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Anal. Chem. 1984, 56, 427-429
Table 11. Determination of Phosphorus in NBS Steels by Using Slim and Interfering Element Correction P found" P P found Cu found, corrected, certified, sample raw, % % % % 8j
0.098
36 2 a
0.148
0.017 0.486
0.095
0.095
0.044
0.041
Correction factor = 0.213ppm P/ppm Cu.
___l-l 1 _ 1 __ -_ ~ __ -
-
prominent Fe line shown in Figure 8 was used as the side line, and the reference solutions were matrix matched with the samples to contain 1.00% Fe (w/v). The small Fe peak located approximately 0.005 nm to the long wavelength side of phosphorus does not interfere with the analysis as long as both SLIM and matrix matching are applied. Both P and Cu were determined (at 214.91 nm and 327.40 nm, respectively) and the P results corrected for Cu spectral interference; analytical
resulta are shown in Table 11. Phosphorus results are accurate even when Cu is present in 10-fold excess, as in the low alloy steel (362).
CONCLUSION The side line indexing method is a viable alternative to conventional peak search, but it should be applied only when an alternate, interference-free analyte emission line cannot be found. Whenever possible, it is advisable to avoid matrix spectral interference by choice of an alternate line.
LITERATURE CITED (1) Floyd, M. A.; Fassel, V. A,; Winge, R. K.; Katzenberger, J. M.; D'Silva, A. P. Anal. Chem. 1980, 52, 431-438. (2) Nygaard, Danton D.; Chase, Duane S.; Lelghty, David A. Appl. Spectrosc. 1983, 37, 432-435.
RECEIVED for review September 2, 1983. Accepted October 31, 1983.
Optical Sensor for Continuous Determination of Halides Edmund Urbano, Helmut Offenbacher, and Otto S. Wolfbeis* Institut fur Organische Chemie, KF- Uniuersitat, A-8010 Graz, Austria
The first optical sensor for halides and pseudohaildes Is described. It is based on dynamic fluorescence quenchlng of acrldlnlum and qulnoiinlum Indlcators, which were immobilized vla spacer groups onto a glass surface. The sensors are able to indicate the concentration of halides in solution by virtue of the decrease in fluorescence intendty due to the quenching process. The sensltivity toward different halides can-to some extent-be varied by the cholce of the indicator. The method Is increasingly sensitlve on going from chloride to bromide to iodlde. Detection limits are 0.15 mM for iodide, 0.40 mM for bromide, and 10.0 mM for chiorlde. The errors of determination in the concentratlon range from 0.01 to 0.1 M are 1 % for Iodide, 1.5% for bromide, and 3-5% for chloride.
The development of sensors based on immobilized fluorescent reagents is a matter of growing interest (1-4). Sensors offer advantages over conventional solution fluorescence measurements, since they allow the determination of concentrations without significantly perturbing the sample. In addition, they can be used for continuous sensing. Fluorescence-optical sensors are based on three fundamental principles: (a) changes in acid-base equilibria of indicators (2,5);(b) reversible formation of fluorescent chelates (3); (c) dynamic fluorescence quenching ( 4 ) . The first two principles are similar in that they are based on processes that occur in the ground state, whereas the latter occur in the first excited singlet state. In this paper we report the characteristics of a sensor for halides that is based on the dynamic quenching of glass-immobilized heterocyclic indicators 1 and 2, whose structures are shown in the formula scheme. The quenching of quinoline-type fluorophores was first described by Stokes as early as 1869 (6),when he observed
that the fluorescence of quinine in dilute sulfuric acid was reduced after addition of hydrochloric acid or halide ions. The involved process is now known to obey the Stern-Volmer equation
Here, Fo is the fluorescence intensity of a solution in the absence of a quencher, F is the fluorescence intensity in the presence of a quencher, [Q] is the concentration of the quencher, and Kq is the so-called quenching constant (7).K p is the product of the specific quenching constant and the natural fluorescence decay time, but this refinement is without significance for this work. On the basis of the Stern-Volmer equation we have recently developed a fluorimetric method for the determination of one (8) or more halides in solution (9). Since fluorescence quenching of certain heterocyclic indicators by halides is a fully reversible process, it seemed attractive to immobilize the indicators on a solid support to obtain a sensor element. Indicators 1 and 2 were chosen, since they are relatively easily accessible and possess functional groups suitable for immobilization by covalent bonding. In addition, compound 1 in aqueous solution has an overall quenching constant of 9.3 M-l, which makes it a promising candidate for continuous monitoring of chloride in serum, where concentrations usually range from 90 to 110 mM. Glass was preferred over cellulose as polymeric support, because it is not readily attacked by bacteria. CH,CH,CH,SO,
CH,CH,COOH
2 1
0003-2700/84/0358-0427$01.50/00 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
EXPERIMENTAL SECTION The preparation of 3-(9-acridinyl)propionic acid was described by Jensen and Howland (IO). Its quarternization was accomplished by heating 1.0 g with 0.60 g of methyl iodide in a sealed glass tube for 4 h at 130 "C. The product was extracted with three 20-mL portions of hot water and precipitated as the tetrafluoroborate by adding a solution of 1 g of sodium tetrafluoroborate in 10 mL of water to the aqueous extract. 3-(10Methylacridinium-9-y1)propionicacid (1) precipitated in greenish yellow needles of mp 260 OC. Double recrystallization from hot water gave 0.9 g (65% yield) of the pure product. Anal. Calcd for Cl7Hl6BF4NO2(mol wt 317.12): C, 57.82; H, 4.57;N,3.97. Found: C, 57.90; H, 4.50; N, 4.07. The 'H NMR and IR spectra are in agreement with the proposed structure. Synthesis of indicator 2 starting from 6-methoxyquinolineand propanesultone has already been described (11). M o d i f i c a t i o n o f Glass. The specific surface of a 2 X 2 cm glass sheet (as used for microscopy) was fiit enlarged by treatment with 50% hydrofluoric acid for 0.1 h followed by activation with a mixture of nitric acid and sulfuric acid (l:l, v/v) according to established procedures. Chemical surface modification was accomplished by refluxing the glass platelets in a solution of 1 pL of (3-aminopropy1)triethoxysilanein a mixture of distilled toluene (10 mL) and water-saturated toluene (1 mL). Traces of water have a beneficial effect upon the reaction. Tosyl chloride (100 pg) was added as a catalyst. I m m o b i l i z a t i o n P r o c e d u r e . Coupling of indicator 1 to the modified glass surface was achieved with the help of water-soluble 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide(EDC, Sigma Chemical Co.). Fifty milligrams of EDC, dissolved in 1 mL of water, was added over a period of 5 min to a solution of 100 mg of 1 in 10 mL of water containing the activated glass platelets. The solution was rapidly stirred. Coupling was completed within 24 h at room temperature. Nonimmobilized acid was removed by washing with water and acetone. The resulting sensor (referred to as sensor 1)showed green fluorescenceunder the mercury lamp and was ready for use. It is noted that coupling with the help of EDC results in distinctly higher fluorescence intensities of the sensor than coupling via the acid chloride of 1. Compound 2 was transformed into its sulfochloride by reacting 100 mg for 30 min with boiling thionyl chloride (1 mL) in the presence of 1drop of dimethylformamide (DMF). Excess thionyl chloride was evaporated under vacuum and the crystalline residue was dissolved in 10 mL of DMF plus 1drop of dry pyridine. Glass platelets bearing amino groups on the surface were prepared as previously described and placed into the above solution. After 5 h the resulting sensor platelets (referred to as sensor 2) were washed with water and acetone and were then ready for use. The surface showed blue fluorescence when irradiated with the mercury lamp. A p p a r a t u s and Fluorescence Measurements. Fluorescence measurements were performed on an Aminco SPF 500 spectrofluorimeter. For sensor type 1 the excitation was at 357 nm, the emission maximum at 488 nm. For sensor type 2 the respective wavelengths were 350 and 460 nm. The band-passes were 8-10 nm in excitation and 10-15 nm in emission. The signals of the fluorescence sensors were collected at an angle of 40" to the excitation beam at the back of the sensors. A 408 nm cutoff filter was used as a secondary filter to reduce interferences from stray light. The halide solutions were pumped through a flow-through cell, whose one side was the sensor with the indicator layer exposed to the solution. Fluorescence intensity was measured and plotted vs. time data. The halide concentration was varied by changing the aqueous standard solutions. Syntheses.
RESULTS AND DISCUSSION The excitation and emission spectra of the immobilized indicators were found to be almost the same as in solution, thus indicating no electronic interaction between the glass surface and the indicator molecules. The possible interfering effect of pH upon the fluorescence intensity was studied by using phosphate buffers of varying pH in the range 4.2 to 7.4 and was found to be negligible.
6 -methoxyquinolinium based sensor
L ' lo-'
\ IO-'
0
loo
halide conc., M
Flgure 1. Relative fluorescence intensities (in arbitrary unlts) of sensor type 1 (top) and sensor type 2 (bottom) as a function of the halide concentration at 23 OC. Sensor 1 Is highly sensitive toward bromide and iodide but not toward chloride. Sensor 2 is of comparable sensitivity toward chloride but only sllghtly more sensitive toward the other two halides.
Fluorescence decreases with halide concentration as shown in Figure 1. Quenching is increasingly efficient on going from chloride to bromide to iodide. However, the quenching constants are around 10-20% lower than those of the nonimmobilized indicators in solution (8). This reflects the limited mobility of the fluorophores when immobilized and the resulting lower probability of a quenching process. The response toward halides is known to depend slightly upon the ionic strength of the solution (12). This leads to negative deviations of the otherwise linear Stern-Volmer plots a t high halide concentrations above 0.5 M, which, however, is far above physiological concentrations. Thus, this kind of sensor is able to indicate the halide concentration in a continuous way without disturbing the sample. I t offers an alternative to ion-selective electrodes, whose stability (in the halide case) is known to be limited a t present. The amount of indicator bound onto glass was estimated by comparing the fluorescence intensity of the sensor with that of a solution. The sensor signal corresponds to an approximately 1rM solution. We also tried to determine the amount bound by fluorimetry after stripping off the indicators by alkaline hydrolysis. However, both indicators suffered considerable decomposition after treatment with 1N potassium hydroxide, thus rendering a quantitation impossible. The differences in the intensities within a series of sensors were f 2 0 % . The stability of both types of sensors is surprisingly high. Irradiation with the 250-W xenon lamp at around 360 nm a t band-passes of 20 nm resulted in an approximately 15% per hour decrease of the fluorescence signal. The excitation intensity applied in this experiment is around 10 times that of the intensity required for a sensor to be used for practical applications. The bleaching may be overcome in practice by using a reference sensor. An important feature of the indicators used in this work is their large Stokes shift of more than 100 nm. This fact allows the use of cutoff filters rather than interference filters with practically no loss of excitation and emission energy. Secondly, only small interferences from Rayleigh, Tyndall,
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
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A
15
time ( m i n l
Flgure 2. Fluorescence response, response time, and reproducibility of the sensor type 1 signal for aqueous solutions: (A) pure water: (8) 0.1 M potassium iodide: (C) 0.1 M potassium bromide; (D) 0.1 M potassium chloride. Note that the response curves are not symmetric. The signal change from, e.g., A to B is faster than the reverse response. The restoration of the signal is faster by around 10% when water is replaced by a 0.1 M sodium sulfate solution.
and Raman scatter are observed. Sensor 1,containing an acridinium fluorophore, may also be excited at the longest-wave absorption maximum at around 405 nm, albeit at the expense of fluorescence signal due to a lower molar absorption than a t 360 nm. The reversibility of the sensor response was investigated by pumping halide solutions through the sensing unit until the signal was constant and then changing to water or 0.1 M sodium sulfate solution again. The response of sensor 1after passing-by 0.1 M solutions of potassium halides is shown in Figure 2. The average response time for a signal change to be indicated by 95% of its signal is around 40 s. Total fluorescence quenching does apparently not occur, even at halide concentrations of 1M or higher. This does not result from contributions from stray light or background fluorescence, since it is also observed in pure fluid solutions of the indicators. An error analysis was performed with sensor 1which shows higher sensitivity toward halides than sensor 2. Furthermore, the fluorescence quantum yield of its fluorophore (0.95 in solution) is greater than that of the fluorophore of sensor 2
429
(0.54). The detection limit of sensor 1 for halides, defined as the concentration equivalent to four times the standard deviation of the fluorescence signal in water is 0.15 mM for iodide, 0.40 mM for bromide, and 10.0 mM for chloride. The error in the halide determination at concentrations around 1mM is *3% for iodide and &4% for bromide, respectively. In the to 10-1 M concentration range the error decreases to *l% for iodide and f1.5% for bromide. The error for the quantification of chloride at concentrations around 100 mM, which is the level in normal blood, is around 5%. Interferences were studied by using 0.1 M standard solutions of various anions. Sulfite and pseudohalides such as isothiocyanate, cyanide, and cyanate cause also fluorescence quenching and thus lead to erronous results in the halide determination. Quenching by isothiocyanate is as efficient as iodide, while sulfite, cyanide, and cyanate are even less efficient than bromide. Their interferences may be taken into account by making use of the modified Stern-Volmer equation and employing two sensors with different quenching constants (9). However, these ions are usually not present in serum and urine. No interferences were observed with sulfate, phosphate, perchlorate, and nitrate in concentrations up to 1 M. The presence of these ions does likewise not influence the quenching of the sensor by halides in concentrations up to 0.05 M. Registry No. 1+I-, 88326-06-1; 1+BF4-,88326-08-3;2,8390740-8; 3-(9-acridinyl)propionic acid, 88326-05-0; tosyl chloride, 98-59-9; water, 7132-18-5.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (E) (9) (10) (11) (12)
Borman, S. A. Anal. Chem. 1981, 53, 1616A-1618A. Saari, L. A.; Seitz. W. R. Anal. Chem. 1982, 5 4 , 821-823. Saarl, L. A.: Seltz, W. R. Anal. Chem. 1983, 55, 667-670. Stevens, B. US. Patent 3612888, 1972; Chem. Abstr. 1972, 76, 20945. Lubbers, D. W.; Opltz, N. Z . Naturforsch., C : Bloscl. 1975, 30C, 532-533. Stokes, G. G. J . Chem. SOC. 1889, 22, 174-185. Stern, 0.; Volmer, M. Phys. 2. 1919, 20, 183-190. Wolfbels, 0. S.;Urbano, E. Z . Anal. Chem. 1983, 314, 577-581. Wolfbeis, 0. S.; Urbano, E. Anal. Chem. 1983, 55, 1904-1906. Jensen, H.; Howland, L. J . Am. Chem. SOC. 1926, 48, 1988-1990. Wolfbeis, 0. S.; Urbano, E. J . Heterocycl. Chem. 1982, 19, 841-843. Stoughton, R. W.; Roilefson, 0. K. J. Am. Chem. SOC. 1939, 61, 2634-2638.
RECEIVED for review November 1,1983. Accepted December 5, 1983. Financial support by the AVL-Prof.List Ges.m.b.H., Abteilung Mediz. Messtechnik, is gratefully acknowledged.
