2556
Anal. Chem. 1985, 57,2556-2561
Fiber Optical Fluorosensor for Determination of Halothane and/or Oxygen Otto S. Wolfbeis* and Hermann E. Posch Institut fur Organische Chemie, KF University, 8010 Graz, Austria
Herbert W. Kroneis AVL-List GmbH, Postfach 15, 8011 Graz, Austria
A flber optical fluoroescence sensor for measurlng concentrations of the wldeiy used lnhalatlon narcotic halothane In the presence of varying concentratlons of oxygen is presented. I t Is based on dynamlc fluorescence quenching and consists of a highly halothane-sensltive indlcator layer exposed to the sample. Interferences by molecular oxygen are taken Into account by a second, polytetrafluoroethylene-coveredfluorescent indlcator layer highly sensltlve toward oxygen. Haiothane concentrations can be calculated wlth the help of an extended Stern-Volmer relation (eq 10). The two-sensor technlque presented here allows the determination of halothane, or oxygen, or both wlth a preclslon of 1 5 % for halothane and 13.5% for oxygen In the concentration range encountered in practlce. The probe Is practlcally speclflc for the two anaiytes, since other gases present in inhalatlon gases or blood including carbon monoxlde, dlnltrogen monoxlde, or fluorans do not interfere. The method is thought to be applicable to various other quenching analyte couples as well.
THEORY Halothane in Air. Dynamic fluorescence quenching is known to obey the Stern-Volmer equation (eq l),which relates fluorescence intensity Z and quencher concentration [Q]
l o / l - 1= KJQI
with Io being the fluorescence intensity in the absence of a quencher and K, the Stern-Volmer constant. For cases where one fluorophore is dynamically quenched by two quenchers, eq 1 has to be extended (17)to give eq 2 which accounts for the contribution of the second quencher
l o / I - 1 = HKs,[H]
+ 0K8,[0]
Here, [HI and [O] are, respectively, the concentrations of halothane and molecular oxygen and HK,, and OKsvare the respective Stern-Volmer constants. When halothane is added to a gas such as air, the concentration of the second quencher (in this case molecular oxygen) is diminished according to
[O]= ~ Halothane (2-bromo-2-chloro-l,l,l-trifluoroethane) is the inhalation narcotic most frequently used in anesthetics (1). Numerous methods for its determination in breath gas and blood have been described. They are based on quite different analytical techniques such as gas chromatography (2,3),UV ( 4 , 5 ) , mass (6, 71, or NMR spectrometry (81, and surface plasmon resonance (9). A commercially available monitor for anesthetic gases (“EMMA”) is based on the absorption of, e.g., halothane, by a thin oil film. The physical parameter measured in this case is the mass of the film which is monitored with a piezoelectric crystal (10). The Datex anesthetic agent monitor measures halothane (or other anesthetic gases) in air via their characteristic IR absorption. Unfortunately, all of these methods either do not allow continuous determination of halothane or are not suitable for use in probes or catheters. We have taken advantage of the observation that halothane is able to quench the fluorescence of polycyclic aromatic hydrocarbons and have devised a fiber-optical sensor suitable for ita continuous monitoring. Since all indicators were also quenched by molecular oxygen-a process that is frequently observed and can be used for sensing oxygen (1l-l6)-the Stern-Volmer equation has to be modified to take into account multiple quenching. Fluorescence quenching of oxygen-sensitive indicators by halothane has already been noticed (16), a fact that makes all oxygen sensors based on dynamic fluorescence quenching halothane-sensitive and can lead to considerable bias in clinical routine. We report here on the first sensor that is able to probe both oxygen and halothane with practically no mutual interference. Its fiber optical design can be quite similar to that reported by Peterson et al. (16),except that two fibers are required and the mathematics is slightly more complicated.
(1)
(3)
( 1 -[H]/100)
with x being the initial concentration of oxygen (in %) in the gas (air) and [HI the concentration (in %) of halothane. Combination of eq 2 and 3 gives
I,-,/I- 1 = HKs,[H]
+ x0K,,(1
- [H]/100)
(4)
which can be rearranged to give
Io/I- 1 = [H](HKs, - ~ ~ K , , / l 0 0+) xoKSv
+
(5)
Equation 5 describes a straight line of typey = a[H] b, with a slope equal to (HKsv- x°K,,/lOO) and intercept b equal to x°Ksv (see, for instance, Figure 4). Both a and b can be obtained from plots of [HI vs. ( I o / l- 1). The most important consequences of eq 5 are that there is linearity between halothane concentration and (Zo/Z - l), and oxygen does not interfere in fluorometric halothane assays, provided the oxgyen content of the supplying gas remains constant. Halothane at Various Oxygen Concentrations. Unfortunately, the oxygen concentration in both anesthetic gases and blood can vary with time. Thus, inhalation gases for narcosis are composed of variable amounts of oxygen, dinitrogen monoxide, and halothane. In contrast to oxygen and halothane, dinitrogen monoxide does not act as a quencher. Since eq 3 is not valid in these cases, use has to be made of a recently introduced technique for the simultaneous determination of two or more dynamic quenchers (17). For the given problem we made use of two sensors with different sensitivity and different quenching constants for the two quenchers. Sensor A displays a signal a = (Io/Z - l),and sensor B displays a different signal = ( I o / I- 1). The concentration
0003-2700/85/0357-2556$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
of halothane can easily be calculated according to
HKaand HKbare the halothane quenching constants of sensors A and B, respectively, as obtained from a preceding calibration. OKa and OKbare the corresponding constants for oxygen, also obtained by calibration. T o obtain [O]a similar expression can be given (eq 7 )
As a consequence, measurement of fluorescence quenching with two different sensors allows the determination of halothane and/or oxygen simply by measuring CY and 0. The second sensor can be made specific for oxygen by covering it with a thin membrane consisting of polytetrafluoroethylene (PTFE). As a result, the quenching constant for halothane (HKb)becomes zero, and eq 6 is considerably simplified
Simultaneously, eq 7 can be transformed to give
which, of course, is the Stern-Volmer equation again. As will be shown later, the most accurate sensor type consists of two identical sensing membranes, one of which is PTFE-covered. In this case the two quenching constants for oxygen (OK, and O K b in eq 8) become identical, which leads to the final-and very simple-equation that describes the relation between the two sensor signals and halothane concentration
For very accurate determination of halothane a further refinement of eq 9 becomes necessary because the value for the quenching constant for halothane was found to be slightly increasing with the fraction of oxygen being present in the gas (see Figure 3). The effect was only observed when the membrane contained a plasticizer such as dioctyl phthalate, but not with silicone membranes. While this is of little significance for halothane assay in blood with its limited oxygen partial pressure range from 50 to 160 torr, it may be of importance in precise halothane determination in inhalation gases. Since the increase in HK,, is practically linear with oxygen concentration, eq 8 can be modified to account for the effect by introducing a correction factor f,obtained from calibration plots
Typically, f ranges from 0.002 to 0.008, EXPERIMENTAL S E C T I O N Reagents. Halothane (Hoechst, FRG, containing 0.01% thymol as a stabilizer)was used BS obtained. A silicone prepolymer (type E43 from Wacker-Chemie, FRG) was used for the preparation of the silicone membranes. It splits off acetic acid after exposure to humidity from air, thereby forming an optically excellentpolymer. trans-Polyisoprene (density 0.904), polystyrene of typical molecular weight 321 000, and poly(viny1 chloride) (density 1.40) were from Aldrich Chemie (FRG). Decacyclene, fluoranthene, and dibenz[ah]anthracene were used as obtained from Aldrich and Fluka (Switzerland),respectively. Decacyclene was made polymer-soluble by a recently published method (18). Preparation of Sensing Membranes. Silicone membranes were prepared by adding 5.0 g of prepolymer E43 to 5.0 g of a 0.05% solution of the respective indicator in toluene. The mixture was smeared as a 0.1 mm thick film onto the surface of a glass slide. Exposure to ambient humidity over a period of 1-3 days
2557
resulted in solvent evaporation and polymerization, thus giving sensor layers suitable for use. The other membranes were prepared as follows: The polymer was dissolved in an appropriate solvent (isoprene in chloroform, PVC and polystyrene in tetrahydrofuran), and the desired amount of plasticizer was admixed. Then an indicator solution in toluene was added in an amount to make the final concentration of indicator in the membrane 0.05%. A definite volume of this solution was brought onto a confined area of a glass platelet and left for evaporation. The obtained sensor layers were not peeled off, but rather used together with the glass platelet, which can serve as a rigid support. Experimental Setup. An Aminco SPF 500 fluorometer was used in combination with a homemade membrane holder or a bifurcated fiber-optical light guide. In experiments where no use was made of fiber optics, a membrane was tightly attached to a gas flow-through cell. Exciting light from the 250-W xenon lamp was focused onto the membrane from the back (i.e., through the glass support) at an angle of around 40'. The polymer membrane was exposed to the gas stream and the change in fluorescence intensity followed. Halothane was admixed to nitrogen, air, or oxygen in concentrations between 0 and 6% by volume with the help of an evaporator (type VAPOR, from Fa. Drager, FRG). Unfortunately, the instrument has a precision of k:8% only in the 2% halothane range. The gas was guided to the sensing layers via 1 mm i.d. polyethylene tubings. Attention was paid not to make the tubings too long since they tend t o dissolve halothane which may cause a memory effect. In the experiments in which a fiber optic was used, the sensing layer was attached to the tip of a bifurcated fiber optical light guide (with statistically mixed quartz strands in a bundle of length 150 cm and inside diameter of the common end 6 mm, from Moritex Corp., Tokyo). The experimental setup is similar to the one shown in ref 19, except that the tip of the fiber bundle was not immersed into an aqueous sample but rather exposed to the gas stream. Light from the fluorometer xenon lamp was focused into the fiber and fluorescence which was collected by the output bundle was guided to the photomultiplier of the instrument. For the experiments in which one of the two sensors was made halothane-insensitive, the indicator layer was covered with a 12 pm thick membrane of PTFE. Care was taken not to leave air bubbles between membrane and sensor layer. Since it is unlikely that the geometrical arrangement of the sensing unit does affect the method presented here, it may as well be applied to any other oxygen-sensingdevice based on dynamic fluorescence quenching, provided the indicator used is also capable of being quenched by halothane. RESULTS Indicators. A variety of polycyclic aromatic hydrocarbons and related dyes have been tested for use as halothane-sensitive indicators, but none of them was found to be specific. All were also quenched to some extent by oxygen. The results on the quenching experiments with halothane in methanol solution are compiled in Table I. They show that some indicators are not suitable because of their photolability, which was found to be distinctly higher in the presence than in the absence of halothane. This is particularly true for anthracene, which is highly sensitive for halothane, but suffers rapid photodecomposition. Other indicators (entries 4 and 8) are of sufficient sensitivity but lack short-wave excitation. Visible excitation is required in practice when low-cost plastic fibers are to be devised, which are flexible and do not present the risk of breakage in sharp bends when used as invasive catheters. Other fluorophores such as quarternized 6-methoxyquinoline (entry 7 ) ,BMSB (1,4-bis(methylstyryI)benzene,a frequently used fluorophore in scintillation cocktails, entry 31, and POPOP (a related compound) were also studied and found to be quenched with fair efficiency. Unfortunately, their excitation maxima are to far in the UV. As a compromise between sensitivity, stability, and spectral properties, decacyclene can be considered to be the most
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
Table I. Excitation and Emission Maxima, Stokes’ Shifts, Stern-Volmer Quenching Constants for Halothane (in mol-’), and Relative Photostabilities of Polycyclic Aromatic Hydrocarbons in Methanol at Room Temperature ,
entry
indicator
1 2 3 4 5
7 8 9 10
anthracene benzov]fluoranthene BMSBb chrysene coronene decacyclene 6-methoxyquinoliniumion dibenz [a,c]anthracene dibenz[a,h]anthracene fluoranthene
11
perylene
6
(- -)
max excitation, nm 375 385 360 320 340 385 365 340 350 360 410,428
max emission, nm
Stokes’ shift,
Stern-Volmer constant
nm
400 515 420 385 450
25 130 65 110 125 85
450 400 403 465 440-500
__
2.75 0.80 0.65
60
510
photostability”
60 53 105
++
1.56
-
0.17 0.62 0.75 0.81 1.12 1.20 0.39
+
+ ++ + + + ++
means very poor stability, (+ +) means rather good stability. 1,4-Bis(2-methylstyryl)benzene.
1.5 2’o
1 c
B 4
12
8
16
time i m i n )
Figure 2.
Quenching of fluorescence of solubilized decacyclene
(0.05%) in silicone rubber by halothane and oxygen: range A, halothane in pure nitrogen; range B, halothane in air; range C, halothane halothane concentratlon (ml/l) Figure 1. Stern-Volmer plots of the quenching of polycyclic aromatic hydrocarbons (concentrationsbetween 15 and 25 pM) by halothane in methanol at 23 OC. Analytical wavelengths are given in Table I.
suitable candidate. Its absorption maximizes at 380 nm, but extends far into the visible and ends a t around 480 nm. Fluoranthene is even more sensitive but requires UV excitation. Figure 1 shows Stern-Volmer plots of the quenching of polycyclic aromatic hydrocarbons by halothane which are almost linear up to concentrations of 1 M halothane. The experimental lifetime of fluoranthene (which is both stable and efficiently quenched) is 15.2 ns at room temperature ( I I ) , resulting in a rather small bimolecular quenching constant of around 1 X los M-l s-l. Polymers. For the preparation of sensing membranes several polymers were checked for use as highly viscous but gas-permeable solvents for the indicators. No polymer was found that made an indicator specific for one of the two interfering quenchers, though polyisoprene strongly favored quenching by halothane over quenching by oxygen. Poly(Viny1 chloride) (PVC) and polystyrene strongly inhibited quenching, resulting in low sensitivities. Silicone rubber seems to be an ideal support because of its good solubility and high permeability for halogenated hydrocarbons (20). Quenching of decacyclene in silicone by halothane under both nitrogen and air is shown in Figure 2. At 0% halothane the signal is highest, but drops by around 21.3% when 5.5% halothane is admixed. At lower halothane concentrations the signal increases and reaches the initial value again when no more halothane is present. Evidently, the signal changes are fully reversible. With air as a supply gas, fluorescence decreases by around 20.6% with respect to the signal under nitrogen because of quenching by oxygen. Admixing 5.5% halothane diminishes
in oxygen.
-
0.2
-k?
O*’
1
I=:
+
B
i
c
t
25
50 oxygen
75
la3
concentratlon (I)
Figure 3. Effect of oxygen concentration on the quenching constants of polycyclic aromatics in polymer solution: A, dibenz[a ,h]anthracene in polyisoprene containing 50% dioctyl phthalate as a plasticizer; B, fiuoranthene in polyisoprene with 50 % dioctyl adipate; C, fluoranthene in the same polymer as for curve A. No such effect was observed with silicone membranes.
the signal further, this time owing to quenching by halothane. When no more halothane is present, the original signal level is reached again. The response times are a t around 15-20 s for 90% of the total signal change. Polyisoprene is suitable as a polymeric solvent when containing more than 25% of a plasticizer such as dioctyl phthalate. However, an inherent disadvantage of this material is the fact that the quenching constants for halothane vary with the fraction of other gases being present in the gaseous sample. Figure 3 shows that the experimentally determined quenching constants change with increasing oxygen concentration in the gas. No such effect was observed with silicone as a solvent which, however, was
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
Table 11. Experimental Results Obtained with Three Sensor Types Used for Determination of Halothane in Air" % halothane
given
sensor l b
found with sensor zC
sensor 3d
0.00 1.00 2.00 3.00 4.00
-0.01 1.00 2.10 3.01 3.95
-0.07 1.04 2.06 3.05 4.14
0.03 1.04 2.08 3.02 3.86
'*O
2559
I
Concentrations were calculated with eq 5. Consisting of a 50 pm thick membrane (a 0.05% solution of solubilized decacyclene in 1:l polyisoprene-dioctyl phthalate). Consisting of a 45 pm thick membrane (a 0.05% solution of decacyclene in silicone). d A 45 fim thick membrane, consisting of a 0.05% solution of fluoranthene in silicone.
1 .o
2.0
3.0
halothane concentratlon
I
laO 0.8
2 o:
3.0
4; 0
halothane c o n c e n t r a t l m ( % I
Flgure 4. Modified Stern-Volmer graphs obtained by plotting (Io/I 1) vs. halothane concentration under (A) oxygen, (B) air, and (C) nitrogen. The linearity demonstrates the independerlt action of the two quenchers and allows a simple determination of two quenching constants according to eq 5. never used together with a plasticizer. For halothane in air eq 5 can be rearranged to give
[HI= ( l o / l - 1 - 21°K,,)/HK,, - O.2l0K,,
(%)
Figure 5. Same as in Figure 4, but in silicone rubber. Quenching is distinctly more efficient for both halothane and oxygen. Thus, under air (graph B) the slope is 0.0870, but only 0.0804 in Figure 4. Similarly, the intercept is 0.4990 in this figure, but only 0.0838 in Figure 4.
/+
1 :o
4.0
(12)
Typical plots of [HIvs. ( & / I - 1)are shown in Figure 4 and 5. The slope of the lines is equal to (HK,,- x°K,,/lOO), and the intercept is x°KB,. OK,, and HKsvcan be determined by conventional ways via the Stern-Volmer relation, using nitrogen as a supply gas. The graphic method for the simultaneous determination of quenching constants presented here seems to be as simple and allows, in particular, an estimation of whether two quenchers act independently or not. Determination of Halothane in Air. Three types of sensor layers were studied with respect to their ability to sense halothane in air. Their preparation is described in the footnotes of Table 11. First their quenching constants (OK,, and HK,v)were determined from Stern-Volmer plots and from plots according to eq 5 as shown in Figure 4 and 5. For oxygen, they were found to be, respectively, 0.004,0.024, and O.O22%-l. The respective values for halothane are 0.081,0.091, and 0.196%-'. The analytical results obtained with these sensors are summarized in Table 11. Evidently, sensor 2 slightly overestimates the concentration. All three sensors exhibited about the same accuracy with regression coefficients of 0.999 41, 0.999 81, and 0.999 15, respectively. The standard deviation for the determination of 1.5% halothane (a clinically frequently employed concentration) is 10.15% halothane under air. A 4.00% halothane in air mixture was determinable with an accuracy of *0.16%.
Detection limits, defined as the concentration giving a fluorescence signal of 3 times the standard deviation of the background fluctuations under the same conditions, were found to lie between 0.07 and 0.10% halothane. Determination of Halothane and/or Oxygen. In anesthetic practice, halothane is mostly delivered in combination with elevated levels of oxygen. In the initial ("flood") phase of anesthesia, 1-3% halothane is contained in inhalation gases along with 30-50% oxygen, the remainder being dinitrogen oxide. The concentration of halothane is reduced after a while to 0.6-1.0%. Since both oxygen and halothane act as fluorescence quenchers, the fluorometric signal is no longer unambiguous in this case. It becomes therefore necessary to use two sensors with different response. We have applied three types of sensor combinations to determine halothane or oxygen, or both, under varying oxygen levels. Sensor combination 1 consisted of two sensor layers with the same indicator in two different polymeric solvents. Equation 6 and 7 were used to calculate the concentrations from the respective fluorometric signals. Sensor combination 2 consisted of two sensor layers with the same indicator in two different polymeric solvents (see Table 111, footnote b). One layer was covered with polytetrafluoroethylene (PTFE) which is impermeable for halothane. As a result, the second layer was insensitive toward halothane. Equations 9 and 11 were used to calculate the results. Sensor combination 3 consisted of two sensor layers with the same indicator in the same polymeric solvent. One was covered with PTFE to protect it from quenching by halothane. Equations 9 and 10 were applied to calculate the concentrations. The quenching constants (HK,, HKb, OK,, OK,,) and the correction factor f were determined in the calibration procedure. The values are given in the footnotes of Table 111. The results obtained with these sensor combinations are given in the same table showing that at zero oxygen concentration (first five rows) the required precision (*0.1% in the 0-3% halothane range) is provided by all three sensor types, whereas it becomes poorer at 4% halothane. Under air (entries 6-10) sensor combination 1 displays considerably erroneous results for both halothane and oxygen. Sensor combinations 2 and 3 show about the same precision for both, with maximal deviations of f0.2% absolutely. Naturally, the data for oxygen are identical for both sensor combinations because they have the same halothane-insensitive sensor part.
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
Table 111. Experimental Data Obtained in the Simultaneous Determination of Halothane and Oxygen with Three Different Sensor Types
given entry
%H
1 2
0.0 1.0
3 4 5 6 7 8 9
2.0 3.0
4.0
%O 0.0 0.0 0.0 0.0 0.0
found with sensor combination 2b
sensor combination la 70H %O
%H
%O
%H
% 0
0.0
0.0
0.2 0.4 0.6 -0.2 19.8 19.7 19.2 18.6 16.5 96.7 92.7 89.1 84.5 81.3
1.0
0.0 0.0 -0.1 0.0 0.1
0.0 1.1 2.1
0.0 0.0
0.0 1.0
2.0 3.1 4.3 0.2 1.2 2.4 3.5
2.1
2.9 3.8
sensor
combination 3'
3.1 4.1 -0.1
-0.1 0.0
0.1 21.5 0.9 1.0 21.3 2.0 2.0 2.0 21.0 3.0 3.0 3.1 20.6 10 4.0 5.1 3.9 4.2 20.1 11 0.0 100.0 0.5 0.1 -0.1 100.2 12 1.0 99.0 1.1 2.3 0.9 98.0 13 2.0 98.0 2.1 3.8 2.1 95.9 14 3.0 97.0 5.5 3.0 3.0 93.9 15 4.0 96.0 7.1 4.0 4.2 92.9 "Consisting of a sensor A (a 50 pm layer of a 0.05% solution of decacyclene in silicone rubber) and a sensor B (a 50 pm layer of 0.05% decacyclene in polyisoprene/dioctyl phthalate). The following quenching constants were determined: HKA= 0.091%-'; O K A = O.O24%-l; HKB = 0.072%-'; O K B = O.O04%-l. bConsistingof a sensor A (a 50 km thick layer of a 0.05% solution of decacyclene in polyisoprene/dioctyl phthalate), and a sensor B (a 50 pm thick layer of a 0.05% decacyclene solution in silicone, covered with a 12 pm PTFE layer). The following quenching constants were determined: HKA= 0.0725%-'; O K A = 0.004%-'; HKBis zero; O K B = O.O24%-l; factor f (see eq 11) is 0.007. CConsistingof a sensor A (a 50 pm thick layer of a 0.05% solution of decacyclene in silicone rubber), and a sensor B, which is identical with sensor A, except that it was covered with a 12 pm PTFE membrane. The following quenching constants were determined: H K =~ 0.091%-'; OKA= OKB = 0.024%-'. H K is~ zero. 0.0 1.0
21.0 20.8 20.6 20.4 20.2
Under pure oxygen (entries 11-15) sensor combination 1 gives erronous results again. Sensor combinations 2 and 3 are more precise, although they tend to underestimate the oxygen concentration. The precision of the halothane assay is again the same as that of the halothane evaporator and may become better with a more precise vaporizer.
DISCUSSION Quenching of polyaromatics by alkyl or aryl halides has been studied in several cases (21-29), but not with halothane. In fact it has been stated (30) that this anesthetic does not interfere in the fluorometric oxygen sensor. Strong fluorescence quenching was generally observed with bromides and iodides, whereas chlorides are less efficient. This fact is usually interpreted in terms of the so-called heavy-atom effect. Polychlorinated alkanes (with more than one chlorosubstituent a t the same carbon atom) are even weaker quenchers than monochlorides, and fluoroalkanes are practically inert. As a result, among the frequently encountered inhalation narcotics only halothane acts as a dynamic quencher by virtue of the heavy-atom effect of its single bromo atom. Quenching constants for polymer solutions of polycyclic aromatics could not be determined due to the lack of solubility data for halothane. However, it can be anticipated that quenching is less efficient in the polymer owing to a limited mobility of both the dye and the quencher. Silicone can be considered as an almost ideal polymeric solvent since it has high solubility for oxygen (20) and halothane. In addition, the quenching constants do not vary with the fraction of other gases being present, thereby eliminating the need for a correction factor (eq 11). The results obtained *ith sensor combinations 2 and 3 show that it is possible to determine halothane and oxygen over a wide range with sufficient precision. Sensor combination 3 is advantageous over 2 for two reasons: (a) Both sensor layers are prepared in absolutely the same way, except that one of them is covered with PTFE. This situation is desirable when manufacturing layers on a larger scale. (b) Only two quenching constants have to be determined in the calibration procedure. In fact, the lower accuracy of sensor combination 1 is most
-0.1
21.5 21.3 21.0 20.6 20.1 100.2 98.0 95.9 93.9 92.9
probably due to the uncertainties in the determination of four quenching constants, which accumulate to a considerable error. We note that the fluorescence optical method presented here is distinctly more precise a t high oxygen levels than the Clark electrode. The response time (15 to 20 s for halothane, and 10 to 15 s for oxygen, for 90% of the final values) is short enough to allow breath gas analysis. Among the potential fields of applications for the new sensor we consider the following to be most attractive: (a) continuous in vivo monitoring of halothane in blood with fiber optic catheters during operations; (b) continuous monitoring as well as in vitro measurement of oxygen in the presence of halothane which is known to interfere in the simple fluorometric assay; ( c ) continuous monitoring of anesthetic breath gases.
ACKNOWLEDGMENT We thank Y. Morito of the Moritex Corp. (Tokyo) for providing optical fibers. Registry No. 02,7782-44-7;halothane, 151-67-7;decacyclene, 191-48-0;fluoranthene, 206-44-0;benzov] fluoranthene, 205-82-3; coronene, 191-07-1;dibenz[a,c]anthracene, 215-58-7; dibenz[a,hlanthracene, 53-70-3; perylene, 198-55-0; anthracene, 120-12-7; 1,4-bis(2-methylstyryl)benzene, 13280-61-0; chrysene, 218-01-9.
LITERATURE CITED Windholz, M.. Ed. "The Merck Index", 9th ed.; Merck & Co., Inc.: Rahway, NJ, 1976; no. 4455. Ramsey, J. D.; Fiangan, R. J. J . Chromatogr. 1982, 240, 423. Dueck, R.; Rathbun, N.; Wagner, P. D. Anesthesiology 1978, 4 9 , 31. Epstein, H. G.; Redman, L. R. Br. J . Anaesth. 1980, 52, 1155. Blanck, Th. J.; Thompson, M. Anesth. Ana@. ( N . Y . ) 1980, 5 9 , 481. Ozanne, G. M.; Young, W. G.; Mazzei, W. G.; Severinghaus, J. W. Anesthesiology 1981, 5 5 , 62. Ulrlch, R. W.; Bowerman, D. L.; Wittenberg, P. H.; Pierce, A. F.; Schisler, D. K.; Levisky, J. A,; Pflug, J. L. J . Anal. Toxicoi. 1977, 1 , 195. Wyrwicz, A. M.; Schofieid, J. C.; Burt, C. T. Noninvasive Probes Tissue Metab. 1982, 173. Nylander, C.; Liedberg, B.; Lind, T. Sens. Actuators 1982, 3 , 79. Gedeon, A,; Hamilton, K. Proceedings of Kindlund, A,; Lundstrom, I.; the 5Ih Nordic Meeting on Medicinal and Biological Engineering, Linkoping, Sweden, 1981, pp 107-109. Kautsky, H.; Hirsch, A. 2.A/@. Anorg. Chem. 1935, 222, 126 Bergman, I.Nature (London) 1968, 278, 396. Stevens, 6 . US Patent 3612866 (Oct 12, 1971).
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RECEIVED for review April 8, 1985. Accepted June 25,1985.
Determination of Equilibrium Constants by Derivative Spectrophotometry. Application to the pK,s of Eosin Pierre L e d l a i n * and Dominique Fompeydie
Laboratoire de Chimie Analytique, FacultS des Sciences Pharmaceutiques et Biologiques, 4, avenue de I’Observatoire, 75006 Paris, France
Zero crossing derlvatlve spectrophotometry has been used to determine the two pK,s of eosln. This spectrophotornetrlc deterrnlnation Is rather dlfflcult because of the strong overiapplng of both spectra and ionlratlon constants. First, the two pK,s are separated by less than one pH unlt. Second, the molecular form Is colorless while the absorptlon maxima of mono- and dlanionlc forms are found at 519 and 516 nm, respectively. The results are pK,, = 3.25 and pKa2= 3.80 wlth a preclslon f0.05, which suggest that this method can be used to study equlllbrla between compounds whose spectra overlap strongly.
Ionization of compounds sometimes involves minor changes in their electronic spectra. It is thus difficult to determine the corresponding equilibrium constants by using classical spectrophotometric methods. Derivative spectrophotometry seems to be suitable for solving this problem as it improves the detectability of small spectral variations, enhances resolution of multicomponent solutions, and allows the determination of two or several compounds (or forms of the same compound) in a mixture, even if their spectra overlap extensively (1-3). So this method is well adapted to study weak spectral variations resulting from acid-base transformations. We report here the data obtained with this technique for the two pK,s of eosin (Figure 1)whose determination is rather difficult with conventional techniques. Indeed, the molecular form (EH,) is practically colorless while the absorption maxima of mono- (EH-) and dianionic (E2-) forms are very close (516 and 519 nm, respectively), as shown in Figure 2. Moreover, the difference between the two pK,s is only about one unit. Finally, the molecular form precipitates slowly and the dianionic form polymerizes in solution, which shifts the equilibria. These difficulties explain the wide variation for the published values of pK,s (Table I) and the interest of the proposed method.
EXPERIMENTAL SECTION Apparatus. A Gilford 2600 spectrophotometer, with a Hewlett-Packard 7225B plotter was used for spectrophotometric measurements.
Reagents. Eosin was synthesized in our laboratory by a specific method (14) and tribromofluorescein prepared by chromatography (15). Their purity was controlled by TLC on silica gel using a mixture of toluene-acetic acid (65/35 v/v) as eluant. For the determination of pK,s, the working solutions of eosin (concentration = IOm5mol L-l) were prepared with BrittonRobinson buffers (16) in order to reach pH from 2 to 7. Dilute solutions of chlorhydric acid were used to obtain more acidic pH. For the quantitative determination of dyes, various solutions were prepared, with known concentrationsof tribromofluorescein mol L-I) and uncontrolled concentration (from 2 x lo-’ to of eosin, in a pH 7 buffer. Procedure. All measurements were performed at 20.0 f 0.1 “C, within 5 min after the preparation of solutions to avoid precipitation of the molecular form, especially in low pH solutions. Three experiments were performed and, for each one, 30-40 solutions of different pH between 0.5 and 7 were used. The derivative spectra were recorded between 512 and 520 nm for the monoanionic form and 515 and 522 nm for the dianionic form, using a 0.1 nm step and a 0.1 nm slit width. The curves were smoothed by using a “derivative window” of 10 points, which gave the best results to optimize signal-to-noiseratio. Owing to the slight distortion of spectra (17) the exact wavelengths for measurement of derivative values were determined experimentally on the graphs. The values of pK,, and pKa2were calculated from the pH and corresponding concentrations of mono- and dianionic forms of solutions using a Hewlett-Packard 9825A computer and 9872 plotter and the calculation program TOT (18). RESULTS AND DISCUSSION Changes in eosin absorption spectrum with pH are shown in Figure 2. The molecular form (prominent at pH
