Comment pubs.acs.org/ac
Comment on “Partition Coefficient Measurements in Picoliter Drops Using a Segmented Flow Microfluidic Device” Zeqing Bai, Qiaohong He,* Hengwu Chen, and Min Wang The Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Zijin’gang Campus, Hangzhou 310058, China
Anal. Chem. 2009, 81 (4), 1471−1476. DOI: 10.1021/ac801673w
M
arine et al.1 reported a microfluidic method for rapid measurement of the octanol−water partition coefficient (Kow) by using a droplet-based two-phase flow. Because of the large contact area and quick mass transfer offered by the droplet-based two-phase flow, the partitioning of the target species (fluorescein) between the aqueous droplets and octanol carrier reached equilibrium in less than 2 s. The authors deduced an equation (eq 1) to calculate individual partition coefficients (Kows) governed by individual pairs of a droplet and the contacted octanol segment. The mean Kows at varied pHs obtained by their work agreed well with those reported by Grimes et al.2 with the conventional batch method. However, the precision was poor, with the reported coefficients of variance (CV) being as high as 36−49%. The authors attributed the main source of variability to the uncertainty in determining the droplet length for both water and octanol segments (Lw and Lo in eq 1). ⎡ I w,initial − Idark ⎤L Kow = ⎢ − 1⎥ w ⎢⎣ I w,final − Idark ⎥⎦ Lo
Figure 1. Fluorescence intensities of a fluorescein-containing aqueous solution measured after it was extracted with octanol in different phase ratio Vw/Vo. The liquid−liquid extraction was performed in a 20-mL bottle according to ref 3. The total volume of two phases was 9.0 mL. The fluorescence intensity of the aqueous phase was measured with a fluorospectrophotometer. The excitation and emission wavelength were 490 and 520 nm, respectively. The measured fluorescence intensities have been normalized against the initial fluorescence intensity of the aqueous sample solution. Three replicates were examined for each phase ratio Vw/Vo.
(1)
In our opinion, eq 1 that was deduced from an isolated conventional liquid−liquid extraction system1 is not suitable for a microfluidic droplet-based liquid−liquid extraction system. Taking each pair of water droplets and the contacted octanol segment as an independent extraction system is a mistake. Consequently, use of eq 1 to calculate Kow will result in errors. In a conventional liquid−liquid extraction system, the analyte distributes between a given volume of aqueous solution and a given volume of an immiscible organic solvent such as octanol. Either before or after equilibrium be achieved, the analyte is totally involved in the given extraction system, i.e., it cannot move out of the extraction system to the surroundings. We call such an extraction system an isolated extraction system. After equilibrium, the partition coefficient can be calculated by using eq 2 deduced by ref 1: Kow =
co,initial c w,final
⎡ I w,initial ⎤V =⎢ − 1⎥ w ⎢⎣ I w,final ⎥⎦ Vo
droplets are not only segmented by the oil carrier but also surrounded by the wetting layer.4 It is the wetting film that renders all W/O droplets to be connected with each other. As a result, each pair of the water droplet and the contacted oil segment is not an independent extraction system. During the extraction process, the analyte in a water droplet can be transformed to the adjacent oil segments as well as to the wetting film. Moreover, there is a velocity difference between the wetting film and the droplets.5 Thus the contacting boundary between the water and oil carrier renews when droplets move along the extraction channel. Therefore, the analyte in a water droplet can be transported to the next water droplet or even further ones via the mass transferring between the droplets and the wetting film, leading to the analyte dispersion among the droplets. This type of analyte dispersion has been reported by other researchers among stagnant W/O droplets6 and among W/O segments in a flow injection extraction system.7 From this point of view, it is expected that the analyte dispersion via the wetting film would lead to the analyte being
(2)
In such an isolated extraction system, the Iw,initial is unique because the initial analyte concentration in sample solution does not change. On the basis of eq 2, Iw,final increases with the increase of the phase-ratio Vw/Vo. Our experiment confirmed this as shown in Figure 1. In the W/O droplet microfluidic extraction system, the oil carrier usually wets the extraction channel. Thus, a wetting film of the oil carrier is formed on the channel surface, and the water © 2013 American Chemical Society
Published: September 23, 2013 10620
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Comment
Figure 2. continued identical syringe pumps. A home-built confocal laser induced fluorescence (LIF) detection system9 was employed for the detection of droplet fluorescence intensity at the end of the 16-cm-long main channel.
uniformly distributed between the water droplets and the oil segments, provided that the droplet-based extraction finally reach equilibrium. In other words, the final analyte concentration in the aqueous droplets, in turn, the analytical signals generated by the aqueous droplets ought to be independent of the length (volume) of an individual droplet. Figure 4 of ref 1 well demonstrated this expectation. In the profile of the normalized fluorescent intensity (Iw,final/Iw,initial) of the droplets vs the measuring time, the peak-heights for all of the water droplets were at the same level although the water droplet and octanol segment lengths (Lw and Lo) were significantly varied. This expectation has also been confirmed by our experiments where a confocal laser induced fluorescence (LIF) detector was used to investigate the droplet-based liquid−liquid extraction of fluorescein in a hydrophobized glass microchannel. Figure 2 shows the recording traces of fluorescence intensity (expressed as Iw,final/Iw,initial) vs time for the droplet-based flows at varied flow-rate ratio Qw/Qo (Qw and Qo represent the flow rates of aqueous phase and oil phase, respectively). At a specific flowrate ratio, the peak-heights were almost constant despite the significant variation in Lw and Lo. Furthermore, the peakheights increased with the increase of Qw/Qo (Figure 2a−c). The tendency agreed well to the result shown in Figure 1. This clearly demonstrates that the flow rate ratio of water flow to octanol flow, which is actually the phase ratio of the whole droplet-based extraction system, determines the equilibrium concentration of fluorescein in all water droplets. Therefore, the hypothesis that there were thousands of independent extraction units composed of a water droplet and an octanol segment in the droplet-based flow system is fictitious provided that the oil carrier wets the channel. As a result, use of eq 1 to calculate the individual Kow for each pair of water droplet and octanol segment would produce wrong results. The great CV values for the reported Kows were not a result of the uncertainty of measuring the droplet length as ref 1 proposed. Instead, it came from the wrongly hypothesized model. On that account, eq 1 is not suitable for calculating individual Kows for individual droplets. Considering that uniform concentrations of the analyte are equilibrated between all the water-droplets and octanol carrier, eq 3 was proposed to calculate the Kow value, ⎡ I w,initial − Idark ⎤Q Kow = ⎢ − 1⎥ w ⎢⎣ I w,final − Idark ⎥⎦ Q o
Figure 2. Recording traces of fluorescence intensity (normalized against the initial fluorescence intensity Iw,initial of the aqueous sample solution) vs time for the droplet-based flows at a varied flow-rate ratio of water phase (Qw) to octanol phase (Qo). Qw/Qo was (a) 0.5, (b) 1.0, and (c) 2.0. The glass microchip was fabricated as described elsewhere. All microchannels have a depth of about 50 μm and a topwidth of 300 μm. After bonded, the channels were hydrophobilized by octadecyltrichlorosilane solution as described previously.8 The W/O droplets were generated at a T-shaped generator where a fluorescein solution (buffered with pH 6.86 phosphate buffer) was injected into a continuous stream of n-octanol. Qw and Qo were controlled by two
(3)
With eq 3, a partition coefficient of 3.7 ± 0.3 (mean ± s.d., n = 3) was measured for fluorescein at pH 6.86 and a Qw/Qo of 2.0. Here, an individual measurement was carried out by observing the averaged peak-height of fluorescence intensity of water droplets passing the detector within a specified recording time and at constant flow rates (Qw and Qo). Thus, the measured Kow agrees well with that reported by Girimes, and the CV (8%) is much improved in comparison to that reported in ref 1. 10621
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Comment
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Marine, N. A.; Klein, S. A.; Posner, J. D. Anal. Chem. 2009, 81, 1471−1476. (2) Grimes, P. A.; Stone, R. A.; Laties, A. M.; Li, W. Arch. Ophthalmol. 1982, 100, 635−639. (3) OECD Guideline for the Testing of Chemicals, No. 107, Shake flask method, OECD: Paris, France, 1995. (4) Mary, P.; Studer, V.; Tabeling, P. Anal. Chem. 2008, 80, 2680− 2687. (5) Kashid, M. N.; Gerlach, I.; Goetz, S.; Franzke, J.; Acker, J. F.; Platte, F.; Agar, D. W.; Turek, S. Ind. Eng. Chem. Res. 2005, 44, 5003− 5010. (6) Courtois, F.; Olguin, L. F.; Whyte, G.; Theberge, A. B.; Huck, W. T. S.; Hollfelder, F.; Abell, C. Anal. Chem. 2009, 81, 3008−3016. (7) Nord, L.; Karlberg, B. Anal. Chim. Acta 1984, 164, 233−249. (8) Bai, Z. Q.; He, Q. H.; Huang, S. H.; Hu, X. Q.; Chen, H. W. Anal. Chim. Acta 2013, 767, 97−103. (9) Li, Z. M.; He, Q. H.; Ma, D.; Chen, H. W.; Soper, S. A. Anal. Chem. 2010, 82, 10030−10036.
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