Method for Dead Time Determination in Reversed-Phase Capillary Electrochromatography with Methacrylate-Based Stationary Phases 2003, 58, 803–806
Guichen Ping1/ Weibing Zhang1/ Lin Zhang1/ Lihua Zhang1/ Yichu Shan1/ Yukui Zhang1*/ P. Schmitt-Kopplin2 / A. Kettrup3 1
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China; E-Mail:
[email protected] 2 Institute of Ecological Chemistry, GSF-National Center for Environment and Health, Neuherberg, Germany 3 ¨ kologische Chemie und Umweltanalytik, Institut fu¨r Chemie, Technische Universita Lehrstuhl fu¨r O ¨ t Mu¨nchen, 85350 Freising/Weihenstephan, Germany
Key Words Capillary electrochromatography Electroosmotic flow Polymer monolithic column Homologue series Dead time
Summary A method for dead time determination in reversed-phase capillary electrochromatography (RP-CEC) with methacrylate-based monolithic stationary phases has been presented based on homologue series method. The dead times measured with homologue series method and thiourea, a commonly used electroosmotic flow (EOF) marker are compared under various conditions, such as the type and the concentration of organic modifier, ionic strength, and applied voltage. Results show that the former one is larger than the latter one. Under high ionic strength and low applied voltage, EOF velocity measured by thiourea is close to that determined by homologue series method.
Introduction Dead time, t0, corresponds to the time all analytes spend in mobile phase or column residence time of a substance that does not interact with stationary phase [1, 2]. The accurate determination of dead time is important for chromatography [3, 4], which is essential for the correct calculation of retention factor, k, serving as the fundamental parameter for the comparison of retention data and for the interpretation of the physico-chemical phenomena taking place within separation column, especially for fast eluting Short Communication DOI: 10.1365/s10337-003-0105-8 0009-5893/03/12 $03.00/0
the development of CEC, such as tedious packing procedure and frit making. As an alternative to conventional packed columns, monolithic columns have been regarded as the most suitable columns for CEC, which eliminate the need for the frits and hence the problems associated with them could be avoided. So far, silicabased rods [8–10] and polymer-based monolithic stationary phases [11–18] have been reported in the literature. Generally, thiourea is used to measure dead time in reversed-phase CEC (RP-CEC). In this paper, a new approach to determine the dead time on methacrylate-based monolithic columns on the basis of homologue series method has been presented. Furthermore, the effects of operation parameters on the electroosmotic flow (EOF) determined by above mentioned two methods are investigated.
Experimental compounds, and any error in the determination of t0 influences the calculation of k greatly [5, 6]. In addition, the precise calculation of dead time is also significant for the optimization of separation conditions. Capillary electrochromatography (CEC) is usually presented as a hybrid of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), which combines the advantages of HPLC and CE. Accordingly it has attracted extensive attention since it was introduced by Pretorius in 1974 [7]. However, some technical problems retard
Instruments and Materials All CEC experiments were performed on a P/ACE system 5010 with gold software for data acquisition (Beckman, Fullerton, CA, USA). An HPLC pump (Elite Analytical Instrument Ltd, Dalian, China.) was used to flush the monolithic columns with mobile phase for conditioning. Fused-silica capillaries (100 lm I.D. · 375 lm O.D.) were obtained from Yongnian Optic Fiber Plant (Hebei, China). A manual syringe pump was purchased from Unimicro
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where a and b are constants for a given system, and n is the carbon number of homologues. According to the definition of retention factor, k¼
tm t0 t0
ð2Þ
where tm is the retention time of a solute, and t0 is dead time. With the combination of Eqs (1) and (2), tm ln 1 ¼ a þ bn ð3Þ t0
Figure 1. Influence of the type and the content of organic modifier on EOF velocity. 1a and 1b are EOF velocities determined respectively by homologue series method and thiourea with acetonitrile as the organic modifier. 2a and 2b are EOF velocities measured by the homologue series method and thiourea, respectively, in methanol system. Other conditions as shown in Experimental.
technologies (Tongliao, Inner Mongolia, China) to chase bubbles out of the columns.
Chemicals and Buffers Acetonitrile and methanol were of chromatographic grade, and other reagents used in the experiments were of analytical grade. Ethylene dimethacrylate (EDMA) was washed with a 5% (w/v) NaOH solution and with deionized water, and then it was dried by anhydrous magnesium sulfate. Butyl methacrylate (BMA) was distilled in vacuo to remove polymerization inhibitors prior to use. Azobisisobutyrinitrile (AIBN) was purified by recrystallization from methanol. Ultrapure water was obtained by a CLEAR SG (Germany). Stock solutions of 50 mmolÆL)1 phosphate buffer at different pH values were prepared by mixing appropriate concentration of sodium dihydrogenphosphate or hydrogenphosphate solution with phosphoric acid or sodium hydroxide. The mobile phase was made by mixing appropriate volume of organic modifier, stock solution and water. Subsequently, it was degassed in an ultrasonic bath for 15 min. pH values reported were those of stock buffer solutions.
Column Preparation The monolithic columns used in this experiments were fabricated as reported previously [19–21]. Briefly, capillaries
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were treated with 3-(trimethoxysilyl)propyl methacrylate, a bifunctional reagent, prior to polymerization reaction, then polymerization mixture was injected partially into the capillaries. After polymerization, any movements of the stationary phases were not observed in the experiments, indicating that the stationary phases have been bonded to the inner wall of the capillaries via the bifunctional reagent indirectly. Detection window was made immediately after the stationary phase by removing polyimide coating with a razor.
Separation Conditions Before CEC experiments, the columns were first flushed with the mobile phase for 1 h with the HPLC pump. Then the columns were conditioned on the instrument with the mobile phase for another 1 h. Temperature was kept at 25 C and detection wavelength was set at 214 nm. The effective and total length of the capillaries were 20 and 27 cm, respectively.
tm of homologues can be read directly from corresponding electrochromatogram, t0, a and b can be obtained after the regression based on Eq. (3). Therefore, t0 can be achieved with homologue series method. In this experiment, as high as 140,000 theoretical plates per meter for alkylbenzene was obtained [22], which demonstrates that these monolithic columns can work very well. The separation of 7 alkylbenzenes was performed in different mobile phases. Very good linearity of the logk of alkylbenzenes versus carbon number in both acetonitrile and methanol systems with all linear correlations over 0.9996, proves that the separation of neutral species on this methacrylate-based monolithic column is in accordance with reversed-phase retention mechanism. Therefore, homologue series method can be applied here to determine dead time. Since the linear correlations are close to 1, it can be considered that dead times measured with homologue series method offer higher accuracy than those determined by thiourea.
Effect of Type and Content of Organic Modifier on EOF The influence of organic modifier on EOF could be presented by the following equation, u¼A
Results and Discussion Homologue Series Method Based on the principle of chromatographic thermodynamics, the relationship between the retention of alkylbenzene homologues and their carbon number in reversed-phase HPLC is as follows, ln k ¼ a þ bn
ð1Þ
e g
ð4Þ
where u is EOF velocity, A is a constant for a given system, e is permittivity, and g is the viscosity of the mobile phase. Since the ratio of e to g of methanol is lower than that of acetonitrile, relatively low EOF velocity is obtained when methanol is used as organic modifier (Figure 1). Thiourea is a commonly used EOF marker in RP-CEC due to its high molar absorption coefficient and
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weak interaction with hydrophobic stationary phases. It can be seen from Figure 1 that EOF velocity determined with thiourea is higher than that measured with homologue series method. As mentioned above, due to the very good linearity of logk versus carbon number of homologue in both acetonitrile and methanol systems, the dead times determined with homologue series method are considered to be very close to the true values. Therefore, the migration velocity of thiourea is higher than the true EOF velocity, which indicates that thiourea shows the migration behavior of a positively charged solute. Hence, its migration velocity is the sum of EOF velocity and electrophoretic velocity.
Effect of Buffer Concentration on EOF Figure 2a shows the influence of buffer concentration on EOF velocity measured with two methods. The higher ionic strength in the mobile phase, the thinner the thickness of electrical double layer. Therefore, the EOF velocity decreases with the ionic strength. Moreover, the difference in EOF velocity determined by two methods becomes smaller with the increase of the ionic strength, which can be attributed to the fact that the ionic strength affects not only the thickness of electrical double layer of the stationary phase but also the electrophoretic mobility of thiourea. With high ionic strength, the contribution of the electrophoretic velocity to apparent migration velocity of thiourea is low. So the differences in the dead times determined by two methods should be small. The result in Figure 2a is in agreement with our hypothesis that the migration of thiourea in an electric field is of the property of positively charged species.
Effect of pH on EOF The pH values of the mobile phase have a significant influence on EOF, especially when the pH values are close to the pKa of the ionizable groups on the surface of the stationary phase. For ODS, since Ka of silanol is small, and the EOF velocity is dependent on the pH of the mobile phase when it is less than 7. For the monolithic columns presented in this paper, AMPS is Short Communication
Figure 2. Effect of experimental parameters on EOF velocity. The upper line was obtained by thiourea, and lower line was achieved by homologue series method. Other conditions as shown in Experimental.
used as a charged monomer. EOF velocity is nearly independent on the pH over the range of 3–9, due to the Ka of the sulfonic acid on the surface of the stationary phases is much higher than that of silanol, which is the significant advantage over ODS columns. As shown in Figure 2b, the difference in EOF velocity measured with two methods keeps constant over the pH range of 3–9, which shows that the variation of pH does not affect the electrophoretic mobility of thiourea.
Effect of Applied Voltage on EOF From Fig 2c, it could be seen that the difference in EOF velocities by two
methods is related to the applied voltage. The higher applied voltage, the larger difference. Apparent migration velocity of thiourea, uapparent, can be expressed as follows, uapparent ¼ uEOF þ uep
ð5Þ
where uEOF and uep are EOF velocity and the electrophoretic velocity of thiourea, respectively. With the rearrangement of Eq. (5), the following expression could be obtained, uep ¼ uapparent uEOF ¼ lep E
ð6Þ
where lep is electrophoretic mobility of thiourea, and E is electric field strength. Therefore, the difference in EOF velocity measured by two methods is the uep. By
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Figure 3. Linear regression between difference in EOF velocities measured by two methods versus applied voltage.
linear regression, lep is calculated to be 0.000029 cm2Æs)1ÆV)1, and correlation coefficient is over 0.996 (in Figure 3), which further proves the electrophoretic behavior of thiourea on methacrylatebased monolithic columns of CEC. This results demonstrates that the measurement by homologue series methods is more reliable than that obtained from thiourea.
Conclusions A method for dead time determination in RP-CEC based on homologue series method has been presented. The results show that the migration of thiourea has the migration behavior of a positively charged solute, which further proves that thiourea is not suitable EOF marker for accurate determination of dead time.
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Acknowledgement The financial support by both National Natural Foundation of China (No. 20105006) and 973 project of the Ministry of Science and Technology of China (No. 001CB510202) is gratefully acknowledged. The authors are also grateful to Dr. Tao Jiang for the beneficial discussion about monolithic column preparation.
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Received: May 23, 2003 Revised manuscript received: Jul 15, 2003 Accepted: Aug 18, 2003
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