Anal. Chem. 2009, 81, 9048–9054
405 nm Absorption Detection in Nanoliter Volumes Helen Waechter, Klaus Bescherer, Christoph J. Du¨rr, Richard D. Oleschuk, and Hans-Peter Loock* Department of Chemistry, Queen’s University, Kingston, Ontario, K7L 3N6, Canada Analytical UV absorption detection for microfluidic devices, capillary electrophoresis, and even high-performance liquid chromatography is hampered by the small detection volumes, short absorption paths, and the need to sample at a high rate with a stable background and low noise. Fiber-loop ring-down spectroscopy (FLRDS) permits absorption detection of dilute liquid samples in volumes as small as a few nanoliters, while being insensitive to light source fluctuations and permitting a millisecond temporal resolution. We demonstrate a FLRDS based detection scheme that is compatible in dimensions ( 800 nm,18–20 because at these wavelengths there is a large choice of commercially available fiber components, especially fiber-to-fiber couplers with low insertion loss and small coupling fractions (∼1%). Also the silica used in most waveguides exhibits a much larger optical loss at short wavelength and it is commonly believed that these optical losses are dominant over the “desired losses” introduced by the analytes.17 Unfortunately, most substances of interest do not have strong absorption features in the near-infrared region and a system operating at UV wavelengths is strongly preferred. Here, we demonstrate a fiber-loop ring-down detector operating at 405 nm. A low-insertion-loss interface has been designed to enable the coupling of light into the loop and to introduce liquid samples between the fiber loop ends. The ringdown times are measured using phase-shift CRD instead of timedomain CRD to reduce instrument cost and complexity and to allow precise measurements of short ring-down times at high acquisition rates with a duty cycle close to unity.
transmission Ttotal is given by the absorption of the fiber RfL, the absorption due to the sample εsCsd, and the transmission from one fiber end to the other through the sample gap Tgap. τ)
nL nL ) c0 ln(Ttotal) c0(-ln Tgap + RfL + ˜εsCsd)
L is the roundtrip length of the cavity, here identical to the fiber length, n is the refractive index of the cavity medium, c0 is the vacuum speed of light, Rf is the absorption coefficient of the fiber, Cs is the concentration of the absorbing substance with an absorption coefficient ˜εs given with respect to base e (related to the decadic extinction coefficient εs by ˜εs ) εs ln 10). Most cavity ring-down measurements are carried out in the time domain by monitoring the decay of a short laser pulse or light intensity. An alternative is phase-shift CRDS where the phase delay of intensity modulated light between the cavity and the input laser light is measured.21,22 This phase shift, φ, depends on the ring-down time, τ, and therefore on the losses of the cavity. For single exponential decays, the phase shift φ is related to τ by21,22 tan(φ) ) -ωτ
(17) van der Sneppen, L.; Ariese, F.; Gooijer, C.; Ubachs, W. Annu. Rev. Anal. Chem. 2009, 2, 13–35. (18) Loock, H.-P. TrAC, Trends Anal. Chem. 2006, 25, 655–664. (19) Brown, R. S.; Kozin, I.; Tong, Z.; Oleschuk, R. D.; Loock, H.-P. J. Chem. Phys. 2002, 117, 10444–10447. (20) Tong, Z.; Jakubinek, M.; Wright, A.; Gillies, A.; Loock, H.-P. Rev. Sci. Instrum. 2003, 74, 4818.
(2)
where ω is the angular modulation frequency in rad/s. In fiber cavities, multiexponential decays are often observed, due to light traveling in the core and the cladding.19 Sometimes even a third ring-down time (τ ≈ 0) from scattered light needs to be considered. In the time domain, the decaying light intensity is given by a sum of exponential decay functions with decay-times τi and amplitudes ai. N
I(t) )
∑ a exp(-t/τ ) i
i
(3)
i
For multiexponential decays, eq 2 needs to be modified to describe the relationship of the phase shift φ and the different ring-down times τi.23 N
tan φ )
ωaiτi2 2 2 i +
∑ω τ i
N
1
aiτi
∑ω τ i
THEORETICAL BASIS In cavity ring-down spectroscopy, the optical losses, i.e., absorption and scattering, are determined from the average lifetime of a photon in the cavity. The decay time constant (ringdown time) depends only on the losses but not on light intensity, making the technique insensitive to laser power fluctuations. The ring-down time τ is determined by the roundtrip time tR ) Ln/c0 and the total transmission per roundtrip Ttotal. The total
(1)
2 2 i
(4)
+1
In the following, we use eq 4 to fit to the ring-down times and the relative amplitudes of the exponential components. EXPERIMENTAL SECTION Our system consists of a fiber coupled diode laser (405 nm, 30 mW, Nichia, NDHV310APC) which is coupled into a 10 m fiber loop (see Figure 1a) using the custom interface described below. Also at the interface, the fiber loop has a small gap of 190 µm (21) Engeln, R.; von Helden, G.; Berden, G.; Meijer, G. Chem. Phys. Lett. 1996, 262, 105–109. (22) Herbelin, J. M.; McKay, J. A.; Kwok, M. A.; Ueunten, R. H.; Urevig, D. S.; Spencer, D. J.; Benard, D. J. Appl. Opt. 1980, 19, 144–147. (23) Bescherer, K.; Barnes, J. A.; Dias, S.; Gagliardi, G.; Loock, H. P.; Trefiak, N. R.; Waechter, H.; Yam, S. Appl. Phys. B: Laser Opt. 2009, 96, 193–200.
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Figure 1. (a) Setup for UV fiber-loop ring-down spectroscopy. The light of a 405 nm diode laser is coupled into a fiber loop, and by measurement of the phase shift, the absorption of the sample can be determined. (b) Interface for injecting a sample into the fiber loop and for coupling light into the loop. For better alignment, two grooves with the desired angle of 6° are cut into a PMMA plate and the fibers are aligned along the groove. The delivery fiber shines light on the loop fiber end to achieve good coupling. The sample enters the interface through a hole in the top plate, flows along the groove and through the detection gap between the loop fibers, before exiting the interface through the hole below the fiber loop ends. (c) A photo of the interface showing the entrance hole in front of the delivery fiber and (less clearly) the exit hole in the fiber loop gap. The scale bar corresponds to 250 µm.
which is used to introduce a liquid sample. To ensure that the optical losses are due primarily to the analyte and not the fiber, a low loss UV-fiber is used (Polymicro FDP200220240, core diameter 200 µm, cladding diameter 220 µm, absorption coefficient Rf ) 0.011 m-1 at 405 nm). To detect the light intensity in the loop, a photomultiplier tube (PMT, Hamamatsu R955) is mounted on a bend (R ) 8 cm) in the fiber. A large bend radius was chosen to keep the losses in the loop as small as possible, but the intensity of the scattered light is high enough to be readily detected by a PMT detector. The phase shift and the modulated signal intensity were read out simultaneously by a lock-in amplifier (Stanford Research System SRS844, time constant 100 ms) at a sampling rate of either 5 or 10 Hz. The modulation frequency of the laser was set to f ) 1 MHz (ω ) 2πf) for the flow measurements, as the signal-to-noise ratio was determined to be optimal at this frequency. The method by which light is coupled into the fiber loop is critical for the performance of the system. We require a coupler that has a low insertion loss, while transferring at least 0.5% of the intensity into the loop. Insertion losses due to the coupler have to remain under 20% per round trip, because high losses decrease the ring-down time of the system and therefore the sensitivity of the absorption measurement. Efficient coupling to the core of the fiber and not to the cladding is also important, because a larger contribution from the fast decay component (cladding modes) results in a smaller change in the phase shift according to eq 4. There are several ways to couple light into a fiber loop, e.g., using tapered fiber couplers (fused couplers) or side-polished couplers (two adjacent evanescent field blocks). We decided to couple light into the loop by shining it on the fiber end at the sample gap (see Figure 1). This method has the advantage that no additional losses due to the coupler are introduced. For efficient coupling into the fiber core, the light needs to be introduced within the acceptance angle of the loop fiber. Our UVfiber has a numerical aperture of 0.22, corresponding to an acceptance angle of 12.7°. To align the three fiber ends, while allowing for an unobstructed sample flow, we designed a polymethyl methacrylate (PMMA) plate with two grooves cut at the 9050
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desired angle of 6°, one for the loop fiber ends and one for delivery fiber from the laser. Between the two fiber loop ends a 250 µm hole was drilled into the plate to drain the sample. A top plate, also made of PMMA, was used to seal the cell but also contained a 250 µm hole at the power delivery fiber end for sample input (Figure 1b). The sample flows along the groove to the gap in the fiber loop and exits the interface through the hole below the gap. After aligning the fibers, they were secured and sealed with epoxy glue (Summers Optical Lens-Bond J-91). To avoid misalignment while applying epoxy glue, the whole interface was initially filled with UV-curable epoxy before aligning. The channel and holes, through which the sample should later flow, was masked to prevent curing. After alignment, using a red laser as an alignment aid, the epoxy was cured with a UV-lamp for 10 min and the uncured epoxy in the masked part was removed by flushing the interface with isopropanol. Finally, the remaining epoxy was cured fully by exposing it to UV-light for 2 h. The gap width between the loop fiber ends was set to 190 µm, allowing high optical transmission across the gap while retaining a comparably large absorption path length through the sample. In the Supporting Information we describe a mathematical model that helped optimize the gap width.24 With this gap width, the detection volume is calculated to be only 6.0 nL. The entrance and exit holes of the interface were connected to glass capillaries (inner diameter 250 µm) to transport the sample to and from the interface. The samples were injected by a sixport sample injector (Rheodyne) with a sample loop volume of 5 µL at a flow rate of 10 µL/min using a syringe pump. The optimal sample volume was determined by the flow rate and the length of the capillary between the injector and the detector (here, about 40 cm). The sample volume can be reduced considerably when this distance is decreased and/or the flow rate is decreased. Myoglobin (Sigma-Aldrich; horse skeletal muscle, purity >95%), tartrazine (Fluka; purity not specified), and a proposed heterocyclic active pharmaceutical ingredient containing pyrrole, benzodiazepine, imidzole, and piperidinylcarbonyl moieties (supplied (24) See the Supporting Information for a calculation of the optimal gap width. This document can be reached through a link through the online version of this article.
Figure 2. (a) Phase-shift measurements of tartrazine samples in phosphate buffer (pH 7.2) were measured at concentrations from 5 to 1000 µM (buffer flow, 10 µL/min; sample volume, 5 µL). (b) Phase-shift measurements of myoglobin in phosphate buffer (pH 7.2) with concentrations from 100 to 1 µM. (c) Phase-shift measurements of heterocyclic pharmaceutical ingredient provided by Eli Lilly Canada Inc. in HCl buffer (pH 2.0) with concentrations from 1000 to 20 µM. (d) To compare the results from tartrazine, the pharmaceutical ingredient, and myoglobin, the integrated peak areas of the concentration time profiles are correlated to the calculated extinction. As expected, the data of all three substances fall on the same curve.
by Eli-Lilly Canada Inc.; identity or purity not specified) were used without purification. Myoglobin and tartrazine were dissolved in phosphate buffer (pH 7.2; RICCA Chemical Company), whereas the proposed pharmaceutical compound was dissolved in a HCl buffer (pH 2.0; RICCA Chemical Company). Polystyrene microspheres (Fluka; diameter, 5.17 ± 0.10 µm) were supplied in aqueous solution (10% by weight) containing a nonspecified detergent which was diluted in a 1 mM sodium dodecyl sulfate (SDS; Sigma Aldrich) solution. RESULTS AND DISCUSSION Measurements of tartrazine (yellow food dye E102) solutions were performed with nine concentrations ranging from 5 to 1000 µM (see Figure 2a). Tartrazine was chosen as a test substance to characterize the system because it has a strong absorption feature around 400 nm, is nontoxic, and dissolves readily in water. The phase shift (peak height) at each concentration was determined by fitting Gaussian functions to the sample peaks in Figure 2a. As expected from the above equations the phase shift is not linearly correlated to the concentration and the sensitivity is higher at lower concentrations (see Figure 5a). The increase of sensitivity at low concentrations is a useful characteristic of phase-shift ringdown spectroscopy and indeed of all CRD methods (see eq 1). The detection limit was 0.2° (see Figure 2a and 4c) due to drifts in the baseline, which corresponds to 5 µM tartrazine or an absorption of RLOD ) 0.11 cm-1 (with respect to base 10). This corresponds to absorption detection of 30 fmol of sample at a 10 Hz acquisition rate. For comparison, a standard UV-vis detector (e.g., Agilent 1200 series multiple wavelength detector) has a short-term noise of 0.8 × 10-5 absorption units corre-
sponding to a detection limit of 2.4 × 10-5 cm-1 (3σ) assuming a standard flow cell with 1 cm path length and 13 µL volume. Note, however, that the detection volume in such a detector is three orders of magnitude larger and the actual amount of substance that is detected is comparable to that of our system (14 fmol compared to 30 fmol). Also the detection limit of our system was dominated by fluctuations of the baseline on the same time scale as the sample peak widths. For much faster absorption changes, e.g., as observed with microparticle detection (see below), the detection limit was therefore much smaller (standard deviation of the noise in 10 s: 0.007°, compared to 0.07° in 300 s). The upper limit for the concentration (1000 µM) was given by the decrease in light intensity due to absorption between the delivery fiber and the fiber loop. Therefore the dynamic range of our system was about 200. To test the reproducibility of the system, three different samples (10, 50, and 100 µM) were each injected three times in random order (see Figure 4a). The peaks of each concentration show a good reproducibility and a stable baseline. The precision of the system was determined by measuring tartrazine samples with 20 and 5 µM concentration each 10 times (see Figure 4a). The standard deviation of the peak height for a concentration of 20 µM was 2.7% and for 5 µM (detection limit) it was 16%. The ring-down times and the amount of light in the core and cladding were determined by measuring the phase shift at 101 modulation frequencies from 30 kHz to 3 MHz while keeping the concentration constant (see Figure 3a). The phase shift recorded by the lock-in amplifier also contains the offset phase shifts, φ0, introduced by delays in the electronics outside the loop. By Analytical Chemistry, Vol. 81, No. 21, November 1, 2009
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Figure 3. (a) As shown in eq 2 for a single-exponential intensity decay, the tangent of the phase shift depends linearly on the modulation frequency, ω, and the negative slope is identical to the ring-down time, τ. The solid lines are fits to eq 2 using a gradient descent algorithm with fitting parameters Tgap and φ0(ω). (b) The ringdown times, τ, obtained from linear fits of part (a) are related to the tartrazine concentration. The solid line is calculated using eq 1, the known extinction coefficient, and the fitted round trip transmission, Trt ) 0.70.
fitting eq 4 to the total phase shift φtot ) φ + φ0 as a function of concentration for each frequency, we obtained φ0 through extrapolation to infinitely high concentrations. Infinitely high concentration results in ring-down time τi ) 0 and therefore tan(φtot - φ0) ) 0 in eq 2 or 4. At this limiting concentration, the phase-shift φtot recorded by the lock-in amplifier equals φ0.18 The curvature in Figure 3a points to the number and relative weight of the exponential decay functions. The measurements show an almost linear relationship between tan(φtot - φ0) and modulation frequency, indicating the dominance of a single exponential decay. This is consistent with the very short decay times of the cladding modes and their small contribution to the overall signal. In the following, the measurements were evaluated as single exponential decays and the ring-down times at the different concentrations were determined using eq 2 instead of eq 4. The ring-down time was τ ) 142 ns for the phosphate buffer and decreases to 32 ns at a tartrazine concentration of 1000 µM. By fitting of all experimental data to eqs 1 and 2 using an extinction coefficient ˜εs ) 52 177 L/(mol cm) and d ) 190 µm, 9052
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we obtained the round trip transmission through the loop as Trt ) 70% and the frequency dependent offset phase angles φ0(ω). The round trip transmission is the product of the transmission through the gap Tgap and the transmission through the fiber e-RfL. With a fiber length of 10 m and Rf ) 0.011 m-1, the transmission through the gap was Tgap ) 0.77%. While the value for the transmission was lower than desired, it was not unreasonable considering the comparably large gap. For a 190 µm gap, a maximum transmission Tgap ) 92% was calculated from ray optics and using a Gaussian beam profile and Tgap ) 57% for a flat top profile (see the Supporting Information24). We also note that the losses occurring at the interface (8-43% per roundtrip) were comparable or larger than those occurring due to absorption in the fiber material (10%). Proteins with a heme group like myoglobin, cytochrome c, or hemoglobin have a strong absorption features around 400 nm,25-27 and a low detection limits may be expected (see Table 1). The phase shift resulting from myoglobin in phosphate buffer (pH 7.2) was measured using the same experimental settings as before with concentrations from 1 to 100 µM (see Figure 2b). The myoglobin peak was 1.7 times broader than the tartrazine, resulting in a reduced peak height (see Figure 5b). As the concentration and the phase shift are not correlated linearly, the peak integrals as acquired cannot be correlated directly to the extinction of the sample. Instead the phase shift curves in Figure 2a-c) were converted into concentration curves using the fit in Figure 5a). As expected, the integrated peaks in the concentration time profile correlate linearly to the extinction εC for both myoglobin and tartrazine (Figure 2d). Next, a proposed heterocyclic pharmaceutical ingredient (the chemical formula was not disclosed to the authors) was detected in the same setup. The compound has an absorption peak at 450 nm, and at 405 nm the extinction coefficient was determined to be εs ) 5068 L/(mol cm). Using Figure 2a and the LOD absorption of R ) 0.11 cm-1, we expect a detection limit of 22 µM (see Table 1). Six solutions of 20-1000 µM were prepared in HCl buffer with pH 2 (see Figure 2c). A low pH was chosen to increase the solubility of the proposed drug. As expected, the integrated peak areas correlated linearly with the extinction and fell on the same curve as tartrazine and myoglobin (Figure 2d). Finally, detection of spherical polystyrene microparticles (diameter 5.17 µm) was attempted. The microspheres are transparent but they scatter light since their refractive index is high compared to water (npolystyrene ) 1.59). The scattering cross section σ of spherical particles is given by Mie theory σ ) πd2Q,
T ) eσNL
(5)
where d is the particle diameter, N is the number density, and Q is the scattering efficiency coefficient that accounts for the size and shape of the particle. For large (d . λ) spherical particles Q approximates 2. For the microspheres used here, this approximation is valid and a scattering cross section of σ ) 1.68 × 10-6 cm2 is obtained. A detection limit of 1.8 × 108 particles/L is (25) Santos, J. H.; Matsuda, N.; Qi, Z. M.; Yoshida, T.; Takatsu, A.; Kato, K. Anal. Sci. 2003, 19, 199–204. (26) Li, Q. C.; Mabrouk, P. A. J. Biol. Inorg. Chem. 2003, 8, 83–94. (27) Shimojo, K.; Kamiya, N.; Tani, F.; Naganawa, H.; Naruta, Y.; Goto, M. Anal. Chem. 2006, 78, 7735–7742.
Table 1. Detection Limits of Different Analytesa detection limit extinction coefficient ε at 405 nm tartrazine proposed pharmaceutical ingredient myoglobin microspheres (5 µm)
-1
-1
22660 L mol cm 5068 L mol-1 cm-1 145 000 L mol-1 cm-1 7.3 × 10-7 cm2
expected
measured
22 µM 0.78 µM 1.5 × 108 particles/ L
5 µM 20 µM 1 µM
