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Journal of Chromatography A, 1162 (2007) 149–153

Separation and determination of amino acids by micellar electrokineticchromatography coupling with novel multiphoton

excited fluorescence detection

Sheng Chen a,b, Youzhi Xu a, Fei Xu a, Xiaojun Feng a, Wei Du a,Qingming Luo a, Bi-Feng Liu a,∗

a The Key Laboratory of Biomedical Photonics of MOE – Hubei Bioinformatics and Molecular Imaging KeyLaboratory – Division of Biomedical Photonics at Wuhan National Laboratory for Optoelectronics, Department of Systems Biology,

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Chinab CEET of Hubei Normal University, Huangshi 435001, Hubei Province, China

Available online 18 May 2007

bstract

In this article, it was demonstrated that separation and determination of 20 amino acids were accomplished by micellar electrokinetic chromatog-aphy (MEKC) coupling with novel multiphoton excited fluorescence (MPEF) detection method. Different from MPEF achieved by expensive fsaser, continuous wave (CW) diode laser of ultra-low cost was uniquely employed in our MPEF system. Amino acids were fluorescently labeled withuorescein isothiocyanate (FITC), and were subjected to sodium dodecyl sulfate (SDS)-based MEKC separation and CW-based MPEF detection.he result was compared with that by single photon excited fluorescence (SPEF), which indicated that MPEF had the advantages of better mass

etectability and higher separation selectivity over SPEF. Quantitative analysis was performed and revealed linear dynamic range of over 2 ordersf magnitude, with mass detection limit down to ymole level. To evaluate the reliability, this method was successfully applied for analyzing aommercial nutrition supplement liquid.

2007 Elsevier B.V. All rights reserved.

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eywords: Micellar electrokinetic chromatography; Multiphoton excited fluore

. Introduction

Amino acids are the basic units of proteins that constitutelmost all the life forms in the world. Different combination ofmino acids produces proteins with strikingly different proper-ies and functions. They have similar chemical structures androperties, which require highly efficient separation capabilityor analysis. Traditional method usually includes high per-ormance liquid chromatography [1] and gas chromatography2]. Recently, capillary electrophoresis (CE) [3] has devel-ped rapidly, due to advantages of high performance, high

peed, low sample and reagent requirement, and automations well. It has been widely recognized as a method of choiceor analyzing chemical, environmental and biomedical sam-

∗ Corresponding author at: The Key Laboratory of Biomedical Photonics ofinistry of Education, Huazhong University of Science and Technology, Wuhan

30074, China. Tel.: +86 27 87792170; fax: +86 27 87792203.E-mail address: bfliu@mail.hust.edu.cn (B.-F. Liu).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.05.046

e; Amino acids; Laser induced fluorescence

les. Many separation modes based on different mechanismsave been developed, such as capillary zone electrophoresisCZE) [4], micellar electrokinetic chromatography (MEKC)5], capillary gel electrophoresis (CGE) [6] and capillary iso-lectric focusing (cIEF) [7]. Various detection methods haveeen coupled to CE, including UV [4], laser induced fluores-ence (LIF) [8], electrochemistry [9,10], chemiluminescence11–13] and mass spectrometry [14,15], etc., in which LIFs the most widely used detector with respect to its ultraigh sensitivity [16]. For assaying amino acids, two CE sep-ration modes such as CZE [4] and MEKC [17] are oftensed, with UV or LIF detection scheme. Because most ofmino acids are transparent in UV/vis range and nonfluores-ent, derivatization procedure is needed for the detection. Forxample, some dyes like 4-fluoro-7-nitrobenzofurazan (NBD-F)

17], naphthalenedicarboxaldehyde (NDA) [18,19], fluoresceinsothiocyanate (FITC) [20,21], o-phthalaldehyde (OPA) [22,23]nd 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) [24] are fre-uently used for LIF detection.

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50 S. Chen et al. / J. Chroma

Recently, multiphoton excited fluorescence (MPEF) has beenntroduced into CE as a complementary mode of LIF detec-ion [25–30]. Owing to ultra-low detection background andxtremely low detection volume (usually atto-L level), a detec-ion limit in mass of zepto-mole can be readily achieved in

PEF. Furthermore, the feature of spectra excitation of MPEFakes it versatile for analyzing the compounds of different flu-

rescent characters. Consequently, MPEF shows great prospectn the detection of micro/nano scale separations. However, highost of fs mode-locked laser (usually US$ 150,000) quite lim-ts its application in CE area. To use cheaper laser to achieve

PEF will be an interesting topic. Paul et al. [31] tried to use-switched diode laser (usually US$ 6000) instead of fs mode-

ocked laser to analyze some amino acids and proteins. Moreecently, we for the first time constructed a continue wave (CW)-ased MPEF system for CE analysis [29,30], in which muchheaper diode laser (usually US$ 1000) was employed.

In this report, the analysis of 20 amino acids was demon-trated using MEKC with novel MPEF detection method. Aminocids were fluorescently labeled with FITC, and were subjectedo sodium dodecyl sulfate (SDS)-based MEKC separation andW-based MPEF detection. The result was compared with thaty single photon excited fluorescence (SPEF), which indicatedhat MPEF had the advantages of better mass detectability andigher separation selectivity over SPEF. Quantitative analysisas performed that revealed linear dynamic range of over 2rders of magnitude, with mass detection limit down to ymoleevel. To evaluate the reliability, this method was successfullypplied for analyzing a commercial nutrition supplement liquid.

. Experimental

.1. Instrument and procedure

Analyses were carried out on two CE systems. One is a com-ercial CE instrument (P/ACE 5510, Beckman, Fullerton, CA,SA) with an argon ion laser (488 nm) for SPEF detection. Thether one is a homemade system for MPEF detection as reportedreviously [29,30], including an inverted fluorescence micro-cope (CK40, Olympus, Tokyo, Japan), a diode laser source808 nm/1 W; Institute of Semiconductor, Beijing, China), ahotomultiplier (PMT R5070, Hamamatsu, Hamamatsu, Japan)nd a high voltage power supply (0–30 kV; Shanghai Nuclearesearch Institute, Shanghai, China) for electrophoresis. The

aser beam from the diode laser source was reflected by a dichroicirror (E625SP, Chroma, VT, USA) and focused into the capil-

ary column using an oil objective (100×, NA1.25; Olympus).he excited fluorescence was collected using the same objective

nto the PMT through the dichroic mirror and a low-pass filterBG18, Newport, NY, USA). The PMT current was convertednd amplified into a voltage and acquired using a custom-built/D convertor at a sampling rate of 20 Hz. Program for data

cquisition was locally written in Borland C++. The collimator

f the diode laser was manufactured in our laboratory.

In CE-SPEF, a capillary of 57 cm (total length) × 50 cmeffective length) × 375 �m O.D. × 50 �m I.D. was used.amples were injected at a pressure of 34.5 kPa for 5 s

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A 1162 (2007) 149–153

injected volume: 4.36 nL). In CE-MPEF, a capillary of 375 �m.D. × 50 �m I.D. × 57 cm (total length) was used. Samplesere introduced by siphon at 15 cm for 60 s (injected vol-me: 23.5 nL). For new fused-silica capillary (Polymicro, AZ,SA), columns were cleaned with 0.1 M HCl, 0.1 M NaOH andure water sequentially for 10 min, respectively. The capillaryas equilibrated with a running buffer (25 mM borate solution,00 mM sodium dodecyl sulfate and 5% methanol; pH 11.0)or 20 min before experiments. Between runs, the capillary wasinsed with 0.1 M NaOH, pure water and the running bufferequentially for 10 min, respectively, to ensure reproducibility.

.2. Chemicals

Amino acids such as arginine (Arg), cysteine (Cys), methio-ine (Met), glutamine (Gln), 4-hydroxyproline, tyrosine (Tyr),aline (Val), histidine (His), threonine (Thr), leucine (Leu),soleucine (Ile), phenylalanine (Phe), asparagine (Asn), pro-ine (Pro), alanine (Ala), tryptophan (Trp), serine (Ser), glycineGly), lysine (Lys), glutamic acid (Glu) and aspartic acid (Asp),nd FITC were purchased from Sigma–Aldrich (St. Louis,O, USA). NaB4O3·10H2O, boric acid, acetone, hydrochlo-

ic acid, sodium hydroxide and SDS were purchased fromianjing Chemical (Tianjing, China). The nutrition supplement

iquid was a commercial product of Eight-peak Pharmaceuti-al Chemistries (Hubei, China). All reagents were of analyticalrade unless specified otherwise. All solutions were preparedith water purified by the Milli-Q system (Millipore, Bedford,A, USA).

.3. Samples preparation

The standard solutions of amino acids were prepared fromheir stock solution (10 mM in 10 mM borate buffer, pH 11.0).ITC was diluted in acetone at a concentration of 10 mM. Aminocids were derivatized with FITC (five-fold excessive in mole)n dark for overnight at room temperature [20,30]. The commer-ial nutrition supplement liquid was diluted with 25 mM borateuffer solution at a volume ratio of 1:500, and then derivatizedith excessive FITC in dark for overnight at room temperature.

. Results and discussion

Our goal of this work was to establish a CE approach formino acids analysis, coupling with unique CW-based MPEFetection method. To achieve it, the sample that contained 20mino acids (plus 4-hydroxyproline as an internal standard foruantification) was firstly separated by CE and detected by SPEFor a comparison.

.1. MEKC-SPEF

Before CE separation, amino acids had undergone a pre-

olumn derivatization procedure, because they had no nativeuorescence. There were many fluorescent dyes commerciallyvailable for labeling amino acids, such as OPA, NDA, NBD-Fnd FITC, etc. To meet the excitation line of argon ion laser

togr. A 1162 (2007) 149–153 151

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Fig. 2. Electropherogram of standard amino acids by MEKC-MPEF method.Twenty-one FITC-labeled amino acids were excited by a diode laser (808 nm).MEKC separation was carried out in the same conditions as illustrated in Fig. 1.Capillary: 57 cm (total length) × 375 �m O.D. × 50 �m I.D. Peak identity: (1)Arg (20.5 �M); (2) Cys (19.7 �M); (3) FITC (excessive); (4) Met (13.6 �M);(5) Gln (19.5 �M); (6) 4-hydroxyproline (23.4 �M); (7) Tyr (23.5 �M); (8) Val(23.7 �M); (9) His (19.8 �M); (10) Thr (20.3 �M); (11) Leu (20.6 �M); (12) Ile(A(

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S. Chen et al. / J. Chroma

n our system, FITC was chosen. Based on our former expe-ience [29,32] and suggestions of other reports [20], at leastve-fold excessive amount of FITC in mole was required forverall derivatization of the samples.

For CE separation of all these FITC labeled species, MEKChat discriminates molecules based on the differential parti-ioning of the molecules between the aqueous phase and theseudostationary micellar phase was a good choice as previouslyeported by Liu et al. [32]. We then carefully tuned the fac-ors that greatly influenced the separation selectivity of MEKC,uch as electric field strength (EFS), concentration of the run-ing buffer, concentration of the micelle SDS, and the typend concentration of buffer additive. Here, the same type ofuffer electrolyte (borate) and pH value (11.0) was employeds the sample solution. It was found that the separation res-lution was quite dependent of SDS concentration in runninguffer. Higher concentration of SDS led to better separationelectivity. But when SDS concentration was over 100 mM, theeparation resolution became decrease. It might be caused byoule-heating effect. High concentration of SDS could increasehe ionic strength of running buffer, which would in turn increasehe current and produce more heat. To further enhance the sep-ration quality, organic additive (5% methanol) was tried tohange the partition fractions of the samples between SDS phasend aqueous phase. Finally, a buffer (25 mM borate solution,00 mM SDS and 5% methanol, pH 11.0) was chosen. EFS foreparation was controlled at 395 V/cm. Further increasing theFS could shorten the separation time. However, the separation

esolution was significantly decreased. Under these optimizedonditions, all the 21 amino acids together with FITC were sep-rated by MEKC, and detected by SPEF as illustrated in Fig. 1.nfortunately, there were still some peaks in the electrophero-

ig. 1. Electropherogram of the standard amino acids by MEKC-SPEF method.mino acids were labeled with FITC and excited by an argon ion laser (488 nm).EKC separation was carried out in the running buffer (25 mM borate solution,

00 mM SDS and 5% methanol, pH 11.0) with an electric strength of 395 V/cmor separation at 25◦C. Samples were injected at a pressure of 34.5 kPa/5 s. Cap-llary: 57 cm (total length) × 50 cm (effective length) × 375 �m O.D. × 50 �m.D. Peak identity: (1) Arg (20.5 �M); (2) Cys (19.7 �M); (3) FITC (excessive);4) Met (13.6 �M); (5) Gln (19.5 �M); (6) 4-hydroxyproline (23.4 �M); (7) Tyr23.5 �M); (8) Val (23.7 �M); (9) His (19.8 �M); (10) Thr (20.3 �M); (11) Leu20.6 �M); (12) Ile (21.2 �M); (13) Phe (20.4 �M); (14) Asn (19.8 �M); (15) Pro20.1 �M); (16) Ala (22.4 �M); (17) Trp (21.4 �M); (18) Ser (22.5 �M); (19)ly (20.5 �M); (20) Lys (22.7 �M); (21) Glu (23.6 �M); (22) Asp (20.8 �M).

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21.2 �M); (13) Phe (20.4 �M); (14) Asn (19.8 �M); (15) Pro (20.1 �M); (16)la (22.4 �M); (17) Trp (21.4 �M); (18) Ser (22.5 �M); (19) Gly (20.5 �M);

20) Lys (22.7 �M); (21) Glu (23.6 �M); (22) Asp (20.8 �M).

ram not completely resolved, for example, peaks of Tyr andal, Thr and Lue.

.2. MEKC-MPEF

To investigate the possibility of CW-based MPEF detectionor CE analysis of FITC labeled amino acids, all the CE condi-ions that applied in above section were kept for MEKC-MPEFssay. Fig. 2 showed the result. It was true that all the 21 aminocids together with FITC were separated by MEKC and detectedy CW-based MPEF, which proved that MPEF could be achievedy CW-based, ultra-low cost diode laser for the detection oficro-scale separation, for example, CE. It was interesting that

ll the peaks were well resolved, even for the peaks that could note completely separated using MEKC-SPEF. The reason for thishenomenon was yet unknown. It might be concerned with thepecial features of MPEF, for example, its ultra-low detectionolume (usually at atto-L level), because one of contributions tohe peak width in electropherogram was from the detection in

EKC [33]. It indicated that MPEF could also help to enhanceE separation selectivity.

.3. Quantitative data

Based on the analysis in Section 3.2, calibration curves werestablished from seven concentration levels of samples thatxhibited a linear dynamic range of about 2 orders of magni-ude between logarithm of peak heights and logarithm of sampleoncentrations (see Table 1 for details). Detection limits were

lso calculated based on a peak height of three times the base-ine noise as shown in Table 1. For a comparison, the detectionimits by SPEF were also included. The detection volume wasalculated as recommended by a reference [34].

152 S. Chen et al. / J. Chromatogr. A 1162 (2007) 149–153

Table 1Quantitative data

Species Calibration curve Y = AX + B γ Linear range (�M) RSD (%), n = 9 DL (S/N = 3)in SPEF (nM)

DL (S/N = 3) in MPEF

C (�M) Mass (ymole)

Arg Y = 0.867X + 5.91 0.9921 121–1.41 2.43 0.158 0.705 31.7Cys Y = 0.764X + 4.63 0.9916 182–1.92 2.61 0.467 0.605 27.2Met Y = 0.818X + 4.82 0.9907 120–1.96 2.94 0.647 0.981 44.2Gln Y = 0.885X + 6.40 0.9908 221–1.11 2.67 0.202 0.553 24.94-Hpr Y = 0.456X + 2.37 0.9916 389–2.33 1.17 0.141 0.775 34.9Tyr Y = 0.806X + 4.82 0.9913 188–1.51 2.72 0.162 0.754 34.0Val Y = 0.717X + 4.53 0.9905 189–1.54 1.96 0.123 0.514 23.1His Y = 0.459X + 1.16 0.992 282–1.53 1.09 0.234 0.766 34.5Thr Y = 0.693X + 4.35 0.9910 191–1.74 2.03 0.158 0.579 26.1Lue Y = 0.818X + 6.23 0.9940 145–1.08 1.94 0.150 0.361 16.6Ile Y = 0.691X + 4.09 0.9909 181–1.56 1.85 0.370 0.780 35.1Phe Y = 0.713X + 3.76 0.9910 281–2.34 1.89 0.213 0.779 35.1Asn Y = 0.709X + 4.40 0.9910 213–2.22 1.87 0.292 1.11 49.7Pro Y = 0.685X + 3.96 0.9902 233–2.50 2.10 0.281 1.25 56.3Ala Y = 0.781X + 4.63 0.9924 93.3–4.18 2.44 0.588 2.09 94.1Trp Y = 0.772X + 4.03 0.9949 107–4.04 2.16 1.25 2.02 91.0Ser Y = 0.776X + 4.99 0.9913 98.9–2.48 2.53 0.325 1.24 55.9Gly Y = 0.791X + 4.27 0.9920 102–7.34 2.63 1.23 3.68 165Lys Y = 0.459X + 0.92 0.9919 248–8.73 0.97 1.46 4.37 197Glu Y = 0.654X + 4.07 0.9930 109–6.73 1.53 1.25 3.37 151A 2.42 1.91 3.77 170

Y calculated detection volume: 45.0 aL [34]. DL data in SPEF were calculated fromF

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Table 2Quantitative analysis of a commercial nutrition supplement liquid

Species Concentrationmeasured (10−3 M)

Standard concentration(10−3 M)

Error (%)

Arg 10.82 10.95 1.19Gln 38.74 38.68 0.16Tyr 18.99 19.15 0.84Val 6.44 6.21 3.70

sp Y = 0.773X + 4.20 0.9923 125–7.54

: Logarithm of peak height (a.u.); X: logarithm of sample concentration (M);ig. 1, while DL data in MPEF were calculated from Fig. 2.

.4. Analysis of an amino acids supplement liquid product

To evaluate the reliability of this MEKC-MPEF method, aommercial nutrition supplement liquid was analyzed. Fig. 3escribed the electropherogram. Because RSD of migration timef all the species in the sample was within 0.6%, the migra-ion time was used for species identification. Table 2 gave the

etailed results, which revealed that the assayed data was quiteonsistent with the standard value provided by the company forhe product manufactured. The measurement errors were evalu-ted by comparing the measured concentration with the standard

ig. 3. Electropherogram of FITC-labeled amino acids in the commercial nutri-ion supplement liquid product by MEKC-MPEF method. Samples were dilutedn 25 mM borate buffer at a volume ratio of 1:500. MEKC conditions as in Fig. 2.eak identity: (1) Arg, (3) FITC, (5) Gln, (7) Tyr, (8) Val, (11) Lue, (12) Ile,17) Trp, (19) Gly, (20) Lys.

Lue 4.60 – –Ile 10.14 10.12 0.19Trp 14.94 14.58 2.47Gly 8.12 8.03 1.00L

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oncentration provided by the manufacturer. It indicated thathe constructed CE-MEPF was a qualified system for thenalysis.

. Conclusion

In this report, we demonstrated a MEKC method to sepa-ate and determine amino acids, uniquely coupling with MPEFy a diode laser of continuous wave. Amino acids were fluo-escently tagged with FITC, which were further subjected to

EKC separations. LIF detection based on SPEF and MPEFchemes were compared. The result indicated that MPEF had

etter resolution than SPEF, which was believed to be causedy ultra-low detection volume of MPEF that greatly reduced theontribution of the detection to the peak broadening. Althoughhe mass detectability of our system was down to zepto-mole

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evel, the sensitivity in concentration was not so encouraging asPEF-based LIF detection scheme.

cknowledgements

The authors gratefully acknowledge the financial supportsrom National Natural Science Foundation of China (Grant Nos.:0405006, 30570468, 30600134), Program for New Centuryxcellent Talents in University (NCET-05-0644), and Program

or Young Investigators of Huazhong University of Science andechnology.

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