Anal. Chem. 1994,66, 761-764
Sensitivity Enhancement in Capillary/Fiber-Optic Fluorometric Sensors Zhong Yuan Zhu and M. Cecilia Yappert' Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
Fluorometricmeasurements acquired with capillary/fiber-optic sensors offer greater sensitivity and detectability as compared to those obtained with conventional fiber-optic sensors. The sensitivity enhancement has been theoretically derived, and it depends on the relative diameter of the fiber and the capillary and on the length of the capillary relative to the effective depth of the fiber. Fluorescence measurements obtained experimentally with conventional and capillary/double-fiber-optical sensors indicate sensitivity enhancement factors of almost 2 orders of magnitude and improvements in the detection limits of 1 order of magnitude. The study of factors which affect the sensitivity and detectability of fluorometric analysis based on fiber-optic sensors (FOS) is one of the thrusts of our In our previous report,3 we demonstrated that the magnitude of the differential fluorescence signal dF(z) excited and/or collected over a differential sample depth dz by an optical fiber decreases sharply with sample depth and limits the total fluorescence signal F ( m ) , integrated over a infinitely long sample depth, to a relatively small value. To increase the magnitude of the integrated signal, it is necessary to remove this limitation. Komives and Schultz have proposed the use of a mirror4 at the distal end of the sensor to reflect part of the excitation and emission radiations back into the optical fiber. They reported a theoretical enhancement factor of 3.8 for a single fiberoptical sensor. In this report, we discuss the higher sensitivity enhancement obtained with capillary/fiber-optic sensors (C/ FOS). Although several papers have reported the enhancement of fluorescence, Raman scattering, and absorbance signals when the optical fibers were introduced into a capillary t~be,~-lO the significant sensitivity enhancement of C/FOSs over conventional FOSs has not been fully exploited and characterized. In this note we present a theoretical and experimental study of the factors which affect the enhancement in sensitivity and detectability provided by C/FOSs over conventional FOSs. (1) Lal, S.;Yappert, M. C. Appl. Spectrosc. 1991, 45, 1607-1612. (2) Zhu, Z. Y.; Yappert, M. C. Appl. Spectrosc. 1992, 46, 912-918. (3) Zhu, 2. Y.; Yappert, M. C. Appl. Spectrosc. 1992, 46, 919-924. (4) Komives, C.; Schultz, J. S. Talanta 1992, 39, 429-441. (5) Ross, H. R.; McClain, W. M. Appl. Specrrosc. 1981, 35, 439-442. (6) Walrafen, G. E.; Stone, J. Appl. Spectrosc. 1972, 26, 585-589. (7) Schwab, S. D.; McCreery, R. L. Appl. Spectrosc. 1987, 41, 126-130. (8) Deaton, T. Ph.D. Instrumentation and Methodology for Remote Fiber Fluorometry. University of California at Davis, 1984. (9) Fujiwara, K.;Simeonsson, J. B.; Smith, B. W.; Winefordner, J. D. Anal. Chem. 1988.60, 1065-1068. (IO) Wan& W.; Qushe, H.; Wang, T.; Minzhao, F.; Yuanmin, L.; Gouxia, R. Anal. Chem. 1992, 64, 22-25.
0003-2700/94~0368-0761$04.50/0 0 1994 American Chemical Soclely
THEORY Differential and Integrated Fluorescence Signals from a C/FOS. According to our previous studies,2 the value of the differential fluorescence can be described by the following expression: dF(z) = 0.25t~c,CZ(z)S2(~)r-'dY
(1)
where dF(z) refers to the differential fluorescence signal from an element volume dVat a distance z from the tip of the fiber. Z(z) is the intensity of the excitation beam at a depth z, and 0.25Q(z)r-' refers to the normalized solid angle of excitation and collection. Further details and derivations can be found in ref 2. For a conventional FOS, the differential fluorescence signal is dF(z) = F(a)' dz(z
+ zo)-2
(2)
in which F(a)' is a combination of constants and angledependent variables and zo is the effective depth as defined in ref 2. The decrease with the square of the distance (z ZO) clearly points to the factor that limits the sensitivity of fluorescence signals acquired by conventional FOSs. To remove this limitation, the optical fiber can be introduced into a capillary, which is assumed to be a perfect waveguide in which all the radiation can be trapped. If the diameter of the capillary is comparable to the diameter of the optical fibers, then Z(z), 0.25O(z)r-l, and dVin eq 1 can be evaluated according to eqs 3-5, respectively:
+
ZR(z) = ZR(0) = Por-'R-2
(3)
0.25Q(z)r-' = rR2/(4rz;) = 0.25 tan2 a
(4)
dV = r R 2 dz (5) where POabove refers to the total excitation power measured at z = 0. R is the internal radius of the capillary, and a is the semiaperture angle, as shown in Figure 1. By substituting eqs 3-5 for the corresponding terms in eq 1, the differential fluorescence signal can be expressed by dF(z) = 0.25rqe,CZR(0)tan' aR2 dz
(6)
By comparison with eq 2, one can see that the decrease in the intensity by the factor (z + ZO)-~ is no longer affecting the differential fluorescence signal. To consider the attenuation of the fluorescence signal due to sample absorption at the emission and excitation wavelengths, eq 6 should be multiplied by the attenuation term e-(cl+r2)Cz, in which €1 and €2 are the molar absorptivitiesat the excitation and emissionwavelengths. When an optical fiber is introduced into a capillary, only a portion, rro2/rR2,of the total differential fluorescencedF(z) AnatyticaiChemistry, Vol. 66, No. 5, March 1. 1994 761
optical
Eels, depends on the product of two terms. One is the length
sample capillary
--
Aidz Figure 1. Schematic representation of the capillary/fiber-optic fluorometric sensor: ro,fiber core radius; R, capillary internal radius; cy, angle of semiaperture; dz, differential distance.
can be intercepted by the collection fiber. The amount of the signal collected by the fiber in a solution with significant absorbance at the excitation and emission wavelengths can thus be expressed by dF(z) = 0.25ar;qqC tan2 aIR(0)e-(c1+c2)cz dz
s," dF(z) =
0.25ar;q
tan2 aIR(0)q(q + e2)-'(l - e-(c1+c2)czc) (8)
-
-
At an infinitely long sampling depth (z a),(€1+ 4 C z a,e-(cl+c2)cz 0, and Fc(z) reaches a maximum value Fmax: F~~~ =~
--+
= 0.25ar:q
~ ( 0 3 )
tan2 dR(o)q(q
+ e2)-l
(9)
After substitution of eq 9 into eq 8, the fluorescence signal, Fc(zC),from a C/FOS can also be expressed as
When the sample absorbance is negligible, the fluorescence from a C/FOS can be linearly related to the sample concentration or the length of the capillary, as indicated in J'c = Fmax(c1 + 4 C z c
(1 1)
By substituting eq 11 for eq 8, the fluorescencesignal acquired by a C/FOS is related to the sample concentration C, the capillary length zc, and the ratio of the radius of the fiber to that of the capillary (ro/R) as follows: Fc(zc) = 0.25ar;q
tan2 aIR(0)qCzc= o.25qp0 tan2 aq(ro/R)2Czc (12)
SignalEnhancement in a C/FOS. The enhancement factor,
Eels,is defined as the ratio of the signal obtained by a C/FOS to that obtained by a conventionalFOS (without the capillary):
The maximum fluorescence signal collected by a singlefiber-optical sensor in a diluted solution has been described in ref 1 as F s ( a ) = 0.5ar;qt,I~(O,O)zoF(Q)C
(14) in which F ( a ) groups all the angle-dependent variables. If the excitation and collection efficienciesare considered to be unity, and thus F(a)becomes directly proportional to tan2a, then the ratio of eqs 12 and 14 can be expressed as EC/S a
(zc/Zo)(rO/R)2
(15)
Equation 15 demonstrates that the enhancement of the signal, 762
AnalyticalChemistry, Vol. 66, No. 5, March 1, 1994
F ~ I (a ~0.25kiqqPo ) tan2 azoc
(17)
in which ki is a factor that depends on the size and geometrical arrangement of the two optical fibers and is less than unity. By dividing eqs 12 and 17, E C / Dcan be evaluated as
(7)
The total fluorescence signal collected by a fiber from a capillary of length zccan be obtained by integrating eq 7 from z = 0 to z = zc: Fc(zc) =
of the capillary relative to zo (i.e., zC/zo),and the other is the size of the capillary relative to the fiber size ( ~ o / R ) ~ . For a double-fiber-opticalsensor, the enhancement factor, E c ~ Dis, defined as the quotient of the signal from C/FOS and that from a double FOS. The signal obtained by a double FOS can be expressed as3
Equation 18 indicates that the enhancement factor obtained with a capillary/double FOS as compared to the double-fiber sensor (without the capillary) depends on the length of the capillary relative to the fiber depth ZO,the relative diameter of the capillary and the fiber, and the configuration of the sensor (i.e., kj-').
EXPERIMENTAL SECTION The capillaries were made by drawing glass tubing. Before and after drawing the capillaries, the tubing was introduced into 3 M HN03 and then rinsed to ensure the cleanness of the walls and thus minimize scattering. To allow the easy introduction of the double-fiber-optical sensor into the capillary, one end of the capillary was made slightly larger than the rest. All the capillaries were 10 cm long, as measured from the sensor tip. The uniformity of the capillary diameter and the smoothness of the internal and external surfaces affected the enhancement greatly. The diameters of the capillaries used in this research were as follows: (A) 0.28, (B) 0.48, (C) 1.15, (D) 1.6, (E) 2.05, and (F) 3.2 mm. The diameters for capillaries A, B, and D correspond to the diameters of double FOS made with loo-, 200-, and 600pm-core fibers, respectively. Double-fiber-optical sensors of various sizes were constructed. The excitation and the collection fibers were parallel to each other, and there was no additional separation between them. The following optical fibers were used: (1) 100-pmdiameter core; 140-pm-diameter cladding, NA = 0.28; (2) 200-pm-diameter core, 240-pm-diameter cladding, NA = 0.22; and (3) 600-pm-diameter core, 800-pm-diameter jacket, NA = 0.4. In all cases, the fiber length was 200 cm. The monochromator, detector and recorder were the same as those described in ref 1. The experimental setup used in this study is shown in Figure 2. The signals from the conventional double-fiber-optical sensors were taken as reference for comparison. In these measurements, the double FOSs were directly introduced into the sample solution. The experimental setup was the same as that described in ref 2. To evaluate the sensitivity enhancement, eight aqueous solutions of Rhodamine 6G (R6G) ranging in concentration from 0.5 pg/mL to 5 ng/mL were prepared. For each C/FOS, the fluorescence signal of each R6G solution was measured in three independent runs. The reported signals represent the average and standard deviation of the measurements. To test
P; RECORDER
Figure 2. Experimentalsetup for fluorometric measurementswith the capillary/fiber-optic sensor. PMT, photomultiplier tube.
the reproducibility of the measurements, the fiber-optic sensor was pulled out of the capillary and the solution was removed with an aspirator. The sample solution and the sensor were then introduced back into the capillary. The meniscus formed at the distal end of the capillary (away from the FOS) caused large scattering of the excitation beam and greatly increased the background signal. To minimize this problem, the end of the capillary was introduced into a solvent reservoir as shown in Figure 2.
RESULTS AND DISCUSSION Signal Enhancement. As indicated by eqs I5 and 18, an enhancement in the fluorescence signal is expected when a C/FOS is used instead of a conventional FOS (without capillary). In this research, although the capillaries were only partially reflective and did not behave as perfect waveguides, the enhancement was still observed. The experimental comparison is based on the signals obtained with double-fiberoptical sensors with and without capillaries. The calibration curves were obtained with C/FOS made with double-fiber sensors of loo-, 200-, and 600-pm-core fibers. The slopes of the log F vs log C straight lines were unity, within the noise of the measurements. Table 1 lists theexperimental values observed for the signal enhancement and the improvements in signal-to-noise ratio (S/N) and detection limits obtained with capillary/doublefiber sensors as compared to the conventional double-fiber sensors without capillary. Thevalueslisted in Table 1 indicate that the combination of small-diameter capillaries with smaller core optical fibers improves the signal-to-noise ratio, the sensitivityenhancement, and thedetection limit. The different values observed for the enhancement factor can be explained by eq 18, which describes the relationship between the enhancement and the relative sizes of the capillaries and optical
v
-
0.0
I
I
I
I
0.2 0.4 0.6 Fiber Core Diameter (mm)
0.8
Figure 3. Effect of the fiber sire on the relative enhancement obtained with a C/FOS. (-) Solid line was calculated by assuming ,EclD0: (r0)*; (- -1 broken line was calculated by assuming f c l D 0: (ro)2/zo;solid triangles, experimentalenhancement obtained with a caplllary of 2.05mm internal diameter.
-
fibers. According to eq 18, if the same double-fiber-optical sensor is introduced into capillaries of different diameter, then the value of the enhancement should be in inverse proportion to the square of the capillary diameter. As predicted by eq 18, the larger the diameter of the capillary, the smaller the fraction of the total emission that can be intercepted by the collection fiber. If the capillary diameter is kept constant and the size of the fiber-optic sensors is changed, it would seem that the increase of ro would lead to an increase proportional to ro2in the collected emission. However, the theoretical predictions indicate that the signal enhancement is not solely related to the value of ro2,but to the ratio ro2/zo. This relationship and the experimental results are shown in Figure 3. Here the values of ki, which were defined in ref 3, are assumed to be constant for double FOSs made with optical fibers of different sizes. Table 1 also indicates the increase in the background signal observed in CFOSs. The main contribution to the background signal is the scattering of the excitation radiation at the walls of the capillary. The greatest increase in background signal was observed for those CFOSs with the smallest diameter capillaries. For the CFOS made with 0.100-mmcore-diameter
Table 1. C m p a r l m of Analytical Figures of Merit for Fluorometric Signals Obtained with CIFOSI and FOSI'
core diam (mm)
capillary int diam (mm)
enhance. factor
0.100
0.28 1.15 2.05 0.48 1.15 2.05 1.6 2.05 3.2
85 i 13 3.3 f 0.2 1.8 0.3 36 5 5.5 f 0.6 2.6 0.2 21f4 5.9 f 0.4 2.3 f 0.2
0.200 0.600
ECJD~
*
* *
re1 S/N
re1 backgrd'
BcIBD
re1 enhance.d
5.4 f 2 3.5 f 2 1.2 f 0.6 5.0 2 2.4 f 1 1.7 0.8 5.3 3 2.7 1 2.2 f 1
53i5 4.8 f 1 2.2 1 97 10 37 f 5 16 3 8 2 f 10 29 4 8.1 f 2
48 1.8 1 14 2.1 1 3.6 1 0.38
S/N)C/(S/N)D
**
*
* * *
*
re1 R-% 54 3.2 1 18
3.2 1 1.6 1 0.41
obsd improve. in det limit >10 10 1-10 10 1-3 1-3
