. P. Simpson. J. S.Kirby, and M. L. Randoipy, Nature. 199, 243 (1963). (24) R. M. Danziger, K. H. Bar-Eli, and K. Weiss, J. Phys. Chem., 71, 2633 (1967). (25) C. A. Parker, J. Am. Chem. Soc., 83, 26 (1959). (26) W. M. Clark, "Oxidation-Reduction Potentials of Organic Systems", Williams and Wilkins Co., Baltimore, MD. 1960, Chapter 5. (27) C. Bodea and I. Silberg, Adv. Heterocycl. Chem., 9, 322-449 (1968). (26) 0. Tomicek, "Chemical Indicators", Butterworths, London 195 1. (29) H. A. Strobei, "Chemical Instrumentation: A Systematic Approach", 2nd edition, Addison-Wesley, Reading, MA, 1973, pp 285-66. (30) H. L. J. Backstom and K. Sandros, Acta Chem. Scand., 14, 48 (1960). (31) G. N. Lewis, 0. Goldschmid, T. T. Magel, and J. Bigeleisen, J. Am. Chem. Soc., 85, 1150 (1943). (32) G. N. Lewis and J. Bigeieisen, J. Am. Chem. SOC.,85, 1144 (1943). (33) G. W. Ewing, "instrumental Methods of Chemical Analysis", McGrawHili, New York, NY, 1969, pp 62-63.
RECEIVEDfor review June 10, 1974. Accepted October 23, 1974. Financial support for this work was provided by Grant E-384 from the Robert A. Welch Foundation.
NOTES
Theory for Adsorption Re-Equilibration in Reverse Step of Double Potential Step Chronocoulometry of Adsorbed Reactants C. Michael Elliott and Royce W. Murray Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hi//, N C 275 74
Double potential step chronocoulometry is today the method of choice for the study of adsorbed electroactive species ( I ) . For a reducible species, the method consists of allowing the working electrode to come to equilibrium with a solution of the adsorbing species 0 a t a potential E i n i t where no faradaic reaction occurs, then stepping the electrode potential to E f i n a l on the diffusion limiting plateau of the reduction wave of 0, for some time T , while measuring the charge flow, Qf. At time T , the electrode reaction is reversed by returning the potential to E i n i t , and the reverse charge flow, Qr, is measured until t = 27. Double potential step chronocoulometry was introduced by Anson ( 2 ) ,theoretically described by Christie et al. (3),and applied to adsorption by Anson et al. ( 4 ) . The relations for charge during the forward and reverse potential steps are: Qf = Q(t Qb = Q ( t =
908
7)
7)
= KO
+
Qd,
(1)
+
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
+
where K = ~ ~ F A C O ~ ( D O /BT=) ' (/t~-, T ) ' / ~ - t1/2, I'o is the surface excess of 0 a t E i n i t , and Qdl is the difference in double layer charge between E i n i t and Efinal. Data analysis is accomplished by least squares plots of Qf vs. t1l2 and Qr vs. B (using a linearized form of Equation 2) (3), taking the difference between their intercepts to cancel Qdl and extract n F A r o . Double potential step chronocoulometry owes its superiority over the single potential step method (5, 6) to its effective correction for the double layer charge term Qdl, using data from a single experiment, and in a more nearly rigorous form. The term Q d l appearing in Equations 1 and 2 is the change in double layer charge between Einit and E f i n a l with adsorbed reactant being present a t Einit. Any changes in the double layer charge a t E i n i t due to the absorption of 0 are thus automatically taken into account. The double potential step method does include one major assumption, which is that the adsorbed layer, and the appropriate Qdl, becomes instantaneously (or a t least very rapidly compared to T ) re-established upon reverse potential stepping. If such re-equilibrium did not occur,
