ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978 S. S. Hecht, W. E. Bondineil, and D. Hoffman, J. Nafl. Cancer Inst., 53, 1121 (1974). S. S. Hecht, M. Loy, and D. Hoffman, in “Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis”, R. Freudenthal and P. W. Jones, Ed., Raven Press, New York, N.Y., 1976, p 325. K. D. Brunneman and D. Hoffmann, in “Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis”, R. Freudenfhal and P W. Jones, Ed., Raven Press, New York, N.Y., 1976, p 283:G. Mamantov, E. L. Wehry, R. R. Kemmerer, and E. R. Hinton, Anal. Chem., 49 (1977) . - ,86 __ - , R. C.Stroupe, P. Tokousbalides, R. B. Dickinson, Jr., E. L. Wehry, and G. Mamantov. Anal. Chem.. 49, 701 (1977). E. L. Wehry, G.Mamantov, R. R. Kernmere;, H. 0. Brotherton, and R. C. Stroupe, in “Polycyclic Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis”, R. Freudenthal and P. W. Jones, Ed., Raven Press, New York, N.Y., 1976, p 299. R. C. Stroupe, Ph.D. Dissertation, University of Tennessee, Knoxville, Tenn., 1977. E. V. Shpol’skii and T. N. Bolotnikova, Pure Appl. Chern., 37, 183 (1974). C. Pfister, Chem. Phys., 2, 171 (1973). ~
1193
(13) J. J. Dekkers, G. P. Hoornweg, G. Visser, C. Maciean, and N. H. Vekhorst, Chem. Phys. Lett., 47, 357 (1977). (14) G. Mamantov, E. L. Wehry, R. R. Kemmerer, R. C. Stroupe, E. R. Hinton, and G. Goldstein, Adv. Chem. Ser., in press.
RECEIVED for review January 26, 1978. Accepted April 24, 1978. Financial support for this work was derived from a contract with the Electric Power Research Institute (741011-RP-332-1) and a grant from the National Science Foundation (MPS75-05364). Purchase of the FTS-20 Spectrometer was partially supported by the National Science Foundation Research Instrument Grant GP-41711. Portions of the work were presented at the 29th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 2 , 1978.
Corrections for Inner-Filter Effects in Fluorescence Quenching Measurements via Right-Angle and Front-Surface Illumination Richard A. Leese’ and E. L. Wehry” Department of Chemistry, University of Tennessee, Knoxville, Tennessee 379 16
I n measurements of fluorescence quenching, “inner-filter” absorption, by the quencher, of the exciting light or the fluorescence emitted by the fluorophore (or both) can cause large errors in the evaluation of quenching efficiencies. Equations are developed for correction of inner-filter effects In both right-angle and front-surface illumination geometries. Absorption of exciting light only, absorption of fluorescence only, and simultaneous absorption of exciting radiation and fluorescence by the quencher are considered separately for each geometry. An alternatlve procedure for front-surface illumination, wherein quenching data as a function of cell pathlength are subjected to a curve-fitting procedure to produce a “best-fit’’ value for the true quenching efficiency, is also developed. Application of the various procedures to experimental fluorescence quenching data is demonstrated.
Measurements of fluorescence quenching are widely employed in studies of mechanistic photochemistry (1, 2); moreover, the quenching action by one solute, Q, upon the luminescence of a fluorophore, M , has been exploited for analytical purposes ( 3 , 4). “Quenching” is defined as any process by which quencher Q decreases the fluorescence quantum efficiency of fluorophore M; formation of groundstate complexes between M and Q (which does not alter the fluorescence yield of uncomplexed M but does decrease its equilibrium concentration) may also be regarded operationally as a form of fluorescence quenching. The effect of Q upon the fluorescence of M is an attenuation, usually described quantitatively by the Stern-Volmer (SV) equation ( 5 ) : where aF0and aFare the fluorescence quantum yields for M in the absence and presence of Q, respectively, [Q] is the concentration of quencher, and Ksv is the Stern-Volmer Present address, Lederle Laboratories, P e a r l River,
N.Y.
0003-2700/78/0350-1193$01 .OO/O
“constant”, the interpretation of which is discussed elsewhere (1, 5 ) . An important, and often unavoidable, artifact in fluorescence quenching measurements is absorption of the exciting light and/or fluorescence of M by the quencher. Such processes are termed “inner-filter effects” (6);in any treatment of quenching data by the SV equation, inner-filter effects must be carefully distinguished from true quenching, and proper corrections for inner-filter absorption must be made or the calculated Ksv will be subject to significant systematic error. There is no obvious experimental procedure capable of consistent and accurate compensation for inner-filter effects. Measurement of fluorescence, in the presence and absence of Q, for the same set of solutions in rectangular cells of different thicknesses, followed by extrapolation to zero path length (3, has been employed for this purpose in the case of front-surface (8, 9) illumination. In our experience, this procedure often produces nonlinear plots of apparent quenching vs. path length; thus, accurate extrapolation to zero cell thickness cannot be effected. Moreover, it is not obvious that such an approach is applicable to right-angle illumination. Consequently, one must usually resort to mathematical corrections which, it is hoped, reliably correct the observed quenching data for inner-filter effects. Development of such procedures has been described by a number of workers (10-16). The reported corrections, however, are valid only under certain highly specific conditions, and the assumptions made in the derivations are usually not stated explicitly. For example, many corrections for inner-filter absorption seem to implicitly assume that the exciting radiation is monochromatic, which is incorrect for most commercial fluorescence spectrometers. In the present paper, we develop equations which can be used to correct for absorption, by Q, of the exciting light, or the fluorescence of M , or both, in both right-angle and front-surface geometries. Our definitions of right-angle and front-surface illumination are identical with those provided by Parker (8)and Winefordner, Schulman, and O’Haver (9). There are some conceptual similarities in our approach with that previously described by Ehrenberg, 0 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Cronvall, and Rigler ( 2 4 ) for the specific case of absorption by Q of exciting radiation in right-angle geometry.
“monochromaticity” cannot safely be assumed; in that case, a more accurate expression for F’ is
GENERAL CONSIDERATIONS AND ASSUMPTIONS In most cases of fluorescence quenching, the fluorescence spectrum (“intensity” vs. wavelength) of M is unaltered by Q and is thus independent of [Q]. In that case,
F o = kGF0[ M ] J h ”PhofhM , dh
@pFo/@F =
Fo/F
(2)
where F’ and F are the observed fluorescent powers (or “intensities”), at the same excitation and emission wavelengths, in the absence and presence of Q, respectively. The SV equation then becomes
F o / F = ICsv[&]
+1
(3)
In addition to assuming the validity of Equation 2, we assume that self-absorption of fluorescence by M is negligible. This assumption can virtually always be satisfied experimentally by employing a small concentration of M and by proper choice of emission wavelength;while correction procedures have been developed for reabsorption (17-20), correction for simultaneous inner-filter action by M and Q would be very cumbersome. Corrections for changes in refractive index as a function of [Q] are likewise not considered here; if required, the treatment of Rohatgi and Singhal (27) could readily be applied to the equations derived herein. That the SV equation requires measurement of a ratio of fluorescent powers is fortuitous; several quantities (noted in subsequent discussion) which are very difficult to measure accurately in a system exhibiting inner-filter effects either do not have to be evaluated at all (because they occur in F’and F and hence cancel) or can be evaluated in arbitrarily chosen units (because the units cancel). Our general approach consists of multiplication of the observed quenching ratio, F ‘ / F , by a calculated correction factor, P / P :
Ksv[Q] + 1= ( F o / F ) ( F o f / F o=)Fo’/F
(4)
where F“ is the “corrected reference fluorescent power”, i.e., the fluorescent power which would be observed for a solution of M containing a specified [Q] if Q acted as an inner filter but not as a quencher (Ksv = 0). Both and F a r e evaluated for the same [Q] and are thus affected in the same manner by the inner-filter action of Q. Consequently, evaluation of the ratio F”/F “cancels” the inner-filter effect. If Fo‘ can be accurately evaluated, the value of Ksv calculated from Equation 4 is that which would have been observed if Q acted as a quencher but not as an inner filter. Inasmuch as P / F is a measured quantity, correction for the inner-filter effect by this method reduces to development of expressions for F” and F’ (the former of which must be evaluated for each [Q] to be employed in constructing the SV plot). Applications of this procedure to specific situations commonly encountered in fluorescence quenching studies are described in the following section.
SPECIFIC CASES I. Absorption of Exciting Light by Q; Right-Angle Illumination. For dilute solutions of fluorophore M ( A < 0.05 a t the nominal exciting wavelength, A), the fluorescent power observed in the absence of Q is given by (21)
F o = K@’FoPoEM[MI
where AI and X2 are the wavelength extremes of the excitation monochromator bandpass and P: is the incident power per unit wavelength interval. W e assume that @Fo is independent of excitation wavelength within the excitation monochromator bandpass; this assumption may require that the extremes of the bandpass be constrained to lie within a single absorption band of M . Because all quenching data are evaluated as ratios of fluorescent powers, the units of PAo can be arbitrary; P: may thus be measured by use of a quantum counter (22) rather than resorting to the more cumbersome procedure of chemical actinometry. Analogously, the fluorescent power F observed in the presence of Q is expressed as
(7) where PAis the attenuated power per unit wavelength interval within the volume of solution from which fluorescence is measured; in most right-angle spectrometers, emitted light is viewed from only a small portion of the exit cell face (9,14, 23). PAcan be expressed as
where c,Q is the molar absorptivity for Q at X and beffis the average pathlength traveled by exciting radiation in relation to the volume of solution from which fluorescence is measured. Assuming that the exit slit is centered on the fluorescence cell face, beffis simply 0.5b, where b is the nominal thickness of the rectangular cell. Next, the “corrected reference fluorescent power”, F“,is calculated from
(9) Substitution of Equation 8 into Equation 9, followed by division of Equation 9 by Equation 6, yields the correction factor in final form:
Note that, in addition to the molar absorptivities, only PAo need be evaluated in order to calculate the correction factor; this evaluation is conveniently performed via quantum counter (22). It is especially noteworthy that @Fo for M need not be measured, even though it appears in the expressions for both F‘ and F“. For numerical evaluation, it is often convenient to express the integrals in Equation 10 as summations. The range of summation (A, to A,) is incremented by the constant factor Ax,, into N “resolution elements”. Within each of these finite wavelength elements, cAQ, chM, and PAoare calculated as the average value of the respective quantity within that wavelength interval (trapezoidal area approximation). This procedure leads to N
0.5
F o ’ / F = 2 Po(hi)eM(hj)lOi= 0
5 PO(hi)EM(hi)
(5)
where is the source power at A, cM is the molar absorptivity of M at A, and k is a proportionality factor including, inter alia, the instrumental “geometry factor”. Unfortunately, for many commercial fluorescence spectrometers, the spectral bandpass of the excitation monochromator is such that
(6)
i= 0
where Xi= hl and
+ i Ah,,
fQ(hi)b[Q1
I
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Ahex = (hz - h,)/N (13) Multiplication of the observed F‘/F by the correction factor so determined, for each value of [Q], yields a valid measure of the quenching action of 8. 11. Absorption of Fluorescence by Q; Right-Angle Geometry. We assume that Q absorbs the fluorescence of M , but not the exciting light. If the excitation beam is centered on the face of a rectangular cell of width w, then the average pathlength traversed through the solution by the monitored fluorescence is 0.5 w. In the absence of Q, assuming no self-absorption of fluorescence by M , the measured fluorescent power F‘ is given by
F o = k J ~ 3 h 4 E ~ o Bdh xR,
where x is the displacement, in cm, of a particular element of area within the sample from the front face of a rectangular cell of length b, and all other symbols have the same meanings as in Section I. Evaluation of the integral yields I
A value of P for each [Q]is calculated by substitution of Equation 19 into Equation 9. Equation 6 is directly applicable to calculation of t;O in this case. Thus, for the correction factor, we have
(14)
where X3 and X4 are the wavelength limits of the emission monochromator bandpass, EAois the true emitted power per unit bandwidth of M a t wavelength A, B x (22) is the power transmitted at wavelength X by the emission monochromator, and RA (22) is the photomultiplier response factor. Next, a value for P is obtained by calculating the extent of attenuation of EAodue to inner-filter absorption by Q within the emission monochromator bandpass: Fo‘ = kJh,h4EhOBhRh10-‘h Q weff[Q1 dh =
k J ~ 3 h 4 E ~ o B ,10R h0.5ehQw[Q]
1195
dh
(15)
where w,ff, the effective average pathlength of emitted light, is (as noted above) assumed to equal half the cell width, w. The correction factor is obtained by division of Equation 15 by Equation 14 for each value of [Q].As for Equation 10, evaluation of the correction factor may be simplified by expressing it in summation form. If one divides the emission monochromator bandpass, X4 - As, into M equal elements of width Ahx,,, then, using j as the index for the summations, the correction factor is
EE0(hj)B(hj)R(hj)
where x is the displacement of a particular element of area within the sample from the front face of a rectangular cell of path length b. Integration of Equation 2 1 yields
(16)
If the emission monochromator bandpass is not large, then the variation of the photomultiplier response factor, RA,with wavelength may be sufficiently small that it can be assumed constant without significant inaccuracy. In that case, RAneed not be evaluated because the functional form of the correction factor is such that it cancels. 111. Absorption of Exciting Light and Fluorescence by Q; Right-Angle Geometry. Derivation of a rigorous equation for simultaneous absorption of exciting radiation and fluorescence of M by Q is exceedingly cumbersome. Our procedure in this case is therefore simply to apply the correction factors in Equations 11 and 16 in a sequential manner:
(FU/F),~(FB’/F)B,(Fa‘/Fo), = Ksv[Q]i1
If desired, Equation 20 can be expressed in terms of summations of finite wavelength intervals, rather than in integral terms. It is essential to note that Equation 20, and all subsequent expressions for front-surface illumination, implicitly assume that the pathlength of exciting light through the sample to a n y element of area within the sample is identical with that traversed within the sample by observed fluorescence from that same area element. Care is required in the performance of front-surface fluorescence measurements to ensure the validity of this assumption. V. Absorption of Fluorescence by Q; Front-Surface Geometry. When Q absorbs fluorescence of M but not the exciting light, assuming a front-surface arrangement in which emission is monitored with equal efficiency from all depths of solution, the following attenuation (or scaling) factor can be calculated for each wavelength element of the emission monochromator bandpass:
(17)
where (Eb’/F),, and (FO’/PFB,,, are obtained from Equations 11 and 18,respectively, and (Fe/F),b,d is the observed ratio of fluorescent powers for a specific [Q]at the nominal ex. citation and emission wavelengths and bandpasses for which the Correction factors have been evaluated, IV, Absorption of Exciting Light by 8;Front-8urface Geometry, If the fluorescence of M is viewed with equal efficiency from all depths of the solution as measured from the front cell face, the average attenuated intensity of exciting radiation Q € any particular wavelength X is given by
For each concentration of Q, F‘ is calculated from Equation 1 4 and F“‘ is evaluated from
FO’= Jh,‘4ShEhoB~Rh dX
(23)
where SAis obtained from Equation 22 and the other symbols have the same meanings as in Section 11. The correction factor €or this case therefore is given by
F Q ’ / F o= s ~ , ~ ‘ S h E h ~ B h dX/lh,hqEhoBhRh Rh dX (24)
VI, Absorption of B o t h Exoiting L i g h t a n d Fluoresoenoe by 8; Front-Surfaoe Geometry. This correction proceeds very similarly to that described in Case V, with inclusion of an additional term in the attenuation factor to describe absorption of exciting light, As in Case V, we write
FO’= /h,hkThEhQBhR~ dX
(25)
where TAis the attenuation factor, given by
TA / o b l O ” A e s 10-A@mX &
(26) In Equation 21, A,, is the absorbance per centimeter of exciting radiation by Q, integrated over the bandpass of the excitation monochromator:
llS6
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
A,,, the absorbance per centimeter of fluorescence by Q, integrated over the bandpass of the emission monochromator, is calculated from
A,, = log [Jh,h4EhoBhRh d h / l h l h 4 S x E ~ ’ Bd~hRI ~ (28)
W
E:
where SAis defined by Equation 22 and the other terms have the same meanings as in Section 11. Integration of Equation 26 yields
10- {Aex + A,
Th =
2.303A,
+
jb1 2.303Aex
I
The correction factor is then given by
F o ’ / F o= Jh,h4ThEhoBhRh dX/Jh3h4EhoBhRh dX (30) Alternatively, the correction factors for absorption of exciting light (Equation 20) and fluorescence (Equation 24) can be determined separately, and the overall correction factor then calculated via Equation 17.
VII. An Alternative Procedure for Front-Surface Illumination. If the pathlength of solution through which exciting light and fluorescence must pass is negligible, then absorption must also be negligible. This is the rationale for attempts t o correct for inner-filter effects in front-surface geometry by extrapolation to zero cell thickness (7) but, as noted previously, this procedure often fails to yield a linear plot. The procedure outlined below was designed to generate a reasonable functionality with a variable parameter which can be fitted to the data and employed in objectively estimating the intercept of a plot of fluorescence quenching data as a function of cell thickness. We treat here the specific case of absorption of exciting light only by Q; other cases could be developed by analogy. At any nominal excitation wavelength, the observed fluorescent power is proportional to the incident power. Assuming that emission is monitored with equal efficiency from all volume elements of the sample, then, in general where Ftotis the total detected fluorescent power and P, is the excitation power incident upon a volume element situated at a distance x from the front face of the cell. In the presence of Q, P, can be expressed in terms of the unattenuated incident power, Pxo,and the concentration of Q:
p,
= pxOIO-EQIQIX
(32) Substitution of Equation 32 into Equation 31 yields an expression for the fluorescent power of the solution containing a specific [ Q ] : (33) Similarly, for the reference solution ([Q] = 0), one obtains F o = k@~oJobp,o d,X = k @ ~ ’ P ’ b
(34)
Substitution of Equations 33 and 34 into the SV equation (Equation 3) yields
(35) where KsVobsdis the Stern-Volmer “constant” observed in the presence of inner-filter absorption. The “true” Stern-Volmer
400
440
480
520 X lnml
560
600
640
I
Figure 1. Electronic absorption spectra of lumiflavin (-, left inner ordinate), Ni(I1) (- - -, left outer ordinate),and Co(I1) (.-.,left outer ordinate);and fluorescence spectrum of lumiflavin (-. -, right ordinate), all in aqueous HC104 (pH 4.0)
constant is defined by Equation 1. Substitution of Equation 1 into Equation 35 yields
In applying Equation 36 to real data, one begins by choosing a value of [Q]corresponding to the initial region of a SV plot. KsVobsdis measured for a minimum of three different cell thicknesses. The product Q [ Q ] is treated as a variable parameter, and it is varied until self-consistent values of Ksv are obtained for each of the sets of (Ksv,b)data. Note that this procedure does not explicitly consider the wavelength dependence of P or Po. If all front-surface measurements are made under identical conditions except for cell pathlength, it should not be necessary to explicitly consider the wavelength dependence of P or Po in this correction procedure.
APPLICATIONS TO REAL QUENCHING DATA In Figure 1are shown absorption and fluorescence spectra of lumiflavin (7,8,10-trimethylisoalloxazine), the fluorophore in a study (24) of quenching of flavin fluorescence in aqueous solution by transition-metal ions; also shown are the absorption spectra of two quenchers, Ni(I1) and Co(I1). Lumiflavin fluorescence was normally excited at a nominal wavelength of 420 nm (with an excitation monochromator bandpass, X2 - XI, of 25 nm). Fluorescence was measured a t a nominal wavelength of 545 nm, with the limits of the emission monochromator bandpass, X4 - As, also being 25 nm. These “baseline-to-baseline” monochromator bandpasses were obtained using an Aminco-Bowman spectrofluorometer equipped with three 2-mm entrance slits and three 4-mm exit slits. The lumiflavin concentration was such as to produce an absorbance of 0.05 or less in a 1-cm cell a t the nominal excitation wavelength; the concentrations of quenchers ranged from 0.02 to 0.30 M. The results of the various corrections are compared with each other and with the values for in Table I. The close agreement observed between the “front-surface’’ and “right-angle” corrections in this and other systems is regarded as evidence for the essential validity of the assumptions made in deriving the various correction equations. The inner-filter effect for Ni(I1) is absorption of exciting radiation, while that for Co(I1) is absorption of fluorescence (the extent of absorption of exciting light by Co(I1) was sufficiently small in these experiments to be neglected). The discrepancies between “uncorrected” and “corrected” Ksv values indicate clearly the need for inner-filter corrections in fluorescence quenching studies whenever the absorption spectrum of Q overlaps either the excitation or emission
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Table I. Comparison of Corrected and Uncorrected K g v Values Q K S V , uncorraib K w , RA, c o r f K w , FS, c o r f Ni(I1) 6.14 ( b = 2 mm) 5.97 (Eq.1 0 ) 5.86 (Eq.20) 5.88 (Eq.36) 6.58 ( b = 5 mm) 7.08 ( b = 10 mm) Co(I1) 29.3 ( b = 10 mm) 23.8 (Eq.16) 23.9 (Eq.24) a Units = L/mol; RA = right-angle; FS = front-surface. Measured by FS illumination ( b = cell thickness).
_
_
~
spectrum of M , even when (as in the present example) the quenchers are very weak absorbers and front-surface illumination is employed. Moreover, unless relatively highresolution spectrometric instrumentation is employed, the nonmonochromaticity of the incident light and the fluorescence (for any nominal monochromator setting) must be considered. In the present examples, the "corrected values" of Ksv for front-surface geometry were 6.06 and 21.8 for Ni(I1) and COW),respectively, in the absence of explicit consideration of nonmonochromaticity in computation of the correction factors. Comparison of these values with those listed in Table I reveals the magnitude of the errors which can be incurred if the finite monochromator bandpasses are not considered.
LITERATURE CITED (1) P. J. Wagner, in "Creation and Detectlon of the Excited State", A. A. Lamola, Ed., Marcel Dekker, New York, N.Y., 1971, Vol. l A , pp 179-212.
1107
P. Froehilch and E. L. Wehry, In "Modern Fluorescence Spectroscopy", E. L. Wehry, Ed., Plenum Press, New York, N.Y., 1976, Vol. 2, pp 319-433. E. Sawlckl, T. W. Stanley, and H. Johnson, Mlkrochlm. Acta, 178 (1965). 0. G. Gullbault, "Fluorescence: Theory, Instrumentatlon, and Practlce", Marcel Dekker, New York, N.Y., 1967, pp 349-352. W. M. Vaughan and G. Weber, Blochemlstry, 9, 464 (1970). C. A. Parker, "Photoluminescence of Solutions", Amerlcan Elsevler, New York, N.Y., 1968, pp 220-222. J. E. Martln and A. W. Adamson, Theor. Chlm. Acta, 20, 119 (1971). Reference 6, p 221. J. D. Wlnefordner, S. G. Schulman, and T. C. O'Haver, "Luminescence Spectrometry In Analytical Chemistry", Wlley-Intersclence, New York, N.Y., 1972, p 82. E. A. Burshtein, B/ophyslcs, 13, 520 (1968). L. Brand and B. Wltholt, Methods Enzymol., 11, 809 (1967). F. Y.-H. Wu, S . C . Tu, C.-W. Wu, and D. B. McCormick, Biochem. Biophys. Res. Commun., 41, 381 (1970). J. N. Demas and A. W. Adamson, J . Am. Chem. Soc., 95,5159 (1973). M. Ehrenberg, E. Cronvall, and R. Rlgler, FEBS Lett., 18, 199 (1971). J. S. Franzen, I. Kuo, and A. E. Chung, Anal. Blochem.,47, 426 (1972). G. W. Kinka and L. R. Faulkner, J . Am. Chem. SOC.,98,3897 (1976). K. K. Rohatgl and G. S. Singhal, Photocbem. Photoblol., 7, 361 (1968). V. A. Mode and D. H. Sisson, Anal. Chem., 46, 200 (1974). J. E. Gill, Appl. Spectrosc., 24, 588 (1970). J. N. Demas and G. A. Crosby, J . Pbys. Chem., 75, 991 (1971). Reference 8, p 20. W. H. Melhuish, J . Opt. SOC. Am., 52, 1256 (1962). W. E. Ohnesorge, Anal. Chlm. Acta, 31, 484 (1964). R. A. Leese, J. T. Stoklosa, S. Georghlou, and E. L. Wehry, unpublished results.
RECEIVED for review October 11, 1977. Accepted April 21, 1978. Research supported in part by the National Institute of General Medical Sciences, Grant GM-20805. Financial support to R.A.L. in the form of a University of Tennessee Non-Service Graduate Fellowship is acknowledged.
Metallic Phase Analysis of Multicomponent Systems Using a Potassium Cuprochloride-Tartaric Acid Leach T. C. Hughes" and Phillp Hannaker Department of Geology, School of Earth Sciences, University of Melbourne, Parkville, Victoria 3052, Australia
A procedure for the analysis of the metalllc phase in multiphase systems Is described, wlth elemental determlnatlon by flame atomlc absorptlon spectrometry. A potasslum cuprochlorlde-tartaric acid phase selective leach is employed at room temperature to dissolve the metalllc phase In the presence of sulfldes, oxides, sillcates, and carbonaceous rlch material. Partlal attack of the oxldes, ZnO, MgO, and CdO, was observed and these oxldes, when present, were therefore removed by an lnltlal treatment wlth a potasslum dlchromate-tartaric acid solution. The method can be applied to geologlcal and industrial materials to determlne the elements BI, Cd, Co, Fe, I n , Mg, Mn, NI, Sb, Sn, and Zn when present In the metalllc phase. The complete analytlcal procedure Is reported and the effect of the metalllc phase leachant on nonmetalllc material is fully tabulated.
Preferential leaching techniques have long been used by analytical chemists for the determination of elemental distributions in multiphase systems. Inorganic speciation studies, therefore, appeared to be possible using efficient phase selective leachants such as potassium cuprochloride (KCuClJ (I), together with concentration measurements by flame 0003-2700/78/0350-1197$01 .OO/O
atomic absorption spectrometry (AAS). This paper details investigations made into the use of specific leachants for the analysis of metallic components in complex inorganic multiphase materials. Many geologically derived samples (e.g. chondritic meteorites) consist of compacted agglomerates of metallic, sulfide and silicate material, with some specimens also containing carbides and phosphides. The analysis of each phase of these complex materials is required for geochemical and cosmochemical studies ( 2 ) . Industrially, the analysis of slags and ores for their metal content is important in both ferrous and nonferrous metallurgy. Modern materials research has produced many multiphase products which contain metallic components. The determination of the composition of the metallic phase present in these materials is important for both production and quality control. A leaching agent must be capable of selectively attacking and quantitatively extracting elements present in one particular phase. Thus, for the study of the composition of a metallic phase in a multiphase system, the rate of dissolution of nonmetallic components such as oxides, sulfides and silicates must be negligible, with the particular leachant used, when compared to the observed solubilization of the metallic phase constituents. A general metallic phase leaching agent must therefore be capable of quantitatively extracting ele0 1978 American Chemical Society
