2380 Xiaoxia Liu Miaomiao Tian Mohamed Amara Camara Liping Guo Li Yang Faculty of Chemistry, Northeast Normal University, ChangChun, Jilin, P. R. China

Received February 5, 2015 Revised April 27, 2015 Accepted May 14, 2015

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Research Article

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 ␮M (for asparagine) and 2.0 ␮M (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses. Keywords: Capillary electrophoresis / Online derivatization / Optically gated injection / Sequential analysis / UV/vis DOI 10.1002/elps.201500066

1 Introduction Due to its unique advantages, such as high efficiency and sensitivity, fast analysis, low sample volume requirement and so on, CE has been widely applied in a variety of research fields [1–5]. Nowadays, CE has been developed not only for high-performance separations of complex samples [6, 7], but also as a versatile platform for online measurements, e.g. reaction dynamics studies [8,9], combinatorial and pharmaceutical screening [10, 11], process stream [12], and high-throughput enzyme/inhibition assays [13–15]. In particular, time-resolved sequential analysis based on CE technique has attracted increasing research interest during recent years [16–19]. With sequential CE analysis, online monitoring the enzyme reaction from the beginning toward the end with high temporal resolution has been achieved, which is valuable for better understanding the enzyme functions in metabolism and finding their use in clinical diagnostics.

Correspondence: Professor Li Yang, Faculty of Chemistry, Northeast Normal University, ChangChun, Jilin 130024, P. R. China E-mail: [email protected] Fax: +86-431-85099762

Abbreviations: AA, amino acid; Asn, asparagine; Asp, aspartic acid; OG, optically gated; OPA, O-phthaldehyde; ␤-ME, ␤mercaptoethanol  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

One promising method for time-resolved sequential analysis is integrating the optically gated (OG) injection with CE separation and detection. Since it was first reported by Monnig and Jorgenson [20] in 1991, OG injection has been proved to be a fast, serial, and reproducible sample injection method and has been successfully applied for ultrafast separation in capillaries as well as in microchips [21–25]. In general, OG injection is coupled with LIF detection (OG-LIF). To achieve OG-LIF, the fluorescently labeled sample is continuously drawn into the capillary via EOF and electrophoretic mobility of the sample. A laser beam is unevenly split into two beams, i.e. a high-power beam for continuously photobleaching the sample near the capillary entrance, and a low-power beam for LIF probing at downstream. Upon periodically shutting off the bleaching beam, sample injection is accomplished with introducing a narrow unbleached plug, followed by CE separation and LIF detection. Because the injection can be precisely controlled by an electronic shutter with high repetition, OG-LIF has been successfully applied for time-resolved sequential online analysis of ␤-galactosidase-catalyzed reaction in microchips [26] and trypsin cleavage reaction and inhibition in capillaries [15]. Here, we show that OG injection is also capable to couple with commercial-available UV-Vis detection for sequential CE analysis. Unlike that in OG-LIF, the laser in the OG-UV/vis Colour Online: See the article online to view Figs. 1-6 in colour.

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approach is only used for injection, thus complicated optical setup for beam splitting is not required. By combining online derivatization with OG-UV/vis, we investigated the performance of the method for CE analyzing a standard mixture of amino acids (AAs). High reproducible sequential CE analysis was obtained. With the application of the OG-UV/vis method, the L-asparaginase-catalyzed enzyme reaction was online monitored from the beginning to the end of the reaction with the temporal resolution of 12 s by automatically and simultaneously detecting the substrate consumption and the product formation. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays.

2 Materials and methods 2.1 Chemicals AAs, asparagine (Asn), and aspartic acid (Asp), were purchased from DingGuo Biotech. (Beijing, China). L-asparaginase (250 U/mL) from Escherichia coli was purchased from Megazyme International Ireland (Ireland). Poly diallyldimethylammonium chloride, O-phthaldehyde (OPA), and ␤-mercaptoethanol (␤-ME) were purchased from Sigma Chemical (St. Louis, MO). All other reagents were of analytical grade and were used without further purification. All solvents and solutions were filtered using 0.22 ␮m membrane filters prior to use. 2.2 Sequential OG-UV/vis CE analysis Figure 1 shows the schematic diagram of our home-built OGUV/vis system for sequential CE analysis, which consists of online AAs derivatization, OG injection, CE separation, and UV/vis detection. Two fused-silica capillaries with total length of 12 and 25 cm (75 ␮m id, 365 ␮m od, Hebei Yongnian Optical Fiber Factory, China) were coaxially aligned and passed through a sample vial at a distance of 10 ␮m between the smooth ends. The 12-cm capillary was used for sampling OPA/␤-ME solution, while the 25-cm capillary was used for OG-UV/vis analysis. Online derivatization of AAs sample occurs at the interface of the two capillary ends in the sample vial. As the high voltages in the buffer vial (V1 ) and the sample vial (V2 ) were applied, the AA samples in the sample vial were driven in the capillary by EOF and online derivatized by OPA/␤-ME that was initially contained in the running buffer. The derivatized AAs were then OG injected and detected by a commercial UV/vis detector (Sapphire 600 CE, Czech Republic) at downstream of the 25-cm capillary.

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The 375 nm output of a semiconductor laser (LBX-375, Oxxius, France) with the laser power of 20 mW was applied for photobleaching the sample. The laser beam was focused by a 20 mm focal length fused-silica plano-convex lens (L) onto the 25-cm capillary at the position of 11.5 cm distance from the sample vial. Sequential OG injection was accomplished by periodically opening the photobleaching beam using a computer-controlled fast electronic shutter (S) (Uniblitz310B; Vincent Associates, Rochester, NY). The UV absorption of the photobleached plug sample decreases in comparison to the background (i.e. unbleached sample in the capillary), resulting in a negative peak in the electropherogram (see details in the following section). The distance between the bleaching beam and UV/vis detecting point, i.e. the CE separation distance, was 3.5 cm. The UV detection wavelength was set at 340 nm. The sample buffer in the sample vial was 30 mM borate buffer (pH 9.0). The running buffer was prepared by dissolving 1.7 mg of OPA in a mixture of 17 ␮L methanol and 1.7 ␮L of ␤-ME and diluting to 1.7 mL with a 30 mM borate buffer (pH 9.0), resulting in a final concentration of 7.5 mM OPA in the buffer. All capillaries used in the experiments were coated with poly diallyldimethylammonium chloride on the surface of the capillary for the reversed EOF. Before CE analysis, the capillaries were successively pressure-rinsed with distilled water for 3 min and the CE running buffer for 5 min, and the sample vial was rinsed with distilled water and the sample buffer using a syringe. In the enzyme assay experiments, the substrate Asn was initially put into the sample vial. When the L-asparaginase enzyme was added to initiate the enzyme reaction, the substrate, Asn, and the product, Asp, were simultaneously detected online as a function of the reaction time to achieve the CE enzyme assay.

2.3 Traditional Off-line CE enzyme assay The reaction mixture (380 ␮L) contained a 30 mM phosphate buffer (pH 9.0) and the substrates (Asn) of different concentrations. Reactions were initiated by the addition of 20 ␮L of 0.25 U/mL L-asparaginase into the mixture. Aliquots of 50 ␮L were periodically removed from the reaction mixture, then the vials were put into boiling water for 10 min to terminate the enzyme reaction [27]. The CE running buffer was a 30 mM borate buffer (pH 9.0) containing 7.5 mM OPA/ ␤-ME. The sample was injected at −16 kV for 2 s. The substrate consumption and product formation were measured at a separation electric potential of 571 V/cm. The total

-V2

-V1

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Sample vial capillary

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S Buffer vial

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Figure 1. Schematic diagram of the OG-UV/vis setup for sequential CE analysis. See text for details.

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Figure 2. (A) The UV/vis absorption spectra of Asp, Asn, OPA-Asp, OPA-Asn, and OPA/␤-ME without irradiation by the photobleaching laser beam. (B and C) The change of the UV/vis absorption of OPA-Asp (B) and OPA-Asn (C), after being irradiated by the 20-mW photobleaching beam for different time in 0–180 s.

length of the separation capillary (75 ␮m id, 365 ␮m od) was 55 cm with an effective length of 46 cm.

3 Results and discussion 3.1 Performance of online derivatization and OG-UV/vis CE analysis In order to show the feasibility of coupling the OG injection with the UV/vis detection, we initially investigated the change of the UV/vis absorption spectra of the test samples upon irradiated by the photobleaching laser beam. Standard AAs sample (Asp and Asn), their derivatives (OPA-Asp and OPAAsn), as well as OPA/␤-ME were analyzed in these experiments. Each test sample of 150 ␮L was contained in a glass tube (3.0 mm id and 1.8 cm length). The 375 nm laser with a power of 20 mW was used as the UV exposure source and irradiated the sample along the tube axis. The samples were diluted to 2.0 mL before measuring with a Hitachi U-2000 spectrophotometer. Figure 2A shows the measured UV/vis spectra of AAs, derivatized AAs, and OPA/␤-ME without irradiation by the photobleaching laser beam. The UV/vis spectra of both derivatized AAs (OPA-Asp and OPA-Asn) exhibit an obvious absorption peak centered around 340 nm, while those of AAs and OPA/␤-ME do not. After being irradiated by the photobleaching laser beam, the absorption changes significantly. As shown in Fig. 2B and C, the 340 nm absorption continuously decreases as the irradiation time is increased from 0 to 3 min, for both OPA-Asp and OPA-Asn. The decreasing of the absorption is attributed to the change of the chemical structure of chromophore upon photobleaching with the strong laser light [28, 29]. Thus, in the case of OGUV/vis CE analysis, a negative peak appears once a plug of photobleached sample reaches the UV/vis detector operated at 340 nm. Several key factors that may affect the performance of the OG-UV/vis CE analysis have been investigated. The pH value of the borate running buffer was kept at 9.0, considering both the derivatization efficiency and enzyme activity in the study.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The effect of running buffer concentrations was investigated in the range of 20–50 mM. The greater separation resolution of Asn and Asp was obtained when the buffer with the higher concentration was used. However, the analysis time is also increased as the running buffer concentration is increased due to the reduced EOF. The concentration of the running buffer was kept to be 30 mM, in which both the baseline separation and fast analysis were obtained. The sensitivity of the UV detection of the OG-injected plug is proportional to the photobleaching efficiency of the analytes, which depends on the opening time of the electronic shutter (i.e. injection time) and the laser power. Figure 3A shows that the measured peak height of photoproduct vacancy of either OPA-Asn or OPA-Asp increases as the photobleaching laser power is increased. Since we did not observe a significant increase of background noise at 20 mW laser power compared to that at lower laser 10 mW laser power, the photobleaching laser power was kept at 20 mW for the following experiments. It should be mentioned that the wavelength of the laser used here (375 nm) is at the edge of the 340 nm absorption band of the OPA derivatives (see Fig. 2A), thus the photobleaching efficiency would be much less than that using 350 nm laser as reported in [24]. Figure 3B and C show the peak height and peak width as a function of the sample injection time for OPA-Asn and OPA-Asp. The results show that longer injection time favors more sensitive detection. On the other hand, longer injection is not desirable for rapid and efficient separations, as shown in Fig. 3B and C the increasing peak width with the injection time. With a compromise of detection sensitivity and separation efficiency, the injection time was set to be 1.0 s for the successive OG-UV/vis CE analysis. Since AAs have no absorption at UV/vis range (see Fig. 2A), derivatization is required for the OG-UV CE analysis. In this study, online derivatization was achieved using OPA/␤-ME as the reagent and the setup as shown in Fig. 1. Experimental conditions have been optimized for efficient and fast online derivatization. In Fig. 4A, we show the effect of the voltage V2 in the sample vial on the UV www.electrophoresis-journal.com

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Figure 3. (A) Dependence of the peak height of photoproduct vacancy of OPA-Asp and OPA-Asn on the photobleaching laser power. The OG injection time was kept at 1.0 s. (B and C) Effect of the OG injection time on the peak height (B) and peak width (C) of OPA-Asp and OPA-Asn. The power of the photobleaching laser was 20 mW. Sample was a mixture of 0.5 mM Asp and Asn. Running buffer was 30 mM borate buffer (pH 9.0) containing 7.5 mM OPA. The voltage V1 in the buffer vial and V2 in the sample vial were −8 kV and −7 kV, respectively. UV/vis absorption was measured at 340 nm. Each data point in the plot is an averaged result of three repeatable measurements.

Figure 4. (A) Electrophoregram of sequential CE analysis using OG-UV/vis with different V2 values as labeled in the figure. The averaged peak height of five serial OG injections for each V2 value is shown in the inset. (B) Dependence of the peak height on the concentration of OPA in the running buffer. The injection time was 1.0 s and the power of the photobleaching laser was 20 mW. Other conditions were the same as those in Fig. 3.

absorption of the analytes. The voltage V1 in the buffer vial was kept constant at −8 kV. For each V2 value, we performed five serial OG injections. The averaged peak height (n = 5) of each sample at different V2 is presented in the inset of Fig. 4A. It was found that the baseline absorbance did not increase as V2 value was in the range of 0 kV (red line) to 5.0 kV, since AAs samples could not be induced into the separation capillary and no derivatized AAs were formed. It can be seen that the baseline absorbance and peak height of the photoproduct vacancy as well as the separation resolution of AAs increases as the V2 value is increased. This indicates that higher V2 value results in greater flow of AAs in the sample vial to the separation capillary, thus the derivatized products is increased. As the V2 value is larger than 7 kV, the peak height starts to increase gradually. Thus, 7 kV was chosen for the OG-UV CE analysis in the study. Figure 4B presents the measured peak height for different concentrations of derivate reagent (OPA/␤-ME)  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

in the running buffer. The peak height increases linearly with the OPA/␤-ME concentration in the range of 2.5– 5 mM, and then remains almost unchanged as the OPA/␤ME concentration is higher than 5 mM. The effect of the distance between the two capillaries on the derivatization efficiency was also investigated. No significant differences were observed regarding the sample injection amount, the separation resolution, and efficiency, for the distance at 10, 30, and 70 ␮m. A distance of 10 ␮m was chosen for use in our system. 3.2 Sequential analysis for the standard mixture of Asn and Asp Figure 5 presents a typical electropherogram for the 20 sequential analyses of a 0.5 mM mixture of standard Asn/Asp samples under the optimal conditions. Sequential CE analysis was achieved by opening the electronic shutter with the duration of 1.0 s in every 12 s, which periodically created www.electrophoresis-journal.com

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Figure 5. Electrophoregram of 20 sequential CE analysis of 0.5 mM standard Asn and Asp mixture. The V2 value was 7 kV and the OPA concentration in the running buffer was 7.5 mM. Other conditions were the same as those in Fig. 4.

a photobleached plug that electrophoretically migrated toward the UV/vis detection point. This results in a series of two negative peaks with 12-s interval appeared in the electropherograms, as showed in Fig. 5. Quantitative analysis of OG-UV/vis CE experiments was achieved by measuring the height of the negative peaks in each sequence. Within each sequence, the migration time of Asn is 3.8 s ahead of that of Asn due to different apparent mobilities of these two standard samples. These two peaks of the standard samples were used to identify the peaks of the substrate and the product in the sequential CE enzyme assay. Reproducibility of the sequential online OG-UV/vis CE analysis is also demonstrated in Fig. 5. Each sequence in the figure corresponds to a repeating CE run because standard samples of constant concentration are OG injected and separated without any physical disturbance. The results show excellent reproducibility of the present method, with RSD (n = 20) of 2.23, 2.57, and 0.70% for peak height, peak area, and migration time, respectively. The calibration curve of the peak height versus concentration for each sample was measured in the concentration range of 0–3.0 mM. The peak height of Asp and Asn show a linear dependence on the concentration in the range of 0–1.0 mM (for Asp, y = 12.481x + 0.066, R2 = 0.996; for Asn, y = 13.032x + 0.052, R2 = 0.999). The LOD for Asp and Asn was calculated as 5.0 ␮M and 2.0 ␮M, respectively. We should mention here that the peaks of Asn and Asp in the electropherogram have a little tailing and asymmetric. We have found that the peaks become more symmetric if the OG injection time is decreased, however, the detection sensitivity is also reduced. Note that the sensitivity of UV/vis detection is less than that of LIF detection. With a compromise of detection sensitivity and separation efficiency, the injection time was set to be 1.0 s in this study. The theoretical plate numbers for Asn and Asp were determined to be 2.8 × 103 and 3.1 × 103 , respectively. It is well known that the efficiency in theoretical plate numbers increases as the injection time decreases in CE analysis. For OG-LIF, the efficiency is in the range of 103 –105 depending on different analytes, with typical injection time of 20–600 ms. The efficiency of the present OG-UV/vis method is indeed comparable to that of OG-LIF, considering the much longer injection time (1.0 s) used in the study.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 6. Evolution of the substrate and product peaks as a function of reaction time of L-asparaginase-catalyzed reaction. The peak heights of Asn and Asp in each sequence were indicated in the figure as the circle point, demonstrating the consumption of the substrate and formation of the product as the enzymatic reaction progresses. See text for details. The inset shows the measured Michaelis–Menten diagram obtained from the sequential CE enzyme assay.

3.3 Sequential analysis for L-asparaginase-catalyzed reaction L-Asparaginase-catalyzed

reaction that converts Asp to Asn was investigated using the sequential OG-UV/vis CE analysis. Figure 6 shows the evolution of the substrate and product peaks as a function of reaction time. A total of 0.5 mM Asn was initially contained in the sample vial as the substrate. “Time zero” in the electropherogram was set to the time at which the enzyme L-asparaginase was added to the sample vial to initiate the reaction. Once the reaction is started, the substrate Asn and the product Asp are online derivatized, OG injected, then CE separated, and finally UV/vis detected. Each sequence in the figure is related to one time point of the enzyme reaction. As shown in the figure, the intensity of the substrate Asn gradually decreases as the reaction time is increased, while that of the corresponding product peak gradually increases. Thus, the enzyme reaction is online monitored every 12 s from the beginning toward the end of the reaction by simultaneously measuring the substrate consumption and the product formation. Eventually, the peak intensities of the substrate and the product remain unchanged, indicating the enzyme reaction in the sample vial has completed. By measuring the peak height of the substrate and product at each time point (i.e. in each sequence of Fig. 6) and based on the calibration curve, the evolution of the concentration of the substrate or the product is obtained as a function of reaction time, thus the initial reaction rate is determined. The concentration of the substrate (Asn) decays from 0.5 to 0.05 mM, and that of the product (Asp) grows from 0 to 0.43 mM. The initial reaction rate determined by the substrate consumption (0.107 mM/min) is almost identical to that determined by the product formation (0.120 mM/min). The Km values of Asn for L-asparaginase-catalyzed reaction were obtained from nonlinear fitting of the Michaelis–Menten www.electrophoresis-journal.com

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diagrams, as shown in the inset of Fig. 6. Sequential OG-UV/ vis CE runs were carried out with several different concentrations of the substrate. At each concentration, the reaction rate was presented by substrate consumption. Each data point in the figure is an averaged value of three repeatable measurements. The Km and Vmax values for Asp were determined to be 0.25 mM and 0.24 mM/min, respectively. To check the results of our sequential assay, traditional off-line enzyme assays were performed for the L-asparaginase reaction. The Km values were determined to be 0.16 mM, which are in good agreement with the results of our sequential method, indicating that the presented method did not affect the enzymatic activity and kinetics.

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[2] Jesus Lerma-Garcia, M., Zougagh, M., Rios, A., Curr. Anal. Chem. 2014, 10, 184–196. [3] Saiz, J., Koenka, I. J., Thanh Duc, M., Hauser, P. C., Garcia-Ruiz, C., Trend Anal. Chem. 2014, 62, 162– 172. [4] Wang, X., Li, K., Adams, E., VanSchepdael, A., Electrophoresis 2014, 35, 119–127. [5] Zhao, X., Qu, F., Wang, Y., Wang, X., Chin. Chromatogr. 2014, 32, 1–6. [6] Grochocki, W., Markuszewski, M. J., Quirino, J. P., Electrophoresis 2015, 36, 135–143. [7] Zhao, S. S., Zhong, X., Tie, C., Chen, D. D. Y., Proteomics 2012, 12, 2991–3012. [8] Alhusban, A. A., Breadmore, M. C., Guijt, R. M., Electrophoresis 2013, 34, 1465–1482.

4 Concluding remarks In conclusion, we present in this study an alternative application of OG injection for sequential CE analysis using commercial-available UV-Vis detection. Although the detection sensitivity of UV/vis detection is less than that of LIF detection, the OG-UV/vis method is more convenient in optical setup that does not need to split the laser beam for OG injection and LIF detection, as in commonly used OG-LIF method. We show that the OG-UV/vis method in combination of online derivatization is capable to achieve CE analysis of AAs and L-asparaginase-catalyzed enzyme reaction in real time. The feasibility of the OG-UV/vis CE analysis is attributed to the change of the chemical structure of chromophore upon photobleaching with the laser beam, leading to decreasing absorption of the analytes. By analyzing a standard mixture of AAs, we show that the sequential OG-UV/vis CE method exhibits high reproducibility with RSD of 2.23, 2.57, and 0.70% (n = 20) for peak heights, peak areas, and migration times, respectively, as well as the low LOD of 5.0 ␮M (for Asp) and 2.0 ␮M (for Asn). By automatically and simultaneously measuring the substrate consumption and the product formation, the L-asparaginase enzyme reaction was online monitored from the beginning to the end with temporal resolution of 12 s. The Michaelis constants were also obtained, which are in good agreement with those of traditional off-line enzyme assays. Our study indicates that the OG-UV/vis method is valuable for time-resolved sequential online CE analysis of chemical reactions in a variety of research fields. This work is supported by the National Natural Science Foundation of China (grant nos. 21175018 and 21475019), Jilin Provincial Science and Technology Development Foundation (grant no. 20120431). The authors declare no competing financial interest.

5 References [1] Breadmore, M. C., Tubaon, R. M., Shallan, A. I., Phung, S. C., Keyon, A. S. A., Gstoettenmayr, D., Prapatpong, P., Alhusban, A. A., Ranjbar, L., See, H. H., Dawod, M., Quirino, J. P., Electrophoresis 2015, 36, 36–61.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated ...
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