REVIEW

Recent advances in on-line concentration and separationof amino acids using capillary electrophoresis

Tai-Chia Chiu

Received: 29 January 2013 /Revised: 23 February 2013 /Accepted: 8 March 2013 /Published online: 29 March 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract This article highlights recent methodological de-velopments in the on-line concentration and separation ofamino acids and their enantiomers using capillary electro-phoresis. Sections are dedicated to recent contributions toon-line concentration strategies such as field-amplified sam-ple stacking, large-volume sample stacking, dynamic pHjunction, transient isotachophoresis, sweeping, and the com-bination of two methods. The main applications, advan-tages, and limitations of these procedures in the biological,food, and pharmaceutical fields are addressed. Comprehen-sive tables listing on-line techniques for the concentrationand separation of amino acids and their enantiomers, cate-gorized by the stacking strategies used, background electro-lytes, sample matrix, limit of detection, and enhancementfactor, are provided.

Keywords Amino acids . Capillary electrophoresis . Chiralanalysis . On-line concentration . Preconcentration

AbbreviationsBGE Background electrolyteCE Capillary electrophoresisCZE Capillary zone electrophoresisEOF Electroosmotic flowFITC Fluorescein 5-isothiocyanateGEITP Gradient elution isotachophoresisHP-β-CD Hydroxypropyl-β-cyclodextrinHPLC High-performance liquid chromatographyITP Isotachophoresis

LIF Laser-induced fluorescenceLOD Limit of detectionLVSS Large-volume sample stackingMS Mass spectrometryNDA Naphthalene-2,3-dicarboxaldehydePEO Poly(ethylene oxide)RSD Relative standard deviationSDME Single drop microextractionSDS Sodium dodecyl sulfateTB Tris(hydroxymethyl)aminomethane–borate

Introduction

Amino acids are organic compounds that contain amino andamine groups, and are the basic units of proteins. They playmajor roles in metabolic processes, neurotransmission, andlipid transport, and their quantitative analysis in biologicalsystems is becoming increasingly important in disease diag-nosis and in elucidating nutritional influences on physiology[1–8]. Amino acids are also precursors for the synthesis ofhormones and low molecular weight nitrogenous substances,which have significant biological importance [1, 2]. Aminoacids have important physiological functions and they playessential roles in the physiology of organisms. They are pres-ent at trace levels in biological fluids and tissues, but thecomplex matrices in biological samples make them difficultto analyze. Trace analysis of amino acids is, therefore, re-quired in several fields, including the clinical diagnosis ofcongenital errors in amino acid metabolism, biomedical re-search, bioengineering, and food science [9–13].

The development of methods for amino acid analysis hasclosely paralleled development of analytical tools in recentyears. The most frequently used methods have been chromato-graphic and electrophoretic techniques [14–21]. Some efficientmethods, such as high-performance liquid chromatography

Published in the topical collection Amino Acid Analysis with guesteditor Toshimasa Toyo'oka.

T.-C. Chiu (*)Department of Applied Science, National Taitung University,684, Section 1, Chunghua Road,Taitung 95002, Taiwane-mail: tcchiu@nttu.edu.tw

Anal Bioanal Chem (2013) 405:7919–7930DOI 10.1007/s00216-013-6906-1

(HPLC) [14–17] and capillary electrophoresis (CE) [19–21],coupledwith detection systems includingUV–vis absorption orlaser-induced fluorescence (LIF) detectors or mass spectrome-try (MS), have been reported. However, HPLC is time-consuming, and requires large samples and large amounts ofreagents. In contrast, CE has attracted considerable interestbecause of its high separation resolution and efficiency, fastanalysis, versatility for numerous analytes, and extremely small(nanoliter) sample injection volume. CE is, therefore, a power-ful separation tool, and it has been used in various fields ofchemistry [22–24], biology [25–28], medicine [29], forensicscience [30], and pharmaceutics [31, 32]. UV absorbance de-tection, fluorescence detection, and mass spectrometry are con-ventionally used for detecting amino acids after CE separation[20, 21, 24]. Fluorescence detection is particularly good atdetecting low concentrations of analytes compared with theother two types of detection. However, most amino acids arenot fluorescent and need to be labeled with a fluorescentreagent before analysis. The disadvantages of CE–MS instru-ments include that they are usually expensive, and that manycommon low-volatility buffers used in CE are not suitable foranalysis by CE–MS. A major disadvantage of CE–UV instru-ments is their low sensitivity, caused by the short optical pathlength for on-line UV detection and the low sample injectionvolumes.

Improvements in the sensitivity of detection after CE sepa-ration continue to be of great interest. There are at least twoalternative strategies for detector improvements now availablefor enhancing sensitivity. Several on-line preconcentrationmethods for CE have been developed and have been wellreviewed [33–40]. CE has also been coupled with various off-line extraction and cleanup methods, such as solid phase orliquid phase extraction, to enhance trace analysis sensitivity[41–44]. However, these techniques introduce more steps intothe CE procedure, decreasing its speed and increasing the riskof analyte losses. The most commonly used enrichment strate-gy is on-line preconcentration, or stacking, to increase theinjected amount of analytes using hydrodynamic (or pressur-ized) or electrokinetic methods. A number of on-line concen-tration strategies, including large-volume sample stacking(LVSS) [36, 37], field-amplified sample stacking [36, 37],sweeping [45–47], transient moving chemical reaction bound-ary [48], anion- or cation-selective exhaustive injection [36,37], dynamic pH junction [49, 50], and transientisotachophoresis (ITP) [36, 37], have been demonstrated. Theprinciples of several on-line sample preconcentration tech-niques are briefly described here, but off-line techniques arenot. A combination of two on-line sample preconcentrationstrategies can further improve sensitivity.

The aim of this article is to highlight recent developmentsin CE on-line preconcentration techniques for amino acidsand their enantiomers, especially those techniques that in-volve novel stacking methods and analysis of chiral amino

acids, in which case their applications are also mentioned.The first part of the article focuses on the basic principles ofpreconcentration, recent developments, and applications inamino acid analysis, and the second part of the article isfocused on the analysis of chiral amino acids. The analyticalconditions for the on-line concentration of amino acids andtheir enantiomers are summarized in Tables 1 and 2,respectively.

Principles of on-line preconcentration techniques

Most on-line CE concentration techniques are based on thechange in analyte velocity between the sample zone and theseparation solution zone. The change in analyte migrationvelocity is caused by the different electric field strengths inthe sample zone and the separation solution zone or thechange in effective charge on the analyte. Field-amplifiedsample stacking is easy to achieve by simply optimizing thecompositions of the sample solution and the separationsolution or the background electrolyte (BGE), mainly interms of the concentrations or electrical conductivities ofeach solution, leading to different electric field strengths[33]. A large volume of sample is injected into the capillary,hydrodynamically, or electrokinetically. When the voltage isapplied, sample ions migrate rapidly because the electricfield is higher in the sample zone than in the separationsolution. The migration velocity of the sample ions de-creases when they pass through the boundary between thesample zone and the separation solution zone because of thelower electric field strength in the latter, and the ions are,therefore, concentrated into a narrow zone. One drawback ofthis approach is that it is suitable for low-conductivitysamples with simple matrices to provide the necessary con-ductivity difference between the sample and the BGE. Thus,its applicability is limited to low-conductivity samples, orrequires that the sample is diluted with water or an organicsolvent to a level at which it can be stacked [69]. Anotherdrawback is that the sample injection is limited to about 5 %of the capillary volume. Exceeding this will result in bankbroadening due to the mismatch of the local electroosmoticvelocities in the BGE and the sample zone and diffusion ofthe analytes [70]. Despite these limitations, it is the mostfrequently used on-line concentration strategy.

The dynamic pH junction technique is based on a pHdifference between the sample zone and the separation so-lution [49, 50]. The technique uses significant changes inthe sample ion ionization states or electrophoretic mobilitiesat different pH values. The pH values of the sample andseparation solutions are chosen so that the analyte mobilityin each solution is significantly different; therefore, theanalytes stack at the interface between low and high pH. Itis also possible for the pH of the sample solution to match

7920 T. Chiu

Tab

le1

Sum

maryof

experimentalcond

ition

sfortheon

-lineconcentrationandseparatio

nof

aminoacids

Metho

dAminoacids

Matrix

BGE

Detectio

nLOD

EF

Reference

LVSS–MEKC

Leu,Tyr,Val,His,Gln,Thr,

Asn,Ser,Ala,Gly,Cys,

Glu,Asp

Red

wine

200mM

TB(pH

9.0)

containing

10mM

SDSand0.6%

PEO

(8MDa)

LEDIF

495nm

0.30

–2.76

nM50–80

0[51]

CAF-IEF–

ITP–ITP

Arg,His,β-Ala

Birch

leaves

LE:0.03

Mpo

tassium

acetate(pH

8.3)

containing

0.2%

(w/w)HMPC

Con

ductivity

App

roximately

naon

molar

1,00

0,00

0[52]

TE:0.01

3M

acetic

acid

GEITP

Asp,Gly,Ser,Val

Stand

ard

LE:50

mM

citratebalanced

topH

8.3with

Tris

Fluorescence51

5nm

ND

300–10

0,00

0[53]

Sam

ple:

10nM

concentrationof

each

amino

acid

and5%

(v/v)am

pholytemixture

in0.5M

TBbu

ffer

pH-m

ediated

stacking

–tITP

Pro,Val,Thr,Ile,Leu,Glu,

Met,His,Phe,Arg,Tyr

Desulfovibrio

vulgaris

lysates

LE:am

mon

ium

hydrox

ide(12.5%)in

methano

landwater

(20:80

,v/v)

FT-ICRMS

0.1–

2.0μM

20–30

0[54]

TE:form

icacid

(4M)in

methano

landwater

(20:80

,v/v)

Sweeping

–MEKC

Leu,Glu,Pro,Ser,Lys,His,Phe

Salivaand

greentea

0.05

%(v/v)acetic

acid

(pH

4.5)

and50

mM

copp

ersulfate

UVat

254nm

100–

500nM

ND

[55]

GEITP–CZE

Asp,Glu,Gly,Ala,Ser,Val

Stand

ard

LE:50

mM

citratebalanced

with

Tris(pH

8.5)

LIF

500nm

0.2–

2.0pM

ND

[56]

TE:25

0mM

TB(pH

8.5)

SDME–CZE

Arg,Lys,His,Ala,Gly,Glu,Asp

Stand

ard

20mM

tetraborate(pH

9.2)

LIF

520nm

ND

190–35

0[57]

MAB–ACE

His

Urine

50mM

Na 2HPO4–NaH

2PO4(pH

6.0)

containing

1.0mM

NaC

lUVat

208nm

43ng

/mL

48[58]

Transient

trapping

–

MEKC

Leu,Ile,Val,Phe

Stand

ard

29mM

phosph

atebu

ffer

(pH.5)containing

25mM

SDSand10

%(v/v)methano

lLIF

516nm

5–80

0pM

106–12

5[59]

Dyn

amic

pHjunctio

n–CE

Tyr

Urine

1.0M

form

icacid

(pH

1.8)

UVat

200nm

19nM

68[60]

Phe

54nM

33

CM-SDME–CE

HPhe,Trp,Phe,Tyr

Urine

100mM

phosph

atebu

ffer

(pH

2.0)

UVat

200nm

70–50

0nM

120

[61]

ACEaffinity

capillary

electrop

horesis,BGEbackgrou

ndelectrolyte,

CAF-IEFcarrier-am

pholyte-free

isoelectricfocusing,CEcapillary

electrop

horesis,CM

carriermediated,

CZEcapillary

zone

electrop

horesis,EFenhancem

entfactor,FT-ICRMSFou

rier

transform

ioncyclotronresonancemassspectrom

etry,G

EITPgradient

elutionisotacho

phoresis,H

MPChy

drox

ypropy

lmethy

lcellulose,

HPhe

homop

heny

lalanine,ITPisotacho

phoresis,LEleadingelectrolyte,

LEDIF

light-emitting-diod

e-indu

cedfluo

rescence,LIF

laser-indu

cedfluo

rescence,LOD

limitof

detection,

LVSS

large-

volumesamplestacking

,MABmov

ingaffinity

boun

dary,M

EKCmicellarelectrok

inetic

chromatog

raph

y,ND

notdeterm

ined,PEO

poly(ethyleneox

ide),SD

MEsing

ledrop

microextractio

n,SD

Ssodium

dodecylsulfate,TBtris(hyd

roxy

methy

l)am

inom

ethane–b

orate,TEterm

inatingelectrolyte,tITPtransientisotacho

phoresis,Tristris(hyd

roxy

methy

l)am

inom

ethane

Preconcentration of amino acids and their enantiomers using CE 7921

Tab

le2

Sum

maryof

experimentalcond

ition

sfortheon

-lineconcentrationandseparatio

nof

chiral

aminoacids

Metho

dAminoacids

Matrix

BGE

Chiralselector

Detectio

nLOD

EF

Reference

pH-m

ediated

stacking

andsw

eeping

Tyr,Thr,Asn,Phe,His,

Glu,Met,Ala,Arg,Asp,

Ile,Ser,Trp,Gln,Val,

Leu,Lys

Brain

microlysate

ofarctic

grou

ndsquirrel

25mM

phosph

ate(pH

2.0)

containing

2wt%

sulfated

β-CD

Sulfatedβ-CD

LIF

490nm

0.2–

0.3nM

~100

[62]

LVSS–MEKC

Val,Ile,Leu

Urine

andplasma

20mM

TB(pH

9.0)

containing

150mM

SDS,50

mM

HP-β-CD,

and0.5%

w/v

PEO

(8MDa)

HP-β-CD

LEDIF

488nm

0.18

–0.22

nM18

3–31

6[63]

LVSS–MEKC

Asp,Thr,His,Val,Ile,Leu

CSF,

soymilk

,andbeer

150mM

TB(pH

9.0)

containing

150mM

SDS,60

mM

HP-β-CD,

and0.6%

w/v

PEO

(8MDa)

HP-β-CD

LEDIF

488nm

0.24

–0.25

nM10

0–110

[64]

SDME–CE

Leu,Ala,Glu,Asp

Stand

ard

80mM

borate

containing

12mM

β-CD

and18

mM

STC

β-CD

andSTC

LIF

520nm

1–3pM

3,00

0–6,80

0[65]

tMCRB

Glu,Phe,Tyr,Pro,Trp

Stand

ard

BGE1:

2.3M

acetic

acid

(pH

2.1)

(+)-(18-Crown-6)-

2,3,11,12-

tetracarbo

xylic

acid

UVat

200nm

andC4D

2μM

10[66]

BGE2:

BGE1+

10mM

(+)-(18-

crow

n-6)

2,3,11,12-tetracarbo

xylic

acid

LVSEP–ASEI–CE

Val,Met,Phe,Leu,

Ser,Ala

River

water

100mM

Tris–ph

osph

ate(pH

6.0)

con

taining2mM

vancom

ycin

Vancomycin

UVat

214nm

0.38

–2.10

ng/m

L~1

,000

[67]

LVSEP–CZE

Arg,Met,Leu

Urine

40mM

borate

buffer

(pH

9.5)

containing

30mM

SDSand10

mM

γ-CD

γ-CD

LIF

520nm

ND

1,00

0–1,30

0[68]

ASE

Ianion-selectiveexhaustiv

einjection,

CD

cyclod

extrin,CSF

cerebrospinalfluid,

HP-β-CD

hydrox

ypropy

l-β-cyclodextrinLV

SEPlarge-vo

lumesamplestacking

with

anelectroo

smotic

flow

pump,

STCsodium

taurod

exycho

late,tM

CRBtransientmov

ingchem

ical

reactio

nbo

undary

7922 T. Chiu

that of the separation solution, in which case a plug ofsolution with a different pH is introduced in front of thesample, and the analytes stack at the interface between thesample and the plug. This type of stacking is suitable forsamples with high conductivity or a pronounced biologicalmatrix. The pH effect is used to create a low-conductivitysample zone. Weak electrolytes are selectively focusedusing the pH dependence of their effective mobilities. Thehydrodynamically injected sample has a pH significantlydifferent from that of the BGE, and the analytes enter theBGE by electromigration across the pH junction, wheretheir effective mobilities change abruptly (usually decreas-ing strongly) and they are focused into a narrow zone. Asimilar preconcentration technique is called pH-mediatedstacking, and this also uses a moving chemical reactionboundary. A low-conductivity sample zone is created by thetitration of the BGEs with a strong acid or base from thesample zone. Charged analytes travel at a high velocitythrough the resulting low-conductivity zone until they reachthe high-conductivity BGE, where they slow down and accu-mulate at the interface between the sample and the BGE. Thesample is electrokinetically injected into the capillary, andcounterions from the BGE migrate into the sample zone. Aplug of strong acid or base is electrokinetically introduced intothe capillary, where it titrates the BGE counterions in thesample zone and decreases its conductivity. A low samplepH and high BGE pH is the most frequently used dynamic pHjunction system, although it is vital to recognize that theseconditions are heavily dependent on the set of analytes andtheir pKa [49].

ITP is a CE separation mode in which analytes areseparated in successive adjoining zones in decreasing elec-trophoretic mobility order [71, 72]. The principle of ITP canbe applied to preconcentration in capillary zone electropho-resis (CZE), which is named as transient ITP [36, 37]. Theseparated analyte peaks give a stepwise electropherogram,which is different from CZE peaks. In ITP, the sample isintroduced between the leading electrolyte solution and theterminating electrolyte solution. The leading electrolyte hasa higher electrophoretic mobility and the terminating elec-trolyte has a lower electrophoretic mobility than any of theanalyte ions. When the voltage is applied, the concentrationof each separated zone is automatically adjusted to thatdetermined by the concentration of the ions in the precedingzone. The most powerful advantages of transient ITP is itsability to concentrate trace analytes in the presence of largeconcentrations of matrix ions, and this can yield enhance-ments of more than 106-fold [73].

Sweeping uses the interactions between a pseudostationaryphase or complexing agent in the separation buffer and thesample in a matrix that is devoid of the additives [45–47]. Theaccumulation is based on chromatographic partitioning, com-plexation, or any interaction between analytes and the additives

during electrophoresis. Sweeping is a powerful on-line concen-tration technique owing to its versatility and effectiveness in thestacking of both neutral and charged analytes. The analytesmust have a strong affinity for the pseudostationary phase orcomplexing agent for preconcentration to be optimized bysweeping.

On-line concentration and separation of amino acids

Dynamic pH junction

Tak et al. [60] showed that in-capillary stacking of Phe andTyr can improve sensitivity. Using a dynamic pH junction,good resolution (the resolution between Phe and Tyr is 1.4)was obtained for Phe and Tyr dissolved in water using asample injection volume of 196 nL (10 % of the capillaryvolume). The limits of detection (LODs) were 0.036 and0.049 μM for Phe and Tyr, respectively. The optimizedprocedure for urine samples was a 1.7-nL injection of 12.5 %ammonium hydroxide, followed by a 196-nL injection ofurine spiked with Phe and Tyr. Tak et al. were able toanalyze as little as 54 nM Phe and 19 nM Tyr in urine,and found good linearity (R2>0.96) for a concentrationrange of 10–175 μM. The relative standard deviation(RSDs) for the peak area and migration time were less than7.5 % and 0.75 %, respectively, for Phe and Tyr.

Gradient elution ITP

Gradient elution ITP (GEITP) is a novel format forperforming simultaneous capillary ITP enrichment and elec-trophoretic separation that was described by Shackman andRoss [53]. They investigated the effects of a number ofparameters, including the initial counterflow rate, electricfield, and leading electrolyte concentration, on the GEITPenrichment process. Greater than 10,000-fold enhancementsof the LODs and sensitivity were achieved in a run time ofless than 2 min, giving low picomolar LODs using arc lampillumination and low-cost CCD detection. Specific enrich-ment and separation examples were demonstrated, includingsmall dye molecules (6-carboxyfluorescein, fluorescein),DNA (fluorescein-labeled single-stranded DNA of 30 cyto-sine bases), amino acid mixtures (Asp, Gly, Ser, and Val),and protein mixtures (green fluorescent protein andDiscosoma striate red fluorescent protein).

Sweeping

Jiang et al. [55] reported a CE method based on coordinationinteractions between amino acids and Cu(II) ions and an on-line sweeping technique for the direct analysis ofunderivatized amino acids (Leu, Glu, Pro, Ser, Lys, His,

Preconcentration of amino acids and their enantiomers using CE 7923

and Phe). Figure 1 shows a schematic diagram of thecoordination-sweeping enrichment technique. The freeanalytes in the sample zone are neutral or slightly charged,so their effective mobility is very low (Fig. 1a). After thevoltage has been applied, copper ions, the transition ions,which are highlymobile, migrate into the sample zone quicklyand form complexes with the free analytes. The mobility ofthe complexes is generally much greater than the mobility ofthe free analytes. Therefore, the free analytes in the samplezone can be significantly concentrated at the interface betweenthe sample zone and the BGE zone (Fig. 1b). On the basis ofdynamic complexation, the amino acids are swept by thecopper ions and can further improve the sensitivity of thetechnique. A 50 mM CuSO4, 0.05 % acetic acid aqueoussolution (pH 4.5) was used as the BGE. The LODs for theamino acids ranged from 0.1 to 0.5 μM, and all analytes werelinear (with R2>0.99) over concentrations of two orders ofmagnitude. The repeatability of the technique was illustratedby the RSD being less than 3 % for both the migration timeand the peak height (n=5). Amino acids were analyzed withacceptable sensitivity in human saliva and green tea.

Meng et al. [58] demonstrated a similar concept usingnickel(II) ions, and selectively detected His. The sample wasdiluted in the BGE, 1.0 mM NaCl in 50 mM Na2HPO4–NaH2PO4 buffer (pH 6.0), and the capillary was filled withthe sample. From the BGE, 2.0 mM Ni(II) was used in theanodic and cathodic vials. The selectivity of this approachfor His when 19 other amino acids (Trp, Cys, Ala, Arg, Glu,Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr, Val, Asp,Asn, and Lys) were present is illustrated in Fig. 2a, whichshows that there was a single His–Ni(II) complex capturepeak if only His was present in the full capillary, indicatingthat good focusing was achieved. Figure 2b shows a broadand flat peak when the other 19 main amino acids (exclud-ing His) were present in the full capillary, and Fig. 2c showsthe combination of the His–Ni(II) complex capture peakfrom Fig. 2a and the flat peak of the other 19 amino acidsfrom Fig. 2b. These results reveal that the technique selec-tively isolated His in a complex matrix containing 19 other

amino acids under the given conditions. The LOD for Hiswas 43 ng/mL, which was a 48-fold enhancement on theLOD determined (by peak height) using CZE. The approachwas used to determine His in three raw human urine samplesto illustrate its applications, and relative errors of less than10 % were found between the results obtained using thismethod and the results obtained using the standard analyti-cal method (HPLC with postcolumn derivatization).

Chiu and Chang [51] demonstrated the analyses of 13naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized ami-no acids using a stacking and separation micellar electroki-netic chromatography method in the presence ofpoly(ethylene oxide) (PEO). The stacking and separationmechanism is illustrated in Fig. 3. The capillary was filledwith 2.0 M tris(hydroxymethyl)aminomethane–borate (TB)buffer (pH 10.0) containing sodium dodecyl sulfate (SDS)(Fig. 3, panel a), and a mixture of NDA derivatives of aminoacids was hydrodynamically injected into the capillary fromthe anodic end for a specified time (Fig. 3, panel b). ThePEO solution entered the capillary from the anodic end oncea high voltage was applied. SDS micelles migrating fromthe 2.0 M TB buffer zone interact with, and therefore sweep,the negatively charged NDA–amino acid derivatives, whichhave lower electrophoretic mobilities than the SDS micellesin the sample zone (Fig. 3, panel c). Both SDS micelles andNDA–amino acid derivatives migrate against the electroos-motic flow (EOF). The NDA–amino acid derivatives arepicked up by the SDS micelles when the micelles migratethrough the sample zone, and become stacked because ofthe decrease in the electric field and the increase in viscosity(Fig. 3, panel d). The optimum conditions were 2.0 M TBbuffer (pH 10.0) containing 40 mM SDS to fill the capillary,deionized water to dilute the samples, and 200 mM TBbuffer (pH 9.0) containing 10 mM SDS to prepare the0.6 % PEO separation buffer. The LODs for the NDA–amino acid derivatives (at a signal-to-noise ratio of 3)ranged from 0.30 to 2.76 nM. Sensitivity enhancements of50–800 times, compared with standard injection, wereachieved. This approach was applied to the determinationof amino acids in red wine.

Transient trapping

Sueyoshi et al. [59] successfully used transient trapping topreconcentrate amino acids (Val, Ile, Leu, and Phe) byhydrophobically labeling the analytes with three commercialdyes, fluorescein 5-isothiocyanate (FITC), succinimidyl es-ters of Alexa Fluor 488, and BODIPY FL-X. The transienttrapping principle is illustrated in Fig. 4. The micellar solu-tion (29 mM phosphate buffer at pH 5.3 containing 25 mMSDS and 10 % v/v methanol) and the sample solution(negatively charged derivatized amino acids in 34 mMphosphate buffer, pH 5.2) were successively introduced into

Fig. 1 Coordination sweeping. A free analytes, BGE backgroundelectrolyte,Mn+ metal ions, SZ sample zone. (Reprinted from [55] withpermission)

7924 T. Chiu

the capillary filled with the background solution (34 mMphosphate buffer, pH 5.2) without SDS (Fig. 4a), and thenthe separation voltage was applied. The analytes in thesample zone migrate toward the micellar zone with a fastEOF. When the analytes reach the boundary betweenthe sample solution and micellar solution zones (S/Mboundary), they are strongly incorporated into the micellesand trapped near the S/M boundary because of the effect ofmicellar diffusion from the micellar solution to the samplesolution zone. Therefore, the analyte cannot penetrate the

micellar solution zone and is focused at the S/M boundaryas an extremely narrow band (Fig. 4b). At the same time, theconcentration of micelles gradually decreases as the lengthof the micellar solution zone increases because of the diffu-sion, the difference in the velocities of micelles located neareach end of the M zone, and the difference in electrophoreticmobility between the micelles and the surfactant monomers.As a result, the interactions between the analytes and themicelles also decreases, allowing the analytes trapped at theS/M boundary to be released into the micellar solution zone

Fig. 2 Selective isolation ofHis rather than the other 19main amino acids by themethod of moving affinityboundary formed with 2.0 mMNi(II) in the anodic vial and Hisand/or 19 other amino acids inthe whole capillary. (Reprintedfrom [58] with permission)

Fig. 3 Evolution of stackingand separation of naphthalene-2,3-dicarboxaldehyde (NDA)–amino acid derivatives bycapillary electrophoresis–light-emitting-diode-inducedfluorescence in the presence ofelectroosmotic flow (EOF) andpoly(ethylene oxide) (PEO)solutions. SDS sodium dodecylsulfate, TB tris(hydroxymethyl)aminomethane–borate.(Reprinted from [51] withpermission)

Preconcentration of amino acids and their enantiomers using CE 7925

in the order of their hydrophobicity (Fig. 4c, d). Conse-quently, the analytes are separated by the difference in theirrelease times. After being released, the analytes migratetoward the cathode (Fig. 4e). The labeled amino acids werewell concentrated and separated using transient-trappingmicellar electrokinetic chromatography, and a 160-fold en-hancement of the LOD was achieved, with an LOD of5.0 pM for labeled Phe.

Single drop microextraction

A sensitive method for the analysis of zwitterionic amino acidsusing single drop microextraction (SDME), coupled in-linewith CE by attaching a drop to the tip of a capillary, wasdeveloped by Park et al. [57]. Acidic analytes from the donorphase can be extracted into the organic layer and then back-extracted into the acceptor phase using a two-layer drop madeof an aqueous basic acceptor phase covered with a thin organiclayer. Amino acids without a charged side chain werederivatized with 4-fluoro-7-nitro-2,1,3,-benzoxadiazole to con-vert them into carboxylic acids. These derivatized amino acidsare predominantly neutral and are selectively concentrated intothe basic donor phase. With this selective SDME, up to 350-fold enrichment of Ala of was achieved within 5 min, and basicamino acids were left in the acidic donor phase. Choi et al. [61]developed carrier-mediated SDME coupled in-line with CE forthe sensitive analysis of amino acids. The end surface of a

capillary inlet tip was made hydrophobic so that a drop couldbe formed and stably attached to the tip (each day or asneeded). After it had been washed with ethanol for 3 min,the capillary inlet tip was immersed in a coating solution forabout 5 s and then dried for 10 min to complete the conden-sation reaction. The coating solution was a mixture of 5 % v/voctadecyltrimethoxysilane and 0.1 % v/v acetic acid in etha-nol. Nonane-1-sulfonic acid was added, as a carrier, to anacidic sample donor solution to form neutral ion pair com-plexes with amino acids. A schematic of the procedure for thistechnique is shown in Fig. 5. The capillary was filled with arun buffer, and the acceptor phase was injected into the cap-illary (Fig. 5a). Octanol was then injected (Fig. 5b). Then, thecapillary inlet was transferred to the sample solution (donorphase) and a backpressure was applied to form a drop of theacceptor phase covered with octanol (Fig. 5c). During extrac-tion, a backpressure was applied to prevent changes in thedrop size (Fig. 5d). After extraction, the concentrated acceptorphase was injected into the capillary (Fig. 5e). The capillaryinlet was then placed in a run buffer vial and electrophoresiswas conducted (Fig. 5f). With a 20-min SDME with agitationof the donor phase, enrichment factors of up to 120 wereachieved for four aromatic acids (homophenylalanine, Trp,Phe, and Tyr), giving LODs of 70–500 nM. The linear dy-namic ranges for the peak areas were 1–100 μM, with R2>0.9959. The RSDs (intraday) for the migration times and peakareas were 0.01–0.04 % and 2.0–3.7 %, respectively.

Fig. 4 The principle oftransient trapping. νS,S, νS,M, νS,BGS, and νS,B are the apparentvelocities of the samples in thesample solution (S), micellarsolution (M), backgroundsolution (BGS), and near theboundary between the samplesolution and micellar solutionzones, respectively. νmc andνEOF are the apparent velocityof the micelle and theelectroosmotic velocity,respectively. (Reprinted from[59] with permission)

7926 T. Chiu

Furthermore, because the pretreatment effectively excludesthe sample matrix, it was applicable to a human urine samplecontaining 100 mM NaCl.

Combination of different on-line samplepreconcentration strategies

Although each on-line sample preconcentration strategy isuseful, the recent development of combinations of two ormore techniques can lead to further improvements in sensi-tivity. The combination of carrier-ampholyte-free isoelectricfocusing with ITP mobilization and conductivity detectionin ITP mode was demonstrated for the preconcentration andseparation of three amino acids (His, Arg, and β-Ala) byProcházková et al. [52]. The first step was to continuouslydose amino acids, from an infinite-volume reservoir, byelectromigration to the column, where a sharp, stationaryneutralization reaction boundary is created in between theacidic and basic primary electrolytes. Amino acids forwhich the pI decreases because of the pH difference areselectively focused. In the second step, the focused zonesare mobilized and amino acids are accumulated. The migra-tion mode is changed from carrier-ampholyte-free isoelec-tric focusing to ITP, and the analytes start to migrate towardthe analytical capillary. In the third step, amino acids aretransferred into the analytical column, which is equippedwith a conductivity detector, and are detected in the newleading electrolyte. Up to 100-fold improvement in thefocusing of amino acids in the column and nanomolar LODscan be achieved using this technique. This approach wasused to analyze free amino acids in a leaf extract.

Baidoo et al. [54] demonstrated the use of a combination ofpH-mediated stacking and transient ITP coupled with Fouriertransform ion cyclotron resonance MS to improve the overalldetection of cationic metabolites in the bacteriumDesulfovibrio vulgarisHildenborough. This method providedgood separation efficiency, reproducibility, and linearity. TheLODs for selected amino acids (Ala, Ser, Pro, Val, Thr, Ile,Leu, Asp, Lys, Met, His, Phe, Arg, and Tyr) were between0.10 and 1.99 μM.

The combination of GEITP and CZE in a single column toprovide good analyte resolution and sensitivity was developedby Davis et al. [56]. Figure 6 shows the steps involved inperforming GEITP–CZE. During the initial stage (Fig. 6,panel a) the sample and terminating electrolyte are present atthe microcolumn inlet at a negative potential (for anionicanalyses). The leading electrolyte is dispersed into the samplereservoir but the relatively high bulk counterflow is reduced.GEITP is initiated outside the microcolumn (Fig. 6, panel b),and an ionic interface that focuses the analyte between theleading electrolyte and the terminating electrolyte is formed(Fig. 6, panel c). GEITP enrichment continues while the bulkflow is reduced further until the enriched zones are introducedinto the separation column (Fig. 6, panel c). Once the enrichedzones are on-column, the applied potential and pressure areterminated (Fig. 6, panel d), and the inlet solution is replacedwith the leading electrolyte. The electric field is reinitiated anda static hydrodynamic flow attained to ensure the analytesmigrate toward the detector (Fig. 6, panel e). These conditionsare equivalent to those in transient ITP, and polarity inversionis not required. It is believed that refocusing during transientITP reduces diffusional band broadening during the solutionswitching stage. Separation proceeds by CZE once the inlet

Fig. 5 Carrier-mediated singledrop microextraction.(Reprinted from [61] withpermission)

Preconcentration of amino acids and their enantiomers using CE 7927

leading electrolyte breaches the ITP stack, until the zonesmigrate past the detector (Fig. 6, panel f). This approach wasshown to achieve femtomolar LODs for amino acids (Asp,Glu, Gly, Ala, Ser, and Val), and separations were achieved ina short-length microcolumn without the need for spacing ions.Six amino acids were separated, and LODs as low as 200 fMwere achieved using a capillary format with a total analysistime of 11 min.

On-line concentration and separation of chiral aminoacids

The importance of separating chiral amino acids arises fromthe need to take advantage of the different biological functionsand activities associated with each enantiomer. It is widelyaccepted that D-(R)-amino acids and L-(S)-amino acids havedifferent biological properties. Therefore, the separation ofamino acid enantiomers is recognized as being of great im-portance in many research fields, such as chemistry, biology,

medicine, and pharmacology [74–76]. Adding chiral selectorsto the BGE has made CE a reliable and promising alternativeto gas chromatography and HPLC [77–79]. The analysis ofchiral amino acids by CE and its practical applications havebeen described in recent reviews [80–84]. However, there arequite a few studies of strategies for improving the enantiomer-specific sensitivity.

A CE–LIF method for the chiral separation and stackingof 13 NDA-derivatized amino acids with 2 wt% sulfated-β-cyclodextrin in 25 mM phosphate buffer at pH 2.0 and avoltage of −30 kV was demonstrated by Kirschner et al.[62]. The LODs for NDA–DL-Ser and NDA–DL-Glu were0.2 and 0.3 nM, respectively. This method has been used toquantify DL-amino acids sampled from the brains of livearctic ground squirrels. Calibration curves were constructedfor detection at low micromolar concentrations.

Sweeping

Tseng et al. [63] used a discontinuous system for stackingand separating three NDA–amino acid enantiomers (DL-Val,

DL-Ile, and DL-Leu). The capillary was filled with100 mM TB (pH 9.0) containing 150 mM SDS and50 mM hydroxypropyl-β-cyclodextrin (HP-β-CD), and theseparation buffer was 20 mM TB (pH 9.0), 150 mM SDS,50 mM HP-β-CD, and 0.5 % w/v PEO. The LODs for theamino acid enantiomers were in the range 0.18–0.22 nM.This method showed potential for the determination of DL-Leu in urine and plasma samples. Lin et al. [64] reported asimilar technique, filling the capillary with 150 mM TB(pH 9.0) containing 150 mM SDS and 60 mM HP-β-CD,and using 150 mM TB (pH 9.0) containing 150 mM SDS,60 mM HP-β-CD, and 0.6 % w/v PEO in the buffer vials.This approach enhanced the sensitivity for D-Asp and L-Asp100-fold and 110-fold, respectively, compared with normalinjection, and it was applied to the determination of DL-Aspin cerebrospinal fluid, soy milk, and beer.

Single drop microextraction

A highly sensitive method for analyzing four chiral aminoacids (DL-Leu, DL-Ala, DL-Glu, and DL-Asp) using CE–LIFdetection coupled with in-line SDME was demonstrated byLiang et al. [65]. A drop of a basic aqueous acceptor phase(80 mM sodium borate, pH 9.6) covered with a thin organiclayer was formed at the tip of a capillary by simple combi-nations of the CE system sample handling procedures.FITC-derivatized amino acids in an acidic donor solution(0.1 M HCl) were enriched into the drop through the organiclayer (octanol). The organic drop was then hydrodynamical-ly injected into the capillary at 6 psi for 13 s, and the FITC-derivatized amino acids were extracted into the basic

Fig. 6 The stages to perform gradient elution isotachophoresis–capil-lary zone electrophoresis (CZE). LE leading electrolyte, TE terminatingelectrolyte, tITP transient isotachophoresis. (Reprinted from [56] withpermission)

7928 T. Chiu

acceptor drop through the octanol layer for 10 min, drivenby the pH difference between the BGE (80 mM boratebuffer, pH 9.3, containing 12 mM β-cyclodextrin) and18 mM sodium taurodeoxycholate. FITC was used as alabeling agent and as a modifier to aid the extraction ofzwitterionic amino acids by blocking the amino group andincreasing the hydrophobicity. Several-thousand-fold en-richments were achieved, giving LODs as low as 1–3 pM,with 10 min extraction when a microstirrer was fitted to theCE instrument.

Transient moving chemical reaction boundary

Anouti et al. [66] demonstrated the use of a transient movingchemical reaction boundary for the on-line preconcentrationof native amino acid enantiomers (DL-Phe and DL-Thr) inheart-cutting 2-D CE with multiple contactless conductivitydetection points. The sample matrix was 0.8 M ammoniumformate (pH 8.56) and the BGEwas 2.3M acetic acid (pH 2.3)in the first dimension of the heart-cutting 2-D CE. The seconddimension was run with the same electrolyte, but containing(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. DL-Phe and

DL-Thr were separated from a mixture of 22 native aminoacids with an LOD of 2 μM for L-Thr.

LVSS with an EOF pump

Wang et al. [67] demonstrated a highly sensitiveenantioseparation method for six 9-fluorenylmethylchloroformate derivatized amino acids (DL-Val, DL-Ala, DL-Phe, DL-Ser, DL-Met, and DL-Leu) using CE with LVSS withan EOF pump and anion-selective exhaustive injection. TheBGE was 100 mM tris(hydroxymethyl)aminomethane–phos-phate (pH 6.0) and the chiral selector was 2 mM vancomycin.The LODs for the chiral amino acids were in the range 1.07–2.10 ng/mL.

Kawai et al. [68] achieved the chiral separation of threeFITC-derivatized amino acids (DL-Arg, DL-Met, and DL-Leu) using CE with LVSS with an EOF pump. The BGEwas 40 mM borate buffer (pH 9.5) containing 30 mM SDSand the chiral selector was 10 mM γ-cyclodextrin. Theenhancement factors for Arg, Met, and Leu were 1,000,1,100, and 1,300, respectively.

Conclusions

In this article we have reviewed recent developments in on-lineconcentration and separation approaches for the highly sensi-tive and fast analysis of amino acids and their enantiomersusing CE methods. A large number of diverse methods havebeen developed for biological applications, demonstrating that

there is great interest in CE from scientists who are interested inanalyzing amino acids and their enantiomers. It has been dem-onstrated that CE is a suitable technique for the quantificationof amino acids and their enantiomers, even in complicatedsample matrices. Expansion of the analyte spectrum and im-provements in quantification limits, to the picomolar range orlower, still pose great challenges. Finally, there is a need forsimple, robust, automated sample preparation to offer high-speed and high-throughput analysis.

Acknowledgment This work was supported by the National ScienceCouncil of Taiwan under contract NSC 101-2113-M-143-001.

References

1. Wu G (2009) Amino Acids 37:1–172. Elango R, Ball RO, Pencharz PB (2009) Amino Acids 37:19–273. Garibotto G, Sofia A, Saffioti S, Bonanni A, Mannucci I, Verzola

D (2010) Clin Nutr 29:424–4334. Brasse-Lagnel CG, Lavoinne AM, Husson AS (2010) Biochimie

92:729–7355. Lee W-T (2011) Brain Dev 33:745–7526. Ohide H, Miyoshi Y, Maruyama R, Hamase K, Konno R (2011) J

Chromatogr B 879:3162–31687. Friedman M, Levin CE (2012) Amino Acids 42:1553–15828. Billard J-M (2012) Amino Acids 43:1851–18609. Perry M, Li Q, Kennedy RT (2009) Anal Chim Acta 653:1–22

10. Kaspar H, Dettmer K, Gronwald W, Oefner PJ (2009) AnalBioanal Chem 393:445–452

11. Waldhier MC, Gruber MA, Dettmer K, Oefner PJ (2009) AnalBioanal Chem 394:695–706

12. Atanassova SS, Panchev P, Ivanova M (2010) Amino Acids38:1277–1282

13. Piñero M-Y, Bauza R, Arce L (2011) Electrophoresis 32:1379–1393

14. Cserháti T (2007) Biomed Chromatogr 21:780–79615. Ilisz I, Aranyi A, Pataj Z, Péter A (2012) J Pharm Biomed Anal

69:28–4116. Miyoshi Y, Koga R, Oyama T, Han H, Ueno K, Masuyama K, Itoh

Y, Hamase K (2012) J Pharm Biomed Anal 69:42–4917. Ilisz I, Aranyi A, Pataj Z, Péter A (2012) J Chromatogr A

1269:94–12118. Schurig V (2011) J Chromatogr B 879:3122–314019. Siri N, Lacroix M, Garrigues J-C, Poinsot V, Couderc F (2006)

Electrophoresis 27:4446–445520. Poinsot V, Carpéné M-A, Bouajila J, Gavard P, Feurer B, Couderc

F (2012) Electrophoresis 33:14–3521. Desiderio C, Iavarone F, Rossetti DV, Messana I, Castagnola M

(2010) J Sep Sci 33:2385–239322. Viglio S, Fumagalli M, Ferrari F, Bardoni A, Salvini R, Giuliano S,

Iadarola P (2012) Electrophoresis 33:36–4723. Castro-Puyana M, García-Cañas V, Simó C, Cifuentes A (2012)

Electrophoresis 33:147–16724. Geiger M, Hogerton AL, Bowser MT (2012) Anal Chem 84:577–

59625. Subirats X, Blaas D, Kenndler E (2011) Electrophoresis 32:1579–

159026. Petr J, Maier V (2012) Trends Anal Chem 31:9–2227. Rabanes HR, Guidote AM Jr, Quirino JP (2012) Electrophoresis

33:180–195

Preconcentration of amino acids and their enantiomers using CE 7929

28. Ramautar R, Somsen GW, de Jong GJ (2013) Electrophoresis34:86–98

29. Espada A, Molina-Martin M (2012) Drug Discov Today 17:396–40430. Pascali JP, Bortolotti F, Tagliaro F (2012) Electrophoresis 33:117–

12631. Suntornsuk L (2010) Anal Bioanal Chem 398:29–5232. Pioch M, Bunz S-C, Neusüß C (2012) Electrophoresis 33:1517–

153033. Simpson SL Jr, Quirino JP, Terabe S (2008) J Chromatogr A

1184:504–54134. Malá Z, Gebauer P, Boček P (2011) Electrophoresis 32:116–12635. Šlampová A, Malá Z, Pantůčková P, Gebauer P, Boček P (2013)

Electrophoresis 34:3–1836. Breadmore MC, Dawod M, Quirino JP (2011) Electrophoresis

32:127–14837. Breadmore MC, Shallan AI, Rabanes HR, Gstoettenmayr D,

Keyon ASA, Gaspar A, Dawod M, Quirino JP (2013)Electrophoresis 34:29–54

38. Chen Y, LüW, Chen X, TengM (2012) Cent Eur J Chem 10:611–63839. ALOthman ZA, Dawod M, Kim J, Chung DS (2012) Anal Chim

Acta 739:14–2440. Wen Y, Li J, Ma J, Chen L (2012) Electrophoresis 33:2933–295241. Ramautar R, Somsen GW, de Jong GJ (2010) Electrophoresis

31:41–5442. Xie H-Y, He Y-Z (2010) Trends Anal Chem 29:629–63543. Ramautar R, de Jong GJ, Somsen GW (2012) Electrophoresis

33:243–25044. Kohler I, Schappler J, Rudaz S (2013) Anal Bioanal Chem

405:125–14145. Aranas AT, Guidote AM Jr, Quirino JP (2009) Anal Bioanal Chem

394:175–18546. Silva M (2011) Electrophoresis 32:149–16547. El Deeb S, Iriban MA, Gust R (2011) Electrophoresis 32:166–18348. Cao C-X, Fan L-Y, Zhang W (2008) Analyst 133:1139–115749. Kazarian AA, Hilder EF, Breadmore MC (2011) J Sep Sci

34:2800–282150. Ptolemy AS, Britz-McKibbin P (2008) Analyst 133:1643–164851. Chiu T-C, Chang H-T (2007) J Chromatogr A 1146:118–12452. Procházková B, Glovinová E, Pospíchal J (2007) Electrophoresis

28:2168–217353. Shackman JG, Ross D (2007) Anal Chem 79:6641–664954. Baidoo EEK, Benke PI, Neusüss C, Pelzing M, Kruppa G, Leary

JA, Keasling JD (2008) Anal Chem 80:3112–312255. Jiang X, Xia Z, Wei W, Gou Q (2009) J Sep Sci 32:1927–193356. Davis NI, Mamunooru M, Vyas CA, Shackman JG (2009) Anal

Chem 81:5452–5459

57. Park Y-K, Choi K, Badjah-Hadj-Ahmed AY, ALOthman ZA,Chung DS (2010) J Chromatogr A 1217:3357–3361

58. Meng J, Zhang W, Cao C-X, Fan L-Y, Wu J, Wang Q-L (2010)Analyst 135:1592–1599

59. Sueyoshi K, Hashiba K, Kawai T, Kitagawa F, Otsuka K (2011)Electrophoresis 32:1233–1240

60. Tak YH, Somen GW, de Jong GJ (2011) Anal Bioanal Chem401:3275–3281

61. Choi J, Choi K, Kim J, Badjah-Hadj-Ahmed AY, ALOthman ZA,Chung DS (2011) J Chromatogr A 1218:7227–7233

62. Kirschner DL, Jaramillo M, Green TK (2007) Anal Chem 79:736–743

63. Tseng W-L, Hsu C-Y, Wu T-H, Huang S-W, Hsieh M-M (2009)Electrophoresis 30:2558–2564

64. Lin K-C, Hsieh M-M, Chang C-W, Lin E-P, Wu T-H (2010)Talanta 82:1912–1918

65. Liang G, Choi K, Badjah-Hadj-Ahmed AY, ALOthman ZA,Chung DS (2010) Anal Chim Acta 677:37–42

66. Anouti S, Vandenabeele-Trambouze O, Cottet H (2010)Electrophoresis 31:1029–1035

67. Wang Z, Liu C, Kang J (2011) J Chromatogr A 1218:1775–177968. Kawai T, Koino H, Sueyoshi K, Kitagawa F, Otsuka K (2012) J

Chromatogr A 1246:28–3469. Liao H-W, Lin S-W, Wu U-I, Kuo C-H (2012) J Chromatogr A

1226:48–5470. Huhn G, Pyell U (2010) J Chromatogr A 1217:4476–448671. Malá Z, Gebauer P, Boček P (2013) Electrophoresis 34:19–2872. Bahga SS, Santiago JG (2013) Analyst 138:735–75473. Jung B, Bharadwaj R, Santiago JG (2006) Anal Chem 78:2319–

232774. Viglio S, Fumagalli M, Ferrari F, Iadarola P (2010) Electrophoresis

31:93–10475. Prokhorova AF, Shapovalova EN, Shpigun OA (2010) J Pharm

Biomed Anal 53:1170–117976. Kitagawa F, Otsuka K (2011) J Chromatogr B 879:3037–309577. Kalíková K, Riesová M, Tesařová E (2012) Cent Eur J Chem

10:450–47178. Ward TJ, Ward KD (2012) Anal Chem 84:626–63579. Tsioupi DA, Stefan-van Staden R-I, Kapnissi-Christodoulou CP

(2013) Electrophoresis 34:178–20480. Sánchez-Hernández L, García-Ruiz C, Marina ML, Crego AL

(2010) Electrophoresis 31:28–4381. Lu H, Chen G (2011) Anal Meth 3:488–50882. Mangelings D, Heyden YV (2011) Electrophoresis 32:2583–260183. Zhang H, Qi L, Mao L, Chen Y (2012) J Sep Sci 35:1236–124884. Jáč P, Scriba GKE (2013) J Sep Sci 36:52–74

7930 T. Chiu