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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
3 m L of reagent, sulfide concentrations as high as 24 m g / L (as Na2S) were shown not to interfere with t h e recovery of inorganic and organically bound mercury added to distilled water. T h e interference from higher concentrations of sulfide can be eliminated by using larger quantities of brominating reagent but some depression of the subsequent mercury signal may be obtained ( 2 ) . LITERATURE CITED (1) H. Agemian and A. S. Y. Chau, Anal. Chem., 50, 13 (1978).
(2) B. J. Farey, L. A. Nelson, and M. G. Rolph, Ana/yst(London),103, 656 (1978).
Brian J. Farey L. Andrew Nelson* Metropolitan Water Services Thames Water Authority 177 Rosebery Avenue London, ECIR 4 T P , U.K. RECEIVED for review July 24, 1978. Accepted September 11. 1978.
Limitations of Synchronous Luminescence Spectrometry in Multicomponent Analysis Sir: Synchronous excitation spectrofluorimetry was introduced by Lloyd ( I ) and developed as a technique for fingerprinting complex forensic samples (2). I t has also been applied t o t h e characterization of oil pollution samples ( 3 ) . More recently, simultaneous variation of the excitation and emission wavelengths a t a predetermined wavelength separation (AX) has been used to identify specific polyaromatic hydrocarbons (PAH) in a synthetic mixture ( 4 ) . An effort to utilize this approach in our laboratory immediately revealed that only a fortuitous combination of substances, concentrations, and fluorescence quantum efficiencies will provide reliable qualitative and quantitative data. Conventional fluorimetric procedures are required to reveal interferences t h a t may mask t h e presence of one or more components in a mixture. In t h e event t h a t masking is not complete, these same interferences would preclude any quantitative measurements.
(a1
EXPERIMENTAL Apparatus. A laboratory-constructed spectrofluorimeter was used to obtain all spectra. The components used are described in Table I. The monochromator stepping motors were operated individually by controlling their respective scan drivers in the internal mode or simultaneously using an external pulse to both scan drives. Chemicals. All of the compounds used were commercially available and were used without further purification.
303 403 Naveieigth :nm)
500
Figure 1. Synchronous fluorescence spectrum of mixture of polyaromatic hydrocarbons (PAH). AX = 3 nm. (a) Five-component PAH mixture. I. Perylene, 0.06 p g / m L ; 11. Anthracene, 1.0 p g / m L ; 111.
RESULTS AND DISCUSSION
Phenanthrene, 40.0 pg/mL; I V . Fluorene, 24.0 pg/mL; V . Pyrene, 40.0 p g / m L . (b) Four-component PAH mixture. I. Perylene, 0.06 pg/mL; 11. Anthracene, 1.O p g / m L ; 111. Phenanthrene, 40.0 p g / m L ; I V . Fluorene, 24.0 p g / m L
T h e synchronous fluorescence spectrum from a mixture of five randomly selected PAHs is shown in Figure l a (AX c 5 n m was found to be optimum in terms of spectral discrimination and resolution; AXs I 4 n m resulted in severe stray light problems and AX > 5 nm resulted in a loss in selectivity). Peak identification was based on spectra derived from pure solutions of t h e individual compounds. I t should be noted that the largest peak (I1 + 111) is due mainly to an anthracene concentration of 1 pg/mL with only a small contribution from 40 pg/mL of phenanthrene which has its strongest peak a t the same wavelength. T h e secondary phenanthrene peak also has some contribution from pyrene. I t is evident t h a t qualitative surveys can be in error because of wavelength coincidences a n d / o r weak fluorescence signals. Most notable in Figure l a is the lack of any fluorene peak 0 where a strong signal was observed for a pure a t ~ 3 0 nm,
solution of this compound. An emission spectrum of the mixture (used to obtain Figure l a ) obtained with a fluorene excitation wavelength resulted in a pyrene emission spectrum with a small phenanthrene component. making it apparent that any fluorene emission was being efficiently masked. The synchronous spectrum of a mixture containing no pyrene is shown in Figure l b and the strong fluorene signal obtained confirmed that the depressive interference was due primarily to pyrene. T h e extent to which the pyrene concentration affects the fluorene emission intensity is presented in Table 11. It is apparent that as long as the ratio of concentrations of fluorene to pyrene exceeds -36, the error in the fluorene signal will be less than 10%. Concentrations of the PAH components other than those in Table I1 or Figure 1 gave similar results and conclusions.
0003-2700/78/0350-2148$01 O O / O
c 1978 American Chemical Society
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Table I. Components for a Spectrofluorimeter Operable in the Normal or Synchronous Mode component
model
1. source 150 W Xe arc lamp 2. excitation monochromator (spectral bandwidth = 4 n m ) 3. stepping motor with scan control 4. sample compartment with lens 5. emission monochromator (spectral bandwidth = 4 n m ) 6. same as 3 7 . detection system photomultiplier Phl power supply electrometer 8. X-Y recorder 9. pulse generator
R- 15 1-7 A
Eimac Division of Varian
J-Y H10 UV
American ISA Inc
STMC-4
American ISA Inc.
-_-
American ISA Inc.
J-Y H10 V
American ISA Inc.
--1P28 EU-42A 610 BR Plotamatic 7 1 5 M with 7T Time Base
RCA Heath Co. Keithley MFE
180
Wavetek
Table II. Quenching of Fluorene Fluorescence by Pyrene (Fluorene Present at 1 2 wg/mL)
wrene, wdmL
relative synchronous fluorene signal ( A A = 5 nm)
0.00 0.04 0.40 4.0 40.0
100 91 90 54 2
company
It is now apparent that t h e loss of spectral information resulting when the synchronous technique is used increases the probability of error. Qualitative data indicating the presence of a component a t a relative high concentration may be missing or weak signals may be completely obscured and even a partial overlap between emission and absorption bands of compounds in the mixture could result in inconclusive quantitative results. It should be stressed that similar effects occur in conventional fluorimetry but generally stand out when excitation and emission spectra are measured and, therefore, can be accounted for by variation in experimental conditions, dilution techniques, separation procedures, etc. These findings do not detract from the value of synchronous fluorescence as a fingerprinting tool, but certainly caution should be exercised when applying the method to unknown
samples for multicomponent quantitative analysis. In the latter case, t h e fluorogram approach of Rho and Stuart ( 5 ) in which intensity data for all excitation and emission wavelengths is obtained would be preferable. In addition, synchronous fluorescence spectrometry does not negate the need for conventional excitation and emission fluorescence spectra.
LITERATURE CITED (1) (2) (3) (4) (5)
J. 8.F. Lloyd, Nature(London), Phys. Sci., 231, 64 (1971). J . B. F. Lloyd, Chem. B r . , 11, 442 (1975). P. John and J. Soutor. Anal. Chem., 48, 520 (1976). Tuan Vo-Dinh. Anal. Chem.. 50, 396 (1978). Joon H. Rho and J. L. Stuart, Anal. Chem., 50, 620 (1978).
' On leave from the Department of Chemistry, Ohio University, Athens, Ohio.
Present address, Procter and Gamble, Industrial Chemicals Division, Sharon
Woods Technical Center, Cincinnati, Ohio 45241.
H. W. Latz' A. H. Ullman*
J. D. W i n e f o r d n e r * Department of Chemistry University of Florida Gainesville, Florida 32611
RECEWED for review June 16, 1978. Accepted August 31. 1978. This work was exclusively supported by NIH GM-11373-15.
