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Wei Wang Lihong Ma Fenzeng Yao Xiuli Lin Kaixuan Xu Key Lab of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial, Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, Fujian, P. R. China

Received May 14, 2014 Revised August 27, 2014 Accepted September 5, 2014

Research Article

High-speed separation and detection of amino acids in laver using a short capillary electrophoresis system A high-speed separation method of capillary MEKC with LIF detection had been developed for separation and determination of amino acids in laver. The CE system comprised a manual slotted-vial array (SVA) for sample introduction that could improve the separation efficiency by reducing injection volume. Using a capillary with 80 mm effective separation length, the separation conditions for amino acids were optimized. Applied with the separation electric field strength of 300 V/cm, the ten amino acids could be completely separated within 2.5 min with 10 mol/L Na2 HPO4 –NaOH buffer (pH = 11.5) including 30 mmol/L SDS. Theoretical plates for amino acids ranged from 72 000 to 40 000 (corresponding to 1.1–2.0 ␮m plate heights) and the detection limits were between 25 and 80 nmol/L. Finally, this method was applied to analyze the composition of amino acids in laver and eight known amino acids could be found in the sample. The contents of five amino acids, tyrosine, glutamic acid, glycine, lysine, and aspartic acid that could be completely separated in real sample were determined. The recoveries ranged from 82.3% to 123% that indicated the good reliability for this method in laver sample analysis. Keywords: Amino acid / High speed capillary electrophoresis / Laser-induced fluorescence / Laver DOI 10.1002/elps.201400246

1 Introduction CE is a widely used technique for sample separation. It contains some advantages including fast separation, high resolution, and less sample consumption, which makes it advantageous for sample analysis [1, 2]. Among its merits, fast separation is attractive to analysts because it can greatly shorten the analysis time compared with other similar methods. High-speed capillary electrophoresis (HSCE) can perform separations within a very short time (⬍3 min) [3]. Highspeed separations can also be performed with microfluidicchip electrophoresis [4, 5]. However, fabrication and manipulation of a microfluidic-chip are complicated compared with the application of capillary electrophoresis. HSCE can be accomplished by an applied ultrahigh electric field on a very short capillary (⬍5 cm) [6]. But a high electric field may result in the increase of Joule heat in the capillary that can lead to band broadening or even generation of air bubbles.

Correspondence: Dr. Wei Wang, Key Lab of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial, Key Lab of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, Fujian, P. R. China E-mail: [email protected] Fax: +86-591-22866135

Abbreviations: HSCE, high-speed capillary electrophoresis; NTP, number of theoretical plate

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Using the same electric field, a shorter length of capillary is advantageous for HSCE. On the other hand, a shorter capillary causes a limited number of theoretical plates (NTP) that is disadvantageous for sample separation. Therefore a shorter separation channel and a high NTP are essential for HSCE. The length of the sample plug, or sample injection volume, has a great effect on the column efficiency. A narrow sample plug produces narrower sample peaks that can greatly improve the NTP. Therefore, HSCE can be accomplished in a short capillary injected with narrow sample plug. Fang et al. [7] proposed a SVA system that could introduce nanoliter quantities of sample into the channels. In 2009, their further work developed a picoliter-scale spontaneous sample injection technique for HSCE [8]. These techniques had been successfully applied in sample separation, including amino acids [9], proteins [3], and DNA segments [10], using CZE, MEKC, and CGE modes. The injection process was controlled by a computer-programmed translational platform automatically. Based on their former work, we fabricated a manual SVA sample introduction device to perform spontaneous sample injection for analysis of amino acids in laver. The device is made with readily available glass slides and sample switching is performed on a manual platform that can be performed in most laboratories. The brown seaweed laver is a popular marine alga in the cuisine of many countries [11]. Laver is nutritious because it contains abundant amino acids, such as glutamic acid, Colour Online: See the article online to view Figs. 1 and 2 in colour.

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alanine, aspartic acid, tyrosine, etc. Therefore analyzing the composition of amino acids is an important way to assess the nutritional quality of laver. Traditionally, the amino acids could be separated by the means of IEC [12], HPLC, [13] and so on. Separation of amino acids by classical IEC was based on the mechanism of sample affinity to the ion exchanger. HPLC was also a good alternative to IEC for amino acids separation because of its relatively high efficiency for sample analysis [14–16]. Compared with all these means, HSCE analysis is much quick with less sample consumption that makes it very suitable for analysis of these compounds. Amino acids were derivatized with FITC to obtain fluorescence. Owning to the high sensitivity of LIF detection, even little amount of injected amino acids sample could be detected sensitively that made HSCE analysis possible. In this paper, the HSCE system, containing manual SVA spontaneous injection device, was applied to separate ten dominant amino acids in laver. The HSCE greatly shortened the separation time and the method was successfully applied to analysis the contents of amino acids in laver.

2 Materials and methods

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adjusted with 2 mol/L NaOH. Ten amino acids were dissolved, respectively, with water to obtain stock solutions of 1.00 × 10−2 mol/L. All of the stock solutions were stored at 4°C and diluted to the desired concentration with running buffer before use. FITC was dissolved with acetone to obtain stock solution of 1.00 × 10−2 mol/L. The amino acid derivatization was performed by mixing 10 ␮L amino acids (1.00 × 10−2 mol/L), 30 ␮L FITC (1.00 × 10−2 mol/L), and 60 ␮L 10 mmol/L borax buffer solution and then reacting for 12 h. The standard sample solutions were prepared by mixing the labeled amino acid solutions and diluting with running buffer to desired concentration. The extraction process of amino acids from laver was performed as follow: 2.0 g laver sample was pulverized and added with 50 mL 70% v/v ethanol. The solution was water bathed (100°C) for 15 min and then distilled in rotary evaporator at 40°C. The remains was dissolved with proper volume citric acid buffer (pH 2.2), centrifuged and diluted with water to obtain the sample solution containing amino acids. Before derivatization, the solution was adjusted to pH 9.2 with NaOH solution. Then the solution was derivatized with FITC and diluted with running buffer for separation.

2.1 Chemicals and materials 2.2 Apparatus Ten amino acids, Arginine (Arg), Leucine (Leu), Phenylalanine (Phe), Asparagine (Asn), Alanine (Ala), Glycine (Gly), Tyrosine (Tyr), Lysine (Lys), Glutamic acid (Glu), and Aspartic acid (Asp) were purchased from Aladdin company (Shanghai, China); FITC was obtained from Sigma company (St. Louis, MO, USA). The laver was purchased from Jinjiang Sanyuan Food Co. (Fujian, China). All the other reagents were analytical grade and the water used in this experiment was Milli-Q water (18.2 M⍀/cm). The running buffer consisted of Na2 B4 O7 (1025 mol/L) and SDS (1535 mol/L) with pH values from 10.0 to 12.0

The diagrammatic sketch of HSCE is shown in Fig. 1. The system was mainly composed of a manual SVA sampling device, a 100 mm long capillary with 50 ␮m diameter and 80 mm effective separation length, a high voltage supply (Shandong Normal University, China) and a LIF detector (Shandong Normal University, China) having excitation wavelength 473 nm and emission wavelength 525 nm. The main differences of SVA sampling device from that of reported [3] were that the buffer and sample reservoir were fixed on a manual slip platform and all the device was made with easily available glass

Figure 1. The diagrammatic sketch of HSCE.

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Figure 2. The conical tip of capillary.

slides. This design could facilitate the device available in almost all labs. Three plastic centrifuge tubes were used as buffer and sample reservoirs. The bottoms of centrifuge tubes were cut to form 1 mm width and 2 mm deep slots. Then, three centrifuge tubes were fixed on the surface of a slip platform (made with a piece of glass slide) that was fixed on an orbit. The platform could be moved forward and backward by hand. The capillary was fixed on the glass. The tip of capillary for sampling had been sharpened with a handheld grinder coupled with 2000 mesh sandpaper to be a conical (see Fig. 2). Conical tip could greatly reduce the sampling volume to picoliter-scale during spontaneous sampling [8]. Moving the platform could make the capillary tip pass through the slots of reservoirs. At the other end of the capillary, a length of capillary (10 mm) was uncoated and placed over a hole (diameter 5 mm) that was drilled as the window for LIF detection. A chromatography workstation (model HW-2000, Qianpu Software, Shanghai, China) was used to record electropherograms.

Figure 3. Electropherogram of repeated injections of FITC. Experimental conditions: FITC concentration, 1.00 × 10−5 mol/L; Running buffer, 10 mmol/L borax buffer containing 30 mmol/L SDS (pH 11.5); Effective separation length, 80 mm; Separation electric field strength, 300 V/cm.

3 Results and discussion 3.1 Performance of the HSCE system In order to examine the performance of the HSCE system, FITC was selected as a sample to run in the capillary. The electropherogram for 15 consecutive injections of 1.00 × 10−5 mol/L FITC is shown in Fig. 3. The RSD of the peak height and migration time were 3.86 and 0.78% (n = 15), respectively. The reproducibility of the manual HSCE system was similar to that derived from the system coupled with a computer-programmed translational platform [8, 10]. However, the HSCE system with manual translational platform is rather easily fabricated that meant the stable manual HSCE system having the potential of wide application. 3.2 Optimization of analysis conditions 3.2.1 Effects of pH value

2.3 Procedure Before separation, the capillary was rinsed with 0.05 mol/L HCl, 0.05 mol/L NaOH, water, and running buffer for 3 min, respectively. Then, the capillary was filled with running buffer and the capillary tip was stay in the slot of buffer reservoir A to run running buffer by high voltage. Spontaneous sample injection was then performed by pushing the platform forward to make the capillary tip pass through the sample reservoir slot. When the capillary tip touched the sample solution in slot, the sample was injected spontaneously by siphon action. The capillary tip was immersed into the solution of reservoirs about 0.8 mm depth at the moment. Sequentially, the platform was pushed forward to immerse the capillary tip in the buffer of slot of reservoir B. Then, the high voltage was applied on the reservoirs to perform separation. The moving speed of platform to perform sample injection was about 6 cm/s that could be calculated with a timer. So, all the manual injection process could be accomplished within 1 s. The separated sample peaks were record by the chromatography workstation.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The amino acids were hard to be separated under CZE mode. But it could be separated under MEKC mode when SDS concentration was over 15 mmol/L. The study showed that pH value had much greater effect on the separation. So, the effect of pH value on the separation was studied firstly with SDS concentration maintaining 20 mmol/L. As shown in Fig. 4, the migration time was prolonged with the increase of pH value from 10.0 to 12.0, and all of the amino acids could be observed within 3 min. The amino acids could not be completely separated when the pH value was below 11.5. Furthermore, the peak heights of compounds increased gradually with the increase of pH value in the range 10.011.5. So, pH 11.5 was found to be the optimum pH value for the running buffer. 3.2.2 Effects of SDS concentration All of amino acids could be separated when SDS concentration was over 15 mmol/L. The migration time of the analytes was prolonged with the increase of SDS concentration from www.electrophoresis-journal.com

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Figure 4. Electropherograms of the effect of pH value on separation. 1, Arg; 2, Leu; 3, Phe; 4, Asn; 5, Ala; 6, Gly; 7, Tyr; 8, Lys; 9, Glu; 10, Asp; 11, FITC; Experimental conditions: each amino acid concentration, 1.00 × 10−5 mol/L; Running buffer, 10 mmol/L borax buffer containing 20 mmol/L SDS; Effective separation length, 80 mm; Separation electric field strength, 300 V/cm. (A) pH 10.0, (B) pH 10.5, (C) pH 11.0, (D) pH 11.5, (E) pH 12.0.

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Figure 5. Electropherograms of the effect of SDS concentration on separation. 1, Arg; 2, Leu; 3, Phe; 4, Asn; 5, Ala; 6, Gly; 7, Tyr; 8, Lys; 9, Glu; 10, Asp; 11, FITC; Experimental conditions: each amino acid concentration, 1.00 × 10−5 mol/L; Running buffer, 10 mmol/L borax buffer (pH 11.5); Effective separation length, 80 mm; Separation electric field strength, 300 V/cm; SDS concentrations, (A) 15 mmol/L, (B) 20 mmol/L, (C) 25 mmol/L, (D) 30 mmol/L, (E) 35 mmol/L.

15 to 35 mmol/L (See Fig. 5). Changing of SDS concentration had limited effect on the sample separation. When the SDS concentration was 30 mmol/L, the resolutions between the peaks of Leu, Phe, and Asn became better than lower concentrations’ and the migration time was relatively short (⬍2.5 min). So, 30 mmol/L was selected as the optimal concentration of SDS.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Migration time/min Figure 7. Electropherograms of the effect of separation electric field strength on separation. 1, Arg; 2, Leu; 3, Phe; 4, Asn; 5, Ala; 6, Gly; 7, Tyr; 8, Lys; 9, Glu; 10, Asp; 11, FITC; Experimental conditions: each amino acid concentration, 1.00 × 10−5 mol/L; Running buffer, 10 mmol/L borax buffer containing 30 mmol/L SDS (pH 11.5); Effective separation length, 80 mm; Separation electric field strengths, (A) 450 V/cm, (B) 400 V/cm, (C) 350 V/cm, (D) 300 V/cm, (E) 250 V/cm.

3.2.3 Effects of buffer concentration The effect of buffer concentration on separation was studied by using 5, 10, 15, 20, and 25 mmol/L borax solution containing 30 mmol/L SDS (pH 11.5). The migration time for last compound was obviously prolonged from 1.9 to 5.7 min with www.electrophoresis-journal.com

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the increase of borax concentration from 5 to 25 mmol/L (see Fig. 6). Furthermore, the peak height of analytes decreased quickly with the increase of borax concentration. So lower concentrations were benefit for detection sensitivity and highspeed separation. However, too low borax concentration also resulted in poor resolution. When the concentration was below 10 mmol/L, the peaks of Tyr and Lys were overlapped. So, 10 mmol/L borax buffer was chosen for the subsequence experiment.

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Separation electric field strength mainly affected the migration time and peak resolution during separation. The effect of separation electric field strength, between 250 and 450 V/cm, on separation was investigated and the result is shown in Fig. 7. It was obvious that higher voltage was favorable to shorten the migration time. However, too high a voltage led to the decrease of resolution (Fig. 7A and B). In order to save analysis time and obtain higher separation resolution, 300 V/cm was selected as the optimal separation electric field strength. Under the optimum conditions, the electropherogram of separation for the mixture of ten amino acids is shown in Fig. 8A. It was obvious that the ten analytes could be separated within 2.5 min. Theoretical plates for amino acids ranged from 72 000 to 40 000 (corresponding to 1.1– 2.0 ␮m plate heights) that showed high column efficiency of the HSCE.

3.2.5 Linear relationship and detection limit In order to determine the linearity of the ten analytes, a series of concentrations of mixed solutions were tested under the optimized conditions. The detection limits were calculated on the basis of an S/N ratio of 3 and the results are listed in Table 1.

Figure 8. Electrophoregrams of amino acid mixture and laver extract separation. 1, Arg; 2, Leu; 3, Phe; 4, Asn; 5, Ala; 6, Gly; 7, Tyr; 8, Lys; 9, Glu; 10, Asp; 11, FITC; U, unknown peak; (A) Electrophoregrams for amino acid mixture separation; (B) Electrophoregrams for laver extract separation; Experimental conditions: each amino acid concentration, 1.00 × 10−5 mol/L (in A); Running buffer, 10 mmol/L borax buffer containing 30 mmol/L SDS (pH 11.5); Effective separation length, 80 mm; Separation electric field strength, 300 V/cm.

3.3 Application to sample detection in laver According to the procedure described above, amino acids were extracted from laver and separated under the optimized condition. A typical electropherogram for the laver samples is shown in Fig. 8B. Eight amino acids, namely Phe, Asn, Ala, Gly, Tyr, Lys, Glu, and Asp, were found in the laver sample. But Phe, Asn, and Ala were hard to be separated in the real sample. So, the contents of Gly, Tyr, Lys, Glu, and Asp in dry laver were determined and the results were 0.5463, 1.5883, 0.2426, 0.6227, and 0.2117 mg/g, respectively. The study showed that the laver was rich of Gly, Tyr, and Glu, among which Glu brought about the palatable taste of laver. In order to verify the reliability of the measuring method, the recovery experiment was performed under the optimal condition and the results are shown in Table 2. The recoveries

Table 1. Regression equations, correlation coefficients, and detection limits

Compound

Regression equation

Correlation coefficient

Linear range (␮mol/L)

Detection limit (nmol/L)

Arg Leu Phe Asn Ala Gly Tyr Lys Glu Asp

y = 4.93 × 102 x + 49.9 y = 2.92 × 102 x + 13.9 y = 3.25 × 102 x + 24.1 y = 2.24 × 102 x + 17.2 y = 3.09 × 102 x + 25.0 y = 2.91 × 102 x + 12.2 y = 4.26 × 102 x + 28.0 y = 1.52 × 102 x + 8.38 y = 1.62 × 102 x + 11.3 y = 1.10 × 102 x + 27.1

0.9943 0.9921 0.9930 0.9921 0.9933 0.9953 0.9939 0.9905 0.9930 0.9923

0.05–20 0.05–15 0.05–15 0.05–10 0.05–10 0.05–10 0.05–20 0.5–15 0.5–15 0.5–15

25 30 30 50 40 40 25 80 80 80

Y is the peak height (␮V); X is the concentration of analytes (␮mol/L).

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Table 2. Recoveries of amino acids

Amino acids

Added (␮mol/L)

Found (␮mol/L)

Recoveries (%)

RSD (%) (n = 3)

Gly

1.00 1.50 2.00 1.00 1.50 2.00 1.00 1.50 2.00 1.00 1.50 2.00 1.00 1.50 2.00

1.18 1.76 2.28 1.12 1.61 1.68 0.823 1.25 1.65 1.11 1.44 2.46 1.14 1.46 1.99

118 117 114 112 107 84.0 82.3 83.3 82.5 111 96.0 123 114 97.3 99.5

2.51 4.04 3.98 3.24 2.32 3.01 4.03 3.83 4.32 3.55 4.45 3.61 4.61 3.67 3.67

Tyr

Lys

Glu

Asp

were in the range of 82.3123%, indicating that the method is reliable.

4 Concluding remarks In this work, a high-speed electrophoresis system equipped with a manual SVA sample introduction device was applied to separate and detect the amino acids in laver. The study showed that the system is convenient and stable with good reproducibility. The injection method could obtain high separation efficiency. Although the injected sample volume was limited, the system still showed high sensitivity for sample detection. All of analytes could be separated within 2.5 min in a short capillary with 80 mm effective separation length. The contents of Gly, Tyr, Lys, Glu, and Asp in dry laver were determined and the results showed that the laver was rich of Gly, Tyr, and Glu. The recoveries of amino acids were between 82.3 and 123%, indicating that the method was reliable.

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The project was supported by National Natural Science Foundation of China (No. J1103303).

The authors have declared no conflict of interest.

5 References [1] Frost, N. W., Jing, M., Bowser, M. T., Anal. Chem. 2010, 82, 4682–4698. [2] Geiger, M., Hogerton, A. L., Bowser, M. T., Anal. Chem. 2012, 84, 577–596. [3] Lin, Q-Hu., Cheng, Y-Q., Dong, Y-N., Zhu, Y, Pan, J-Z., Fang, Q., Electrophoresis 2011, 32, 2898–2903. [4] Li, X., Xiao, D., Ou, X-M., McCullm, C., Liu, Y-M., J. Chromatogr. A 2013, 1318, 251–256. [5] Zhang, P., Nan, H., Lee, M-J., Kang, S. H., Talanta 2013, 106, 388–393. [6] Monnig, C. A., Jorgenson, J. W., Anal. Chem. 1991, 63, 802–807. [7] Du, W-B., Fang, Q., He, Q-H., Fang, Z-L., Anal. Chem. 2005, 77, 1330–1337. [8] Zhang, T., Fang, Q., Du, W-B., Fu, J-L., Anal. Chem. 2009, 81, 3693–3698. [9] Li, Q., Zhang, T., Zhu, Y., Cheng, Y-Q., Lin, Q-H., Fang, Q., Electrophoresis 2013, 34, 557–561. [10] Cheng, Y-Q., Yao, B., Zhang, H-D., Fang, J., Fang, Q., Electrophoresis 2010, 31, 3184–3191. ´ M. M. B., J. Chro[11] Salgado, S. G., Nieto, M. A. Q., Simon, matogr. A 2006, 1129, 54–60. [12] Ertingshausen, G., Adler, H. J., Reichler, A. S., J. Chromatogr. A 1969, 42, 355–366. [13] Bhushan, R., Agarwal, R., Biomed. Chromatogr. 1998, 12, 322–325. [14] Fekkes, D., J. Chromatogr. B 1996, 682, 3–22. ´ [15] Ilisz, I., Aranyi, A., Pataj, Z., Peter, A., J. Chromatogr. A 2012, 1269, 94–121. ´ [16] Ilisz, I., Aranyi, A., Peter, A., J. Chromatogr. A 2013, 1296, 119–139.

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High-speed separation and detection of amino acids in laver using a short capillary electrophoresis system.

A high-speed separation method of capillary MEKC with LIF detection had been developed for separation and determination of amino acids in laver. The C...
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