Anal. Chem. 1980, 52, 769-771
thermodynamic parameters of acid dissociation reactions, these depend to a great extent on the nature of the functional group involved. For as close a match as possible, one must compare acids of the same charge type (neutral acids such as HCOOH, RSH behave quite differently than cationic acids such as RNH3+,R2NH2+,and R,NH+), and preferably having the same functional group and similar pK values. For a series of ten thiols, including mercaptoacetic acid, P-mercaptopropionic acid, o-mercaptobenzoic acid, 2-mercaptoethanol, 3-mercapto-l,2-propanediol, 2-acetylaminoethanethiol,as well as the simple aliphatic thiols, ethanethiol, 1-methylethanethiol, and 1,l-dimethylethanethiol, whose average value of pK = 10.2 (S= 0.78) the average AH" is 6.0 kcal/mol ( S = 0.48) and average A S o = -27 eu ( S = 4.5) ( I O ) . These compounds provide good comparison with the dithizones with the important exception that they are considerably weaker acids than dithizone (pK = 4.7). Examination of a large number of acids of several related groups indicates that, within a given c h u g e type, the entropy change is far less sensitive to p K variation than is the enthalpy change ( I O ) . Hence, a reasonable estimate of the ASo for the dithizones would be -27 eu (perhaps *lo). From the pK value of dithizone, which gives the equivalent AGO value of 6.5 kcal/mol, this would imply a W" of' dissociation of -2 kcal/mol, with an estimated error of f3.0 kcal/mol (based on f 1 0 eu). This is far below the value of A H o of 15.8 for the overall reaction which indicates a surprisingly highly negative AH" for the distribution process (aqueous organic) (- -18 f 3 kcal/mol). Furthermore, this kind of situation would seem to apply to all of the substituted dithizones, though to a somewhat lesser extent with the p-chloro and o-tolyl derivatives. Analogously, the AS" for distribution of dithizone also may be estimated as -35 eu. This highly unfavorable entropy would appear to indicate that either the dithizone molecule has much more rigid structure in the organic phase than in water, or that it is capable of ordering the organic solvent around it to a far greater extent than it does with water. Both the enthalpy and entropy factors would be consistent with specific dithizonechloroform interaction. This merits further investigation. T h e reaction rate constants for the formation of the nickel dithizonates, NiDz', a t 25 "C are in good agreement with those previously determined ( I ) . It is interesting to note that the large negative activation entropy observed in all cases is the dominant factor affecting the rate. While this may justify our interest in entropy, the relative constancy of AS* (excluding the o-tolyl ligand, it averages as -29 f 3 eu) certainly minimizes its role in the substituent rate enhancement. Rather, the fl, unusually small for all of the systems studied. is seen
-
769
to decrease with increasing substituent size (rather than electronegativity) and, thus, to account for the observed rate enhancement. In the case of the o-tolyl ligand, which may be best compared with the p-tolyl ligand, we note a significant increase in which is in the right direction for the kind of adverse steric hindrance expected with the 2-methyl group. The effect on the rate constant is small because of a compensating rise in AS*.Such compensations of enthalpies and entropies have been observed in metal chelate equilibrium ( 1 1 ) and would seem to be a fairly general phenomenon. While the reaction rate constants for the nickel dithizonates reported here are in general agreement with other nickel ligand substitution reactions ( I ) , the activation enthalpies and entropies of the relatively few other nickel chelation systems studied are considerably different from those seen here. For a series of diverse ligands including oxalate, malonate, glycine, 2,2-bipyridyl, and 1,lO-phenanthroline, the formation of the 1:l complexes exhibited flvalues of 13 to 15 kcal/mol and A S * values of 0 to +14 eu (12). The very low flexhibited in the systems reported here may well indicate that the formation of the 1:1 chelate is fairly complex and may involve a preassociation step having a A H of the order of -8 to -10 kcal/mol, which would largely compensate for the expected value of 13 to 15. Whether the similarities in the AGt accompanied by large differences in SY and A S * are attributable in large measure to sulfur-containing ligands in general or to dithizone-like ligands in particular, awaits further study. Although we do not have any definitive answers, further examination of the kinetics of other S-containing ligands would seem to be of value not only to analytical chemistry, but for a better understanding of mechanisms of chelation reactions. Such work is underway in our laboratory. ~
LITERATURE CITED Oh, J. S.; Freiser, H. Anal. Chem. 1967, 3 9 , 295. Honaker, C. B.;Freiser, H. J . Phys. Chem. 1962, 66, 127. McClellan. B. E.; Freiser, H. Anal. Chem. 1964, 36, 2262. Joy, R.; Orchin, M. J . Am. Chem. SOC. 1959, 8 1 , 305. Carter, S. P.; Freiser, H. Anal. Chem. 1979, 5 1 , 1100. Bamberger, E.; Padova. R.; Ormerod, E. Ann. 1920, 4 4 6 , 260. Busev, A. I.; Bazhanova, L. A. Russ. J . Inorg. Chem. 1961, 6, 1416. Hubbard, D. M.; Scott, E. W. J . A m . Chem. SOC.1943, 65, 2390. Math, K. S.; Fernando, Q . ; Freiser, H. Anal. Chem. 1964, 3 6 , 1762. Christensen, J. J.; Hansen, L. D.; Izatt, R. M. "Handbook of Proton Ionization Heats and Related Thermodynamic Quantities"; John Wiley & Sons: New York, 1976. Johnston, W. D.; Freiser, H. Anal. Chim. Acta 1954, 1 1 , 201. Wilkins, R. G. Acc. Chem. Res. 1970, 3 , 408.
RECEIVED for review November 26, 1979. Accepted January 17, 1980. This work was conducted with financial assistance from the National Science Foundation.
CORRESPONDENCE Selective Analysis of Binary Fluorophor Mixtures by Fluorescence Polarization Sir: There is considerable interest in the problem of analyzing mixtures of fluorophors with overlapping spectra without having to resort to a separation ( I ) . Recently, it has been proposed that differences in polarization can be used t o resolve binary fluorophor mixtures (2). In fact, this has 0003-2700/80/0352-0769$01.00/0
been widely practiced by biochemists and immunochemists who have used fluorescence polarization to study the binding of small fluorophors to proteins (3, 4). The binary mixture in these systems involves two forms of the same fluorophor, free and bound to a protein. $2 1980 American Chemical Society
770
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
I I
In work done to date, the measured parameter has been the polarization, p , which is defined:
I,, - I
PM
I
‘ F
,
P
T o measure p , a sample is excited with polarized light. One then measures the components of the emission which are parallel and perpendicular t o the polarization plane of the excitation radiation. These are designated Ill and I - , respectively. T h e polarization, p , is independent of concentration. In studies of binary mixtures, the procedure has been to relate the observed polarization to the fraction of one of the fluorophors (e.g., fraction of total fluorophor bound to protein). This requires knowing the polarizations of both forms of the fluorophor as well as knowing the total amount of fluorophor by a n independent method ( 3 ) . We wish to point out and discuss an alternative approach for using fluorescence polarization to analyze binary mixtures. T h e new approach makes it possible to observe a signal that is directly proportional to the concentration of one component of t h e mixture and independent of the concentration of the other component. It is necessary only to know the polarization of the unmeasured component. This approach is illustrated experimentally using mixtures of 2’,7’-dichlorofluorescein and rhodamine B.
THEORY We will assume a mixture of two components, A and B, which differ in polarization. Furthermore, we will assume that the concentrations of A and B are in a range where fluorescence intensity is directly proportional to concentration, Le., no “inner filter” effects. The ratio of I , to I,,depends on the polarization for both A and B. The relationship of this ratio t o the polarization is calculated by rearranging Equation 1:
where the subscript refers to component. Since fluorescence intensity is proportional t o concentration:
where CA is the concentration of A, and K.4 is the proportionality constant. Analogous expressions exist for component B, of course. Experimentally, with a mixture of A and B, one measures t h e total intensities parallel and perpendicular to the excitation radiation, defined Zlr and ZIT,respectively. These total intensities will represent the sum of the contributions of A and B:
I I ~=TI~IA + I I ~=B KACA+ KBCB
T o selectively determine A in the presence of B, we measure and both I l i T and ILT.Then we multiply IIT by IIIB/IIB subtract from TIT. This causes the two CB terms to be equal so the resulting difference depends only on CA:
D
PF-
IPNI diagram of instrument for measuring fluorescence polarization, S = source, M = monochromator, P = polarizer, C = sample cell, F = filter, and PM = photomultiplier Figure 1. Block
3:
4 0
3
061
c a 041
01 35c
’
’
400
4 50
50C
L
5 50
600
65 0
WAVELENGTH . n m ”
Excitation and emission spectra for 2’,7‘dichlorofluorescein in glycerol Figure 2.
Clearly one can also selectively measure B in the presence of A by the same approach. If A and B have the same polarization, the (1- ( I - A / ~ l , A ) ’ ( z l i B / z I B ) ) term equals zero and this approach fails. The greater the difference in polarization, the larger this term is.
EXPERIMENTAL Reagents. Mixtures of 2’,7’-dichlorofluorescein and rhodamine B in glycerol were prepared by appropriately diluting stock solutions of 2’,i’-dichlorofluorescein and rhodamine B in glycerol. The high viscosity of glycerol minimizes depolarization of fluorescence due to molecular rotation during the lifetime of the excited state. Instrumentation. Figure 1 shows our homebuilt instrument for measuring fluorescence polarization. The source is a 10000 lumen tungsten-halogen lamp from Edmund Scientific. The excitation wavelength is selected by an Aminco grating monochromator. There are two detection channels for simultaneous measurement of Ill and I _ . The emission wavelengths are selected by a filter. Conventional film polarizers are used. Photomultiplier currents are amplified by a current-tovoltage operational amplifier and introduced directly to a DEC ?VIINC-11computer system for data analysis. Procedures. The relative gains of the two detection channels are determined by orienting the excitation polarizer horizontally so that both channels measure I,. Blank values for both channels are determined using pure solvent with the excitation polarizer reoriented vertically. The polarization of the unwanted component is determined using a pure solution of this component. Both blank values, relative gain, and polarization of the unwanted component are entered into the computer and are appropriately accounted for in subsequent calculations. Instrumental Settings. Figure 2 shows excitation and fluorescence spectra for 2’,7’-dichlorofluorescein along with the transmittance characteristics of the cutoff filters used with both detection channels. All mixtures were excited at 420 nm. Figure 3 shows identical information for rhodamine B. These conditions have been contrived to yield significant spectral overlap while maintaining a significant difference in the polarizations of the two components at the excitation wavelength. Excitation spectra were measured on the fluorescence polarization spectrometer using only one detector with the polarizers removed. Fluorescence emission spectra were measured on a Perkin-Elmer 204 spectrofluorometer. Rather than presenting complete spectra, we show only spectra Over the wavelength range
77 1
Anal. Chem. 1980, 52, 771-773
01
350
400
450
500
WAVELENGTH
5 50
GOO
6 50
1 nm 1
Flgure 3. Excitation and emission spectra for rhodamine B in glycerol. T is t h e transmittance of the cutoff filters used for all measurements
Table I. Signals for Varying Concentrations of Rhodamine B ( C R , B . )in the Presence of Fluorescein’
0 5.1 x lo-1.01 x
2.00 X 2.98 x 3.79 x
1.370 1.919 1.829 2.046 2.435 2.611
0.814 1.339 1.509 2.004 2.593 3.0‘74
as the conventional approach based on measuring “ p ” . I t should, however, be pointed out that measurements of p are not affected by inner filter effects, and therefore are possible in absorbing solutions where the new approach will fail. The 2’,7’-dichlorofluorescein-rhodamineB mixtures were contrived to illustrate a new idea. In fact, we believe that this approach w i l l be of greatest value for binary mixtures involving two forms of the same fluorophor. For example, consider the reaction of fluorescein isothiocyanate (FITC) with a protein to yield a protein-fluorescein conjugate.
0.026 0.379 0.760 1.392 2.013 2.663
1.34 x 1.33 x 1.44 X 1.48 x 1.42 X
M a Fluorescein concentrations are roughly 2 X For this with some variation from solution to solution. ) 1.72 where R is set of data, the value of R ( I , ( F / I L Fwas the relative gain of the two detection channels and IiiF/I-F are the relative values for 11, a n d I - determined using a solution containing only fluorescein.
relevant to the measurements described in the text. The y-axis scale refers to the transmittance of the cutoff filters. The emission and excitation spectra are in relative units. RESULTS AND DISCUSSION Calibration curves for both rhodamine B and 2’,7’-dichlorofluorescein were prepared in the presence of each other. In all cases, response was linear as long as analyte concentration was low enough to avoid inner filter effects. Table I shows a typical set of raw data and how it is analyzed to yield concentration. In the chosen example, the response to varying amounts of rhodamine B was measured in the presence of fluorescein. The ratio of the rhodamine B concentration to the difference signal corrected for relative gain stays essentially constant. This shows that response is proportional to rhodamine B concentration with an intercept going through zero. T h e possibility of utilizing differences in polarization to selectively measure one component of a binary mixture has been demonstrated. The new approach yields concentration directly and does not require as much additional information
FITC
+ protein
-
protein-F
This reaction will lead to significant change in polarization with very little change in spectral characteristics. T h e concentration of protein-fluorescein conjugate can be selectively measured in the presence of excess FITC by our method. We hope to investigate some systems of this type in the future. We do not recommend this approach when the two components of a mixture have differing spectra and can be resolved by making measurements a t more than one set of excitation and emission wavelengths. Spectral resolution offers better signal-to-noise ratios since measurements are made a t wavelengths affording maximum selectivity for each component. Also, to get significant polarizations for small molecules, it is necessary that they have either short fluorescence lifetimes or long rotational relaxation times which require high viscosity. We would also like to point out that this approach can be readily implemented on conventional fluorescence instruments merely by adding two polarizers. On a one-detector instrument, Ill and I , must be measured sequentially rather than simultaneously as on our two-detector instrument. This is simply a matter of convenience. Polarizers for visible light are trivial in cost while polarizers for UV light cost a few hundred dollars. LITERATURE CITED Johnson, D. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1977, 4 9 , 747A. (2) Bozhel’nov, E. A , ; Gribkov, V. I . ; Tropina, L. P.; Fakeeva, 0.A . Russ. J . Anal. Chem. 1978, 32, 1594. (3) Dandliker, W.: Dandliker, J.; Levison, S. A , ; Kelly, R. J.; Hicks, A. N.; White, J. U. In “Methods of Enzymology”, Vol. XLVIII, Hirs. C. H. W., Timasheff, S. N., Eds.; Academic Press: N ew York. 1978; p 380 ff. (4) Dandliker, W. 6.;d e Saussure, V. A . Immunochemistry, 1970, 7, 7 9 9 . (1)
P a u l M. Roemelt A n t h o n y J. L a p e n W. Rudolf Seitz* Department of Chemistry University of New Hampshire Durham, New Hampshire 03824
RECEIVED for review October 1, 1979. Accepted January 8, 1980.
Determination of Peroxides by High Performance Liquid Chromatography with Amperometric Detection Sir: The chemistry of organic peroxides has found widespread technological usefulness. The interesting and useful features of this chemistry, however, also pose a significant safety hazard in the storage and handling of these compounds. As a result of the combination of these factors, a considerable research effort has been devoted to the analysis of compounds containing the peroxide bond. This is reflected in the large 0003-2700/80/0352-0771 $ O l . O O / O
number of approaches available for this purpose ( I ) . Physical, instrumental, chemical reduction, and colorimetric methods have all been developed. T h e wide range of physical and chemical properties among the various classes of peroxides has been largely responsible for this proliferation. The various methods can be loosely grouped in two categories. Experiments designed to separate peroxide mixtures have been 0 1980 American Chemical Society
