On-column labeling technique and chiral ligand-exchange CE with zinc(II)-L-arginine complex as a...

Short Communication

On-column labeling technique and chiralligand-exchange CE with zinc(II)-L-argininecomplex as a chiral selector for assayof dansylated D,L-amino acids

A novel on-column labeling method of amino acid (AA) enantiomers by using dansyl

chloride (Dns-Cl) has been explored combined with chiral ligand-exchange CE (CLE-CE)

technique and UV detection. Efficient labeling was achieved by sequential injection of

buffer, Dns-Cl, AA enantiomers, Dns-Cl and buffer at 0.2 psi for 10.0, 3.0, 24.0, 3.0, and

10.0 s, respectively. After injection, the sandwich sections were allowed to react at room

temperature for 35.0 min. With this procedure, successful on-column labeling and CLE-

CE separation of 17 pairs AA enantiomers have been achieved with a buffer of 100.0 mM

boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and 6.0 mM L-Arg at pH 8.4,

giving nine pairs fully enantioresolved with resolution in between 2.0 and 5.1. CLE-CE of

some individual and mixed pairs was also demonstrated, much the same as using pre-

column labeling. As validated by both artificially prepared solutions and serum samples,

this new method was shown to be applicable to the quantitative analysis, with a linear

range between 14.0 mM and 3.7 mM, correlation coefficient above 0.99 and recovery in

between 90.4% and 111.7%. It was also demonstrated that the migration time-

temperature based curve allows for temperature determination in CE by using this new

method.

Keywords:

Dansyl amino acid / Enantioseparation / Ligand-exchange CE / On-columnlabeling / Zinc complex DOI 10.1002/elps.200800753

1 Introduction

Many studies of amino acids (AAs), which are often

considered to be the most important and ubiquitous group

of chiral compounds known, have been made to elucidate

their biological function and metabolism. While L-AAs seem

to be more prevalent in nature, D-AAs have been reported to

be widely distributed in many living organisms ranging

form bacteria to mammals, and sometimes have been

indicated a negative symptom, aging or diseases [1–5].

Chiral analysis of AAs is thus remarkably important for

studying the life science, biotechnology and many other

related issues.

The tremendous expansion chromatographic methods

with different separation strategies, such as gas chromato-

graphy, high-performance liquid chromatography and CE

[6–10], have been described in the literature for their ability

to detect D,L-AAs. These methods were usually carried out by

pre-column and post-column derivatization techniques in

order to enhance detection sensitivity or selectivity.

However, these methods usually require some extraction

procedures in order to remove excess reagents in pre-

column derivatization, and also require special chemical

reactor devices for labeling in post-column derivatization.

These extra requirements have been complicated in D,L-AAs

derivatization methods. Therefore, developing new deriva-

tization methods of D,L-AAs are imminently required.

In recent years, although the on-column derivatization

method by using the inlet of a separation capillary tube as a

reaction chamber was developed into a new derivatization

technique [11] as the alternative method to the pre- and post-

derivazation method for CE, and many labeling chemical

reagents are applicable to chiral separation of AAs or amines,

only a few labeling reagents [12–17] are applicable to maintain

a high resolution with on-column derivatization [18, 19].

Kennedy and co-workers [20] developed a method to determine

D- and L-aspartate in microdialysis samples obtained from

rats by on-column derivatization with o-phthaladehyde

and b-mercaptoethanol in chiral CE. The on-column derivaza-

tion approach of D, L-carnitine with 9-fluoroenylmethyl chlor-

oformate using CE technique has been explored by Mardones

et al. [21].

Li Qi1,2

Gengliang Yang1

1College of PharmaceuticalSciences, Hebei University,Baoding, P. R. China

2Beijing National Laboratory ofMolecular Science; Laboratoryof Analytical Chemistry for LifeScience, Institute of Chemistry,Chinese Academy of Sciences,Beijing, P. R. China

Received November 17, 2008Revised February 9, 2009Accepted February 9, 2009

Abbreviations: AA, amino acid; CLE-CE, chiral ligand-exchange CE; Dns-Cl, dansyl chloride; Rs, enantioresolution

Correspondence: Professor Gengliang Yang, College of Pharma-ceutical Sciences, Hebei University, Baoding 071002, P. R. ChinaE-mail: [email protected]:186-312-5079788

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 2882–28892882

The on-column technique (or called on-site in-capillary

derivatization) is an attractive and special technique because it

not only can greatly minimize the consumption of samples and

labeling reagents, but also can greatly reduce the operation cost

and improving the precision of analyzing nano-molar or micro-

molar samples. Therefore, the on-column labeling is a very

useful technique for the analysis of minute sample, such as

serum or human saliva samples. However, to the best of our

knowledge, the on-column labeling has not been explored in

chiral ligand-exchange CE (CLE-CE) up to now. This induced us

to consider the exploration of the on-column derivatization

technique by using dansyl chloride (Dns-Cl) as labeling reagent

combined with the CLE-CE method. A key step of high

performance CLE-CE assay is the on-column derivatization that

makes the analytes of interest compatible with high sensitivity

UV detection. The exploration has led to some effective and

promising results, of which Dns-Cl was shown applicable to the

on-column derivatization and chiral separation of AAs with

CLE-CE technique.

Dns-Cl is a cheap and the most widely used derivatiza-

tion reagent [22, 23], which has high sensitive UV absor-

bance from 200–280 nM. Under alkaline conditions, it can

react not only with primary amines but also with secondary

amines, applicable to all common AAs. Recently, the

potential of CLE-CE system [24] has been demonstrated for

Dns-AAs enantioseparation by using Zn-complex as chiral

selector in our laboratory [25]. The main purpose of this

study was to extend the on-column derivatization technique

and combining CLE-CE with UV detection to the enantio-

separation of Dns-AAs with very limited sample volume,

without evident loss of enantioresolution (Rs). For demon-

stration, 17 pairs of Dns-AA enantiomers have been chosen

and tried. Successful on-column labeling and CLE-CE

separation of each pair of Dns-AA have been conducted,

giving nine pairs with Rs from 2.0–5.1, and eight pairs with

lower Rs from 0.9–1.2. Surprisingly, although the new

method decreased the Rs of some Dns-AAs, it maintained or

even enhanced the chiral resolution of seven pairs Dns-AA

enantiomers compared with pre-column labeling methods

(Table 1). Some mixed Dns-AAs were also successfully

resolved, demonstrating that the method is potentially

adaptable to the analysis of some complicated chiral

samples. This has also been confirmed by the measurement

of AAs in serum samples.

2 Materials and methods

2.1 Chemicals

All D- and L-AA standards and Dns-Cl were purchased from

Sigma Chemical (St. Louis, USA). Tris, lithium carbonate,

zinc sulfate, boric acid and other chemicals were all of

analytical reagent grade from Beijing Chemical Factory

(Beijing, China).

2.2 Preparation of buffer and sample solutions

All solutions were prepared in tripe distilled water produced

by a distillation apparatus model SZ-93 (Yarong Biochem-

ical Instrument , Shanghai, China) and stored at 4.01C. CE

Table 1. Comparison of the on-column labeling method with the pre-column labeling via Rs and peak efficiency (N, theoretical plates)

measured from various AAsa)

Analyte Rs ND/104 NL/104

Pre-column On-column Pre-column On-column Pre-column On-column

Ala 3.4 2.5 22.6 47.8 26.9 32.4

Asn 2.7 2.4 19.8 30.0 17.6 25.9

Asp 1.3 1.1 14.7 3.6 12.7 5.9

Cys 2.6 5.1 8.7 5.4 3.8 6.7

Glu 1.0 2.0 4.4 18.8 8.3 12.1

Ile 3.0 1.2 23.2 20.5 24.7 20.0

Leu 1.6 1.2 26.6 30.1 25.9 34.6

Lys 1.7 4.2 16.9 3.2 16.2 4.0

Met 4.2 4.2 23.1 33.9 20.6 26.2

Orn 1.4 4.5 15.7 9.0 10.6 6.4

Phe 1.2 1.0 22.6 19.2 21.3 24.5

Pro 0 0 3.4 8.0 34 8.0

Ser 4.2 5.0 20.0 23.9 18.3 19.9

Thr 2.1 2.7 24.6 22.4 25.1 24.7

Trp 0.9 1.2 2.6 23.9 2.9 24.1

Tyr 1.4 1.1 23.9 21.7 24.1 18.8

Val 0.6 0.9 9.6 2.6 16.6 11.3

a) The subscript denotes the D- or L-form. All the data were the mean from experiments run in triplicate using the condition described in

Sections 2.4 and 2.5.

Electrophoresis 2009, 30, 2882–2889 CE and CEC 2883


running buffers, unless stated otherwise, were composed of

5.0 mM ammonium acetate, 100.0 mM boric acid, 3.0 mM

ZnSO4 � 7H2O and 6.0 mM L-Arg, adjusted to pH 8.4 with

Tris. Before use, all the running buffers were filtered

through a membrane filter with 0.45 mm pores and degassed

by sonication for 2.0 min.

Standard stock solutions of D- and L-AAs were prepared

in 40.0 mM lithium carbonate buffer (adjusted to pH 9.5

with 0.1 M HCl) at a final concentration of 2.0 mg/mL.

Working solutions were diluted from the stock solutions

with 40.0 mM lithium carbonate by 10–104-fold. Dns-Cl

solution was freshly prepared by dissolving 4.0 mg Dns-Cl

in 2.0 mL acetone.

2.3 Serum sample pretreatment

Serum, S-1 and S-2, from healthy human volunteers were

collected in 1.5 mL vials and kept in ice for 30.0 min.

Samples in vials were centrifuged at 5000 rpm for 15.0 min

and the supernatant was further depreoteinized by mixing it

with acetonitrile at the volume ratio of 1:2. Then the

resultant was centrifuged at 5000 rpm for 15.0 min. The

supernatant was divided into aliquots of 400 mL, blown to

dryness by N2 and stored at �20.01C. Before analysis, the

residue was re-dissolved in 200 mL 40.0 mM lithium

carbonate.

2.4 On-column labeling of AAs

A capillary was first sequentially rinsed with methanol,

water, 1.0 M NaOH and water for 10.0 min each. Before

each injection, the capillary was sequentially rinsed with

0.1 M HNO3, water, 0.1 M NaOH, water and running

buffer for 2.0 min each. Then the capillary was filled with

running buffer and a sandwich injection in the

order of running buffer, Dns-Cl reagent solution, sample,

Dns-Cl reagent solution and running buffer (Fig. 1A),

conducted at 0.2 psi for 10.0, 3.0, 24.0, 3.0, 10.0 s each

section. To avoid contamination, the inlet tip of the

capillary was cleaned by dipping it into water for 5.0 s in

between the injections. The injected sample sandwich was

reacted at the end of the capillary inlet for 35.0 min

(Fig. 1B). Separation was then started at �20.0 KV (Figs. 1C

and D).

It should be mentioned that 10.0 s running buffer

injection at last section was especially necessary; otherwise,

the separation would be broken off easily, which might be

caused by the acetone in Dns-Cl solution. In addition, to

avoid the double peaks of one D-AA or L-AA component

showing up, the mixing method, usually used in on-

column labeling by applying an electric field (o3.0 KV)

across the injection sections could not be used in this

study.

2.5 Pre-column labeling of AAs

AAs were dansylated according to literature [26]. Briefly, an

aliquot of 100 mL AAs in a 0.5 mL vial was mixed with

200 mL of 40.0 mM lithium carbonate buffer and 100 mL

labeling solution of Dns-Cl. The mixed solution was

allowed to react at room temperature for 35.0 min

[25]. After addition of 5 mL 2.0% ethylamine to terminate

the reaction, the reacted solution was either directly

injected for CE separation or kept at 4.01C for future

analysis.

2.6 CE

Electrophoretic experiments were conducted using P/ACE

model 5000 (Beckman Coulter , CA, USA). Unless stated

otherwise, separations were performed at 20.01C in an

uncoated fused-silica capillary (Yongnian Optical Fiber

Factory, Hebei, China) of 50.0 mm i.d.� 57.0 cm (50.0 cm

effective). Before injection, the bare fused-silica capillary was

sequentially rinsed with 0.1 M HNO3, water, 0.1 M NaOH,

water and running buffer for 2.0 min each. Dimethyl

sulfoxide was used to mark the EOF. Samples were

separated at �20.0 kV and detected by UV absorption at

214 nm. The data were acquired at 4 Hz. Peaks were

identified by spiking relative standard AAs in sample

solutions. The peaks with increased height were considered

to be the targets.

Figure 1. Schematic procedure for on-column labeling and CLE-CE separation of D,L-AAs. The sample is injected betweenlabeling reagent Dns-Cl plugs (A). After these injections, thesandwich sections are mixed, and the labeling reaction (B) isallowed to happen at room temperature. After the voltage-freereaction, enantioseparation is started (C) and the separated D,L-Dns-AAs moved to the detector window (D).

Electrophoresis 2009, 30, 2882–28892884 L. Qi and G. Yang


3 Results and discussion

According to the previous studies, Zn(II)-based CLE-CE [24,

25] was adopted and our previously explored [25] running

buffer of 5.0 mM ammonium acetate, 100.0 mM boric acid,

3.0 mM ZnSO4 � 7H2O and 6.0 mM L-Arg was tried in this

study by rechecking the critical parameters including the

molar ratio of central ion-to-ligand, buffering reagent, buffer

concentration and pH, aiming at further improving the

resolution and shortening the separation time. Fortunately,

the purpose was achieved just by adjusting the running

buffer pH at 8.4. It should be noted that the label-free D,L-

AAs (Table 1) became negative-charged Dns-D,L-AAs after

dansylation; thus, the CLE-CE separation was studied by

using reverse polarity.

3.1 Optimization of on-column labeling

Figure 1 highlights the general principle of on-column

labeling for chiral AA analysis using CLE-CE method, and

Rs of AA adducts occur within a single capillary during

electromigration. In order to obtain a better on-column

labeling performance, the injection sequence of sample and

labeling reagent, injection time and the reaction time, which

were expected to the key factors, have been evaluated and

optimized. The injection sequence was found to be the first

important factor because mixing of the derivatization

reagent and the analytes is caused by the differences in

the moving velocities between the reagent and analytes in a

capillary column during CLE-CE. Note that AAs should be

injected after Dns-Cl, otherwise, lower peak area would

result (Fig. 2a). This is because Dns-Cl migrates faster than

AAs, quickly moving away from the frontal of AAs plug

once the voltage is applied. Hence, to make AAs come into

contact with Dns-Cl, Dns-Cl solution should be injected

ahead of AAs (Fig. 2b). Figure 2c depicts that the tested four

pairs of AAs obtain the biggest peak area if another section

of Dns-Cl is placed behind the sample zone. It was found in

the study that the running buffer plug should be introduced

ahead and behind the sample and Dns-Cl plugs (Fig. 2c).

The running buffer plug will basify the Dns-Cl while they

migrate through AAs, which is crucial to label amines with

Dns-Cl and to keep the steady current in CE when the

separation starts.

The amount of AAs and Dns-Cl injected largely impacted

the labeling reaction and the Rs of D,L-AAs. As known, enough

Dns-Cl is required to fully label a target solute, but in the case of

on-column labeling, the amount of AAs has to be well

controlled in order to maintain the CE efficiency and Rs as

much as possible. In this study, the amount of AAs was opti-

mized together with the two 1.5 mM Dns-Cl plugs using a

sample plug of 0.2 mM AAs injected at 0.2 psi from 6.0 to

60.0 s. As expected, the Rs of D,L-Dns-Met and D,L-Dns-Ser

increased very fast with the injection time of AAs and gradually

came to a plateau as the injection time of AAs reached 24.0 s or

longer (Fig. 3). Meanwhile, the Rs of D,L-Dns-Asn or D,L-Dns-Ala

was more than 2.0 when the injection time of AAs less than

30.0 s. However, the injection time of AAs should be kept under

30.0 s in order to acquire high reproducibility and Rs.

Comparatively, running buffer and Dns-Cl plug length impac-

ted much less on the Rs and reproducibility than AAs.

In addition to the injection sequence and injection time, it

is important to examine the on-column reaction time. For

optimization, the on-column reaction time of four AAs was

tested from 5.0 to 50.0 min and the resulted peak area was

measured (Fig. 4). Although not only for faster reacting AAs,

such as Ala and Asn, but also for slower reacting AAs, such as

Figure 2. Effect of injection order on the on-column labelingyield denoted by the peak area of AAs (0.2 mM). On-columnlabeling was achieved by injecting the running buffer, Dns-Cl(1.5 mM), sample (0.2 mM, each solute), Dns-Cl (1.5 mM) andrunning buffer at 0.2 psi for 10.0, 3.0, 24.0, 3.0, 10.0 s, respec-tively, followed by voltage-free reaction at room temperature for35.0 min. Separation was performed at �20.0 KV. The runningbuffer was composed of 100.0 mM boric acid, 5.0 mM ammo-nium acetate, 3.0 mM Zn(II) and 6.0 mM L-Arg, adjusted to pH 8.4with solid Tris. Injection sequence: (a) B-D-S-B; (b) B-S-D-B;(c) B-D-S-D-B. B: buffer; S: sample; D: Dns-Cl. Capillary: 50 mmi.d.� 57.0 cm (50.0 cm effective); Temperature: 20.01C; UV detec-tion: 214 nm.

Figure 3. Dependence of Rs on the amount of D,L-AAs injectedfor on-column labeling with Dns-Cl. For other conditions refer toFig. 2 (c).



Ser and Met, the peak area increased with the on-column

reaction time, plateaux of peak area for the four AAs all reached

at 35.0 min. This is reasonable, considering that the tested AAs

usually need at least 35.0 min reaction time [25, 26] in pre-

column derivatization.

It is especially difficult in a tiny tube to fully mix the

sections injected; the electric field-induced mixing method was

first tried. Once an electric field of 18.0–35.0 V/cm was applied

to the capillary for 30.0 s, a small excessive peak, which paral-

leled each D-AA or L-AA main peak, resulted. To avoid the

phenomenon, then another mixing method has been tried just

by remaining Dns-Cl and AAs plugs in capillary when electric

filed was free. Thus, the best mixing effect has been achieved

(Fig. 2C). As a consequence, 35.0 min of on-column reaction

time was adopted, and the separation of five individual pair D,L-

AA (Fig. 5A-E) and the baseline separation of four pairs mixed

D,L-AAs (Fig. 5F) were successfully obtained.

It should be noted that diffusion is a key factor affecting

the separation of Dns-AAs and indeed exists in the on-

column labeling process, not only for the analytes, but also

Figure 4. Effect of on-column derivatization time on the labelingyield denoted by the peak area of D,L-AAs. For other conditionsrefer to Fig. 2.

Figure 5. Electropherogrammeasured from individualpair of D,L-AA (A–E) and thefour pairs of mixed D,L-AAs(F) for on-column labelingwith Dns-Cl using the CLE-CE method. Other condi-tions were the same asthat in Fig. 2 (c). Peak iden-tity: (1) D-Leu, (1’) L-Leu; (2)D-Ala, (20) L-Ala; (3) D-Ser, (30)L-Ser; (4) D-Lys, (40) L-Lys.



for the Dns-Cl. We presumed that if the labeling reaction

rate was greater than the rate of diffusion of the analyte

molecules, the product could be obtained virtually at the

separation between the plugs. The experimental results

indicated that sharp peaks of Dns-AAs could be obtained

even though diffusion existed in the capillary, which further

confirmed our hypothesis.

3.2 Determination of the temperature in capillary

The migration time of D,L-AA enantiomers are sensitive to

temperature of capillary and the Rs are also affected by Joule

heat generated during CE. Therefore, the reliable use of CE

required a means of accurately measuring the temperature

inside the capillary during the course of CE. Temperature in

capillaries was usually measured with a number of spectro-

scopic techniques, such as backscattering of light [27],

Raman spectroscopy [28] of hydrogen bonds.

The developed CLE-CE method combining on-column

labeling of D,L-AAs with Dns-Cl and the CE instrument (P/ACE

model 5000) has been explored for studying the dependence of

migration time on temperature. The migration time, which

shortened with increasing temperature, changed drastically with

the temperature of the capillary (Fig. 6). These changes reflected

temperature dependencies of the EOF. This dependence can be

exploited as a curve for determining an unknown temperature

during CE with any CE instrument by using the same running

buffer, the same samples and the same other conditions. We

applied the migration time–temperature determination method

to study the migration time of D,L-Dns-Ala and D,L-Dns-Asn in a

CE instrument cooling off the capillary with ambient air. We

found the temperature in the capillary during CLE-CE was

36.670.61C (Fig. 6A) for D,L-Dns-Ala and 36.071.31C

(Fig. 6B) for D,L-Dns-Asn, respectively, when the ambient air

temperature was 20.01C, which was similar to the case

mentioned in reference [28]. These results indicated that the

non-spectroscopic approach to determining temperature in

CLE-CE was available.

3.3 Quantitation of AAs

To further reveal the features of the new method, quantitation of

D,L-AAs in human serum samples was conducted. The linearity

and recovery for D- and L-AAs were evaluated according to the

method described in Section 2. The standard working equations

were constructed from the standard D- and L-AAs between peak

area (y) and concentration (x) as listed in Table 2, giving linear

range between 0.014 and 3.7 mM with correlation coefficient of

all above 0.99. The recovery of the method determined by

spiking 45.0 mM of standard D,L-AAs into serum samples was

from 90.4 to 111.7% (the last column in Table 2).

The reproducibility of the developed method was

determined by five injections of mixed D,L-AA standard

solutions artificially prepared. The run-to-run RSD of

migration time was less than 1.9% and that of peak area less

than 3.6%.

Figure 6. Dependence of migration time of (A) D,L-Ala and(B) D,L-Asn on the temperature in capillary. The on-columnlabeling technique with Dns-Cl and other conditions were thesame as that in Fig. 2 (c).

Table 2. Quantitation features of CLE-CE measured from on-column labeling D,L-Dns-AAs with the same conditions as in Table 1

Aas Working equation Range/10�3 M r2 a) LODa)/10�6 M Mean recoverya)(%)

S-1 S-2

D-Glu y 5 193.111118.0x 0.014�3.4 0.994 9.5 94.471.3 90.472.6

L-Glu y 5 205.011020.0x 0.014�3.4 0.995 9.5 91.974.5 111.771.7

D-Asp y 5 207.311376.3x 0.015�3.7 0.997 8.3 107.872.0 101.171.9

L-Asp y 5 256.111266.3x 0.015�3.7 0.998 8.3 98.074.8 94.374.1

a) y is the peak area, x the concentration of D,L-Dns-AAs, r2 the linear correlation coefficient and LOD the limit of detection. The recovery

was averaged over three measurements with the supernatants of serum as a background.



In order to demonstrate the utility of the new method in

detecting D,L-AAs in biological samples, the real human

serum samples, S-1 (Fig. 7A) and S-2 (Fig. 7B), were then

analyzed. Figure 7 depicts the typical electropherograms

related to sample S-1 and S-2. Table 3 shows that only L-Glu

and L-Asp were found in both S-1 and S-2. The type and

their content are similar [29–32] to those in serum samples

analyzed by other methods. It should be mentioned that the

quantification of the other AAs in serum or dialysis

samples, such as D,L-Ala [30], D,L-Phe [33] or D,L-Ser [34],

was possible with the presented method if those D,L-AAs

peaks (as shown in Fig. 5) would not overlap with the

unknown peaks in serum samples. In addition, further

study for the quantification of the D.L-AAs in urine or saliva

samples by using the proposed method is in progress.

4 Concluding remarks

A new strategy for integrating on-column labeling with CLE-

CE and UV detection was developed for enantioselective

analysis of micro-molar levels of AAs that lack intrinsic

chromophores. The method was simple, effective, economic

and applicable to the determination of temperature in

capillary and the quantitative analysis of chiral AAs in

serum samples. It is important to correctly introduce the

sample between two Dns-Cl plugs for efficient on-column

labeling. The reaction time and the amount of D,L-AAs

introduced seriously impacted labeling yield and CE Rs, and

should be optimized. After optimization, the method was

applicable to the direct analysis of all 17 pairs of AA

enantiomers. Meanwhile, the on-column labeling technique

did not seriously reduce the peak efficiency and Rs. As a

result, nine pairs were baseline-resolved, with seven pairs

partially separated, which is similar or parallel to pre-

column labeling method. Surprisingly, the method yielded

even better separation of Cys, Glu, Lys, Orn, Ser, Thr, Trp

and Val enantiomers than the pre-column labeling.

We gratefully acknowledge the financial support from NSFC(No. 20875091 and No. 20675084), Ministry of Science andTechnology of China (No. 2007CB714504), and ChineseAcademy of Sciences. We also thank Professor Yi Chen and Dr.Zhenpeng Guo for their kind assistance in capillary temperaturemeasurement.

The authors have declared no conflict of interest.

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Figure 7. Electrophero-grams measured from theserum samples of S-1 (A)and S-2 (B), the spikedsample S-1 with D-Asp (C)and the spiked sample S-2with D-Glu (D). The on-column labeling techniquewith Dns-Cl and otherconditions were the sameas that in Fig. 2 (c).

Table 3. AAs determined in serum samplesa)

Sample S-1/mM S-2/mM

D-Glu –a) –a)

L-Glu 27.971.4 29.871.9

D-Asp –a) –a)

L-Asp 21.671.6 24.770.8

a) The measuring conditions were the same as in Table 2; the

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On-column labeling technique and chiral ligand-exchange CE with zinc(II)-L-arginine complex as a...

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