Anal. Chem. 2001, 73, 3502-3505
On-Line Incorporation of Cloud Point Extraction to Flow Injection Analysis Qun Fang, Ming Du, and Carmen W. Huie*
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong
The on-line incorporation of cloud point extraction (CPE) to flow injection analysis (FIA) is demonstrated for the first time. The technical difficulties of inducing the cloud point phenomenon, separating the surfactant-rich phase from the aqueous phase, and detecting trace amounts of analyte(s) in the presence of the highly scattering surfactant medium in an on-line FIA system were resolved by the following: (1) mixing the sample solution containing the analyte(s) and CPE surfactant with an appropriate salting-out agent, (2) using a collection column to entrap the analyte-containing surfactant aggregates, and ( 3) employing the peroxyoxalate chemiluminescence reaction for the sensitive and selective determination of the analyte(s) in the presence of surfactant micelles. The figures of merit for the determination of coproporphyrin in pretreated urine samples were as follows: precision, 1.12.2% (RSD); limit of detection, 2.0 µg/L; and the calibration curve was linear from 46 to 2319 (µ/L (r ) 0.9996). Flow injection analysis (FIA) has proved to be a powerful tool for automatic/rapid sample pretreatment and nonchromatographic separations prior to determination of the analyte(s) by a variety of detection methods.1 The on-line coupling of FIA and a nonchromatographic separation technique, such as dialysis, gas diffusion, liquid-liquid extraction, or solid-phase extraction, is relatively straightforward and allows for an increase in the sensitivity or selectivity of FIA-based analytical systems or both.2 Aqueous solutions of certain surfactants display the so-called cloud point phenomenon in which the aqueous surfactant solution (surfactant above the critical micelle concentration) suddenly becomes turbid as a result of a decrease in the solubility of the surfactant in water.3 This clouding phenomenon is usually induced by an increase in temperature; however, the addition of saltingout agents has also proven to be an effective approach.4 For sample pretreatment/preconcentration, the cloud point method provides an attractive alternative to the use of relatively large volumes of toxic and expensive organic solvents (as in liquid-liquid extraction), since the analytes can be collected in very small volumes of surfactant-rich phase. * To whom correspondence should be addressed: (e-mail) cwhuie@ net1.hkbu.edu.hk; (fax) 852-2339-7348. (1) Ruzicka, J.; Hansen, E. Anal. Chim. Acta 1975, 78, 145. (2) Valcarcel, M.; Luque de Castro, M. D. Flow Injection Analysis: Principle and Applications; Ellis Horwood: Chicester, U.K., 1987. (3) Hinze, W. L.; Pramauro, E. CRC Crit. Rev. Anal. Chem. 1993, 24, 133. (4) Horvath, W. J.; Huie, C. W. Talanta 1992, 39, 487.
3502 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
Cloud point extraction (CPE) has been shown to be an effective sample pretreatment approach for improving sensitivity and selectivity prior to FIA as well as high-performance liquid chromatography (HPLC) analysis.5,6 Moreno Cordero and co-workers6 were the first to recognize the advantages of combining CPE with FIA; however, sample preconcentration using the cloud point methodology was performed off-line in their experiments. The ability to perform CPE on-line in a FIA system has never been reported and, if shown technically feasible, should represent an important development, since this would lead to a significant decrease in the number of manually operated procedures. In this note, the on-line incorporation of CPE to FIA is reported for the first time. The technical difficulties involved were resolved by employing the following methodologies: (1) the complexity of incorporating a heating device within the FIA system was avoided by employing an appropriate salting-out agent (e.g., (NH4)2SO4) to induce cloud point phase separation on-line; (2) the difficulty of the on-line collection of the surfactant-rich phase after phase separation (normally accomplished by the use of a centrifuge during off-line CPE extraction) was resolved with the use of an on-line column packed with a suitable filtering material, followed by subsequent elution of the analyte-containing surfactant aggregates with an appropriate solvent such as acetonitrile or water; and (3) the problems associated with the spectroscopic determination of the analyte(s) in the presence of highly scattering surfactant aggregates were minimized by employing the peroxyoxalate chemiluminescence (CL) reaction to induce light emission for sensitive and selective detection. The analytical performances of the on-line CPE/FIA system were evaluated by using hematoporphyrin as a model test compound, and the usefulness of this new method was demonstrated for determination of coproporphyrin in pretreated urine samples. EXPERIMENTAL SECTION Apparatus. A model FI-2100 FIA system (Haiguang Instrument Co., Beijing) equipped with two variable-speed peristaltic pumps and an eight-channel injection valve was employed for the on-line CPE/FIA experiments. The schematic of the flow injection manifold is shown Figure 1. To collect the surfactant-rich phase after induction of the cloud point phenomenon, a glass tube with an effective length of 1.3 cm and inner diameter of 3 mm (volume, 100 µL) packed with a suitable filtering material, such as cotton (5) Moreno Cordero, B.; Pe´rez Pavo´n, J. L.; Garcı´a Pinto, C.; Ferna´ndez Laespada, M. E. Talanta 1993, 40, 1703. (6) Ferna´ndez Laespada, M. E.; Pe´rez Pavo´n, J. L.; Moreno Cordero, B. Analyst 1993, 118, 209. 10.1021/ac010103f CCC: $20.00
© 2001 American Chemical Society Published on Web 06/13/2001
Figure 1. Schematic diagram of the CPE/FIA system. Key: P1 and P2, peristaltic pumps; ST, sample solution (hematoporphyrin standard and nonionic surfactant, Triton X-114 or -100); SA, salting-out agent (e.g., ammonium sulfate solution); ES, eluting solvent (organic solvent and/or water); TCPO: bis(2,4,6-trichlorophenyl) oxalate solution (2 mmol/L); H2O2, hydrogen peroxide solution (5%); LD, luminometer; V, valve; X, channel blocked; C, collection column; KR, knotted reactor; W1, W2, and W3, waste.
or glass fiber, was used as the collection column within the FIA system. To enhance mixing of the analyte(s), surfactant, and the eluting solvent after the collection column, a knotted reactor (KR) as described by Fang7 was employed. A model LS50B luminescence spectrometer (Perkin-Elmer, Norwalk, CT) was used for CL measurements by setting the wavelength of the emission monochromator at 620 nm. A homemade detection flow cell was produced by coiling a 50-cm-length PFE tubing (0.75-mm i.d., 1.5mm o.d.) into a spiral disk with an overall diameter of ∼3 cm. Reagents. Hematoporphyrin and coproporphyrin standards were obtained from Porphyrin Products (Logan, UT). Stock solutions (100 mg/L) of these two porphyrins were prepared by first dissolving the standard with 200 µL of NaOH solution (0.1 M) and then diluting with deionized water (Millipore, MA). In preparing the CL reagents, the bis(2,4,6-trichlorophenyl) oxalate solution (TCPO, 2 mmol/L) was prepared by dissolving an (7) Fang, Z. L. Flow Injection Separation and Preconcentration; VCH Publishers: Weinheim, 1993.
appropriate amount of TCPO in ethyl acetate. The working hydrogen peroxide solution (5%) was prepared by diluting stock H2O2 (35%) with acetonitrile. Ammonium sulfate and other salt solutions were prepared by dissolving the particular salt (analytical reagent grade) in deionized water. The salt solution was filtered before use. The surfactants, Triton X-100 and Triton X-114, were obtained from Fluka (Buchs, Switzerland) and used without further purification. HPLC grade acetonitrile and analytical reagent grade ethyl acetate were obtained from Arcos (Geel, Belgium). All solvents and working solutions were degassed for 5-10 min before use. Procedures. The on-line incorporation of CPE to FIA was performed as follows: In the first stage of the operation (Figure 1a), the sample solution, containing the porphyrin and the surfactant, was first loaded into a FIA manifold. A time-based sampling mode was employed in which the sample flow rate and loading time were used to govern the amount of sample solution loaded into the FIA system. Typically, a sample solution flow rate of 2 mL/min and loading time of 80 s were used, representing the loading of ∼2.7 mL of the sample solution. After sample injection, the sample solution merged with the salt solution to induce separation of the sample solution into two distinct phases: one containing molecules of porphyrin solubilized within surfactant aggregates and the other containing mostly water molecules. This mixture then passed through the collection column, which allowed for the surfactant-rich phase containing the porphyrin to be collected inside the column, while the aqueous phase (containing the salt and other components) passed through the column. When Triton X-114 was used as the CPE surfactant, the sample solution was cooled in an ice bath prior to sample loading. In the second stage (Figure 1b), the elution solvent passed through the column, carrying the porphyrin and surfactant molecules in the direction of the detector. Prior to the detector, the eluting flow merged with the premixed peroxyoxalate reagents in a mixing tee, and afterward, the CL signals were recorded as the chemically excited porphyrin molecules in the surfactant medium passed through the homemade detection flow cell. In all experiments, the concentrations of TCPO and H2O2 were 2 mmol/L and 5%, respectively. Enhancement Factor. For the present CPE/FIA system, estimation of the extent of the on-line preconcentration of the test compound (named hereafter as the enhancement factor (EF)) was carried out according to an off-line method described by Fang.7 The EF was estimated using the equation: EF ) 2Ae/As; where Ae and As is the absorbance of the collected solutions with and without preconcentration, respectively. The absorbance wavelength was set at the peak maximum of hematoporphyrin at 395 nm. Pretreatment of Urine Samples. For the deproteinization of the urine sample, 20 mL of the sample was treated with 40 mL of acetonitrile and 1 mL of 1 M NaOH. The sample mixture was then thoroughly shaken and allowed to stand for two distinct layers to form. The lower aqueous layer was subsequently drawn off and centrifuged. Prior to loading of the deproteinated urine sample solution into the CPE/FIA system, the sample solution was adjusted to a pH of ∼5.5 with 5 M HCl. Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
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Figure 2. Effects of surfactant (Triton X-114) concentration on the CL intensity and EF. Test compound, hematoporphyrin standard (1 mg/L); salt concentration, 2 mol/L (NH4)2SO4; filtering material, cotton; eluting solvent, acetonitrile/water (95/5, v/v); other conditions as in Figure 1.
RESULTS AND DISCUSSION Effects of Salting-Out Agents. In studying the effects of salt concentration on the CL intensity and EF under the following conditions: sample solution, 1 mg/L hematoporphyrin and 0.1% Triton X-114; filtering material, cotton; and eluting solvent, acetonitrile/water (95/5, v/v), it was found that an increase in (NH4)2SO4 concentration caused a concomitant increase in the magnitude of both of these parameters, which clearly demonstrated the effectiveness of using an appropriate salting-out agent to induce CPE for the preconcentration of the test compound prior to CL detection. An optimum salt concentration of 2.0 M (NH4)2SO4 was chosen because, at higher concentrations, occasional blockage of FIA channels occurred and the intensity of EF and CL increased only slightly after 2.0 M. In addition to (NH4)2SO4, NaCl and Na2SO4 were also tested for their effectiveness as saltingout agents in the CPE/FIA system. When compared to 2.0 M (NH4)2SO4 (EF ) 18), the EF for NaCl and Na2SO4 at the same concentration was found to be somewhat lower, about 13 and 14, respectively. Effects of Surfactants. Figure 2 shows the variation in CL intensity and EF as a function of Triton X-114 concentration. It can be seen that both of these parameters reached a maximum near 0.1% surfactant concentration. At concentrations higher than 0.1%, the decrease in the magnitude of these two parameters can be explained in part by an increase in the volume of the surfactantrich phase within the collection column as the percentage of the surfactant-rich phase increases, leading to a dilution of the extracted hematoporphyrin. Using another common CPE surfactant, Triton X-100, but with a cloud point temperature (Tc ) ∼70 °C) that is significantly higher than that of Triton X-114 (Tc ) ∼25 °C), the EF values obtained for on-line CPE/FIA of hematoporhyrin (1 mg/L) were found to be quite similar, 18.1 and 15.4, respectively, for the same concentration (0.1%) of Triton X-114 and Triton X-100, using 2.0 M (NH4)2SO4 as the salting-out agent. Using these CPE conditions, parts a and c of Figure 3 show that the average CL intensity (n ) 4) obtained from the chemical excitation of hematoporphyrin using these two common nonionic surfactants were almost identical. Also, parts b and d of Figure 3 show that, in the absence of the filtering material in the collection column, the CL intensity of the 3504 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
Figure 3. FIA peaks obtained with the presence (a, c) and without the presence (b, d) of filtering material (cotton) in the collection column. CPE surfactant for (a) and (b): 0.1% Triton X-114; (c) and (d) 0.1% Triton X-100. Test compound, hematoporphyrin standard (1 mg/L); salt concentration, 2 mol/L (NH4)2SO4; other conditions as in Figure 2. Table 1. Effects of Filtering Material on Enhancement Factor (EF), Relative Chemiluminescence (CL) Intensity, and Precision (RSD) filtering material
dry wt (mg)
EF
CL intensitya
RSD (%, n )7)
cotton glass wool nylon fiber
25 14 23
18.1 19.3 14.8
2.18 2.25 2.03
1.3 2.4 3.8
a
The average CL intensity of the blank was ∼0.008.
test compound decreased dramatically using Triton X-114 and Triton X-100 as the CPE surfactant, respectively. These results clearly demonstrated the preconcentation effect of the cloud point phenomenon within the present CPE/FIA system and the effectiveness of using the collection column for the filtration of the analyte-containing surfactant-rich phase. Collection Column. A homemade column packed with different filtering materials was employed to carry out phase separation after salt-induced CPE by entrapping larger size surfactant aggregates and allowing smaller size components within the aqueous medium to pass through, including the salt and water molecules, as well as certain amounts of the unretained test compound, surfactant monomer, and smaller size surfactant aggregates. Using this column, we were able to achieve relatively high EF, CL intensity, and reproducibility for the on-line CPE/ FIA of the test compound as shown by the data in Table 1. When compared to glass wool and nylon fiber, cotton was found to provide the best reproducibility and thus was chosen as the filtering material within the collection column for further evaluation of the analytical performances of the present on-line CPE/ FIA system. Elution of the Surfactant-Rich Phase. To remove the analyte-containing surfactant aggregates within the filtering material, the FIA system is switched to the second stage (Figure 1b) in which an appropriate solvent is used for the elution of the analyte(s) and surfactant aggregates off the collection column. The effects of using three different solvents as the elution solvent were examined under the following conditions: sample solution, 1 mg/L hematopoprhyrin and 0.1% Triton X-114; filtering material,
cotton; and salt, 2 mol/L (NH4)2SO4. The average CL intensity (n ) 4) for acetonitrile/water (95/5, v/v), ethyl acetate, and water were about 2.3, 0.3, and 0.2, respectively. The acetonitrile/water solvent system clearly provided the highest CL intensity (the small percentage of water was found to reduce the formation of bubbles in the system). Analytical Performances. The relative standard deviation (RSD) for the CL detection of more than 40 consecutive injections of the hematoporphyrin standard was ∼2.7%, with a sample throughput rate of 30 samples/h. The relative CL intensity, covering the concentration range of hematoporphyrin between 100 and 1000 µg/L, was found to be linear with following calibration equation: y ) 0.0024x + 0.0061 (r ) 0.9992), and the limit of detection (LOD) was calculated to be ∼1.3 µg/L (based on S/N ) 3). The experimental conditions were as follows: surfactant concentration, 0.1% Triton X-114; salt concentration, 2 mol/L (NH4)2SO4; filtering material, cotton; and eluting solvent, acetonitrile/water (95/5, v/v). Real Samples. The applicability and reliability of the present methodology for the analysis of analytes in real samples was demonstrated for the CPE/FIA of coproporphyrin in urine samples using extraction and detection conditions identical to those employed for the test compound. Table 2 shows the comparisons of coproporphyrin contents and recovery data obtained between a solvent extraction method and the present CPE/FIA method. It should be noted that the solvent extraction method employed for the comparison study has already been validated for the determination of urinary coproporphyrin in clinical laboratories.8 As shown in Table 2, good agreements between the two methods were obtained for the determination of coproporphyrin contents in three normal urine samples, and these values are well within the coproporphyrin range reported for normal adult population.8 Also, similar recovery values, in the range between about 93 and 108%, were obtained between the two methods when the urine samples were spiked with various amounts of coproporphyrin standard. To further examine the applicability and reliability of the present method for real sample analysis, the reproducibility of FIA peaks for the consecutive injection of urine samples was found to be similar to that of standard samples. For example, the RSD for the CL detection of normal urine samples from two healthy males (number of injections (n), 19 for each sample) was about (8) Fernandez, A. A.; Henry, R. J.; Goldenberg, H. Clin. Chem. 1966, 12, 463.
Table 2. Comparison of Solvent Extraction and CPE/ FIA Methods for the Determination of Urinary Coproporphyina solvent extraction method
CPE/FIA method
urine sampleb
content (µg/L)
spiked (µg/L)
% recovery
content (µg/L)
spiked (µg/L)
% recovery
1
46.8 48.1
3
80.1
104 94.7 103 104 108 97.1 96.6 104
47.8
2
30 90 150 30 120 180 120 180
30 90 150 30 120 180 120 180
105 92.7 108 94.1 95.3 106 101 97.3
50.5 82.0
a A validated solvent extraction method8 based on absorbance measurements of coproporphyrin at the Soret band (∼400 nm) was used in the present comparison. The coproporphyrin contents and recovery data reported for both solvent extraction and CPE/FIA method in the above table represent average values from three individual measurements, and the calibration plots for both methods were obtained using the standard addition method. b The urine samples were collected from three healthy male individuals. For the CPE/FIA experiments, the urine samples were deproteinated prior to loading of the samples into the CPE/FIA system (see Experimental Section for details).
1.1 and 2.2%, respectively. For the consecutive injection (n ) 24) of normal urine spiked with 200 µg/L coproporphyrin standard, the RSD was also good, ∼1.1%. For the CPE/FIA of coproporphyrin in the urine matrix, the relative CL intensity, covering the range of 46-2319 µg/L, was found to be linear with the following standard addition equation: y ) 0.0026x + 0.6052 (r ) 0.9996), and the LOD of coproporhyrin present in the urine matrix (after deproteinization) was ∼2.0 µg/L (based on S/N ) 3). ACKNOWLEDGMENT Financial support from the Research Grant Council of the UGC (HKBU 2056/98P) and from HKBU a Faculty Research Grant (FRG/97-98/II-06) is gratefully acknowledged. The authors thank Professor Zhaolun Fang (Flow Injection Analysis Research Centre, Northeastern University, Shenyang, China) for providing valuable comments on the manuscript. Received for review January 23, 2001. Accepted May 3, 2001. AC010103F
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