- Email: [email protected]
Development and Evaluation of Capillary Electrophoresis Based on Coordination Interaction
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 34, Issue 9, September 2006 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2006, 34(9), 1219−1222.
RESEARCH PAPER
Development and Evaluation of Capillary Electrophoresis Based on Coordination Interaction Wei Weili1, Chen Zhitao1,2, Shi Kaiyun2, Yuan Lin2, Jiang Xuemei2, Xia Zhining1,2, * 1
Department of Pharmaceutics, Institute of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China
2
College of Bioengineering, Chongqing University, Chongqing 400044, China
Abstract:
In nonaqueous capillary electrophoresis (CE), the electrostatic interactions between additives and analytes can offer
additional separation mechanism. Similarly, the dynamic coordination interactions between metal ions and analytes can also be possible of providing separation selectivity. In this work, a new CE mode with applying coordination interaction as separation motivity was developed and it was defined as coordination interaction capillary electrophoresis (CICE). The CICE method was established by using seven arylamides as model compounds and Cu2+, Ni2+, Zn2+ as additives of running medium. The effect of operating conditions such as pH value, nature of metal ion, concentration, and separation voltage were investigated. The conditions of running medium for the separation of arylamides were optimized as 3 mM Cu2+ and 0.1% (v/v) acetic acid in water. The quantitative reliability of CICE, as given by linear range of concentration, correlation coefficient and detection limit, was presented. The potential application of CICE in analysis of real sample was validated by separation of degenerative o-phenylenediamine sample. In addition, good separation results of four sulfonamide compounds were obtained with CICE, demonstrating the great possibilities of CICE as a new, selective, and efficient CE separation mode.
Key Words: Coordination interaction; Arylamides; Capillary electrophoresis; Cu2+; Sulfonamides
1
Introduction
Most transition metal ions are Lewis acids that can accept lone pair electrons from electron-donating ligands. And this process is called coordination interaction of center ions, mostly transition ions, and ligands. Numerous organic compounds can donate lone pair electrons, thus they can coordinate with metal ions. Generally, the coordination between metal ions and different substances is diverse (the coordination difference can be expressed by the different formation constant Kf). As a broadly existent interaction, coordination has been applied to analytical science in many areas, such as ligand exchange chromatography (LEC). Some kinds of transition metal ions, based on resins, have been employed as the stationary phase, both in high performance liquid chromatography (HPLC)[1] and gas chromatography
(GC)[2], for the separations of various substances, which can coordinate with metal ions. Furthermore, the coordination interaction has also been utilized in capillary electrophoresis (CE). The applications mainly include two aspects, which are ligand exchange chiral separation[3,4] and metal ions resolution[5,6]. In the former applications, expensive chiral pure enantiomer is essential for achieving chiral selectivity. Moreover, in the separation of metal ions, the coordination interaction plays an important role in amplifying the mobility difference of metal ions through the formation of the coordinate complex. On the other hand, compounds can also be separated by CE by adding transition metal ions into the running buffer. It was hypothesized that the mobility differences may be induced by different coordinate interactions between the transition ion and the compounds which act as ligands. The separation of
Received 30 December 2005; accepted 4 June 2006 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20375051) and the Foundation for Young University Teachers by the Ministry of Education of China (Educational Department [2002]123). Copyright © 2006, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
ephedrine and pseudoephedrine has been achieved by Zheng et al.[7] by adding Cu2+ into the CE running buffer. Li et al.[8] separated a series of alcohols in non-aqueous CE where In ions were selected as the coordination center ions. Although the coordination interactions have been used for the separation of electron-donating compounds in a few studies, there is still none of the methodological research in point. Therefore, the separation can be carried out in a new CE mode, which for the first time, has been defined as coordination interaction capillary electrophoresis (CICE). The aim of this article is to demonstrate the possibility of CICE as a new CE mode. Using seven arylamides as model compounds, several electrophoresis parameters such as pH value, the nature of metal ions, concentration, and the high voltage applied for the separation, were optimized. The quantitative and quantitative reliability of the method and the quality of separation, as given by resolution, are presented.
2 2.1
Experimental Instrument and reagents
Electropherograms were obtained on a self-assembled CE system including: 0–30 kV DC power supply; 190–900 nm UV-Vis spectrometer; photomultiplier tube detector (Beijing Seven Star Instrument Co. Ltd., China). Instrument control and data acquisition were performed with HW-2000 Chromatographic Workstation (Nanjing Qianpu Software Co. Ltd., China). The uncoated fused silica capillary (Hebei Yongnian Ruifeng Chromatographic Implements Ltd., China) was of 50 μm i.d., 375 μm o.d., and had a total length of 65 cm (52.5 cm to the detector). The temperature of the capillary was nearly the same as the room temperature, about 25℃. Sulfadiazine, sulfamethoxazole, sulfaguanidine are raw medicines (the purity ≥ 99%, determined by HPLC, obtained from Southwest Synthesis and Pharmacy Co. Ltd.). The other reagents benzidine (BZ), o-phenylenedialmine (o-PED), 1-naphthylamine (1-NPA), sulfanilamide (SA), o-nitroaniline (o-NA), p-nitroaniline (p-NA), and m-nitroaniline (m-NA) were of analytical-reagent grade. Doubled-distilled water was used for preparation of solutions except samples. Naphthalin was selected as the electro-osmotic flow (EOF) marker. 2.2
detection and 254 nm for sulfonamides. The sample was injected by electro-injection for 5 s, applying a voltage of 20 kV. The separation voltage was 20 kV
3
Results and discussion
3.1
Influence of the acidity of the running buffer
It is well known that the transition metal ions hydrolyze in aqueous solutions, and the extent of this reaction is dependent on the pH value of the solution. One of the most important effects of hydrolysis is that the coordination center is no longer entirely in the form of a metal ion. This has the effect of reducing the availability of the coordination center for other ligands. Therefore, the acidity of the running buffer is important and should be controlled easily. The o-NA, p-NA, m-NA, and SA were selected as model compounds to investigate the effect of acidity, under the condition that Cu2+ was used as the center ion. As shown in Fig.1, the resolutions were notably improved with an increase in the amount of HAc. After the volume percentage of HAc reached a proper value (0.2%, v/v), the resolutions were slightly changed and might even be decreased. The forgoing phenomena was mainly because the HAc could adjust the acidity of the CE running buffer and affect the extent of hydrolysis of Cu2+. When there was no HAc, the resolutions were nearly zero because the Cu2+ was hydrolyzed to a great extent and existed mainly as the species of Cu(OH)2. And when a little amount of HAc (0.01%) was added to the buffer, the Cu2+ was remarkably restrained and the resolutions were greatly increased. However, after 0.2% of HAc was added, the contribution to the resolutions was small and might even have
Procedure Fig.1
Different transition metal sulfate water solutions were used as CE running buffers, and the pH values were adjusted by adding the proper amount of acetic acid (HAc). All the sample solutions were prepared in methanol. To obtain reproducible and uniform inner walls of fused-silica capillaries, the capillaries were rinsed sequentially with 0.1 M NaOH, water, and running medium for 20 min before the experiment. Absorbance at 233 nm was monitored for arylamides
Dependence of resolution on acidity of running medium running mediums: 2 mM Cu2+-H2O
Curves: 1. Rs of p-NA and SA; 2. Rs of SA and o-NA; 3. Rs of o-NA and m-NA.
the opposite effect. The reason for this was that the hydrolysis of Cu2+ was mostly restrained and the copper mainly existed as the species of Cu2+. And the resolutions might even be
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
reduced owing to the serious hydrolysis of analytes under very strong acidic conditions. According to the dissociation constant of Cu(OH)2 (Kd = 2.2 × 10–20, 18–25 ℃ , ionic strength I = 0), a proper acidity of running buffer is about pH 4.0 (0.2% HAc). 3.2
Effect of the metal ions on separation
Three arylamides, namely o-NA, p-NA, and m-NA, were selected as model compounds. Three transition metal ions (Cu2+, Ni2+, Zn2+) were tested alone at a 3 mM concentration, and the same amount of HAc was added. As can be seen in Fig.2, no separation of o-NA and m-NA was observed in the presence of Zn2+. Partial separations were obtained in the cases of Cu2+ and Ni2+. This means, compared to Zn2+, Cu2+ and Ni2+ are better separation selectors. Actually, in CICE the separation of components was closely related to the coordination ability and hydrolysis extent of transition metal ions. According to the literature[9], Zn2+ had the weakest coordination ability among the three selected metal ions. And the Cu2+ and Ni2+ had a similar coordination ability and hydrolysis extent, thus the separations were almost the same. In view of the less expensive and easier to obtain copper reagents, CuSO4 was selected as the running buffer additive in the following experiments found to increase with increasing Cu2+ concentration for the arylamides of o-NA, p-NA, m-NA, and SA. Complete or nearly complete separations were observed at a concentration of 2 mM. However, over the scale of 2 mM, resolutions were found to increase slowly or even kept still with increasing Cu2+ concentration. Moreover, EOF velocity was reduced with increasing concentration, thus the analysis time was prolonged. Therefore, 2 mM of Cu2+ was chosen for the following experiments which were without clear indications.
When Cu2+ solutions with high concentrations were used as running buffers, several problems, such as serious hydrolysis, strong background absorbance, and high electric current would be induced. Therefore, the effect on the separation of the Cu2+ concentration over the 1–4 mM was investigated in the presence of 0.1% HAc (shown in Fig.3). Resolutions were found to increase with increasing Cu2+ concentration for the arylamides of o-NA, p-NA, m-NA, and SA. Complete or nearly complete separations were observed at a concentration of 2 mM. However, over the scale of 2 mM, resolutions were found to increase slowly or even keep still with increasing Cu2+ concentration. Moreover, EOF velocity was reduced with increasing concentration; thus the analysis time was prolonged. Therefore, 2 mM of Cu2+ was chosen for the following experiments which did not have clear indications.
Fig.3 Dependence of resolution on Cu2+ concentration Running medium: all running mediums contain 0.1% HAc. Curves: 1. Rs of p-NA and SA; 2. Rs of SA and o-NA; 3. Rs of o-NA , m-NA and m-NA.
Fig.4 Electrophorogram of arylamides under optimized condition Capillary: i.d. 50 μm, total length 62 cm, effective length 49.5 cm. Running medium: 3 mM Cu2+-0.1%HAc-H2O. Other conditions as Sec. 2.1. Peaks: 1. BZ; 2. o-PED; 3. 1-NPA; 4. p-NA; 5. SA; 6. o-NA; 7. m-NA. Fig.2 Electrophorogram of m-, o-, p-NA of three transition metal ions as the coordination center Curves:1. 3.0 mM CuSO 4 -0.1%HAc-H 2 O; 2. 3.0 mM NiSO 4 0.1%HAc-H2O; 3. 3.0 mM ZnSO4-0.1%HAc-H2O. Peaks: a. p-NA; b. o-NA; c. m-NA .
3.3
Influence of Cu2+ concentration
Fig.4 was the CICE separation of seven arylamides. The efficiency of the peaks was high and the separations were complete, except part of the separation of o-NA and m-NA. There are many reports about the CE separation of arylamides, but usually few with both high efficiency and satisfactory separation. A CZE separation with a rather complex running buffer, which is composed of β-CD, urea, isopropyl, and
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
phosphates, using voltage gradient variety, was achieved by Chen et al.[10]. But problems such as complexity, little peak capability, and peak tailing still existed. On comparison, CICE was more convenient, more efficient, with high selectivity and available to analyze complex systems. 3.4
Influence of appling voltage
It is well known that the applying voltage can affect the efficiency, resolution, and running time. Thus the effect of voltage was investigated over the 15–28 kV range. It was observed that the running times were shortened, efficiencies were improved, and resolutions weakly decreased (because of Joule heat) with applying voltage increasing. With a comprehensive consideration of factors such as resolution,
efficiency, and analysis time, 20 kV was chosen for all the following experiments. 3.5
Quantitative evaluation of this method
Under the optimized condition of 3 mM CuSO4-0.1%HAc water solutions, quantitative analysis of this method was evaluated based on seven arylamides which are BZ, o-PED, 1-NPA, SA, o-NA, p-NA, and m-NA (shown in Table 1). As shown in Table 1, the linearity of analyte concentrations and detector signal responses are good (all the r2 are around 0.99), and the detection limits are lower than 0.30 mM. This means the CICE method can be applied to some analytical areas for routine analysis.
Table 1 Quantitative analysis results of seven model compounds Linear regression equation
Linear range
Correlation coefficient
Detection limit
(A-C)
(mM)
2
(r )
(S/N = 3, mM)
BZ
y = 84716x+26492
0.1–5.0
0.9906
0.02
o-PED
y = 30899x+12550
0.4–6.3
0.9882
0.12
1-NPA
y = 16684x+5864.1
0.4–6.3
0.9941
0.16
SA
y = 29357x+4881.9
0.3–5.0
0.9978
0.25
p-NA
y = 71163x+3075.9
0.4–6.3
0.9988
0.20
m-NA
y = 38122x+8543.6
0.4–6.3
0.9986
0.24
o-NA
y = 84716x+26492
0.4–5.0
0.9960
0.30
Compounds
3.6 3.6.1
Applications Analysis of degenerative o-PED
to confirm whether they are degenerated, is of great importance. A degenerative o-PED sample was analyzed with CICE under optimized conditions. Fig.5 represented that several impurity peaks were observed. This indicated that the CICE method had the potential of real sample analysis. 3.6.2
Fig.5 Electrophorogram of degenerative o-phenylenediamine Samples: 1, o-PED; 2, o-PED laid for three days.
The o-PED is a very important intermediate product of chemical engineering. It is lapsable and should be kept under very strict conditions. Thus, the analysis of o-PED products,
Analysis of sulfonamides
Sulfonamides are antibacterial compounds commonly used to prevent and treat diseases, in medical and veterinary practices. The separation and monitoring of these analytes have drawn much attention[11]. Because all sulfonamide molecules have one or more N atoms, which are electron-donators, it is especially proper to analyze sulfonamides with CICE. Under the condition of 3 mM Cu2+-0.2% HAc, the separation of SA, sulfadiazine, sulfamethoxazole, sulfaguanidine and the EOF marker naphthalin was obtained (as shown in Fig. 6).
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
8(3): 52–55 [3]
Schmid M G, Laffranchini M, Dreveny D, Gübitz G. Electrophoresis, 1999, 20: 2458–2461
[4]
Zhao Y F, Liu C H, Zhang H L, Wang X, Chen L R, Li Y M. Chinese J. Anal. Chem., 2005, 33(7): 955–957
[5]
Padarauskas A, Olšauskaite V, Paliulionyte V. Anal. Chim. Acta, 1998, 374: 159–165
[6]
Zhang L Y, Zhang W B, Duan J C, Zhang Y K. Chinese J. Anal. Chem., 2005, 33(3): 305–308
Fig.6 The CICE Electrophorogram of SAs
[7]
faguanidine; 2. sulfamethoxazole; 3. SA; 4. sulfadiazine; 5.
[8]
naphthalin, EOF marker.
Li S, Weber S G. Journal of the American Chemical Society, 2000, 122: 3787–3788
[9]
References
Zheng Y N, Xie T Y, Mo J Y, Wei S L. Chem. J. of Chinese Universities, 2002, 23: 199–202
Capillary: the same as Fig.4. Other conditions as 2.1. Peaks: 1. sul-
Harris D C. Quantitative Chemical Analysis. Third edition. New York: W. H. Freeman and Company, 1991: 285–288
[10] Chen X, Chen W, Li M J, Yi C Q, Wang X R. Chinese J. Anal. [1]
Ersoz M, Vural U S, Yildiz S. Sep. Sci. Technol., 1995, 30: 3555–3557
[2]
Zhu D C. Journal of Huaihai Institute of Technology, 1999,
Chem., 2001, 29(7): 760–764 [11] Yang H F, Wang Z H, Ding T H. Chinese Journal of Analytical Instruments, 1998, 1: 52–55
Cite this article as: Chin J Anal Chem, 2006, 34(9), 1219−1222.
RESEARCH PAPER
Development and Evaluation of Capillary Electrophoresis Based on Coordination Interaction Wei Weili1, Chen Zhitao1,2, Shi Kaiyun2, Yuan Lin2, Jiang Xuemei2, Xia Zhining1,2, * 1
Department of Pharmaceutics, Institute of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China
2
College of Bioengineering, Chongqing University, Chongqing 400044, China
Abstract:
In nonaqueous capillary electrophoresis (CE), the electrostatic interactions between additives and analytes can offer
additional separation mechanism. Similarly, the dynamic coordination interactions between metal ions and analytes can also be possible of providing separation selectivity. In this work, a new CE mode with applying coordination interaction as separation motivity was developed and it was defined as coordination interaction capillary electrophoresis (CICE). The CICE method was established by using seven arylamides as model compounds and Cu2+, Ni2+, Zn2+ as additives of running medium. The effect of operating conditions such as pH value, nature of metal ion, concentration, and separation voltage were investigated. The conditions of running medium for the separation of arylamides were optimized as 3 mM Cu2+ and 0.1% (v/v) acetic acid in water. The quantitative reliability of CICE, as given by linear range of concentration, correlation coefficient and detection limit, was presented. The potential application of CICE in analysis of real sample was validated by separation of degenerative o-phenylenediamine sample. In addition, good separation results of four sulfonamide compounds were obtained with CICE, demonstrating the great possibilities of CICE as a new, selective, and efficient CE separation mode.
Key Words: Coordination interaction; Arylamides; Capillary electrophoresis; Cu2+; Sulfonamides
1
Introduction
Most transition metal ions are Lewis acids that can accept lone pair electrons from electron-donating ligands. And this process is called coordination interaction of center ions, mostly transition ions, and ligands. Numerous organic compounds can donate lone pair electrons, thus they can coordinate with metal ions. Generally, the coordination between metal ions and different substances is diverse (the coordination difference can be expressed by the different formation constant Kf). As a broadly existent interaction, coordination has been applied to analytical science in many areas, such as ligand exchange chromatography (LEC). Some kinds of transition metal ions, based on resins, have been employed as the stationary phase, both in high performance liquid chromatography (HPLC)[1] and gas chromatography
(GC)[2], for the separations of various substances, which can coordinate with metal ions. Furthermore, the coordination interaction has also been utilized in capillary electrophoresis (CE). The applications mainly include two aspects, which are ligand exchange chiral separation[3,4] and metal ions resolution[5,6]. In the former applications, expensive chiral pure enantiomer is essential for achieving chiral selectivity. Moreover, in the separation of metal ions, the coordination interaction plays an important role in amplifying the mobility difference of metal ions through the formation of the coordinate complex. On the other hand, compounds can also be separated by CE by adding transition metal ions into the running buffer. It was hypothesized that the mobility differences may be induced by different coordinate interactions between the transition ion and the compounds which act as ligands. The separation of
Received 30 December 2005; accepted 4 June 2006 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 20375051) and the Foundation for Young University Teachers by the Ministry of Education of China (Educational Department [2002]123). Copyright © 2006, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
ephedrine and pseudoephedrine has been achieved by Zheng et al.[7] by adding Cu2+ into the CE running buffer. Li et al.[8] separated a series of alcohols in non-aqueous CE where In ions were selected as the coordination center ions. Although the coordination interactions have been used for the separation of electron-donating compounds in a few studies, there is still none of the methodological research in point. Therefore, the separation can be carried out in a new CE mode, which for the first time, has been defined as coordination interaction capillary electrophoresis (CICE). The aim of this article is to demonstrate the possibility of CICE as a new CE mode. Using seven arylamides as model compounds, several electrophoresis parameters such as pH value, the nature of metal ions, concentration, and the high voltage applied for the separation, were optimized. The quantitative and quantitative reliability of the method and the quality of separation, as given by resolution, are presented.
2 2.1
Experimental Instrument and reagents
Electropherograms were obtained on a self-assembled CE system including: 0–30 kV DC power supply; 190–900 nm UV-Vis spectrometer; photomultiplier tube detector (Beijing Seven Star Instrument Co. Ltd., China). Instrument control and data acquisition were performed with HW-2000 Chromatographic Workstation (Nanjing Qianpu Software Co. Ltd., China). The uncoated fused silica capillary (Hebei Yongnian Ruifeng Chromatographic Implements Ltd., China) was of 50 μm i.d., 375 μm o.d., and had a total length of 65 cm (52.5 cm to the detector). The temperature of the capillary was nearly the same as the room temperature, about 25℃. Sulfadiazine, sulfamethoxazole, sulfaguanidine are raw medicines (the purity ≥ 99%, determined by HPLC, obtained from Southwest Synthesis and Pharmacy Co. Ltd.). The other reagents benzidine (BZ), o-phenylenedialmine (o-PED), 1-naphthylamine (1-NPA), sulfanilamide (SA), o-nitroaniline (o-NA), p-nitroaniline (p-NA), and m-nitroaniline (m-NA) were of analytical-reagent grade. Doubled-distilled water was used for preparation of solutions except samples. Naphthalin was selected as the electro-osmotic flow (EOF) marker. 2.2
detection and 254 nm for sulfonamides. The sample was injected by electro-injection for 5 s, applying a voltage of 20 kV. The separation voltage was 20 kV
3
Results and discussion
3.1
Influence of the acidity of the running buffer
It is well known that the transition metal ions hydrolyze in aqueous solutions, and the extent of this reaction is dependent on the pH value of the solution. One of the most important effects of hydrolysis is that the coordination center is no longer entirely in the form of a metal ion. This has the effect of reducing the availability of the coordination center for other ligands. Therefore, the acidity of the running buffer is important and should be controlled easily. The o-NA, p-NA, m-NA, and SA were selected as model compounds to investigate the effect of acidity, under the condition that Cu2+ was used as the center ion. As shown in Fig.1, the resolutions were notably improved with an increase in the amount of HAc. After the volume percentage of HAc reached a proper value (0.2%, v/v), the resolutions were slightly changed and might even be decreased. The forgoing phenomena was mainly because the HAc could adjust the acidity of the CE running buffer and affect the extent of hydrolysis of Cu2+. When there was no HAc, the resolutions were nearly zero because the Cu2+ was hydrolyzed to a great extent and existed mainly as the species of Cu(OH)2. And when a little amount of HAc (0.01%) was added to the buffer, the Cu2+ was remarkably restrained and the resolutions were greatly increased. However, after 0.2% of HAc was added, the contribution to the resolutions was small and might even have
Procedure Fig.1
Different transition metal sulfate water solutions were used as CE running buffers, and the pH values were adjusted by adding the proper amount of acetic acid (HAc). All the sample solutions were prepared in methanol. To obtain reproducible and uniform inner walls of fused-silica capillaries, the capillaries were rinsed sequentially with 0.1 M NaOH, water, and running medium for 20 min before the experiment. Absorbance at 233 nm was monitored for arylamides
Dependence of resolution on acidity of running medium running mediums: 2 mM Cu2+-H2O
Curves: 1. Rs of p-NA and SA; 2. Rs of SA and o-NA; 3. Rs of o-NA and m-NA.
the opposite effect. The reason for this was that the hydrolysis of Cu2+ was mostly restrained and the copper mainly existed as the species of Cu2+. And the resolutions might even be
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
reduced owing to the serious hydrolysis of analytes under very strong acidic conditions. According to the dissociation constant of Cu(OH)2 (Kd = 2.2 × 10–20, 18–25 ℃ , ionic strength I = 0), a proper acidity of running buffer is about pH 4.0 (0.2% HAc). 3.2
Effect of the metal ions on separation
Three arylamides, namely o-NA, p-NA, and m-NA, were selected as model compounds. Three transition metal ions (Cu2+, Ni2+, Zn2+) were tested alone at a 3 mM concentration, and the same amount of HAc was added. As can be seen in Fig.2, no separation of o-NA and m-NA was observed in the presence of Zn2+. Partial separations were obtained in the cases of Cu2+ and Ni2+. This means, compared to Zn2+, Cu2+ and Ni2+ are better separation selectors. Actually, in CICE the separation of components was closely related to the coordination ability and hydrolysis extent of transition metal ions. According to the literature[9], Zn2+ had the weakest coordination ability among the three selected metal ions. And the Cu2+ and Ni2+ had a similar coordination ability and hydrolysis extent, thus the separations were almost the same. In view of the less expensive and easier to obtain copper reagents, CuSO4 was selected as the running buffer additive in the following experiments found to increase with increasing Cu2+ concentration for the arylamides of o-NA, p-NA, m-NA, and SA. Complete or nearly complete separations were observed at a concentration of 2 mM. However, over the scale of 2 mM, resolutions were found to increase slowly or even kept still with increasing Cu2+ concentration. Moreover, EOF velocity was reduced with increasing concentration, thus the analysis time was prolonged. Therefore, 2 mM of Cu2+ was chosen for the following experiments which were without clear indications.
When Cu2+ solutions with high concentrations were used as running buffers, several problems, such as serious hydrolysis, strong background absorbance, and high electric current would be induced. Therefore, the effect on the separation of the Cu2+ concentration over the 1–4 mM was investigated in the presence of 0.1% HAc (shown in Fig.3). Resolutions were found to increase with increasing Cu2+ concentration for the arylamides of o-NA, p-NA, m-NA, and SA. Complete or nearly complete separations were observed at a concentration of 2 mM. However, over the scale of 2 mM, resolutions were found to increase slowly or even keep still with increasing Cu2+ concentration. Moreover, EOF velocity was reduced with increasing concentration; thus the analysis time was prolonged. Therefore, 2 mM of Cu2+ was chosen for the following experiments which did not have clear indications.
Fig.3 Dependence of resolution on Cu2+ concentration Running medium: all running mediums contain 0.1% HAc. Curves: 1. Rs of p-NA and SA; 2. Rs of SA and o-NA; 3. Rs of o-NA , m-NA and m-NA.
Fig.4 Electrophorogram of arylamides under optimized condition Capillary: i.d. 50 μm, total length 62 cm, effective length 49.5 cm. Running medium: 3 mM Cu2+-0.1%HAc-H2O. Other conditions as Sec. 2.1. Peaks: 1. BZ; 2. o-PED; 3. 1-NPA; 4. p-NA; 5. SA; 6. o-NA; 7. m-NA. Fig.2 Electrophorogram of m-, o-, p-NA of three transition metal ions as the coordination center Curves:1. 3.0 mM CuSO 4 -0.1%HAc-H 2 O; 2. 3.0 mM NiSO 4 0.1%HAc-H2O; 3. 3.0 mM ZnSO4-0.1%HAc-H2O. Peaks: a. p-NA; b. o-NA; c. m-NA .
3.3
Influence of Cu2+ concentration
Fig.4 was the CICE separation of seven arylamides. The efficiency of the peaks was high and the separations were complete, except part of the separation of o-NA and m-NA. There are many reports about the CE separation of arylamides, but usually few with both high efficiency and satisfactory separation. A CZE separation with a rather complex running buffer, which is composed of β-CD, urea, isopropyl, and
WEI Weili et al. / Chinese Journal of Analytical Chemistry, 2006, 34(9): 1219–1222
phosphates, using voltage gradient variety, was achieved by Chen et al.[10]. But problems such as complexity, little peak capability, and peak tailing still existed. On comparison, CICE was more convenient, more efficient, with high selectivity and available to analyze complex systems. 3.4
Influence of appling voltage
It is well known that the applying voltage can affect the efficiency, resolution, and running time. Thus the effect of voltage was investigated over the 15–28 kV range. It was observed that the running times were shortened, efficiencies were improved, and resolutions weakly decreased (because of Joule heat) with applying voltage increasing. With a comprehensive consideration of factors such as resolution,
efficiency, and analysis time, 20 kV was chosen for all the following experiments. 3.5
Quantitative evaluation of this method
Under the optimized condition of 3 mM CuSO4-0.1%HAc water solutions, quantitative analysis of this method was evaluated based on seven arylamides which are BZ, o-PED, 1-NPA, SA, o-NA, p-NA, and m-NA (shown in Table 1). As shown in Table 1, the linearity of analyte concentrations and detector signal responses are good (all the r2 are around 0.99), and the detection limits are lower than 0.30 mM. This means the CICE method can be applied to some analytical areas for routine analysis.
Table 1 Quantitative analysis results of seven model compounds Linear regression equation
Linear range
Correlation coefficient
Detection limit
(A-C)
(mM)
2
(r )
(S/N = 3, mM)
BZ
y = 84716x+26492
0.1–5.0
0.9906
0.02
o-PED
y = 30899x+12550
0.4–6.3
0.9882
0.12
1-NPA
y = 16684x+5864.1
0.4–6.3
0.9941
0.16
SA
y = 29357x+4881.9
0.3–5.0
0.9978
0.25
p-NA
y = 71163x+3075.9
0.4–6.3
0.9988
0.20
m-NA
y = 38122x+8543.6
0.4–6.3
0.9986
0.24
o-NA
y = 84716x+26492
0.4–5.0
0.9960
0.30
Compounds
3.6 3.6.1
Applications Analysis of degenerative o-PED
to confirm whether they are degenerated, is of great importance. A degenerative o-PED sample was analyzed with CICE under optimized conditions. Fig.5 represented that several impurity peaks were observed. This indicated that the CICE method had the potential of real sample analysis. 3.6.2
Fig.5 Electrophorogram of degenerative o-phenylenediamine Samples: 1, o-PED; 2, o-PED laid for three days.
The o-PED is a very important intermediate product of chemical engineering. It is lapsable and should be kept under very strict conditions. Thus, the analysis of o-PED products,
Analysis of sulfonamides
Sulfonamides are antibacterial compounds commonly used to prevent and treat diseases, in medical and veterinary practices. The separation and monitoring of these analytes have drawn much attention[11]. Because all sulfonamide molecules have one or more N atoms, which are electron-donators, it is especially proper to analyze sulfonamides with CICE. Under the condition of 3 mM Cu2+-0.2% HAc, the separation of SA, sulfadiazine, sulfamethoxazole, sulfaguanidine and the EOF marker naphthalin was obtained (as shown in Fig. 6).
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Fig.6 The CICE Electrophorogram of SAs
[7]
faguanidine; 2. sulfamethoxazole; 3. SA; 4. sulfadiazine; 5.
[8]
naphthalin, EOF marker.
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