I CORRESPONDENCE Relative Effect of Molecular Absorption on Atomic Absorption and Atomic Fluorescence Sir: Atomic fluorescence as an analytical technique does not compete favorably with atomic absorption in many practical situations. Often this is due to the fact that when flame atom reservoirs are used, atomic fluorescence suffers relative to atomic absorption in the respect that flame noise and scattering effects are relatively more important, and flame gases may quench atomic fluorescence. For a given analysis, however, there are several reasons why one might wish to consider atomic fluorescence. These include the possibilities of greater sensitivity; greater linear dynamic range; less need for a high dispersion monochromator; and the ability to do simultaneous multielement analysis. Often unrecognized is the possibility that spectral matrix interference effects may be less severe with atomic fluorescence. It is this last -point on which I wish to comment here. A misconception exists as to the relative effect of matrix molecular -absorption on atomic fluorescence, and incorrect statements have been made in the literature ( 1 ) . Many excellent, detailed articles have been published dealing with expressions for fluorescence flux (2-5). It is not the purpose of this paper to give a general theory, but rather to consider the relative effects of matrix molecular absorption on the determination of mercury concentration by atomic absorption and resonance atomic fluorescence of the 2536.5-A line. It is expected, however, that results will serve as a useful guide to the magnitude of concentration errors to be expected for a given degree of molecular absorption in many other practical analyses.
MONOCHROMATOR
Figure 1. O p t i c a l
system considered
by the molecular absorber, since those atoms fluorescing nearest the detector will suffer no molecular absorption, and the maximum (by atoms furthest from the detector) will be 2% absorption. Thus, the total interference cannot exceed 3% in atomic fluorescence. Thus, for this example, atomic absorption shows an interference effect =30 times greater than atomic fluorescence. Consider, for simplicity, the experimental arrangement shown in Figure 1, and let us assume a perfectly monochromatic light source providing a radiant flux of photons cm-2 sec-l incident w o n the homoeeneouslv distributed sample contained in a sample volume of ab2 cm3. In the case of atomic absorption with a hollow cathode line the halfewidth of the Source is sufficiently smaller than the absorption half-width (assuming atmom spheric pressure) of the absorption line so that one need not consider the variation of absorptivity with wavelength. The flux which passes through the sample, 9,is given by Beer,s law. Y
DISCUSSION This paper will demonstrate that absorption
Of light by a molecuIar interferent causes a much greater concentration error in atomic absorption analyses than it does in atomic fluorescence. Before proceeding with a more rigorous discussion, the following rationale may prove helpful in understanding. Suppose mercury vapor and a volatile organic which absorbs at 2536 A are both present in a vapor sample at such concentration that they each have an- absorbance of 0.01 (approximately 98% transmittance). The interference effect due to molecular absorption will have caused an error in the concentration determination by atomic absorption of 100%. Assuming no collisional interactions or molecular fluorescence, the molecular absorber may affect the mercury fluorescence in two ways(1) by decreasing the flux available for absorption by mercury atoms, and (2) by absorption of the fluoresced radiation. The first of these must cause an error of much less than 2%, since 98% of the light incident was passed by the molecular absorber. In like manner, only some 1%of the light fluoresced by the mercury atoms could be absorbed
D Winefordner and R. C. Elser. Anal. Chem., 43, ( 4 ) , 24A (1971), Table 1 1 , p 28A. H. P. Hooymayers, Spectrochim. Acta, Part B, 23, 567 (1968). P. J . T . Zeegers and J. D. Winefordner, Spectrochim. Acta, Part 6, 26, 161 (1971). J. D. Winefordner, V . Svoboda, and L. J . Cline, Grit. Rev. Anal. Chem., 1, 233 (1970) J D Winefordner, S. G. Schulman, and T. C. O'Haver. "Luminescence Spectrometry in Analytical Chemistry," Wiley, New York, N . Y , 1972
(1) J
(2) (3) (4)
(5)
@( X )
@oe-k~~~x
(1)
where 2.3 k l is the absorptivity (cm2 molecules-l) and c1 is the concentration of absorbing atoms (molecules ~ m - ~ ) . It might be noted that the flux, a, is, in general, a function of frequency as well as x. If one adds to this sample a molecular species also capable of absorbing the source radiation to some degree and also assumed to be homogeneously distributed, the combined effects will be @,,
2=
(POe-~(ki~i+kzcz)
(2)
since absorbances are additive. In this equation, 2.3 k 2 and c 2 are the absorptivity and concentration of the molecular interferent. Because of the addition of the molecular absorber, will be decreased from the numerical value it had in Equation 1. Thus, in atomic absorption, the absolute error caused by the absorbance of a molecular contaminant is directly proportional to c2, the concentration of that contaminant. Now, in the corresponding atomic fluorescence case depicted by Figure l, a quite different situation exists. Consider first the situation with no molecular interferent. The A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 6, M A Y 1974
797
~~
~
~
Table I. Theoretical Error Caused by Matrix Molecular Absorption in Atomic Fluorescence and Atomic Absorption Ratio of absorbance of molecular interferent to absorbance of analyte
Percent of incident light absorbed by analyte
Percent error in Percent error in concentration concentration calculated by calculated by atomic fluorescence atomic absorption
0 04 0 004 0 0004 0 4 0 04 0 004
1000 100 10 1000 100
10
4 3
1000
1 0 1 10 1
0 44
10 1 01 10 1
0 01 0 10
0 1
1 0 5 0
0 044
19 2 2 0 22
01
10 100 10
1000 100 10
flux of fluorescent photons d F emanating from the point dxdy, depends on the number of photons absorbed by the analyte atoms -d@. Since is the flux incident on this element, one may use Equation 1 to obtain dF. Then, since d F is a function of both x and y, dF(x, y > = K~~c,,klcle-h~'~'"-J'dxdy (3) Of the quantities not previously defined, a and b are defined by Figure 1 and the proportionality constant, K , includes such atomic constants as the fluorescence quantum yield, and such geometrical constants as the solid angle of acceptance of the detection system (among many others). We may lump these together into K as we are interested only in the relative effect, and they will cancel in the ratio. Since we are considering resonance fluorescence, and since the analyte concentration will be very small for most practical cases, we may consider the fs ration discussed by Winefordner, Schulman, and O'Haver (6) to be near unity. Not considered are prefilter or postfilter effects (7), which also should be negligible in the ratio. Equation 3 does take account of both the continuous decrease of intensity of fluorescence due to the absorption of source light (in the x direction) and the decrease of fluorescence due to reabsorption of fluoresced light (in t h e y direction). Substituting for +(, from Equation 1:
F,
=
k'kb
K~oklcle-klclxe-k~cl(a-Y)dxdy
Integrating and simplifying gives
(4) As before, if we now consider the effect of adding a molecular species which absorbs at the analyte wavelength, the molecular species will absorb both the incident and the fluorescent light, giving
F,
E
~
~
n
~
b
~
~
,
~
,
c
,
e
- e~ - ( h~ l c l~+ k "2 c >t K o~ - y zi
~dxdy z ~ ~
~
which integrates to give
(6) Reference 5 , p 86. (7) Reference 5,p 83.
798
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 6, M A Y 1974
We are now in a position to compare the relative error caused by a molecular interferent in atomic absorption and atomic fluorescence. To accomplish this, we calculate c 1 in Equations 1 and 2, take their difference and divide by c1 to give a relative error in the atomic absorption case. For the atomic fluorescence case, it is not possible to solve Equations 4 and 5 for c1 exactly. Although the power series expansion of the exponential terms does provide an approximate solution, it is sufficient for present purposes to assume direct proportionality between F1 and c1 (linear calibration curve), so that the relative error in F is identical to the relative error in c. For most atomic fluorescence analyses, this assumption is experimentally valid over a concentration range of several orders of magnitude. We then calculate FI from Equation 4, subtract F2 from Equation 5 , and divide by F1 to give the relative error in the atomic fluorescence case. This treatment assumes that less than a few percent of the incident light is absorbed, since higher order effects (such as the refluorescence of absorbed fluorescent light) are considered negligible. This requirement of low absorbance also ensures the linear calibration curve assumption mentioned previously, and corresponds to the concentration range where atomic fluorescence should be used analytically. As an example of these calculations let us assume that a = b (square cross-section in Figure 1) and calculate the error as described above under conditions where the molecular interferent absorbs 0.1, 2, and 10 times as many photons as the analyte atoms. The results of these calculations are shown in Table I, for values of percent incident light absorbed by the analyte varying from 0.01 to 5%.
CONCLUSIONS Since absorbance is directly porportional to concentration, the relative absorbance values in column 2 of Table I correspond to errors of lo%, loo%, and 1000% for analytical determinations performed by atomic absorption as shown by column 4. For example, if the absorbance of the molecular interferent is equal to that of the analyte, the concentration calculated by atomic absorption will be in error by a factor of two, or 100%. The corresponding errors for atomic fluorescence are shown in the third column. Here, as can be seen, the error becomes significant only when the molecular interferent absorbs a sizable percentage of the incident light. In fact, it might be emphasized that if c1 = 0, atomic absorption spectroscopy will still give an error signal if c;! # 0, whereas atomic fluorescence gives none (unless the molecular interferent either scatters or fluoresces). I t should be emphasized that the above treatment assumes that the molecular interferent does not itself fluoresce, collisionally quench, or scatter the incident radiation. These are separate problems, and may be treated separately theoretically. Furthermore, they may be expected to be negligible in many practical situations. The conclusions of Table I have been verified in a semiquantitative way in the author's laboratory by purposely introducing benzene vapor into the gas stream of the Hatch and Ott system for mercury determination ( 8 ) . It was found, for example, that a concentration of benzene vapor sufficient to produce an error of 300% in the atomic absorption determination caused no noticeable error when the mercury concentration was determined by atomic fluorescence. This is a finding of some practical importance, as molecular absorption interference has been reported for (8) W R Hatch and W L Ott Ana/ Chem 40, 2085 (1968) (9) H L Kahn At Absorptlon Newslett 7,40 (1968) (10) D C Manning A t Absorption Newslett 9. 109 (1970) (11) Ron L Windham Ana/ Chem 44,1334 (1972)
this analysis (9-11). It seems probable t h a t other such examples will appear as the number of practical applications of atomic fluorescence increases. In summary, it has been shown theoretically and experimentally t h a t atomic fluorescence spectroscopy enjoys a considerable advantage over atomic absorption spectroscopy insofar as the error associated with matrix molecular absorption is concerned. Since the error values tabulated in Table I are thought t o be in the range of many analytical applications of atomic fluorescence spectroscopy, it is hoped t h a t Table I will find use with atomic fluorescence
spectroscopists for making rough estimates as t o the magnitude of the concentration error to be expected for a given degree of matrix molecular absorption interference. C . David West Department of Chemistry Occidental College Los Angeles, Calif. 90041 Received for review October 19, 1972. Accepted January 17, 1974.
I AIDS FOR ANALYTICAL CHEMISTS Simple and Inexpensive Temperature Controlled Spectrophotometric Cell Holder R. L. Wilson and J. D. Ingle, J r . l Department ofChemistry, Oregon State University. Corvallis, Ore. 9733 1
The temperature of the solutions in the sample cells of molecular absorption or fluorescence spectrometers must be carefully controlled to make precise and accurate equilibrium- or kinetics-based measurements. Temperature regulation in the range of * O . l to kO.01 "C is often required ( I ) . Most commercial spectrometer manufacturers provide controlled temperature sample cell holders as accessories and a number of authors (1-4) have described the construction of thermostatable cell holders. In this paper. the construction and performance of a new simple and inexpensive controlled temperature cell holder is discussed. This cell holder is constructed to be part of a fluorometric reaction rate monitoring system, although it is easily adapted to other applications. Temperature stability is particularly critical in fluorometric reaction rate measurements because of the significant temperature dependence both of rate constants and of fluorescence parameters such as the quantum efficiency. The temperature stability of solutions and the time required for them t o reach eyuilibrium are comparable to recently described cell holders (1-5). Compared to previous thermostatable cell holders. the described holder has the following advantages: simple and inexpensive construction, immediate capability for use in fluorescence or absorbance measurements, and ability t o remove and replace the sample cell with ease.
GENERAL COXSIDERATIONS A temperature controlled cell holder is basically a block through which is circulated water from a thermostated constant temperature bath. The cell holder must have To whom correspondence should be addressed. P. D. Feil, D . G Kubler. and D . J. Wells, Jr.. Ana/. Chem.. 41, 1908 (19 6 9 ) . Theodore Weichselbaum, Raymond E. Adams, and Harry E. Mark, Jr , A n a / . Chem.. 4 1 , 1913 (1969) Harry L. Pardue and Pedro A. Rodriguez, A n a / . Chem.. 39, 901 (1967). Paul H . Bell and C. R. Stryker. Science. 105,415 (1947). J. D . Ingle. Jr.. Ph.D. Thesis, Michigan State University. East Lansing, Mich.. 1971
provision for securely holding a spectrophotometric cell. The ability of the holder to bring solutions rapidly to a desired temperature and to maintain a constant temperature is dependent on the thermal contact between the holder and the cell and the flow rate and temperature stability of the thermostated water. Two basic approaches have been used for construct ion of spectrophotometric cell holders. In one approach. the sample cell is readily removed from the holder. while the other approach involves sealing the sample cell in the holder. The former approach allows easy cleaning of the cell and use of the cell in other applications. but often has poor thermal contact. The latter approach provides good thermal contact but does not possess the convenience of the first design approach. One variation of the first approach used by one of us (5) is basically a hollow metal block with a square well in the center for insertion from the top of a standard 1-cm square spectrophotometric cell. Appropriate windows are located in the sides of the block for absorbance or fluorescence measurements. The square well must be larger than the sample cell for easy insertion and to prevent scratching. Because of this. the cell position is not completely secure and the thermal contact between the glass cell walls and metal lilock is not good. It was also noted that the outside dimensions of standard 1-cm cells varied among different manufacturers and even among cells of the same manufacturer. Some cells w o i ~ l d not fit into the hole. while others were somewhat loose. Many of the temperature controlled holder> provided hy manufacturers of spectrophotometers are basically t h e same design discussed above, except the well in which the cell is inserted is made larger and springs are placed on the inside walls of the well. The springs securely hold cells of slightly varying outside dimensions. The disadvantage is that the contact area of the glass cell walls with the thermostatable metal block walls is considerably diminished. Recently. two temperature controlled cell holders for absorption measurements have been reported that PO,.yqess excellent characteristics. The designs involve sealing the sample cell into a block in a way that provides efficient A N A L Y T I C A L C H E M I S T R Y , VOL. 46,
NO. 6.
M A Y 1974
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