ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
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Solvent Effects on Fluorescence Decay Times and Quantum Yields of Atabrine and Its Homologues L. J. Cline Love,” Linda M. Upton, and Arthur W. Ritter I11 Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
Evaluation of the analytical utility of selected excited state properties of molecules for chemical analysis was performed. The effects of molecular structure and microenvironment on the singlet excited state properties of an atabrine-based homologous series and the parent compound were determined. The ultraviolet and visible spectra, the fluorescence spectra, fluorescence lifetime, and quantum yields were determined. These quantities were examined in different polarity solvents and for both the acid and the base forms of the molecular species. These excited state properties are used l o illustrate what molecular properties permit differentiation between similar species. By selecting certain solution conditions, it may be possible to improve the sensitivity and selectivity of fluorescence analysis through use of the variations in fluorescence lifetimes and quantum yields.
T h e luminescent properties of molecules, such as absorption, fluorescence and phosphorescence spectra, and quantum yields have been studied extensively in t h e past (1-6). Improvement in instrumentation in recent years has enabled t h e routine study of luminescence lifetimes as well, with acceptable levels of precision and accuracy (7-9). Studies of all of these excited state characteristics present a more complete picture of the solvated excited state, a chemical entity whose properties can be quite different from the solvated ground state’s properties (10, 11). In some cases, molecules which are nondifferentiable in their ground states may be differentiated through study of their excited states (12). Excited state studies may enable the analyst to have a better idea of the conformation of the excited state species and t o understand the behavior of the species as it interacts with its environment. With t h e knowledge of how to selectively perturb the behavior of a species through its environment, the analyst can manipulate the molecule to produce a solvated species which is more analytically useful. Through understanding how the matrix affects the analyte, its behavior may be better predicted and a matrix selected to improve the quality of analysis rather than hinder and interfere with the integrity of t h e results. Phosphorescence and fluorescence spectra, along with triplet state lifetimes, have been used to distinguish between several groups of molecular species by Winefordner and co-workers (13-17). Fluorescence lifetimes have not been used for identification and quantitation of molecular species, although mixtures of similar fluorophores have been analyzed, in one instance, through deconvolution of their fluorescence lifetimes (18). The objectives of this work were to determine which excited state properties allow differentiation between similar species and which environmental changes accentuate these differences. A further objective was to attempt to understand why the chemical species behave as they do. This understanding of chemical behavior would then allow better control of analytically important variables. 0003-2700/78/0350-2059$01 O O / O
Specifically, this paper presents an example of how the structure and environment affect the excited state of a homologous series related to atabrine. shown as species I in Figure 1. The series of structures studied were those derived from species 11, where n = 1 to n = ’7. Also, the parent compound, shown as species I11 in Figure 1, was studied. All of these compounds have superimposed absorption and fluorescence spectra in acid, the usual medium used for analysis (19). A fluorescence lifetime of 3.5 ns (20) and a quantum yield of 0.04 (21)have been determined for atabrine in 0.1 N hydrochloric acid. T h e environmental variables studied were pH, solvent dielectric constant, and solvent hydrogen-bonding ability. The characteristics measured were ultraviolet-visible absorption and fluorescence spectra, fluorescence quantum yields, and fluorescence experimental lifetimes. The fluorescence lifetime, T , is defined as the time required for the fluorescence intensity to decay by the factor l / e . Calculation of the natural lifetime, T,,,from the fluorescence quantum yield, 4, and the experimental lifetime as shown in Equation 1, To
= l/hf =
T / @
(1)
where k f is the rate constant of decaq through fluorescence, provides a better understanding of the excited state as it compares with the ground state. T h e use of both T and T,, allows separation of the effects due i o t h e excited state intrinsic radiative deactivation rate from those due to other physical processes such as radiationless deactivation ( 6 , 2 2 ) . These lifetimes are related as shown in Equation 2,
where hl is the rate constant of internal conversion and h2 is the rate constant of intersystem crossing from the lowest excited singlet state to the lowest excited triplet state. By measurement of all of these parameters, the effects of the immediate environment of the fluorophore on several of its photochemical and photophysical properties may be observed.
EXPERIMENTAL Apparatus and Procedures. A Beckman Acta I11 spectrophotometer was used to record all absorbance spectra. Precision of the measured absorption wavenumber maxima was *0.005 X lo4 cm-’. Uncorrected fluorescence spectra were obtained using a laboratorq constructed spectrofluorometer consisting of a 200 W Hanovia mercury light source, two GCA McPherson Series 700 scanning monochromators, a Hamamatsu red-enhanced photomultiplier tube along with conventional optics and electronic recording readout. Precision of the measured fluorescence wavenumber maxima was zk0.02 x IO4 cm Fluorescence lifetimes were measured using the time correlated single photon technique (TCSP) (7). A listing of the major components of the laboratory constructed nanosecond fluorescence spectrometer is given in Table I. The flashlamp used was a free running air discharge operating at approximately 20 kHz with a pulse width at half height of approximately 2.5 ns. The excitation wavelengths were isolated with a monochromator using C 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
2
I
CI
m
P
Figure 1. Structure of atabrine and related species: I, atabrine (base form); 11, homologous series n = 1 to n = 7 (base form): 111, parent compound (base form);I V , homologous series (acid form); V, proposed excited state species (acid form) Table I. Time Correlated Single Photon Spectrometer Components Ortec model 9352 nanosecond lamp flashlamp Jobin Yvon model H 20 V excitation monochromator emission filters Corning sample Laboratory Constructed compartment photomultiplier RCA 8850 electronic modules Ortec model 457 time-to-amplitude converter START channel: Ortec model 9352 signal pickoff Ortec model 436 discriminator STOP channel: Ortec model 454 timing amplifier Ortec model 463 constant fraction discriminator Ortec model 425 delay Heath model EU 8 0 1 counter “single photon” monitor multichannel Packard model 115 analyzer parallel-to-serial Packard model 70 converter Teletype high speed, early design tape punch Interdata model 7 / 1 6 equipped with minicomputer TTY and dual floppy _ _ _ disc adjustable slits (2-8 mm) and the appropriate filters were used to select the emission wavelength interval. A rhodamine B quantum counter (8 g/L in ethylene glycol) was used to correct for the wavelength dependence of the photomultiplier tube and the error in the lifetime measurements which would have resulted from the wavelength dependence ( 2 3 ) . The ratio of the number of lamp flashes to the number of single photons observed in a given time period was monitored to ensure that the instrument was truly operating in the “single photon” mode ( 7 ) . A variable aperture was placed in front of the photomultiplier and adjusted to produce a 1 to 5% data observation rate. This requires that 95-9970 of the lamp flashes do not produce a single fluorescence photon in the nanosecond range observation time window.
The determination of a decay rate required recording both the flashlamp time dependent emission profile and the decay profile of the sample. The data were accumulated in 200 channels of a multichannel analyzer until 2 x lo4 counts were accumulated in the peak channel. This count was chosen because it provides adequate signal-to-noise ratio to permit reliable deconvolution ( 2 4 ) . The number of counts per channel were converted from parallel-binary information to serial-ASCII code and punched onto a paper tape for hardcopy storage. These data were transferred to a laboratory 16-bit minicomputer where a reiterative convolution (25) and a PLOT and PLOT-LOG programs were used to analyze the data. The precision of the experimental measurement and the convolution procedure overall was f 0 . 3 ns. Fluorescence quantum yields were determined by the comparative method using a value of 0.51 for the quantum yield of quinine bisulfate in 0.1 N sulfuric acid (26). The precision of the quantum yield measurements was &lo% for the lowest quantum yield measured and decreased with increasing values of the quantum yield. The solution concentrations used in all of the quantum yield and lifetime measurements were 1 X 10-5M or less. The excitation wavelength used was 336 nm. Reagents. The solvents methanol, chloroform, and heptane (spectral quality) and ethyl acetate were obtained from Fisher Scientific and were used without further purification or deoxygenation. Hydrochloric acid and the glacial acetic acid used to make the 4% acid/methanol and 4% acid/chloroform solutions were obtained from Fisher Scientific. Freshly distilled water was used for all aqueous solutions. Solvent blanks were checked for absorbance and fluorescence interferences. Reagent grade quinine bisulfate was obtained from Fisher Scientific and Aldrich. It exhibited single exponential decay behavior when measured with the TCSP technique and was used without further purification. Similarly, the solvents were all found to give single exponential fluorescence decay when they were used to dissolve the quinine bisulfate or other fluorescence standards such as anthracence. The rhodamine B (reagent grade) and the ethylene glycol used in making the quantum counter solution were also obtained from Fisher Scientific. The homologous series related to atabrine, shown in Figure 1, species 11, where n = 1to n = 7 were obtained from G. Downing of Merck, Inc., as the dihydrochlorides along with a sample of atabrine, shown as species I. The parent compound 3-chloro7-methoxy-9-aminoacridine, and a second sample of atabrine were obtained from Sterling-Winthrop Co. as the dihydrochlorides. All of the salts were converted to the free bases by extraction from 1 N NaOH into ethyl acetate. The purity of each hydrochloride compound was greater than 99.770, as determined by phase solubility analysis. RESULTS AND DISCUSSION T h e structures of the compounds studied for both the free base (11) and the acid (IV) forms are shown in Figure 1. T h e absorption spectra suggest that species I1 is present in t h e nonaqueous solvents used, as well as in aqueous solutions a t p H greater than 9, and species IV is present in solutions of pH 1 to 9. This agrees with the results obtained by Schulman and Capomacchia for atabrine (I) and the parent compound (111) (27). All of the compounds studied were found to fluoresce in the 440 to 510 n m spectral range when excited with radiation at 336 nm. R e s u l t s for Species 11. Measurement of the energies of transition through absorption, ljar and the fluorescence, uf, maxima reveals that a decrease in solvent polarity causes an increase in u, and vf. These results are shown in Table 11. The m e a u r e of polarity used is the dielectric constant, t, a quantity which increases as the solvent polarity increases (28). For example, in going from methanol, having a t value of 33, to heptane, having e equal to 2, t h e transition energies of compound I11 increase from 2.410 X lo4 cm-’ t o 2.525 X lo4 cm-’ at its absorbance maximum. The fluorescence maximum of compound I11 also increases from 2.11 X lo4cm-’ to a value of 2.25 X lo4 cm-’. These results indicate a n excited state which is more polar than the ground state and one that is stabilized when placed
ANALYTICAL CHEMISTRY, VOL.
Table 11. Effect of Solvent Polarity on t h e Transition Energies of Species I1 methanol chloroform ethyl acetate heptane ( E = 33)" ( E = 6)b ( E = 6) ( E = 2) compound vaC vfd v, vf v, vf v, vf 111 2.410 2.11 2.427 2.14 2.439 2.14 2.525 2.25 I 2.381 2.01 2.463 2.03 2.433 2.08 2.525 2.14 2.08 -2.03 - 2.370 2.07 -2.14 E, n= 2 II, 2.410 2.02 2.427 2.06 2.451 2.06 2.475 2.16 n= 7 a E is the dielectric constant, a measure of solvent polarThe chloroform contains ethanol and the E is adity. justed accordingly. The uncertainty in v,, the absorption maximum in wavenumbers is iO.005 x l o 4 em-'. The uncertainty in v f , the fluorescence maximum in wavenumbers is iO.02 x 1 0 4 c m - ' . Units of v, and v f are (x lo4 em-'). in a polar environment (29). This fact, along with the findings of Schulman and Capomacchia t h a t t h e exocyclic nitrogen loses electron density upon absorption (27),would suggest assigning the absorption transition of species I1 to a charge transfer (CT) from the exocyclic nitrogen to the ring system (30). T h e effect of structural variations within the homologous series may be seen from this data. T h e fluorescence energy maxima, vf, are lowered upon addition of the first two donating carbon groups. Further addition of carbon groups does not affect t h e value of vf, probably because t h e groups are too distant to inductively donate t o t h e nitrogen. There is no appreciable difference in t h e energies of the excited and ground states due to increased chain length. It is instructive t o compare the fluorescence energy maximum of compound 111, the parent, t o t h e energies of the other compounds. Generally, the values for compound I, compound I1 (n = 2) and compound I1 ( n = 7) are the same as one another but lower than those for t h e parent, compound 111, without any donating carbon groups. Donating carbon groups should stabilize an excited state where t h e nitrogen is electron deficient. This does not imply t h a t the absolute energy of stability of one molecule is greater or less than another molecule, because the energy values of the ground and excited states are not necessarily the same. T h e lifetime and quantum yield data support these conclusions and are presented in Table 111. Both the experimental lifetime, 7, a n d the natural radiative lifetime, T ~ decrease with decreasing polarity of the solvents. For example, for the compound 11, n = 7 , in going from an environment of methanol to chloroform to ethyl acetate to heptane (decreasing polarity), the T decreases from 7.6 ns to 5.9 ns to 4.2 ns to 2.1 ns, respectively. In t h e case of this particular series of compounds, it appears that excited states which are closer in
,
a
18.7 6.9 10.4 7.2 7.9 8.7 7.5 7.5 7.6
0.540 0.111 0.120
35 63
11, n = 1 87 11, n = 2 0.100 72 11, n = 3 11, n = 4 11, n = 5 11, n = 6 0.130 58 0.090 84 11, n = 7 average 73 Units of T and r 0 are nanoseconds. The
13.0 4.9 8.3 10.4 6.6 6.8
5.8 5.9 5.9 error
0.436 0.142 0.210 0.144 0.195 0.165 0.174 0.155 0.065
2061
energy t o their ground states have ,horter natural lifetimes, T ~ . T h e decrease in the average 7" frzm 7 3 ns to 33 ns as the polarity of the solvent system decre,ses may reflect the difference in polarity of the excited statt compared to the ground state. Referring t o Equation 1, it is this decrease in T~ which accounts for some of the decrease in the experimental T values. The lifetime and quantum yield data also reflect the change in structure as the carbon chain attached to the exocyclic nitrogen is lengthened. Addition of the first two carbon chain methylene groups attached to a diethvl amine moiety, increases T~ from a value of 35 ns for the parent, compound 111, to an average value of 73 ns for the series in methanol. This information, again, shows an increase in 70 with a n increase in excited singlet state stabilization, possibly due to carbon donation by t h e first added groups. However, t h e experimentally observed lifetimes decrease upon addition of the first few methylene groups. This may be seen by comparing the T value of 11.2 ns for the parent compound I11 in ethyl acetate with the value of 8.6 ns for the compound 11, n = 1. This is probably a direct result of an increase in the vibrational deactivation because of the increase in the vibronic mode degrees of freedom. T h e magnitude of the rate constant, k l , would be larger in that case since vibrational deactivation is one possible mode of the internal conversion process. T h e observed lifetimes from about two or three methylene groups and above remain essentially constant with increasing chain length and the natural lifetimes also show no obvious trends. This can be seen by comparing compounds 11, n = 3 and n = 7 , in values of observed and natural lifetimes, T and T ~ for , each solvent system. This suggests t h a t further addition of the methylene groups has little or no effect on the radiative transition probability, nor does it increase the excited state's radiationless deactivation. This conclusion can be explained on t h e basis of two observations. First, the chain on the exocyclic nitrogen is not in conjugation with the chromophore and, therefore, does not affect its excited state energy or transition probability with added carbons. Second, a chain attached to the exocyclic nitrogen cannot affect t h e rigidity of the ring system chromophore to any great extent as the carbon chain lengthens. Thus, the vibrational deactivation, the most likely radiationless deactivation process due to increased chain length, probably is not increased as the carbon methylene chain is increased from n = 3 and above. In general, the rigidity of the chromophore determines, predominately, the amount of vibrational deactivation (10). T h e behavior of t h e free base form of all of the species studied may be summarized as follows. T h e homologous series, the parent compound, and atabrine appear to undergo a charge transfer transition involving the exocyclic nitrogen and the fused ring chromophore. T h e excited state is more polar, as compared with the ground state, and is stabilized upon addition of one or two carbon chain methylene groups.
Table 111. Lifetimes and Quantum Yields for Species I1 in Different Polarity Solvents methanol chloroform ethyl acetate b compound T" 0 TO T 0 7 0 T 0 I11 I
50, NO. 14. DECEMBER 1978
30 11.2 0.337 35 4.3 0.088 40 8.6 0.230 72 6.5 0.099 34 4.6 0.104 41 4.5 0.136 33 4.6 0.132 38 4.3 0.106 91 4.2 0.122 48 in T~ reflects both the error in and the
heptane
-
0
T
33 5.9 49 1.9 37 4.4 66 4.1 44 2.1 33 2.0 35 2.0 41 1.9 34 2.1 42 error in 0 .
0
0.289 0.075 0.123 0.111 0.070 0.053 0.055 0.051 0.077
To
20 25 36 37 30 38 36 37 27 33
2062
ANALYTICAL C H t M I S T R Y , VOL. 50, NO. 14, DECEMBER 1978 ~
~ _ _ _ _
Table IY. Effect of Solvent Polarity o n the Transition Energies of Species IV 4% acetic 4% acetic 0 . 1 N HC1 acid/methanol acid/chloro( E = 78)a ( E = 33) form ( E = 6 ) compound uab uf ua "f ua Uf I11 2.433 2.02 2.427 2.03 2.403 1.98 I 2.350 2.00 2.350 1.98 2.331 2.03 11, n = 2 2.342 2.00 2.415 2.22 2.326 --11, n = 7 2.364 1.99 2.364 2.06 2.347 1.99 a E is the dielectric constant, a measure of solvent polarity. The uncertainty in u,, the absorption maximum in wavenumbers is f 0.005 x l o 4cm-I. The uncertainty in uf, the fluorescence maximum in wavenumbers is iO.02 x l o 4 cm-I. Units of u a and u f are ( X l o 4 cm-I). Above n = 2 , no appreciable stabilization is observed. Furthermore, the carbon chain does not appear to cause increased radiationless deactivation beyond n = 2 or 3, i.e., it does not affect the rigidity of the chromophore. Results for Species IV. The energies of transition, U , and uf, are generally not significantly affected by changing from an aqueous/acid mixture to a methanol/acid mixture as shown in Table IV. T h e acetic acid mixtures were all 4% v/v in acid. A decrease in solvent polarity from the acidified methanol ( t = 33) to the acidified chloroform ( t = 6) solvents causes a slight decrease in u,. As can be seen from Table I\', ua for the parent, compound 111, in 0.1 N HC1 is 2.433 X lo4 cm-' and it has the same value in the acidified methanol system. However, the energy of transition, u,, decreases to 2.403 X lo4 cm-' in the acidified chloroform solvent. This implies t h a t nonbonding electrons are involved in this transition since a change to a weakly hydrogen bonding solvent such as chloroform aids the absorption transition while the fluorescence transition is not affected by the solvent in the same way (29). According to pK,* studies on atabrine, the exocyclic nitrogen gains electron density in the excited state (27). T h e transition in acidified water and acidified methanol is thus probably a n - r * transition in the direction of the exocyclic nitrogen (31). A charge transfer cannot take place in water or methanol solvents because the nonbonding electrons on the heterocyclic nitrogen cannot form the T system of species V shown in Figure 1. This is probably because the localization of electrons around the heterocyclic nitrogen is partially retained because of hydrogen bonding by the solvents. In a weakly hydrogen bonding solvent such as chloroform, the electrons on the heterocyclic nitrogen are free to participate in the r system and a charge transfer can take place to the exocyclic nitrogen. This would explain the lower transition energies (I!,) in chloroform listed in Table IV; charge transfer transitions are often of lower energy than n-T* transitions (30). The absorption spectra in chloroform show species IV to be present in the ground state (27). T h e abTable Y.
a
I50
W i l t ,
Ta
@
7 0
b
7
io*
4jO
nTI
Figure 2. Absorbance spectrum of species I V in 4 % acetic acid/ chloroform
IE
P
Figure 3. Charge transfer transition reaction in acetic acid/chloroform proposed for species I V
sorption spectrum given in Figure 2 confirms this fact. It has the same features as an acridone spectrum, as explained by Schulman, indicating the presence of a species with compound IV structure. It is proposed, based on these observations, that the excited state of species IV in chloroform has no double bond to the exocyclic nitrogen; it is broken in the charge transfer transition as shown in Figure 3. Carbon groups would normally be thought to destabilize such transitions as present in species IV through donation. The energies of transition do show that carbon groups aid the absorption transition and not the fluorescence transition. Schulman has suggested that the donating effect would be canceled by the carbon groups disruption of the coplanarity between the nitrogen and the ring system ( 2 7 ) . T h e data support this theory b u t it may also be true that the carbon groups disrupt the solvation of the protonated nitrogen allowing it greater positive character. T h e singlet state observed and natural lifetimes as well as the quantum yield measurements given in Table V support the conclusions from the transition energies study. Increase in the length of the carbon chain in the solvents 0.1 N hydrochloric acid and methanol/acid solvents causes a continuous decrease in the observed lifetime, T, while the natural
Lifetimes and Quantum Yields for Species IV in Different Polarity Solvents 0.1 N HC1 acetic acid/methanol
compound I11 I 11, n = 1 11, n = 2 11, n = 3 11, n = 4 11, n = 5 11, n = 6 11, n = 7 Units of 7 and
InI..
0
20.1 ___ ___ 19.9 0.865 3.2 0.056 57 3.8 0.057 12.7 0.234 54 15.0 0.358 7.4 0.135 6.7 0.090 74 0.089 3.6 0.075 48 4.0 3.3 0.062 2.9 0.027 107 0.057 2.3 0.027 85 2.7 2.1 0.050 1.6 0.013 123 0.068 1.3 0.015 87 2.7 The error in r 0 reflects both the error in 7 0 are nanoseconds.
7 0
acetic acid/chloroform T
0
23 14.0 0.210 67 13.1 0.316 42 10.7 0.265 55 12.7 0.340 45 12.9 0.317 0.398 53 13.4 47 14.3 0.510 0.389 42 13.5 40 14.2 0.222 T and the error in 0.
7 0
67 41 40 37 41 34 28 35 64
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
lifetimes, T ~show , no trend. In the acidified methanol system, t h e observed lifetime decreases from 15.0 ns for compound 11, n = 1, to a value of 2.7 ns for compound 11. n = 7 , while the natural lifetimes are all found to fall around 50 ns. This suggests t h a t the increased chain length causes greater radiationless deactivation of t h e n-T* excited state of species IV, but does not affect the radiative rate or the energy of the excited state. This is shown through both t h e very similar energies of transition, uf, and the similar natural lifetimes, T~~ Of the two possible radiationless deactivation processes, internal conversion, k l , and intersystem crossing, hl, increased chain length most probably would increase k l through vibrational deactivation of the ring system. Phosphorescence studies of spectral energies. intensities, and triplet state lifetimes done in the authors' laboratory show no changes with increased chain length. This lends support to the conclusion, but does not prove, t h a t internal conversion is the primary radiationless deactivation process. In the acidified chloroform system, the increased carbon chain length produces no trends in either T or T~ As was seen in species 11, t h e increased chain length does not affect the deactivation processes of the excited singlet state. This is to be expected if t h e excited state of species IV in acidified chloroform is indeed the species V shown in Figure 3. Species V is very similar to the excited state of species 11. The chain probably cannot affect the rigidity of the chromophore system and, therefore, cannot cause vibrational deactivation of the excited state. In the polar solvents acidified water and acidified methanol, the chain can affect the rigidity of species IV (Figure 1) and can cause increased vibrational deactivation. Other unusual observations made upon these molecular systems may be mentioned. First, no intramolecular exciplex formation was detected such as that seen by E. Chandross e t al., in the case of 9-alkylamino anthracene (32). This could be due to the withdrawing effect of the ring nitrogen. Secondly, there appears t o be no heavy atom effect on these molecules. Acetic acid/aqueous solvent systems gave identical results to those of hydrochloric acid. Chloroform, when used as a solvent, produced no quenching effect. As a result, no solvent quenching effects were probably present. Perhaps the presence of chlorine in the molecular structure itself controls t h e spin-orbit coupling.
CONCLUSIONS From these results, it is found t h a t the best medium for analyzing for one of the members of the atabrine-based series in terms of quantum yield would be an acid-hydrocarbon solvent such as acetic acid/chloroform shown in Table V. This medium produces maximum quantum yield and thus sensitivity for all of the compounds studied. This characteristic could not, however, identify t h e compounds used. Only through the observed lifetimes in an acidic hydrogen bonding solvent can a n individual compound be differentiated from the others in t h e series. A solvent of 0.1 N HCl (aqueous) would be able to distinguish the particular species as shown in Table V. Only in this type of solvent is the primary difference between the members of this atabrine series-the carbon chain length-made t o affect a luminescent property. The chain interacts to decrease the rigidity of the chromophore and thus alter its lifetime with increasing chain length. T h e use of fluorescence lifetimes for qualitative analysis is particularly advantageous in t h a t multicomponent mixtures of t h e series can be separated through computer-aided deconvolution of their fluorescence decay curves (18). There are several conclusions that can be gleaned from this particular study with respect t o t h e qualitative and quantitative analysis of the homologues of the atabrine-based series. (1)Fluorescent lifetimes may be used to distinguish between members of the atabrine-based homologous series under
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certain conditions. Their absorption and fluorescence spectra are superimposed under these same conditions and are useless for qualitative identification. (2) Small changes in an exocyclic group attached to t h e atabrine fluorophore have greater effect on excited state energy if they can influence a n atom which gains or loses electrons in the transition. (3) A substituent which affects t h e rigidity of t h e homologue's fluorophore has a large effect upon its excited state lifetime. T h e conjugated carbon chain substituent in the atabrine series probably enhances t h e amount of internal conversion via vibrational deactivation of the excited state by disruption of the rigidity of the fluorophore, thus decreasing t h e observed lifetime. (4) T o manipulate lifetime differences so that analysis for each of the homologues is possible, the environment can be changed to involve more groups in the excited state structure or t o actually change the transition t h a t is occuring.
SUMMARY One objective of this study was to investigate the analytical utility of selected excited state characteristics in terms of their use to improve both sensitivity and selectivity of analysis for the atabrine-based series. It was shown that via excited state properties it is possible to differentiate between homologues of this series. Another objective was to attempt to understand why the chemical species behave as they do. Work in the authors' laboratory is currently aimed a t investigating a broader class of compounds in order to develop a more general theory of molecular excited state behavior. I t is hoped t h a t these types of studies will ultimately result in a better understanding of the excited state and that this understanding will lead to better analytical control. T h e earlier portions of this paper contain a great deal of mechanistic hypothesizing in addition to the evaluation of analytical usefulness. If one is to ultimately design a theory of how the microenvironment can be used to influence the excited state so that more selective and sensitive analysis can be carried out, it is felt that such mechanistic interpretation is mandatory. These excited state studies should allow more intelligent manipulation of molecules and development of methods of analysis through judicious use of their excited state properties.
ACKNOWLEDGMENT T h e gifts of t h e atabrine-based homologous series from George Downing, Merck, Inc., Rahway, N.J., and the atabrine and parent compounds from Sterling-Winthrop, Rensselaer, N.Y., are gratefully acknowledged. We also thank Rytis Balciunas for t h e phase solubility analyses.
LITERATURE CITED (1) A. Weissler, Anal. Chem., 46, 500R (1974). (2) C. O'Donnell and T. N. Solie, Anal. Chem., 48, 17% (1976). (3) S. Udenfriend, "Fluorescence Assay in Biology and Medicine", Academic Press, New York, 1969. (4) G. Guilbault, "Practical Fluorescence", Marcel Dekker, New York. 1973. (5) J. D. Winefordner, S. Schulman, and T. O'Haver. "Luminescence Spectrometry in Analytical Chemistry", Wiley-Interscience, New York, 1972, Chap. IVB. (6) C. Parker, "Photoluminescence of Solutions' , Elsevier Publishing, Amsterdam, 1968, Chap. 5. (7) L. J. Cline Love and L. A. Shaver, Anal. Chem., 48, 364A (1976). (8) H. Zimmerman, D. Werthemann and K. Kamm, J . Am. Chem. SOC., 96, 439 (1973). (9) W. R. Ware in "Creation and Detection of the Excited State'', Vol. 1. Part A, A. A. Lamola, Ed., Marcel Dekker, New York, 1971, Chap. 5 . (10) T. C. Werner in "Modern Fluorescence Spectroscopy", Vol. 2, E. L. Wehry, Ed., Plenum Press, New York, 1976, Chap. 7. (11) T. C. Werner and R. Hoffman, J . Phys. Chem., 77, 161 (1973). (12) A. Bowd and J. Turnbull, J . Chem. Soc., Perkin Trans. 11, 121 (1977). (13) J. D. Winefordner, Acc. Chem. Res., 2, 361 (1969). (14) J. D. Winefordner, K. Harbaugh, and C. M. O'Donnell. Anal. Chem., 45, 381 (1973). (15) J. D. Winefordner, K. Harbaugh, and C. M. O'Donnell, Anal. Chem.. 46, 1206 (1974). (16) J. Aaron, L. Sanders, and J. D. Winefordner, Clin. Chlm. Acta. 45, 375 (1973).
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(17) J. D. Winefordner, K. Harbaugh, and C. M. O'Donnell, Anal. Chem., 46, 1195 (1974). (18) L. A. Shaver, L. Upton, A. M e r , and L. J. Cline Love, 28th Annual Meeting, PittsburghConference on Anavical Chemistry and Applied Spectroscopy, Cleveland Ohio, 1977, Paper No. 359. (19) L. Roberts, J . Assoc. Offic. Anal. Cbem., 53, 117 (1970). (20) S. Latt, S. Brodie, and S. Munroe. Cbromosoma, 49, 17 (1974). (21) S. Massari, P. Dell'Antone, R. Colonna, and G.Azzone, Biochemistry, 13, 1038 (1974). (22) B. Henry and W. Siebrand in "Organic Molecular Photophysics", Vol. I , J. Birks, Ed., Wiley-Interscience, London, 1973, Chap. 4. (23) W. Ware, C. Lewis, L. Doemeny, and T. Nemzek, Rev. Sci. Instrum., 44, 107 (1973). (24) L. A. Shaver, L. J. Cline Love, J. Habarta, L. Upton, and R. Balciunas, 27th Annual Meeting, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, 1976, Paper No. 94. (25) L. A. Shaver and L. J. Cline Love, Appl. Spectrosc., 29, 485 (1975). (26) J. Demas and G.Crosby, J . Pbys. Cbem., 75, 991 (1971).
(27) S. Schulman and A. Capomacchia, Anal. Cbim. Acta, 77, 79 (1975). (28) D. Sawyer and J. Roberts, "Experimental Electrochemistry for Chemists", John Wiley and Sons, New York, 1974. (29) S. Schulman, Fluoresc. News, 6 (4), 1 (1972). (30) S. Schulman, Fluoresc. News, 7 (5), 33 (1973). (31) S. Babiak and A. Testa, J . Pbys. Cbem., 80, 1882 (1976). (32) E. Chandross and H. Thomas, Cbem. Pbys. Lett., 14, 563 (1972).
RECEILTD for review November 1, 1977. Accepted September 5 , 1978. T h e financial assistance of the Analytical Division of the American Chemical Society through a n award of a 1976-77 full year fellowship t o L.M.U. is gratefully acknowledged. Research support provided by the State of New Jersey under provisions of the Independent Colleges and Universities Utilization Act is also gratefully acknowledged.
Separation of Eight Transition Elements from Alkali and Alkaline Earth Elements in Estuarine and Seawater with Chelating Resin and Their Determination by Graphite Furnace Atomic Absorption Spectrometry H.
M. Kingston," I. L.
Barnes, T. J. Brady, and T. C. Rains
National Measurement Laboratory, Center for Analytical Chemistry, Inorganic Analytical Research Division, National Bureau of Standards, Washington, D.C. 20234
M. A. Champ The American University, Washington, D. C. 200 16
A method is described for determining Cd, Co, Cu, Fe, Mn, Ni, Pb, and Zn in seawater using Chelex 100 resin and graphite furnace atomic absorption spectrometry. The pH of the seawater is adjusted to 5.0 to 5.5 and then passed through a Chelex 100 resin column. Alkali and alkaline earth metals are eluted from the resin with ammonium acetate and then the trace elements are eluted with two 5-mL aliquots of 2.5 M "0,. The difficulties previously encountered with resin swelling and contracting have been overcome. By careful selection of the instrumental conditions, it is possible to determine subnanogram levels of these trace elements by graphite furnace atomic absorption spectrometry. The proposed method has been shown to separate quantitatively the elements desired from the alkali and alkaline earth metals and has been applied in the analysis of trace elements in estuarine water from the Chesapeake Bay and seawater from the Gulf of Alaska.
T h e literature of marine water analysis reflects the considerable difficulty in establishing an accurate and precise method of analysis for trace metals. A seawater matrix defies a simplified approach. For example, specific sampling techniques, container contamination, suspended particulate matter, and analytical techniques have to be considered. I t is beyond t h e scope of this paper to discuss all of these parameters; however, the solving of the analytical problem is of little value unless a representative sample can be obtained, free of contamination and properly stored until analysis. In recent years, methods have been developed to determine trace elements in seawater by X-ray fluorescence ( I ) , neutron
activation ( 2 , 3 ) , spectrophotometry ( 4 ) , anodic stripping voltammetry (5),and atomic absorption spectrometry (6-8). However, each of these analytical techniques requires a preliminary separation. Fabricand et al. (9)reported the direct determination of Cu, Fe, Mn, Ni, and Zn in seawater by atomic absorption spectrometry (AAS) using an air-acetylene flame, but other workers have reported difficulties using their technique because of light scattering and burner clogging. Except for neutron activation analysis and anodic stripping voltammetry, no analytical techniques are currently available for the direct determination of trace elements in seawater a t concentrations below 5 pg L-'. Usually it is necessary to preconcentrate the trace elements from a large volume and separate the transition elements from the alkali and alkaline earth elements. In such sample preparations, the efficiency of concentration, completeness of separation, and total analytical blank become critical to the final instrumental method (10). Preconcentration techniques which have been used are coprecipitation ( I I ) , chelation and extraction ( 1 2 ) , and chelating ion-exchange resin ( I O , 13). Most of these isolation methods require large volumes of chemicals which can lead to high blanks unless the reagents have been carefully purified. Of the presently used preconcentration techniques, Chelex 100 chelating resin has been shown t o be efficient and yields low analytical blanks (14). Applications of Chelex 100 resin for trace metal preconcentration from seawater have been reviewed by Riley and Skirrow (10). Chelex 100 is a strong chelator and removes metal ions from most known naturally occuring chelators in seawater (14-16). T h e resin will not, however, remove metals held in organic and inorganic colloids which can be present even after ultrafiltration. Precautions
This article not subject to U.S. Copyright. Published 1978 by the American Chemical Society
