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Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection
Analytica Chimica Acta 680 (2010) 48–53
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection Haiyan Liu ∗ , Ning Han, Lingyi Zhang, Yiping Du, Weibing Zhang ∗ School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, PR China
a r t i c l e
i n f o
Article history: Received 20 June 2010 Received in revised form 28 August 2010 Accepted 9 September 2010 Available online 8 October 2010 Keywords: Dynamic coating Capillary electrophoresis Interface Chemiluminescence Hemoglobin Haemolysis
a b s t r a c t A dynamic coating capillary electrophoresis coupled with a simplified on-line chemiluminescence detection system was designed and evaluated. In the proposed system, poly-vinylpyrrolidone was used as dynamic coating substance in the separation buffer to reduce the unwanted protein non-specific adsorption, which was first applied in capillary electrophoresis coupling with on-line chemiluminescence detection. In order to avoid complex processing, an ordinary plastic cuvette was modified as a three-way joint. The chemiluminescence reaction conditions and capillary electrophoresis separation conditions were investigated in detail. The results showed that the coated capillary can be injected protein samples at least 30 times continuously with good repeatability. Under optimal conditions, the chemiluminescence relative intensity was linear with the concentration of hemoglobin in the range of 4–1850 g mL−1 and the detection limit was 2.0 g mL−1 (S/N = 3). The relative standard deviation of migration times and peak heights for 40 g mL−1 hemoglobin were 2.5% and 4.1% (n = 11) respectively. Interference of matrix effects was overcome by the calibration according to standard addition methods. Afterwards, the method was validated successfully and was applied to detect the concentration of hemoglobin in the serum of haemolytic patients. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Chemiluminescence (CL) detection, which is one of the most sensitive detection modes for capillary electrophoresis (CE) [1,2], is characterized by inexpensive apparatus and simple optical devices without the need of an excitation light source like fluorescence detection, and thus providing low background with excellent sensitivity comparable to that of laser-induced fluorescence and suitable for on-line detection for CE. This method has great potential applications in various fields, such as biochemical analysis, medicine and environmental research. There are several different designs including end-column [3–8], on-column [9–12], off-column [13,14] based on the site of detection and whether the detection region is isolated from the CE high voltage supply. Coaxial flow is the most popular detection mode in on-column or off-column CE–CL systems due to its simplicity and sensitivity. It usually requires on-line introduction of one or more kinds of CL reagents into the system to achieve chemiluminescence. The CL solution can be delivered either by gravity [8–10,13] or a microsyringe pump [11,12,14] into a three or four-way joint and then reached the mixing point or detection window in most interfaces. The repeatability and separation efficiency
∗ Corresponding authors. Tel.: +86 021 64253977; fax: +86 021 64233161. E-mail addresses: [email protected], lhy [email protected] (H. Liu), [email protected] (W. Zhang). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.09.024
mainly depend on the reaction type, the mixing mode of reagents, the stability of reagent flow and the conditions of electrophoresis. A successful CE–CL system may be determined by a good interface which connects both CE separating and CL detection system. However, a good interface usually requires precise manufacturing process and operational control. On the other hand, the interaction between proteins and silica inner-wall in capillary electrophoresis always degrades the separations in two different ways. Firstly, the adsorption leads to peak broadening and peak distortion and as a consequence, efficiency and sensitivity are hindered. Secondly, the magnitude of the electro-osmotic flow (EOF) becomes unpredictable resulting in poor repeatability of the migration times and low protein recovery rates. So far, different strategies have been proposed to reduce unwanted interactions. These strategies involve either the use of extreme pH values, high ionic strength, and dynamic coatings in the separation buffer, or covalently coating of the silica surface. These strategies have been well described and extensively reviewed [15–22]. Tsukagoshi once applied phosphate buffer (pH 10.8) to the direct detection of biomolecules [7]. However, this procedure is not very suitable for proteins, because choosing an extreme pH may not only cause protein denaturation, but also shorten the life of the capillary. Capillaries covalently coated with polymers, such as polyacrylamide [22], lead to high efficiency and reproducible protein separations in general. However, the preparation of coatings is
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laborious and can be frequently used only within a given pH range and for a limited number of injections. Furthermore, the different CL reactions also have different optimum pH ranges. Therefore, the covalently coated capillaries are not recommended in CE–CL systems. The main advantage of dynamic coatings is that the coating can be easily prepared just by rinsing the capillary with a solution containing the coating that is either a polymer or a low molecular mass compound [16,17]. However, literature about dynamic coating modified CE coupled with CL detection is rare, owing to the fact that many substances such as surfactants and amine can easily introduce interference in CL detection. Hydroxypropylmethylcellulose was successfully used as dynamic coating material in the separation of protein and other biomolecules by Hashimoto et al. [5] and Huang and Ren [23]. However, the migration time of about 30 min seemed to be unsatisfactory for fast measurements in traditional isoelectric focusing [5]. It is known that polyvinylpyrrolidone (PVP) is one kind of linear polymer which is easy to dissolve in water. Its macromolecule solution is colloidal and its viscosity is much smaller than other macromolecule solutions which have the same concentration and relative molecular mass. So it can be easily infused into capillaries and dynamically coated onto capillary wall. As a result, the adsorption of protein can be largely suppressed and the EOF can be decreased. Gao and Yeung [24] once adopted PVP (M = 1,000,000) as a new sieving matrix for DNA separation. Small amount of PVP was used as a buffer additive to modify the capillary wall dynamically in the work of Lu et al. [25]. The column efficiency was found to be increased with the increasing concentration of PVP in the running buffer. However, EOF increased with the pH of buffer and PVP showed UV absorption at 214 nm, which can interfere with the detection of protein in their experiment. In our preliminary experiment, it was found that PVP did not interfere with the CL reaction of luminol. To our knowledge, PVP has yet to be applied for protein analysis in CE combined with CL detection. In order to simplify the manufacturing process of interface in CE–CL system, an ordinary plastic cuvette was used as a three-way interface in this work. It can be easily designed for on-column or offcolumn detection mode according to the system needed. Dynamic coating CE, using PVP in the separation buffer, was applied to reduce the non-specific interaction between protein and capillary wall. The conditions of separation and detection were optimized in detail and hemoglobin (Hb) was chosen to evaluate the proposed CE–CL system. 2. Experimental 2.1. Chemicals and reagents Luminol (Fluka product) was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Human hemoglobin was a product of Sigma (St. Louis, MO, USA). PVP (k30, the relative molecular mass was about 65,000) was a product of Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents were of analytical grade or better. All aqueous solutions were prepared with ultrapure water purified by the Sartorius Arium 611 water purification apparatus (Sartorius, Germany). All solutions were filtered through a 0.22 m membrane filter. The electrophoresis running buffer was 0.015 mol L−1 borate solution containing 5 × 10−4 mol L−1 luminol, 1 × 10−8 mol L−1 Cu2+ , pH 8.9 (the pH of the buffer solution was adjusted with 0.1 mol L−1 hydrochloric acid solution); the CL oxidizer buffer solution was 50 mmol L−1 H3 BO3 solution containing 0.06 mol L−1 H2 O2 , pH 9.6 (the pH was adjusted with 0.1 mol L−1 NaOH solution). The outlet buffer was 0.015 mol L−1 borate solution at pH 8.9 which was used to fulfill the capillary electrophoresis circuit.
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Fig. 1. Schematic diagram of the CE–CL detection system: (1) running buffer reservoir (inlet); (2) Pt electrode; (3) separation capillary; (4) a cuvette containing H2 O2 solution. This cuvette was used as three-way interface; (5) reaction capillary; (6) black box; (7) buffer reservoir (outlet); (8) silicone seal patch; (9) reflector.
2.2. CE–CL apparatus CE–CL detection system was carried out in a laboratory-built system that is illustrated in Fig. 1. A coaxial flow on-column detection mode was applied for the proposed CE–CL experiment. A high-voltage power supply (Unimicro Technology Inc., Shanghai, China) was used for CE. Two sizes of fused-silica capillary (Rui-feng, Yongnian, Hebei, China) were used for separation and detection. The separation capillary (75 m i.d., 365 m o.d. × 38 cm) was vertically inserted into the reaction capillary (450 m i.d. × 13 cm). A 5 cm section at the end of the electrophoresis capillary was inserted into the reaction capillary and its outlet was just positioned at the center of the detection window. The end section of the reaction capillary stretched out the detector and entered an outlet buffer reservoir to complete the circuit. The detection window was formed by burning off 5 mm polyimide coating on the reaction capillary. In order to collect the most intensive CL signals, the detection window was situated just in front of the photomultiplier tube (PMT, CR-105, Hamamatsu, Japan) and a reflector was put on the opposite side. The PMT of the detector was operated at −400 to −900 V. The whole CL detection system was enclosed within a black box. The post-column CL reagents were stored in a 1.50 mL volume of ordinary plastic cuvette, which also served as a joint to connect and fix the separation capillary and reaction capillary. Cu(II) and luminol was added to a separation buffer, and was driven by EOF to move forward to the outlet of the capillary. Hydrogen peroxide was stored in the cuvette mentioned above and delivered by gravity flow through the reaction capillary which was fixed by a silicone seal patch in the bottom and stretched out of a small hole drilled in the bottom of the cuvette. With the reaction capillary in position, the hole was sealed using epoxy glue. Before CE analysis, the cuvette was filled with CL reaction buffer solution. The data acquisition and collection were processed using commercially available software (IFFM-D data analysis system, Xi’an Remax Electronic Science-Tech Co., Ltd, Xi’an, China).
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2.3. Preparation of capillaries The new capillary was rinsed with 0.1 mol L−1 NaOH, 0.1 mol L−1 HCl and water for 15 min respectively, followed by 5% PVP solution for 15 min, and finally with the running buffer for 5 min. To maintain reproducible migration times and CL signal intensity, the capillary was flushed with 0.1 mol L−1 NaOH for 15 min, water for 5 min, 5% (w/v) PVP solution for 15 min, and finally with the running buffer for 5 min every day before the CE–CL experiment. Sample injections were performed by gravity over a period of 25 s from a height of 32 cm, and 7 kV was applied to separation for 300 s with current reading of about 20 A. In order to keep the capillary wall in good condition, 0.1 mol L−1 NaOH was filled in when not in use. 2.4. Sample preparation The human serum sample was donated by the Psychiatric Hospital of the Medical School of Shanghai JiaoTong University. The serum sample was diluted 3 times with electrophoresis running buffer. Calibration was performed according to the standard addition method in order to avoid matrix-influence. This was attained by addition of a different standard Hb solution into the human serum sample in the vial. 3. Results and discussion 3.1. Characteristics of the proposed interface In this work, an ordinary plastic cuvette was used as three-way interface. It is not only served as post-column CL reagents reservoir, but also as a joint to connect and fix the separation capillary and reaction capillary. The end of the separation capillary was inserted into a reaction capillary with a larger diameter. CL reagents were delivered to the mixing point by gravity through the reaction capillary. In this setting, the separation capillary, the reaction capillary and the CL reagents reservoir were vertically arranged, so as to solve the problems caused by bubbles in the reaction capillary. On the other hand, the flow rate of CL reagent through reaction capillary can be controlled by adjusting the height of the solution in the reservoir, which was able to match the flow rate of running buffer in separation capillary. The CL reagents get mixed with Hb from the separation capillary instantly at the mixing point. The mixing mode is simple because it did not require an additional micro-pump and the CL reaction proceeded immediately in the detection window. There is no dead volume in the applied device and the dilution ratio is very little when the flow rates of running buffer and CL reagents were matched, so the strongest CL signals can be detected instantly by PMT. The other merit of the interface is that it can be easily used in on-column or off-column detection mode since the electrode can be inserted in the plastic cuvette, or the outlet solution according to the system needs. On-column detection was applied in this work with a suitable flow rate (15–25 L min−1 selected in different high voltage in this work) of post-column CL reagent. The signals were stable and showed good repeatability, which allowed high sensitivity to be obtained in this system. 3.2. Dynamic coating of the separation capillary In order to suppress the protein adsorption onto the capillary wall, two methods were compared in our study. We first tested an extreme pH in running buffer to diminish the interaction between capillary wall and protein. When the pH of phosphate buffer was larger than 10.8, the repeatability of migrating time and peak shape of Hb was still poor. The possible cause
may be the interaction between silanol groups and Hb still exist at this point. Secondly, PVP was directly resolved in separating buffer and then flushed into the capillary before CE experiments in our study. The effect of the concentration of PVP in buffer solution on the repeatability of migration time and peak shape of Hb were evaluated before and after the capillary was dynamically coated with PVP. It was found that when 1.0–5.0% PVP in buffer was flushed into capillary in advance, the repeatability of migration time and peak shape of Hb was greatly improved. After one flush with 5.0% PVP, the dynamically coated capillary can continuously inject protein sample at least 30 times with good repeatability without any peak broadening or peak distortion. The separation efficiency of CE was modified. The repeatability was investigated by injecting a standard Hb solution 11 times and recording the migration times and peak heights. The relative standard deviation (RSDs) of migration times and peak heights for 40 g mL−1 of Hb were 2.5% and 4.1% (n = 11), respectively. Considering the manual sample injection mode in our CE–CL experiment, the repeatability was greatly improved compared with other methods which need rinsing with buffer or saline solution between each run. The migration time is about 2 min with a 38 cm effective length of capillary, allowing for application in rapid analysis. Additionally, the interference experiment results showed that 5% PVP in buffer solution did not interfere with the present CL detection of Hb. 5% PVP in buffer was adopted in dynamic coating before CE experiment in the following work. 3.3. Optimization of CE–CL conditions In order to achieve sensitive detection and an efficient separation, many CL reaction conditions and separation conditions were investigated such as the concentration of luminol, Cu2+ , and borate in CE running buffer, the concentration of H2 O2 in reaction solution, the pH in CE running buffer and reaction solution, the applied voltage and the mode of injection of sample. 3.3.1. The effect of CL reagent concentration on CL signal intensity CL reagents concentration affects the CL intensity, such as luminol, H2 O2 and Cu2+ . If the concentration is very low, the sensitivity is also low. On the other hand, if the concentration is very high, a quenching effect and inner filter effects of luminescence and other interference reactions may occur. We have evaluated the effect of luminol concentration on the intensity of CL signal between 1 × 10−5 and 1 × 10−3 mol L−1 . The results showed that when the concentration of luminol was increased, the CL intensity enhanced at first but then decreased when the concentration value were higher than 5.0 × 10−4 mol L−1 . Therefore, 5.0 × 10−4 mol L−1 of luminol in running buffer was applied in the following CE–CL experiment. The effect of H2 O2 concentration on the CL intensity was studied between 0.01 and 0.09 mol L−1 . It was indicated that when the concentration of H2 O2 was 0.06 mol L−1 , the strongest CL signal was achieved. It was found in our study that there is a linked enhancement effect between Cu2+ and Hb when the concentration ratio of Cu2+ to Hb is appropriate. As a conjugated protein, Hb consists of four polypeptide subunits and a single heme (iron-porphyrin) as the active center. The iron-porphyrin shows similar catalytic functions to peroxidase [26]. It is reported that both Cu2+ and Hb can enhance the CL intensity of luminol-hydrogen peroxide CL reaction alone [27–29], further more, Cu2+ showed more catalytic effect to luminol-hydrogen peroxide CL reaction when it formed a complex with the biomolecule [7,30]. The following experiment showed that the strongest linked enhancement effect occurred when the con-
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Fig. 3. The calibration curve of Hb. Fig. 2. The effect of the pH in borate running buffer on the CL intensity.
centration of Cu2+ was 1 × 10−8 mol L−1 in the range of 1 × 10−9 to 1 × 10−5 mol L−1 . 1 × 10−8 mol L−1 of Cu2+ in running buffer solution was thus chosen. In our preliminary tests, it also showed that on above selected experimental conditions, the relative CL intensity was proportional with the concentration of Hb. 3.3.2. The effect of CL reaction medium on the CL signal intensity It is well known that luminol can emit strong CL signal in alkaline medium, but no CL signal can be observed under acidic conditions [31]. The pH value in reaction medium is one of the most important factors to the CL intensity. The final pH in reaction medium was determined by both the pH of CL reaction solution and the pH of running buffer. The pH in running buffer solution also affects the separation efficiency. We first optimized the pH in borate running buffer solution while the pH in reaction solution was fixed at 9.6. The effect of pH on the CL intensity and peak shape was investigated over the range of 6.80–10.0. The results (Fig. 2) showed that when the pH in borate buffer was 8.9, the CL signal reached a maximum value and the peak was close to symmetrical while the CL signal peak had a smaller half width. Therefore, a pH of 8.9 was chosen as the optimum pH in running buffer solution. The subsequent experiment proved that when the pH in CL reaction solution was 9.6, CL signal was the strongest in the range of 8.5–10.8. Other separation conditions, such as concentration of borate buffer, the applied voltage and injection mode of sample were also investigated. After a careful study, according to the relative CL intensity and the signal/blank ratio, the experimental conditions were selected as following: the separation capillary was 75 m i.d. × 38 cm; the reaction capillary was 450 m i.d. × 13 cm; the high voltage was 7 kV for CE separation and −500 V for PMT; the CE running buffer was 15 mmol L−1 of borate solution containing 5 × 10−4 mol L−1 luminol, 1 × 10−8 mol L−1 Cu2+ , and the pH was 8.9, which was adjusted using hydrochloric acid; the reaction buffer was 50 mmol L−1 H3 BO3 solution containing 0.06 mol L−1 H2 O2 , and the pH was 9.6, which was adjusted with 0.1 mol L−1 NaOH solution; sample injections were performed by gravity over a period of 25 s from a height of 32 cm; the outlet buffer was 15 mmol L−1 borate solution, at pH 8.9. 3.4. Analytical figures of merit The CE–CL method was validated by examining the linear response and the limits of detection. To test the CL response linearity, a series of Hb standard solutions at concentrations ranging from 2 to 2000 g mL−1 were determined. The result was that under the selected conditions, CL response to the concentration of Hb
solution was linear in the range of 4–1850 g mL−1 (Fig. 3, n = 17, every experimental point was a mean of three determinations) and the detection limit was 2.0 g mL−1 (S/N = 3). When the concentration of Hb is higher than 1850 g mL−1 , the CL response deviates from linearity significantly. The regression equation of calibration curve for Hb was ICL = 37.52 + 0.87C (g mL−1 ) with a coefficient of 0.9968. 3.5. Re-optimization of the concentration of Cu2+ There are multiple kinds of proteins in human serum, such as human serum albumin and serum globulin. These proteins may interfere with the proposed system. The result showed that when blank human serum from healthy volunteer was injected, there was no CL signal. This result indicated that these coexisting proteins did not interfere with CE–CL detection in above conditions but can conjugate with Cu2+ and accordingly affect the conjugation between Cu2+ and Hb. The concentration of Cu2+ should be re-optimized in order to obtain the maximal relative CL intensity and sensitivity. The effect of Cu2+ concentration on the relative CL intensity was studied once more using human serum sample as blank. It was found that the relative CL intensity enhanced greatly at first when the concentration of Cu2+ was between 1 × 10−8 and 1 × 10−7 mol L−1 . The blank signal increased more than the relative CL intensity when the Cu2+ concentration was higher than 1 × 10−7 mol L−1 , which because Cu2+ itself is one of the catalyzers to luminol CL reaction. Hence 1 × 10−7 mol L−1 Cu2+ was adopted in the following standard addition calibrating test. 3.6. The validation of standard addition calibration method and application High sensitivity is one of the main advantages of the CL reaction system, but the possible interference of the matrix in human serum may occur when quantitative analysis is employed. Therefore, calibration was performed by standard addition in order to get an accurate quantification. Additionally, it appears that separate standard addition curves should be made for each human serum under study. The content of Hb in the serum is either less than 500 g mL−1 or between 500 and 1000 g mL−1 for very slight or slight haemolytic patients respectively. The regression equation of standard addition calibration curve was constructed for Hb in the range of 50–500 g mL−1 . The regression equations were ICL = 37.42 + 0.17C (g mL−1 ) with a coefficient of 0.9997 for sample 1 and ICL = 22.89 + 0.13C (g mL−1 ) with a coefficient of 0.9907 for sample 2 respectively.
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H. Liu et al. / Analytica Chimica Acta 680 (2010) 48–53
Table 1 Results of percentage recovery test. Sample
Founda (g mL−1 )
Added (g mL−1 )
Total founda (g mL−1 )
RSDa (%)
Recovery (%)
Sample 1
205.1 205.1 205.1 205.1
50.0 106.7 150.0 200.0
256.0 313.9 355.1 404.7
3.5 3.9 4.1 3.6
101.8 102.0 100.0 99.8
Sample 2
234.8 234.8 234.8 234.8
50.0 106.7 150.0 200.0
285.2 337.4 392.2 431.4
3.7 4.5 3.9 3.6
100.8 96.2 104.9 98.3
a
Average of three determinations.
showed that after the correction of matrix background signal, the determined results of Hb in serum samples from two haemolytic patients were obtained satisfactorily. 4. Conclusions
Fig. 4. The electropherograms of serum sample. (A) The blank serum sample from a slight haemolytic patient; (B) the blank serum sample with additional 50 g mL−1 of standard Hb; (C) the blank serum sample with additional 150 g mL−1 of standard Hb; (D) the blank serum sample with additional 200 g mL−1 of standard Hb.
In this paper, dynamic coating CE coupled with on-line CL detection system was successfully devised for the determination of Hb in human serum. The interface was simplified using an ordinary plastic cuvette. In order to reduce the undesirable protein adsorption onto the capillary wall and improve separation efficiency, 5% PVP in buffer solution was applied in dynamically coated capillary. Hb can be injected at least 30 times in succession with good repeatability. The RSDs of migration times and peak heights for 40 g mL−1 Hb were 2.5% and 4.1% (n = 11), respectively. Finally, the developed method was validated and applied successfully to determine the Hb content in serum samples of haemolytic patients. The proposed method would be promising in broadening the applications of CE–CL system into many fields, such as biomarkers in body fluids, immunoassay and DNA hybridization analysis. Conflict of interest
When human serum from healthy volunteers was injected after triple dilution, no peak was observed in the electropherograms. This indicated that the proposed method was not interfered by existing proteins in serum. The human serum from haemolytic patients was determined directly after triple dilution with borate buffer. The electropherograms of serum sample from one of the haemolytic patients were showed in Fig. 4. It was found that the migration time of Hb was increased from 110 s of standard Hb solution to about 160 s of serum sample from haemolytic patient. There are two potential explanations for this phenomenon. One is that the higher concentration of Cu2+ in separation buffer used in real human serum sample analysis may decrease the EOF. Under pH 8.9 running buffer, EOF causes Hb with negative charge to migrate to the cathode even though the EOF was decreased by dynamic coating with PVP. The second is that the high concentrations of proteins in real human serum sample, such as Human IgG and HSA could be partly adsorbed onto the capillary inner wall causing change in the migration time of Hb. Two human serums from slight haemolytic patients were determined using above standard addition method. The Hb concentrations were found as 615 and 704 g mL−1 in serums and the confidence intervals were 615 ± 57 and 704 ± 67 respectively. Comparision with the results by conventional colorimetric assay, which is 650 and 746 g mL−1 from Psychiatric Hospital, it was found that there is no significant difference between our proposed method and the traditional method with t-test. The accuracy of Hb detection was farther evaluated by the percentage recoveries of Hb after adding Hb standard into serum sample. The results obtained by the proposed method for the determinations of Hb in serum sample are shown in Table 1 and the recovery was satisfactory. The results
The authors have declared no conflict of interest. Acknowledgements The authors are grateful for financial support from the Excellent Youth Founding of East China University of Science and Technology (YJ0157104), National Natural Sciences Foundation of China (No. 20675083) and “973” program (2007CB914102). The authors also hope to thank Dr. Intisar for his helpful discussion in grammar and sentence construction in our manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2010.09.024. References ˜ F.J. Lara, L. Gámiz-Gracia, J.F. Huertas-Pérez, Trends Anal. [1] A.M. García-Campana, Chem. 28 (2009) 973–986. [2] X.H. Ji, Q.F. Xu, Z.K. He, Chin. J. Anal. Chem. 36 (2008) 1579–1586. [3] R. Dadoo, A.G. Seto, L.A. Colón, R.N. Zare, Anal. Chem. 66 (1994) 303–306. [4] N.W. Barnett, B.J. Hindson, S.W. Lewis, S.D. Purcell, Anal. Commun. 35 (1998) 321–324. [5] M. Hashimoto, K. Tsukagoshi, R. Nakajima, K. Kondo, J. Chromatogr. A 852 (1999) 597–601. [6] K. Tsukagoshi, T. Nakamura, R. Nakajima, Anal. Chem. 74 (2002) 4109–4116. [7] K. Tsukagoshi, K. Nakahama, R. Nakajima, Anal. Chem. 76 (2004) 4410–4415. [8] L.H. Guo, B. Qiu, Y.Y. Jiang, Z.Y. You, J.M. Lin, G.N. Chen, Electrophoresis 29 (2008) 2348–2355. [9] Sh.L. Zhao, Ch. Xie, X. Lu, Y.R. Song, Y.M. Liu, Electrophoresis 26 (2005) 1745–1750. [10] W.W. He, X.W. Zhou, J.Q. Lu, J. Chromatogr. A 1131 (2006) 289–292.
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Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection Haiyan Liu ∗ , Ning Han, Lingyi Zhang, Yiping Du, Weibing Zhang ∗ School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, PR China
a r t i c l e
i n f o
Article history: Received 20 June 2010 Received in revised form 28 August 2010 Accepted 9 September 2010 Available online 8 October 2010 Keywords: Dynamic coating Capillary electrophoresis Interface Chemiluminescence Hemoglobin Haemolysis
a b s t r a c t A dynamic coating capillary electrophoresis coupled with a simplified on-line chemiluminescence detection system was designed and evaluated. In the proposed system, poly-vinylpyrrolidone was used as dynamic coating substance in the separation buffer to reduce the unwanted protein non-specific adsorption, which was first applied in capillary electrophoresis coupling with on-line chemiluminescence detection. In order to avoid complex processing, an ordinary plastic cuvette was modified as a three-way joint. The chemiluminescence reaction conditions and capillary electrophoresis separation conditions were investigated in detail. The results showed that the coated capillary can be injected protein samples at least 30 times continuously with good repeatability. Under optimal conditions, the chemiluminescence relative intensity was linear with the concentration of hemoglobin in the range of 4–1850 g mL−1 and the detection limit was 2.0 g mL−1 (S/N = 3). The relative standard deviation of migration times and peak heights for 40 g mL−1 hemoglobin were 2.5% and 4.1% (n = 11) respectively. Interference of matrix effects was overcome by the calibration according to standard addition methods. Afterwards, the method was validated successfully and was applied to detect the concentration of hemoglobin in the serum of haemolytic patients. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Chemiluminescence (CL) detection, which is one of the most sensitive detection modes for capillary electrophoresis (CE) [1,2], is characterized by inexpensive apparatus and simple optical devices without the need of an excitation light source like fluorescence detection, and thus providing low background with excellent sensitivity comparable to that of laser-induced fluorescence and suitable for on-line detection for CE. This method has great potential applications in various fields, such as biochemical analysis, medicine and environmental research. There are several different designs including end-column [3–8], on-column [9–12], off-column [13,14] based on the site of detection and whether the detection region is isolated from the CE high voltage supply. Coaxial flow is the most popular detection mode in on-column or off-column CE–CL systems due to its simplicity and sensitivity. It usually requires on-line introduction of one or more kinds of CL reagents into the system to achieve chemiluminescence. The CL solution can be delivered either by gravity [8–10,13] or a microsyringe pump [11,12,14] into a three or four-way joint and then reached the mixing point or detection window in most interfaces. The repeatability and separation efficiency
∗ Corresponding authors. Tel.: +86 021 64253977; fax: +86 021 64233161. E-mail addresses: [email protected], lhy [email protected] (H. Liu), [email protected] (W. Zhang). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.09.024
mainly depend on the reaction type, the mixing mode of reagents, the stability of reagent flow and the conditions of electrophoresis. A successful CE–CL system may be determined by a good interface which connects both CE separating and CL detection system. However, a good interface usually requires precise manufacturing process and operational control. On the other hand, the interaction between proteins and silica inner-wall in capillary electrophoresis always degrades the separations in two different ways. Firstly, the adsorption leads to peak broadening and peak distortion and as a consequence, efficiency and sensitivity are hindered. Secondly, the magnitude of the electro-osmotic flow (EOF) becomes unpredictable resulting in poor repeatability of the migration times and low protein recovery rates. So far, different strategies have been proposed to reduce unwanted interactions. These strategies involve either the use of extreme pH values, high ionic strength, and dynamic coatings in the separation buffer, or covalently coating of the silica surface. These strategies have been well described and extensively reviewed [15–22]. Tsukagoshi once applied phosphate buffer (pH 10.8) to the direct detection of biomolecules [7]. However, this procedure is not very suitable for proteins, because choosing an extreme pH may not only cause protein denaturation, but also shorten the life of the capillary. Capillaries covalently coated with polymers, such as polyacrylamide [22], lead to high efficiency and reproducible protein separations in general. However, the preparation of coatings is
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laborious and can be frequently used only within a given pH range and for a limited number of injections. Furthermore, the different CL reactions also have different optimum pH ranges. Therefore, the covalently coated capillaries are not recommended in CE–CL systems. The main advantage of dynamic coatings is that the coating can be easily prepared just by rinsing the capillary with a solution containing the coating that is either a polymer or a low molecular mass compound [16,17]. However, literature about dynamic coating modified CE coupled with CL detection is rare, owing to the fact that many substances such as surfactants and amine can easily introduce interference in CL detection. Hydroxypropylmethylcellulose was successfully used as dynamic coating material in the separation of protein and other biomolecules by Hashimoto et al. [5] and Huang and Ren [23]. However, the migration time of about 30 min seemed to be unsatisfactory for fast measurements in traditional isoelectric focusing [5]. It is known that polyvinylpyrrolidone (PVP) is one kind of linear polymer which is easy to dissolve in water. Its macromolecule solution is colloidal and its viscosity is much smaller than other macromolecule solutions which have the same concentration and relative molecular mass. So it can be easily infused into capillaries and dynamically coated onto capillary wall. As a result, the adsorption of protein can be largely suppressed and the EOF can be decreased. Gao and Yeung [24] once adopted PVP (M = 1,000,000) as a new sieving matrix for DNA separation. Small amount of PVP was used as a buffer additive to modify the capillary wall dynamically in the work of Lu et al. [25]. The column efficiency was found to be increased with the increasing concentration of PVP in the running buffer. However, EOF increased with the pH of buffer and PVP showed UV absorption at 214 nm, which can interfere with the detection of protein in their experiment. In our preliminary experiment, it was found that PVP did not interfere with the CL reaction of luminol. To our knowledge, PVP has yet to be applied for protein analysis in CE combined with CL detection. In order to simplify the manufacturing process of interface in CE–CL system, an ordinary plastic cuvette was used as a three-way interface in this work. It can be easily designed for on-column or offcolumn detection mode according to the system needed. Dynamic coating CE, using PVP in the separation buffer, was applied to reduce the non-specific interaction between protein and capillary wall. The conditions of separation and detection were optimized in detail and hemoglobin (Hb) was chosen to evaluate the proposed CE–CL system. 2. Experimental 2.1. Chemicals and reagents Luminol (Fluka product) was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Human hemoglobin was a product of Sigma (St. Louis, MO, USA). PVP (k30, the relative molecular mass was about 65,000) was a product of Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other reagents were of analytical grade or better. All aqueous solutions were prepared with ultrapure water purified by the Sartorius Arium 611 water purification apparatus (Sartorius, Germany). All solutions were filtered through a 0.22 m membrane filter. The electrophoresis running buffer was 0.015 mol L−1 borate solution containing 5 × 10−4 mol L−1 luminol, 1 × 10−8 mol L−1 Cu2+ , pH 8.9 (the pH of the buffer solution was adjusted with 0.1 mol L−1 hydrochloric acid solution); the CL oxidizer buffer solution was 50 mmol L−1 H3 BO3 solution containing 0.06 mol L−1 H2 O2 , pH 9.6 (the pH was adjusted with 0.1 mol L−1 NaOH solution). The outlet buffer was 0.015 mol L−1 borate solution at pH 8.9 which was used to fulfill the capillary electrophoresis circuit.
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Fig. 1. Schematic diagram of the CE–CL detection system: (1) running buffer reservoir (inlet); (2) Pt electrode; (3) separation capillary; (4) a cuvette containing H2 O2 solution. This cuvette was used as three-way interface; (5) reaction capillary; (6) black box; (7) buffer reservoir (outlet); (8) silicone seal patch; (9) reflector.
2.2. CE–CL apparatus CE–CL detection system was carried out in a laboratory-built system that is illustrated in Fig. 1. A coaxial flow on-column detection mode was applied for the proposed CE–CL experiment. A high-voltage power supply (Unimicro Technology Inc., Shanghai, China) was used for CE. Two sizes of fused-silica capillary (Rui-feng, Yongnian, Hebei, China) were used for separation and detection. The separation capillary (75 m i.d., 365 m o.d. × 38 cm) was vertically inserted into the reaction capillary (450 m i.d. × 13 cm). A 5 cm section at the end of the electrophoresis capillary was inserted into the reaction capillary and its outlet was just positioned at the center of the detection window. The end section of the reaction capillary stretched out the detector and entered an outlet buffer reservoir to complete the circuit. The detection window was formed by burning off 5 mm polyimide coating on the reaction capillary. In order to collect the most intensive CL signals, the detection window was situated just in front of the photomultiplier tube (PMT, CR-105, Hamamatsu, Japan) and a reflector was put on the opposite side. The PMT of the detector was operated at −400 to −900 V. The whole CL detection system was enclosed within a black box. The post-column CL reagents were stored in a 1.50 mL volume of ordinary plastic cuvette, which also served as a joint to connect and fix the separation capillary and reaction capillary. Cu(II) and luminol was added to a separation buffer, and was driven by EOF to move forward to the outlet of the capillary. Hydrogen peroxide was stored in the cuvette mentioned above and delivered by gravity flow through the reaction capillary which was fixed by a silicone seal patch in the bottom and stretched out of a small hole drilled in the bottom of the cuvette. With the reaction capillary in position, the hole was sealed using epoxy glue. Before CE analysis, the cuvette was filled with CL reaction buffer solution. The data acquisition and collection were processed using commercially available software (IFFM-D data analysis system, Xi’an Remax Electronic Science-Tech Co., Ltd, Xi’an, China).
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2.3. Preparation of capillaries The new capillary was rinsed with 0.1 mol L−1 NaOH, 0.1 mol L−1 HCl and water for 15 min respectively, followed by 5% PVP solution for 15 min, and finally with the running buffer for 5 min. To maintain reproducible migration times and CL signal intensity, the capillary was flushed with 0.1 mol L−1 NaOH for 15 min, water for 5 min, 5% (w/v) PVP solution for 15 min, and finally with the running buffer for 5 min every day before the CE–CL experiment. Sample injections were performed by gravity over a period of 25 s from a height of 32 cm, and 7 kV was applied to separation for 300 s with current reading of about 20 A. In order to keep the capillary wall in good condition, 0.1 mol L−1 NaOH was filled in when not in use. 2.4. Sample preparation The human serum sample was donated by the Psychiatric Hospital of the Medical School of Shanghai JiaoTong University. The serum sample was diluted 3 times with electrophoresis running buffer. Calibration was performed according to the standard addition method in order to avoid matrix-influence. This was attained by addition of a different standard Hb solution into the human serum sample in the vial. 3. Results and discussion 3.1. Characteristics of the proposed interface In this work, an ordinary plastic cuvette was used as three-way interface. It is not only served as post-column CL reagents reservoir, but also as a joint to connect and fix the separation capillary and reaction capillary. The end of the separation capillary was inserted into a reaction capillary with a larger diameter. CL reagents were delivered to the mixing point by gravity through the reaction capillary. In this setting, the separation capillary, the reaction capillary and the CL reagents reservoir were vertically arranged, so as to solve the problems caused by bubbles in the reaction capillary. On the other hand, the flow rate of CL reagent through reaction capillary can be controlled by adjusting the height of the solution in the reservoir, which was able to match the flow rate of running buffer in separation capillary. The CL reagents get mixed with Hb from the separation capillary instantly at the mixing point. The mixing mode is simple because it did not require an additional micro-pump and the CL reaction proceeded immediately in the detection window. There is no dead volume in the applied device and the dilution ratio is very little when the flow rates of running buffer and CL reagents were matched, so the strongest CL signals can be detected instantly by PMT. The other merit of the interface is that it can be easily used in on-column or off-column detection mode since the electrode can be inserted in the plastic cuvette, or the outlet solution according to the system needs. On-column detection was applied in this work with a suitable flow rate (15–25 L min−1 selected in different high voltage in this work) of post-column CL reagent. The signals were stable and showed good repeatability, which allowed high sensitivity to be obtained in this system. 3.2. Dynamic coating of the separation capillary In order to suppress the protein adsorption onto the capillary wall, two methods were compared in our study. We first tested an extreme pH in running buffer to diminish the interaction between capillary wall and protein. When the pH of phosphate buffer was larger than 10.8, the repeatability of migrating time and peak shape of Hb was still poor. The possible cause
may be the interaction between silanol groups and Hb still exist at this point. Secondly, PVP was directly resolved in separating buffer and then flushed into the capillary before CE experiments in our study. The effect of the concentration of PVP in buffer solution on the repeatability of migration time and peak shape of Hb were evaluated before and after the capillary was dynamically coated with PVP. It was found that when 1.0–5.0% PVP in buffer was flushed into capillary in advance, the repeatability of migration time and peak shape of Hb was greatly improved. After one flush with 5.0% PVP, the dynamically coated capillary can continuously inject protein sample at least 30 times with good repeatability without any peak broadening or peak distortion. The separation efficiency of CE was modified. The repeatability was investigated by injecting a standard Hb solution 11 times and recording the migration times and peak heights. The relative standard deviation (RSDs) of migration times and peak heights for 40 g mL−1 of Hb were 2.5% and 4.1% (n = 11), respectively. Considering the manual sample injection mode in our CE–CL experiment, the repeatability was greatly improved compared with other methods which need rinsing with buffer or saline solution between each run. The migration time is about 2 min with a 38 cm effective length of capillary, allowing for application in rapid analysis. Additionally, the interference experiment results showed that 5% PVP in buffer solution did not interfere with the present CL detection of Hb. 5% PVP in buffer was adopted in dynamic coating before CE experiment in the following work. 3.3. Optimization of CE–CL conditions In order to achieve sensitive detection and an efficient separation, many CL reaction conditions and separation conditions were investigated such as the concentration of luminol, Cu2+ , and borate in CE running buffer, the concentration of H2 O2 in reaction solution, the pH in CE running buffer and reaction solution, the applied voltage and the mode of injection of sample. 3.3.1. The effect of CL reagent concentration on CL signal intensity CL reagents concentration affects the CL intensity, such as luminol, H2 O2 and Cu2+ . If the concentration is very low, the sensitivity is also low. On the other hand, if the concentration is very high, a quenching effect and inner filter effects of luminescence and other interference reactions may occur. We have evaluated the effect of luminol concentration on the intensity of CL signal between 1 × 10−5 and 1 × 10−3 mol L−1 . The results showed that when the concentration of luminol was increased, the CL intensity enhanced at first but then decreased when the concentration value were higher than 5.0 × 10−4 mol L−1 . Therefore, 5.0 × 10−4 mol L−1 of luminol in running buffer was applied in the following CE–CL experiment. The effect of H2 O2 concentration on the CL intensity was studied between 0.01 and 0.09 mol L−1 . It was indicated that when the concentration of H2 O2 was 0.06 mol L−1 , the strongest CL signal was achieved. It was found in our study that there is a linked enhancement effect between Cu2+ and Hb when the concentration ratio of Cu2+ to Hb is appropriate. As a conjugated protein, Hb consists of four polypeptide subunits and a single heme (iron-porphyrin) as the active center. The iron-porphyrin shows similar catalytic functions to peroxidase [26]. It is reported that both Cu2+ and Hb can enhance the CL intensity of luminol-hydrogen peroxide CL reaction alone [27–29], further more, Cu2+ showed more catalytic effect to luminol-hydrogen peroxide CL reaction when it formed a complex with the biomolecule [7,30]. The following experiment showed that the strongest linked enhancement effect occurred when the con-
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Fig. 3. The calibration curve of Hb. Fig. 2. The effect of the pH in borate running buffer on the CL intensity.
centration of Cu2+ was 1 × 10−8 mol L−1 in the range of 1 × 10−9 to 1 × 10−5 mol L−1 . 1 × 10−8 mol L−1 of Cu2+ in running buffer solution was thus chosen. In our preliminary tests, it also showed that on above selected experimental conditions, the relative CL intensity was proportional with the concentration of Hb. 3.3.2. The effect of CL reaction medium on the CL signal intensity It is well known that luminol can emit strong CL signal in alkaline medium, but no CL signal can be observed under acidic conditions [31]. The pH value in reaction medium is one of the most important factors to the CL intensity. The final pH in reaction medium was determined by both the pH of CL reaction solution and the pH of running buffer. The pH in running buffer solution also affects the separation efficiency. We first optimized the pH in borate running buffer solution while the pH in reaction solution was fixed at 9.6. The effect of pH on the CL intensity and peak shape was investigated over the range of 6.80–10.0. The results (Fig. 2) showed that when the pH in borate buffer was 8.9, the CL signal reached a maximum value and the peak was close to symmetrical while the CL signal peak had a smaller half width. Therefore, a pH of 8.9 was chosen as the optimum pH in running buffer solution. The subsequent experiment proved that when the pH in CL reaction solution was 9.6, CL signal was the strongest in the range of 8.5–10.8. Other separation conditions, such as concentration of borate buffer, the applied voltage and injection mode of sample were also investigated. After a careful study, according to the relative CL intensity and the signal/blank ratio, the experimental conditions were selected as following: the separation capillary was 75 m i.d. × 38 cm; the reaction capillary was 450 m i.d. × 13 cm; the high voltage was 7 kV for CE separation and −500 V for PMT; the CE running buffer was 15 mmol L−1 of borate solution containing 5 × 10−4 mol L−1 luminol, 1 × 10−8 mol L−1 Cu2+ , and the pH was 8.9, which was adjusted using hydrochloric acid; the reaction buffer was 50 mmol L−1 H3 BO3 solution containing 0.06 mol L−1 H2 O2 , and the pH was 9.6, which was adjusted with 0.1 mol L−1 NaOH solution; sample injections were performed by gravity over a period of 25 s from a height of 32 cm; the outlet buffer was 15 mmol L−1 borate solution, at pH 8.9. 3.4. Analytical figures of merit The CE–CL method was validated by examining the linear response and the limits of detection. To test the CL response linearity, a series of Hb standard solutions at concentrations ranging from 2 to 2000 g mL−1 were determined. The result was that under the selected conditions, CL response to the concentration of Hb
solution was linear in the range of 4–1850 g mL−1 (Fig. 3, n = 17, every experimental point was a mean of three determinations) and the detection limit was 2.0 g mL−1 (S/N = 3). When the concentration of Hb is higher than 1850 g mL−1 , the CL response deviates from linearity significantly. The regression equation of calibration curve for Hb was ICL = 37.52 + 0.87C (g mL−1 ) with a coefficient of 0.9968. 3.5. Re-optimization of the concentration of Cu2+ There are multiple kinds of proteins in human serum, such as human serum albumin and serum globulin. These proteins may interfere with the proposed system. The result showed that when blank human serum from healthy volunteer was injected, there was no CL signal. This result indicated that these coexisting proteins did not interfere with CE–CL detection in above conditions but can conjugate with Cu2+ and accordingly affect the conjugation between Cu2+ and Hb. The concentration of Cu2+ should be re-optimized in order to obtain the maximal relative CL intensity and sensitivity. The effect of Cu2+ concentration on the relative CL intensity was studied once more using human serum sample as blank. It was found that the relative CL intensity enhanced greatly at first when the concentration of Cu2+ was between 1 × 10−8 and 1 × 10−7 mol L−1 . The blank signal increased more than the relative CL intensity when the Cu2+ concentration was higher than 1 × 10−7 mol L−1 , which because Cu2+ itself is one of the catalyzers to luminol CL reaction. Hence 1 × 10−7 mol L−1 Cu2+ was adopted in the following standard addition calibrating test. 3.6. The validation of standard addition calibration method and application High sensitivity is one of the main advantages of the CL reaction system, but the possible interference of the matrix in human serum may occur when quantitative analysis is employed. Therefore, calibration was performed by standard addition in order to get an accurate quantification. Additionally, it appears that separate standard addition curves should be made for each human serum under study. The content of Hb in the serum is either less than 500 g mL−1 or between 500 and 1000 g mL−1 for very slight or slight haemolytic patients respectively. The regression equation of standard addition calibration curve was constructed for Hb in the range of 50–500 g mL−1 . The regression equations were ICL = 37.42 + 0.17C (g mL−1 ) with a coefficient of 0.9997 for sample 1 and ICL = 22.89 + 0.13C (g mL−1 ) with a coefficient of 0.9907 for sample 2 respectively.
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Table 1 Results of percentage recovery test. Sample
Founda (g mL−1 )
Added (g mL−1 )
Total founda (g mL−1 )
RSDa (%)
Recovery (%)
Sample 1
205.1 205.1 205.1 205.1
50.0 106.7 150.0 200.0
256.0 313.9 355.1 404.7
3.5 3.9 4.1 3.6
101.8 102.0 100.0 99.8
Sample 2
234.8 234.8 234.8 234.8
50.0 106.7 150.0 200.0
285.2 337.4 392.2 431.4
3.7 4.5 3.9 3.6
100.8 96.2 104.9 98.3
a
Average of three determinations.
showed that after the correction of matrix background signal, the determined results of Hb in serum samples from two haemolytic patients were obtained satisfactorily. 4. Conclusions
Fig. 4. The electropherograms of serum sample. (A) The blank serum sample from a slight haemolytic patient; (B) the blank serum sample with additional 50 g mL−1 of standard Hb; (C) the blank serum sample with additional 150 g mL−1 of standard Hb; (D) the blank serum sample with additional 200 g mL−1 of standard Hb.
In this paper, dynamic coating CE coupled with on-line CL detection system was successfully devised for the determination of Hb in human serum. The interface was simplified using an ordinary plastic cuvette. In order to reduce the undesirable protein adsorption onto the capillary wall and improve separation efficiency, 5% PVP in buffer solution was applied in dynamically coated capillary. Hb can be injected at least 30 times in succession with good repeatability. The RSDs of migration times and peak heights for 40 g mL−1 Hb were 2.5% and 4.1% (n = 11), respectively. Finally, the developed method was validated and applied successfully to determine the Hb content in serum samples of haemolytic patients. The proposed method would be promising in broadening the applications of CE–CL system into many fields, such as biomarkers in body fluids, immunoassay and DNA hybridization analysis. Conflict of interest
When human serum from healthy volunteers was injected after triple dilution, no peak was observed in the electropherograms. This indicated that the proposed method was not interfered by existing proteins in serum. The human serum from haemolytic patients was determined directly after triple dilution with borate buffer. The electropherograms of serum sample from one of the haemolytic patients were showed in Fig. 4. It was found that the migration time of Hb was increased from 110 s of standard Hb solution to about 160 s of serum sample from haemolytic patient. There are two potential explanations for this phenomenon. One is that the higher concentration of Cu2+ in separation buffer used in real human serum sample analysis may decrease the EOF. Under pH 8.9 running buffer, EOF causes Hb with negative charge to migrate to the cathode even though the EOF was decreased by dynamic coating with PVP. The second is that the high concentrations of proteins in real human serum sample, such as Human IgG and HSA could be partly adsorbed onto the capillary inner wall causing change in the migration time of Hb. Two human serums from slight haemolytic patients were determined using above standard addition method. The Hb concentrations were found as 615 and 704 g mL−1 in serums and the confidence intervals were 615 ± 57 and 704 ± 67 respectively. Comparision with the results by conventional colorimetric assay, which is 650 and 746 g mL−1 from Psychiatric Hospital, it was found that there is no significant difference between our proposed method and the traditional method with t-test. The accuracy of Hb detection was farther evaluated by the percentage recoveries of Hb after adding Hb standard into serum sample. The results obtained by the proposed method for the determinations of Hb in serum sample are shown in Table 1 and the recovery was satisfactory. The results
The authors have declared no conflict of interest. Acknowledgements The authors are grateful for financial support from the Excellent Youth Founding of East China University of Science and Technology (YJ0157104), National Natural Sciences Foundation of China (No. 20675083) and “973” program (2007CB914102). The authors also hope to thank Dr. Intisar for his helpful discussion in grammar and sentence construction in our manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2010.09.024. References ˜ F.J. Lara, L. Gámiz-Gracia, J.F. Huertas-Pérez, Trends Anal. [1] A.M. García-Campana, Chem. 28 (2009) 973–986. [2] X.H. Ji, Q.F. Xu, Z.K. He, Chin. J. Anal. Chem. 36 (2008) 1579–1586. [3] R. Dadoo, A.G. Seto, L.A. Colón, R.N. Zare, Anal. Chem. 66 (1994) 303–306. [4] N.W. Barnett, B.J. Hindson, S.W. Lewis, S.D. Purcell, Anal. Commun. 35 (1998) 321–324. [5] M. Hashimoto, K. Tsukagoshi, R. Nakajima, K. Kondo, J. Chromatogr. A 852 (1999) 597–601. [6] K. Tsukagoshi, T. Nakamura, R. Nakajima, Anal. Chem. 74 (2002) 4109–4116. [7] K. Tsukagoshi, K. Nakahama, R. Nakajima, Anal. Chem. 76 (2004) 4410–4415. [8] L.H. Guo, B. Qiu, Y.Y. Jiang, Z.Y. You, J.M. Lin, G.N. Chen, Electrophoresis 29 (2008) 2348–2355. [9] Sh.L. Zhao, Ch. Xie, X. Lu, Y.R. Song, Y.M. Liu, Electrophoresis 26 (2005) 1745–1750. [10] W.W. He, X.W. Zhou, J.Q. Lu, J. Chromatogr. A 1131 (2006) 289–292.
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