enantiomeric separations by means of nano-lc

J. Sep. Sci. 2013, 00, 1–24 1

Anna Rocco1,2

Audrius Maruska1∗Salvatore Fanali2

1Department of Biochemistry andBiotechnologies, VytautasMagnus University, Kaunas,Lithuania

2Institute of ChemicalMethodologies, ConsiglioNazionale delle Ricerche, Rome,Italy

Received September 19, 2012Revised October 24, 2012Accepted October 26, 2012

Review Article

Enantiomeric separations by meansof nano-LC

Enantiomers represent a class of compounds extensively investigated since they can showtotally different behaviors when they interact with a chiral environment. Because of theiridentical chemical structure (they differ only in the spatial arrangement of the atoms in themolecule), the separation of optical isomers is a challenging task of analytical chemistry. Sofar employed methods for the separation of enantiomers are mainly based on chromatog-raphy. CE as well was considered as an analytical technique suitable for chiral separations,characterized by high efficiency and low consumption of reagent. Recently, miniaturizationwas introduced in LC to answer the needs to perform analyses in the minimum time, touse the smallest amount of samples and to reduce environmental pollution. Nano-LC repre-sents nowadays a valid alternative to the abovementioned conventional analytical techniques,and can be advantageously exploited for enantiomeric separation especially because it needsminute amounts of the chiral material necessary to carry out enantiomeric separations. Thisreview describes the development and applications of nano-LC in the field of chiral sepa-rations. The data reported in literature show its relevance for the study enantiomers-chiralselectors interaction, as well as for application in pharmaceutical and clinical research.

Keywords: Capillary column / Chiral separation / Enantiomer / Nano-LCDOI 10.1002/jssc.201200886

1 Introduction

Enantiomer separations represent an important topic of re-search in analytical chemistry, since the bioactivity of manymolecules (drugs, pesticides, proteins, etc.) is related to theirchirality.

In the pharmaceutical field, i.e. where a wide number ofdrugs has one or more than one asymmetric centre, only oneisomer can be responsible for the desired activity, while theother may exhibit no therapeutic effects and may be the onlyaccountable for adverse effects, as a consequence of differentstereoselective interactions with biological matrices, such asreceptors. Metabolism of two enantiomers of a certain drugmay be different as well and thus analytical methods are

Correspondence: Dr. Salvatore Fanali, Institute of ChemicalMethodologies, Consiglio Nazionale delle Ricerche, Via SalariaKm 29.300, 00015 Monterotondo, Rome, ItalyE-mail: [email protected]: +390690672269

Abbreviations: CD, cyclodextrin; CDCPC, cellulose tris(3,5-dichlorophenylcarbamate); CDMPC, cellulose tris(3,5-dimethylphenylcarbamate); CLC, capillary LC; CMPA, chiralmobile phase additive; CS, chiral selector; CSP, chiral sta-tionary phase; FITC, fluorescein isothiocyanate; HP-�-CD,hydroxypropyl-�-cyclodextrin; MA, macrocyclic antibiotic;MIP, molecular imprinted polymer; OT, open tubular;Ph-�-CD, perphenylcarbamoylated �-CD; PPAR�, perox-isome proliferator-activated receptor; SFC, supercriticalfluid chromatography; THP, tetrahydropalmatine; TM-�-CD,heptakis(2,3,6-tri-O-methyl)-�-cyclodextrin

required for chiral purity control of pharmaceuticals, phar-macokinetic, and metabolism studies, etc. [1–3].

Analytical separation techniques so far employed to dis-criminate chiral compounds are GC, LC, supercritical fluidchromatography, and electrokinetic techniques ones (mostlyCE) [4–7]. In order to discriminate chiral compounds twostrategies can be used: so called direct and indirect methods.In the second approach, analytes are derivatized with an enan-tiomerically pure reagent resulting in the formation of a pairof diastereomers, which can be afterwards separated in anachiral environment. This approach is less common becauseit requires additional reactions and derivatization can affectthe results of quantitative analysis. To the best of our knowl-edge, no works are reported in literature, concerning the sep-aration in nano-LC of enantiomers by the indirect method.

The direct approach is based on interactions with a chiralselector and results in the formation of transient, noncova-lent diastereoisomer complexes. The chiral selector can beeither bound (by a covalent bond, absorption, or entrapped)to the stationary phase contained in the column, directlybound to the wall of the column, or dissolved in the mobilephase/background electrolyte as chiral additive. The enan-tiomer resolution process is based on different interactions,between analyte enantiomers and the chiral selector (CS),which depend on chiral selector functional groups, asym-metric centers, and in many cases, on steric repulsion. Theinteraction between the analyte and the CS should be fast

∗Additional Correspondence: Professor Audrius Maruska,E-mail: [email protected]

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

2 A. Rocco et al. J. Sep. Sci. 2013, 00, 1–24

and the equilibrium constant for each enantiomer should bediverse to result in different migration time [2, 6, 8].

Recently, miniaturization, a trend of modern analyticalchemistry, was successfully introduced in conventional LC,resulting in the development of micro-LC and later on ofcapillary- and nano-LC (CLC and nano-LC, respectively). Ini-tially, there was no a clear distinction among these minia-turized chromatographic systems. Currently micro-LC, CLC,and nano-LC refer to chromatographic separations performedin capillary columns with internal diameter (id) of 0.5–1 mm,100–500 �m, and 10–100 �m, respectively. Nano-LC offersseveral advantages over conventional LC, e.g. use of smallamount of reagent (i.e. mobile and stationary phases) with aconsequent low environmental pollution, low sample require-ment, good efficiency, short analysis time, and easy couplingwith MS [9–12]. Furthermore, compared with other minia-turized techniques, such as CE and CEC, nano-LC is char-acterized by an easier reliability, reproducibility, universalitythat make it more adequate for industries ruled by regulatoryissues [13].

In case of chiral separations, where expensive chiralstationary phases (CSPs) or chiral mobile phase additives(CMPA) have to be employed, nano-LC results very usefulsince it allows to perform analysis with a very small amountof this costly material.

The aim of this review was to reveal general features ofnano-LC, outline the main aspects of chiral separation, andreport potential and applications of nano-LC for enantiosep-arations.

2 Nano-LC

Nowadays, nano-LC has established itself as a complemen-tary and/or competitive separation technique to conventionalLC, providing a wide number of important applications par-ticularly in pharmaceutical field and “omics” branches of bi-ology [14, 15].

Even if currently different apparatus for nano-LC are com-mercially available, in the past great efforts have been madeto solve technical problems such as nano-flow rate control,gradient performance, required injection volume, and detec-tion [11, 16, 17].

As nano-LC is performed in capillary columns with an idof 10–100 �m, the working flow rate usually is in the orderof a few hundred nanoliters-per-minute. The low flow ratemakes nano-LC suitable for the coupling with MS, and thisaspect markedly contributed to its success in the analyticalfield. Fused silica capillaries are mostly used for fabricationof nano-LC columns.

Capillary columns used in nano-LC can be distinguishedinto three different types: packed, open tubular (OT), andmonolithic columns [17–19].

Packed capillary columns are made with the particulatestationary phases widely used in HPLC. Usually 3–5 �m par-ticles are employed, however recently, particles of smaller size(sub-2 �m), developed to obtain higher efficiency and selec-

tivity, have been also successfully tested in nano-LC. Severalpacked capillary columns are commercially available for bothchiral and achiral separations. They can be prepared in lab-oratory as well, however, the packing procedure requires aconsiderable experience. The entrapment of the stationaryphases in the capillary column is also a challenging task andis obtained in the most of cases with retaining frits at the ex-tremity of the packed section of the column. Inlet and outletfrits are usually prepared in situ by sintering the packed ma-terial with a heated wire. This procedure causes the removalof polyimide coating at the frit zone and makes the columnfragile. Furthermore, a bad sintering can compromise theflow through the column and change the properties of thepacking material where the heating has been applied [20,21].

In OT-columns, the stationary phase is directly boundor adsorbed to the inner wall of the capillary, and for thisreason their id is lower, usually between 10 and 60 �m. Theadsorption of the stationary phase, or modifier, can occur dy-namically (weak interactions, the inner modifier is added tothe mobile phase) or physically (modifier strongly adsorbed).The layer of stationary phase can be also covalently attachedand/or cross-linked. OT-columns are characterized by a lowconvective dispersion, good efficiency, and high permeabil-ity, however, they suffer of poor selectivity and low sampleloading capacity, both due to the very low phase ratio [22,23].

The introduction of non-particulate monolithic stationaryphases in LC and then in miniaturized systems, resulted innew advantages for these techniques. In the capillary format,monolithic column can be easily prepared by in situ polymer-ization and it does not require frits, thus avoiding problemsrelated to the stationary phase packing procedures, frits per-meability, fragility, and manufacture. This kind of columns ischaracterized by high permeability and high efficiency. Thewide availability of monomers permits to obtain columnswith specific chromatographic properties [24–26].

Monolithic columns can be obtained by polymerizationof organic monomers or by sol-gel process in case of silica-based monoliths. Water-soluble comonomers in the presenceof salt as phase separation catalyzing agent, or organic solventsoluble comonomers in the presence of porogen cosolvent,can be used for the polymerization process.

Polymeric mixtures are useful for the preparation ofmolecularly imprinted monoliths, which have been used forseveral applications in the analytical field, including chiralseparations. Molecular imprinted stationary phases are car-ried out by the addition of a template (imprint) moleculein the polymeric mixture. Before starting polymerization,functional groups on the template interact with those onthe monomer(s), and when the reaction begins monomersresult arranged around the template. During the polymer-ization the template, depending on its structure, can react,forming covalent bonds or give rise to secondary interac-tions with other monomers. If the imprinting is success-ful, after the removal of the imprint molecule, the poly-mer network will posses cavities having shape, size, andfunctional groups complementary to those of the template[27, 28].


J. Sep. Sci. 2013, 00, 1–24 Liquid Chromatography 3

2.1 Microdevices

Currently, one of the main requirements in the analyticalfield is the possibility to analyze as many as possible samples,in the minimum quantity and in the shortest time. Further,a great effort is being performed in order to achieve samplepretreatment and analytical separation with the same instru-ments (so-called �-total analysis system). This has broughtto the advent of microdevices, consisting of, e.g. plastic chipthat showed high potential in the analytical field, especially for“omics” sciences [29]. They offer good peak capacity and sen-sitivity, low sample handling, minute sample requirementsand are portable, giving the opportunity to perform field anal-ysis. Mostly, microdevices are used for electrophoretic anal-ysis, since the conditions to realize this kind of separations(liquid media, application of a voltage) are easy to realizefor such small devices [30, 31]. However, the introduction ofmonolithic materials has given a new impetus to the prepa-ration of microdevices for chromatographic purposes. In situpreparation instead of packing procedure and high perme-ability of the media can be considered as the main advantagesthat should make easier to realize chromatographic systemin chip dimensions. At the moment, promising results wereachieved with microchip CEC [32–35]. As an example, Zeng etal. obtained the enantioresolution of FITC-labeled dansyl-d,l-threonine in about 100 s, employing a microdevice based ona monolithic polydimethylsiloxane (PDMS) column deriva-tized with allyl-�-CD [36]. In a more recent work [37], thesame group developed a chip-based enantioselective open-tubular CEC employing BSA-gold nanoparticle conjugates asa CSP. The simultaneous enantioresolution of norephedrineand ephedrine was achieved in less than 300 s.

3 Chiral separations

Chiral compounds are classically defined as not superposableon their mirror image. Chirality does not affect chemico-physical properties of these molecules, with exception ofpolarized light rotation in opposite direction, however, itcan strongly influence activities of enantiomers in a chiralenvironment. For this reason, the stereochemistry of com-pounds is an important tool with regard to interaction be-tween stereoisomers and biological targets, such as receptorsand enzymes, which are at molecular level homochiral [38].

This has resulted in the requirements for chiralcompounds-based products and over the last decades, therehas been a growing interest in the separation of chiral drugs.

The determination of the stereochemical composition orstereochemical purity control are required in pharmaceuti-cal industry by regulatory authorities, such as US Food andDrug Administartion and the European Agency for the Eval-uation of Medicinal Products, which promote those drugsdeveloped as single enantiomer. This kind of inspection iscrucial not only during chiral drug synthesis, but also duringthe determination and monitoring of the drug metabolites fortherapy optimization or toxicological studies. Further, stereo-

chemical studies involve chemical and cosmetic industry, aswell as biological, agrochemical, and environmental analyses[39, 40].

In all these fields, analytical separative methods arerequired, e.g. for the quantitative assessment of the exactcomposition of a racemate or determining the enantiomericexcess (ee) of an enantiomeric pure substance. The latter typeof verification gains in importance since, in 2006, The Inter-national Conference on Harmonisation of Technical Require-ments for Registration of Pharmaceuticals for Human Use(ICH) guidelines included the noneffective enantiomer (dis-tomer) as impurity in case of single enantiomer drug (www.emea.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002823.pdf), [41].

Considering that already at a content of 0.05% the enan-tiomeric impurity should be reported, and identified at 0.1%,it results evident the difficulty to unequivocally evaluate theee, when the content of minor enantiomer has to be evaluatedin the presence of the major enantiomer.

4 Chiral selectors

Enantioseparations in nano-LC are mostly performed em-ploying the same CS already known from HPLC and CE,which have been thoroughly reviewed previously [4,42]. Con-sequently, in this paragraph a short overview of the mainfeatures of the principal classes of CSs used in nano-LC isgiven. It should be again emphasized, instead, that nano-LCallows to perform chiral separations either with a CSP oradding the CS to the mobile phase. The last mode is consid-ered prohibitive for HPLC, where huge volumes of solventare consumed, even if some applications are reported in lit-erature, while is largely exploited in CE. In nano-LC, bothapproaches result advantageous since nano-LC need smallamounts of CSP and offers low mobile phase and selectorconsumption.

Considering this aspect, cyclodextrins (CDs) and CDderivatives represent the ideal chiral selector that can beused as CSP or in the free form as CMPA. CDs form tran-sient diastereomeric inclusion complexes with enantiomersby means of the cavity, while the hydroxyl groups of glu-copyranose units favor the enantiodiscrimination process byhydrogen-bonding interactions. CDs offer high efficiency andselectivity toward a large numbers of molecules. The deriva-tization of the hydroxyl groups on the external rims leads toCD derivatives with higher solubility, different depth of cav-ity, different interaction sites, etc. The use of CDs as CMPAis also justified by the fact that they are no UV-absorbingand they have good solubility in aqueous/polar organic mo-bile phase [43–49]. Another class of CSs, related to CD andconsidered as the most powerful for chiral separations, isrepresented by polysaccharide derivatives (e.g. cellulose oramylose based). Their enantiorecognition capability is deter-mined by the higher order structure of the polymer as well asby functional groups naturally or synthetically occurring onit. Among all polysaccharides derivatives, phenylcarbamate



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edw

ithdi

ffere

ntlo

adin

gof

Chira

celO

Jor

Chira

lpak

ADor

CDM

PC;

100

�m

id×

22.0

cm,5

�m

UV(2

14nm

)10

mM

amm

oniu

mac

etat

e,pH

7.7

inM

eOH

CEC

[80]

trans

-Stil

bene

oxid

e,Tr

oger

’sba

se,

inda

pam

ide,

glut

ethi

mid

e,et

ozol

in,

pipr

ozol

in

Amin

opro

pyls

ilani

zed

sphe

rical

Dais

ogel

(3or

5�

mpa

rticl

edi

amet

er,d

iffer

entp

ore

-60,

120,

200,

1000

,and

2000

A)co

ated

with

poly

sacc

harid

eph

enyl

carb

amat

e;10

0�

mid

×24

.0cm

UV(2

14nm

)2.

5m

Mam

mon

ium

acet

ate

inM

eOH,

pH7.

7CE

C[8

1]

2-(B

enzy

l-su

lfiny

l)ben

zam

ide,

2-(b

enzy

lsul

finyl

)ben

zoic

acid

,ben

zyl

este

r,et

ozol

in,p

ipro

zolin

Amin

opro

pyls

ilani

zed

Dais

ogel

coat

edw

ithdi

ffere

ntam

ount

ofCD

CPC

(4.8

,1.0

,0.5

,w/w

);10

0�

mid

x24

.0cm

,5�

m

UV(2

14nm

)2.

5m

Mam

mon

ium

acet

ate

buffe

r,pH

7.7

inM

eOH

CEC

[85]



Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

Ambu

ceta

mid

e,be

nzyl

2-(b

enzy

lsul

finyl

)be

nzoa

te,e

tozo

lin,C

-met

hylth

alid

omid

e,m

etom

idat

e,m

icon

azol

e,no

mife

nsin

e,no

rges

trel,

omep

razo

le,p

ipro

zolin

,th

alid

omid

e,th

alid

omid

ean

alog

(EM

-12)

Sphe

rical

Dais

ogel

parti

cles

coat

edw

ithdi

ffere

ntam

ount

(5or

25%

)ofC

DCPC

;100

�m

id×

24.0

cm,5

�m

UV(2

14nm

)15

mM

amm

oniu

mac

etat

ein

MeO

HCE

C[8

8]

Pipr

ozol

inAm

inop

ropy

lsila

nize

dan

dna

tive

silic

aco

ated

with

CDCP

C;10

0�

mid

×24

.0cm

,5�

mUV

(214

nm)

2.5

mM

amm

oniu

mac

etat

e,pH

7.7

inM

eOH

CEC

[86]

Pipr

ozol

inAm

inop

ropy

lsila

nize

dsi

lica

diffe

rent

nom

inal

pore

size

(12,

20,3

0,10

0,an

d20

0nm

)CDC

PC;

100

�m

id×

24.0

cm,5

�m

UV(2

20nm

)2.

5m

Mam

mon

ium

acet

ate

inM

eOH

CEC

[87]

Ambu

ceta

mid

e,am

ino-

gute

thiim

ide,

C-m

ethy

l-tha

lidom

ide,

etoz

olin

,gl

utet

him

ide,

Nifu

rtim

ox,t

halid

omid

e,th

alid

omid

ean

alog

EM-3

10,T

roge

r’sba

se,

lora

zepa

m

CDM

PC(C

hira

lcel

OD)(

load

ing

5–20

%)c

oate

don

silic

age

l;10

0�

mid

×24

.0cm

,5�

mUV

(214

nm)

10m

Mam

mon

ium

acet

ate

buffe

ror

10m

MTr

isbu

fferi

nM

eOH

orAC

N[8

2]

trans

-Stil

bene

oxid

e,w

arfa

rin,p

raziq

uant

el,

bend

roflu

met

hiaz

ide,

benz

oin

CTPC

-sili

ca;1

00�

mid

×8

.0cm

,5�

mUV

(214

nm)

Non

acqu

eous

and

acqu

eous

mob

ileph

ases

CEC,

p-CE

C[9

5]

Ambu

ceta

mid

e,am

inog

lute

thim

ide,

etoz

olin

,ni

furti

mox

,nor

gest

rel,

omep

razo

le,

thal

idom

ide

anal

og(E

M12

),Tr

oger

’sba

se

ADM

PC-c

oate

dsi

lica;

100

�m

id×

24.0

cm,5

�m

UV(2

14nm

)5

mM

amm

oniu

mac

etat

ein

ACN

/H2O

(60:

40,v

/v).

CEC

[83]

m-H

ydro

xym

ande

licac

id,3

-hyd

roxy

-4-

met

hoxy

man

delic

acid

,man

delic

acid

,p-

hydr

oxy-

man

delic

acid

,2-p

heny

llact

icac

id,4

-chl

oro-

man

delic

acid

Hept

a-Ty

rmod

ified

diol

silic

a/am

ino

silic

a(3

:1w

/w);

75�

mid

×6.

6cm

,5�

mUV

(195

nm)

50m

Mam

mon

ium

acet

ate,

pH6.

0/H 2

O/M

eOH/

MeC

N(1

0:40

:20:

30,

v/v/

v/v)

CEC

[97]

o-,m

-,an

dp-

nitro

anili

ne,i

ndap

amid

e,2-

phen

ylpr

opio

nald

ehyd

e,tra

ns-2

-ph

enyl

cycl

ohex

anol

,1,2

-cyc

lohe

xane

diol

,1-

(1-n

apht

hyl)

etha

nol,

sec-

phen

ethy

lal

coho

l,Sp

irono

lact

one,

1-ph

enyl

-1,2

-et

hane

diol

,pro

pran

olol

,�-m

ethy

l-1-

naph

thal

ene-

met

hano

l,w

arfa

rin

8-Am

inoq

uino

line-

2-yl

met

hyl-

and

8-am

inoq

uino

line-

7-yl

met

hyl-d

iaza

-18-

crow

n-6-

capp

ed[3

-(2-O

-�-C

D)-2

-hyd

roxy

-pr

opox

y]pr

opyl

sily

lsili

capa

rticl

es;7

5�

mid

×23

.0cm

or18

.0cm

,non

poro

ussi

lica

parti

cles

,1.

5�

m

UV(d

iffer

entw

avel

engt

h)M

ixtu

res

ofAC

N/p

hosp

hate

buffe

r[7

4]

Nor

gest

rel

25%

CDCP

Con

Sphe

rical

Dais

ogel

;100

�m

id×

24.0

cm,5

�m

UV(2

54nm

)5

mM

amm

oniu

mac

etat

ein

MeO

HCE

C[8

9]

Loxi

glum

ide

Hept

a-Ty

rmod

ified

diol

silic

a/am

ino

silic

a(3

:1,

w/w

);75

�m

id×

7.0

cm,5

�m

UV(2

14nm

)10

mM

sodi

umph

osph

ate

buffe

r,pH

6/AC

N(1

:1,v

/v)

CEC

[98]


J. Sep. Sci. 2013, 00, 1–24 Liquid Chromatography 7Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

Mec

opro

p,di

chlo

rpro

p,fe

nopr

opVa

ncom

ycin

-mod

ified

silic

apa

rticl

es;1

00�

mid

x21

cm,5

�m

UV(1

95nm

)50

0m

Mam

mon

ium

acet

ate,

pH4.

5/H 2

O/M

eOH

(5:1

0:85

,v/v

/v)

[101

]

Benz

oin,

war

farin

,pra

ziqua

ntel

,alp

reno

lol,

met

opro

lol,

prop

rano

lol,

Trog

er’s

base

,ra

nazo

line,

�-D

DB1,

�-D

DB2,

�-D

DB3,

drug

cand

idat

es

Met

hacr

yloy

ldie

thyl

enet

riam

inop

ropy

late

dsi

lica

coat

edw

ithce

llulo

se2,

3-bi

s(3,

5-di

met

hyl-p

heny

lcar

bam

ate)

-6-m

etha

cryl

ate;

100

�m

id×

8.0

cm,5

�m

UV(2

14nm

)He

xane

-EtO

H-TH

F(8

0:15

:5,v

/v/v

)or

hexa

ne-E

tOH-

chlo

rofo

rm(8

0:15

:5,

v/v/

v)

CEC

[96]

Mirt

azap

ine,

8-hy

drox

ymirt

azap

ine,

N-d

esm

ethy

lmirt

azap

ine

Vanc

omyc

in-m

odifi

edsi

lica

stat

iona

ryph

ase;

75�

mid

×23

.5cm

,5�

mUV

(205

nm)/

MS

500

mM

amm

oniu

mac

etat

e,pH

4.5/

H 2O/

MeO

H/AC

N,(

1:14

:40:

45,

v/v/

v/v)

[102

]

Alpr

enol

ol,a

teno

lol,

met

opro

lol,

oxpr

enol

ol,

pind

olol

,pro

pran

olol

,2-[(

5′-b

enzo

yl-

2′-h

ydro

xy)p

heny

l]pro

pion

icac

id,2

-[(4′

-be

nzoy

loxy

-2′ -h

ydro

xy)p

heny

l]pro

pion

icac

id,k

etop

rofe

n,in

dopr

ofen

,and

supr

ofen

Vanc

omyc

inm

odifi

edsi

lica

stat

iona

ryph

ase;

75�

mid

×22

.8cm

,5�

mUV

(195

nm)

Basi

cco

mpo

unds

:90%

ACN

orM

eOH/

H 2O/

100

mM

amm

oniu

mac

etat

e,pH

4.5

(90:

5:5,

v/v/

v)ac

idic

com

poun

ds:M

eOH

orAC

N/1

00m

Mam

mon

ium

acet

ate,

pH3.

5or

4.5/

H 2O

(90:

5:5,

v/v/

v)

[99]

Mec

opro

p,fe

nopr

op,d

ichl

orpr

op,fl

ampr

op,

feno

xapr

op,fl

uazif

op,h

alox

yfop

,dic

lofo

p,ci

clop

rofe

n,ca

rpro

fen,

keto

prof

en,

flurb

ipro

fen,

ibup

rofe

n,su

prof

en,

napr

oxen

,2-[(

5′-b

enzo

yl-2

′ -hyd

roxy

)-ph

enyl

]-pro

pion

icac

id,2

-(3′ -c

arbo

xy-

phen

yl)p

ropi

onic

acid

,2-(3

′ -car

boxy

-ph

enyl

)pro

pion

itrile

,2-[(

4′-b

enzo

ylox

y-2′

-hy

drox

y)ph

enyl

]pro

pion

icac

id,2

-(4′ -

isob

utyl

phen

yl)-3

-met

hylb

utan

oic

acid

,2-

(4′ -is

obut

ylph

enyl

)-but

anoi

cac

id,

2-(4

′ -isob

utyl

phen

yl)c

iclo

pent

ylac

etic

acid

CHI-T

BBst

atio

nary

phas

e;10

0�

mid

×23

.0cm

,5

�m

UV(1

95nm

)n-

Hexa

ne/i-

PrOH

/ace

ticac

id(8

9:10

:1,

v/v/

v)[1

07]

2-(6

-Chl

oro-

benz

othi

azol

-2-y

lsul

fany

l)-,

2-(6

-met

hoxy

-ben

zoth

iazo

l-2-y

lsul

fany

l)-,

2-(q

uino

lin-2

-ylo

xy)-,

2-(6

-chl

oro-

quin

olin

-2-

ylox

y)-,

2-(7

-chl

oro-

quin

olin

-4-y

loxy

)-pr

opio

nic

acid

Vanc

omyc

inm

odifi

edsi

lica;

75�

mid

×22

.8cm

,5

�m

UV(2

30nm

)50

0m

Mam

mon

ium

acet

ate,

pH4.

5/M

eOH/

H 2O

(5:6

0:35

,v/v

/v)

CZE

[100

]

Aten

olol

Teic

opla

nin

CSP;

75�

mid

×23

.0cm

,5�

mUV

(205

nm,c

onve

ntio

nal

and

Zce

ll)an

dES

I-MS

500

mM

amm

oniu

mac

etat

e,pH

4.5/

MeO

H/M

eCN

(1:6

0:39

,v/v

/v)

[105

]

Prop

rano

lol,

celip

rolo

l,es

mol

ol,b

isop

rolo

l,at

enol

ol,m

etop

rolo

l,ca

rteol

ol,

clen

bute

rol,

bam

bute

rol,

terb

utal

ine,

salb

utam

ol

Vanc

omyc

inCS

P;10

0�

mid

×25

cm,5

�m

UVDi

ffere

ntm

ixtu

res

ofM

eOH/

i-PrO

H/ac

etic

acid

-trie

thyl

amin

ebu

ffer

p-CE

C[1

34]



Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

Lora

zepa

m,o

xaze

pam

,tem

azep

am,

trans

-stil

bene

oxid

e,et

ozol

ine,

thal

idom

ide

Cellu

lose

tris(

3-ch

loro

-4-m

ethy

lphe

nyl-

carb

amat

e)na

tive

silic

apa

rticl

esco

ated

with

6or

12or

25%

w/w

poly

sacc

harid

ede

rivat

ive

and

(ii)a

min

opro

pyls

ilani

zed

silic

aco

ated

with

the

chira

lsel

ecto

rata

conc

entra

tion

of25

%w

/w;1

00�

mid

×25

cm,5

�m

UV(2

14nm

)Po

laro

rgan

icm

obile

phas

esCE

C[9

0]

Alpr

enol

ol,o

xpre

nolo

l,pr

opra

nolo

l,m

etop

rolo

lVa

ncom

ycin

CSP;

75�

mid

×25

cm,5

�m

UV(Z

cell,

205

nm)

500

mM

amm

oniu

mac

etat

e,pH

4.5/

H 2O/

MeO

H(1

:4:9

5,v/

v/v)

[103

]

Epin

ephr

ine,

isop

rote

reno

l,at

enol

ol,

syne

phrin

e,m

ethi

onin

e�

-nap

hthy

lam

ide,

N-b

enzo

yl-p

heny

lala

nine

�-n

apht

hyl

este

r,N

-ace

tyl-t

rypt

opha

n,5-

(4-

hydr

oxyp

heny

l)-5-

phen

ylhy

dant

oin,

prop

rano

lol,

alpr

enol

ol

Pern

apht

hylc

arba

moy

late

d�

-CD

(CSP

1),

pera

cety

late

d�

-CD

(CSP

2)an

dpe

rmet

hyla

ted

�-C

D(C

SP3)

;100

�m

id×

18cm

,5�

m

UV(2

30nm

)Tr

ieth

ylam

mon

ium

acet

ate/

MeO

Hor

phos

phat

ebu

ffer/M

eOH

mob

ileph

ases

p-CE

C[7

5]

FITC

deriv

ativ

esof

:�-a

min

obut

yric

acid

,ar

gini

ne,p

rolin

e,al

anin

e,le

ucin

e,se

rine,

phen

ylal

anin

e,as

para

gine

,glu

tam

icac

id,

aspa

rtic

acid

Vanc

omyc

inCS

P;75

�m

id×

25cm

,5�

mUV

(205

nm)a

ndIT

-MS

500

mM

amm

oniu

mfo

rmat

e,pH

3.5/

H 20r

/MeO

H(4

:11:

85,v

/v/v

)[1

04]

DNB-

Ala-

N,N

-die

thyl

amid

e,DN

B-Le

u-N

,N-

diet

hyla

mid

e,Z-

Ala-

N-(3

,5-d

imet

hyl)

phen

ylam

ide,

Z-Va

l-N-(3

,5-d

imet

hyl)

phen

ylam

ide,

Z-Le

u-N

-(3,5

-dim

ethy

l)ph

enyl

amid

e,Z-

PhG-

N-(3

,5-d

imet

hyl)-

phen

ylam

ide,

Z-Ph

e-N

-(3,5

-dim

ethy

l)ph

enyl

amid

e

(S)-N

-(3,5

-Din

itrob

enzo

yl)le

ucin

e-N

-phe

nyl-N

-pr

opyl

amid

ebo

nded

silic

a10

0�

mid

×20

cm,

5�

m

UV(2

54nm

)M

ixtu

res

ofn-

hexa

nean

di-P

rOH

cont

aini

ng3–

7%H 2

0v/

vCE

C[1

08]

Flav

anon

e,te

maz

epam

,lop

iraze

pam

,and

etoz

olin

eAm

ylos

etri

s(5-

chlo

ro-2

-met

hylp

heny

l-ca

rbam

ate)

coat

edsi

lica

parti

cles

:(i)

nativ

esi

lica

parti

cles

5�

m,p

ore

size

of10

0nm

coat

edw

ith5,

10,1

5or

25%

w/w

poly

sacc

harid

ede

rivat

ive;

(ii)a

min

opro

pyl-

sila

nize

dsi

lica

with

3,5,

7,10

�m

,por

esi

zeof

100

nmco

ated

with

the

CSat

aco

ncen

tratio

nof

25%

w/w

;(iii

)nat

ive

silic

apa

rticl

es5

�m

,po

resi

zeof

10,3

0,10

0,an

d20

0nm

coat

edw

ithth

eCS

ata

conc

entra

tion

of25

%w

/w;

100

�m

id×

25.0

cm

UV(2

14nm

)50

0m

MN

H 4Ac

,pH

5.5/

H 2O/

MeO

H/AC

N,1

:4:2

5:70

v/v/

v/v

CEC

[93]



Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

Flav

anon

e,2′

-hyd

roxy

-flav

anon

e,4′

-hyd

roxy

-fla

vano

ne,6

-hyd

roxy

flava

none

,7-h

ydro

xy-

flava

none

,4′ -m

etho

xyfla

vano

ne,

6-m

etho

xyfla

vano

ne,7

-met

hoxy

-fla

vano

ne,h

espe

retin

,hes

perid

in,

narin

geni

n,na

ringi

n

Phen

yl-c

arba

mat

e-pr

opyl

-�-C

Dgr

afte

don

tosi

lica;

100

�m

id×

22.0

cm,5

�m

UV(2

05nm

)1%

Trie

thyl

amm

oniu

mac

etat

e,pH

4.5

buffe

rin

MeO

H/H 2

Om

ixtu

res,

pola

rorg

anic

and

norm

alph

ase

mob

ileph

ases

[76]

Phen

ylal

anin

e,ph

enyl

glyc

in,t

yros

ine,

trypt

opha

n(−

)-(18

-Cro

wn-

6)-2

,3,1

1,12

-tetra

carb

oxyl

icac

idbo

nded

toam

ino

prop

ylsi

lica;

100

�m

id×

20cm

,5�

m

UV(2

10nm

)20

mM

bis–

tris

buffe

r/MeO

H(8

0:20

,v/

v)CE

C[1

09]

Hesp

eret

inPh

enyl

-car

bam

ate-

prop

yl-�

-CD

graf

ted

onto

silic

a;10

0�

mid

×22

cm,5

�m

UV(2

05nm

)Tr

ieth

ylam

mon

ium

acet

ate

buffe

r,pH

4.5/

wat

er/m

etha

nol(

1:29

:70,

v/v/

v)[7

7]

FMOC

deriv

ativ

esof

amin

oac

ids:

citru

lline

,py

rogl

utam

icac

id,p

ipec

olin

icac

id,

allo

-isol

euci

ne,o

rnith

ine,

leuc

ine,

valin

e,m

ethi

onin

e,hi

stid

ine,

serin

e,ar

gini

ne,

cyst

eine

,lys

ine,

thre

onin

e,tri

ptop

hane

,gl

ycin

e,al

anin

e,gl

utam

ine,

aspa

rtic

acid

,is

o-le

ucin

e,an

dpr

olin

e,ph

enyl

alan

ine,

glut

amic

acid

,asp

arag

ine

Cellu

lose

tris(

3-ch

loro

-4-m

ethy

lphe

nyl-

carb

amat

e);1

00�

m×

24.0

cm,5

�m

UV(2

10an

d26

0nm

)0.

5M

amm

oniu

mfo

rmat

e,pH

2.5/

H 2O/

ACN

(1:1

9:80

,v/v

/v)

CEC

[91]

Resm

ethr

in,d

inic

onaz

ole,

fenp

ropa

thrin

,�

-cyh

alot

hrin

,�-c

yflut

hrin

,cis

-bife

nthr

in,

met

alax

yl,b

enal

axyl

,hex

acon

azol

e,m

yclo

buta

nil,

tebu

cona

zole

,dic

hlor

prop

,m

ecop

rop,

�-c

yper

met

hrin

,flut

riafo

l,un

icon

azol

e

Cellu

lose

tris(

3-ch

loro

-4-m

ethy

lphe

nyl-

carb

amat

e)an

dce

llulo

setri

s(4-

chlo

ro-

3-m

ethy

lphe

nylc

arba

mat

e)co

ated

inth

eam

ount

of25

%w

/won

toam

inop

ropy

lsila

nize

dsp

heric

alsi

lica

parti

cles

;100

�m

id×

24.0

cm,

5�

m(1

00nm

nom

inal

pore

size

)

UV(2

10±

2nm

)Di

ffere

ntRP

and

norm

alph

ase

mob

ileph

ases

CEC

[92]

Praz

iqua

ntel

,tem

azep

am,t

halid

omid

e,tra

ns-s

tilbe

neox

ide,

Trog

er´s

base

,w

arfa

rin,e

tozo

line

Cellu

lose

tris(

4-ch

loro

-3-m

ethy

lphe

nyl-

carb

anat

e)co

ated

(10%

w/w

)on

(i)na

tive

silic

apa

rticl

esw

ithpa

rticl

esi

zeof

3�

man

d(ii

)cor

e-sh

ells

ilica

with

2.8-

�m

parti

cle

size

(1.9

-�m

nonp

orou

sco

rean

d0.

45-�

mpo

rous

shel

lthi

ckne

ss);

75�

mid

×25

.0cm

UV(2

05nm

)5

mM

amm

oniu

mac

etat

e,pH

4.5/

ACN

(30:

70,v

/v)

CEC

[94]

Mon

olith

icco

lum

nsPh

enyl

alan

ine

One

step

insi

tupo

lym

eriza

tion

ofN

-(2-h

ydro

xy-

3-al

lylo

xypr

opyl

)-L-4

-hyd

roxy

prol

ine;

75�

mid

×26

.0cm

UV(2

23nm

)50

mM

sodi

umdi

hydr

ogen

phos

phat

e/0.

1m

MCu

(II),

pH4.

6[1

23]

Hexo

barb

ital,

benz

oin,

carp

rofe

nCh

irasi

l–De

xm

odifi

edsi

lica

mon

olith

;100

�m

id×

17.0

and

25.0

cmUV

(230

nm)

20m

MM

ESbu

ffer,

pH6/

MeO

H(7

0:30

,v/v

)CE

C[1

10]



Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

DNS

deriv

ativ

esof

leuc

ine,

met

hion

ine,

nor-

leuc

ine,

nor-

valin

e,ph

enyl

alan

ine,

serin

e,th

reon

ine,

trypt

opha

ne,v

alin

e,�

-am

ino-

n-bu

tyric

acid

,p-,

m-h

ydro

xy-

man

delic

acid

,3-h

ydro

xy-4

-met

hoxy

-m

ande

licac

id,4

-hyd

roxy

-3-m

etho

xy-

man

delic

acid

,�-p

heny

llact

icac

id,

p-hy

drox

y-ph

enyl

lact

icac

id,

indo

le-3

-lact

icac

id

L-Pr

olin

amid

e-m

odifi

edsi

lica

mon

olith

;100

�m

id×

26.0

cmUV

(254

nm)

ACN

/0.5

mM

Cu(A

c)2–

50m

MN

H 4Ac

,pH

6.5

(70:

30,v

/v)

CEC

[116

]

DNS-

serin

e,DN

S-try

ptop

han,

DNS-

thre

onin

e,try

ptop

han,

phen

ylth

iohy

-da

ntoi

nse

rine,

4-flu

orom

ande

licac

id,

absc

isic

acid

,men

adio

neso

dium

bisu

lfite

,flu

rbip

rofe

n,3-

phen

ylbu

tyric

acid

,w

arfa

rin,c

hrys

anth

emic

acid

,(1R

,2R)

-an

d(1

S,2S

)-N-m

ethy

lpse

udoe

phed

rine

Phys

ycca

llyad

sorb

edav

idin

onsi

lica

mon

olith

;50

�m

×20

.0or

6.5

cmUV

(200

or21

4nm

)10

mM

phos

phat

ebu

fferc

onta

inin

gdi

ffere

ntco

ncen

tratio

nv/

vof

MeO

H(p

H5.

95)

CEC

[115

]

DNS

deriv

ativ

esof

aspa

rtic

acid

,thr

eoni

ne,

met

hion

ine,

serin

e,le

ucin

e,gl

utam

icac

id,

nor-

leuc

ine,

nor-

valin

e,va

line,

phen

yl-

alan

ine,

�-a

min

o-n-

buty

ricac

id;

hydr

oxyp

heno

llact

icac

id,i

ndol

e-3-

lact

icac

id

L-Ph

enyl

alan

inam

ide/

L-al

anin

amid

e/L-

prol

inam

ide-

mod

ified

silic

am

onol

ith;1

00�

mid

×32

.0cm

UV(2

54nm

)AC

N/1

0–10

0m

MN

H 4Ac

(des

ired

pH)/0

.50

mM

Cu(A

c)2

mix

ture

s[1

17]

DNS

deriv

ativ

eof

serin

e,le

ucin

e,m

ethi

onin

e,va

line,

thre

onin

e,ph

enya

lani

ne,t

rypt

opha

ne,n

or-le

ucin

e,gl

utam

icac

id,a

spar

ticac

id,

�-a

min

o-n-

buty

ricac

id

L-Pr

olin

amid

e,L-

alan

inam

ide,

and

L-ph

enyl

alan

inam

ide

chem

ical

lym

odifi

edsi

lica

mon

olith

;100

�m

id×

26.0

,32.

0,28

.5cm

UV(2

54nm

)Di

ffere

ntm

ixtu

reof

amm

oniu

mac

etat

e/M

eCN

/Cu(

Ac) 2

CEC,

CZE

[118

]

2,2,

2-Tr

ifluo

ro-1

-(9-a

nthr

yl)e

than

ol,b

enzo

in,

2,29

-dih

ydro

xy-6

,69-

dim

ethy

lbip

heny

l,tra

ns-c

yclo

prop

aned

icar

boxy

licac

iddi

anili

de,fl

avan

one,

trans

-stil

bene

oxid

e,pi

proz

olin

,pro

pran

olol

,oxp

reno

lol,

alpr

enol

ol,fl

avan

one

CDM

PCm

odifi

edsi

lica

mon

olith

;100

�m

id×

12.0

or20

.0cm

UVn-

Hexa

ne/2

-PrO

H,90

:10

or80

:20

(v/v

)or

MeC

N/H

2O50

:50

(v/v

)[1

3]

Feno

xapr

opet

hyl,

dicl

ofop

met

hyl,

mec

opro

pm

ethy

l,m

ethy

lthio

hyda

ntoi

npr

olin

e,he

xoba

rbita

l,ca

rpro

fen,

poly

chlo

rinat

edbi

phen

yl13

2

Sol-g

elgl

ued

Chira

-Dex

-sili

capa

rticl

es;1

00�

mid

×20

.0cm

UV(2

30nm

)M

eOH

and

20m

MM

ESbu

ffer,

pH6.

0or

10m

Mso

dium

acet

ate,

pH4.

5m

ixtu

res

p-CE

C[1

11]



Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

Trog

er’s

base

,tra

ns-s

tilbe

neox

ide,

benz

oin,

1,2,

2,2-

tetra

phen

ylet

hano

l,2-

phen

yl-

cycl

ohex

anon

e,2,

2,2-

triflu

oro-

1-(9

-ant

hryl

)eth

anol

,cob

alt(I

II)tri

s(ac

etyl

acet

onat

e),fl

avan

one,

trans

-cyc

lopr

opan

dica

rbox

ylic

acid

dian

ilide

,2,2

′ -dih

ydro

xy-6

,6′ -

dim

ethy

lbip

heny

l

Cellu

lose

2,3-

bis(

3,5-

dim

ethy

lphe

nylc

arba

mat

e)-

6-(3

,5-d

imet

hylp

heny

lcar

bam

ate)

;cel

lulo

se2,

3-bi

s(3,

5-di

chlo

roph

enyl

carb

amat

e)-6

-(3,5

-di

chlo

roph

enyl

carb

amat

e);a

myl

ose

2,3-

bis(

3,5-

dim

ethy

lphe

nylc

arba

mat

e)-6

-(3,5

-di

met

hylp

heny

lcar

bam

ate)

mod

ified

silic

am

onol

ith;1

00�

mid

×12

.0cm

UVn-

Hexa

ne/i-

PrOH

(90:

10v/

v)[1

14]

Trog

er’s

base

,tra

ns-s

tilbe

neox

ide,

benz

oin,

1,2,

2,2-

tetra

phen

ylet

hano

l,2-

phen

yl-

cycl

ohex

anon

e,2,

2,2-

triflu

oro-

1-(9

-ant

hryl

)eth

anol

,cob

alt(I

II)tri

s(ac

etyl

acet

onat

e),fl

avan

one,

trans

-cyc

lopr

opan

dica

rbox

ylic

acid

dian

ilide

,2,2

′ -dih

ydro

xy-6

,6′ -

dim

ethy

lbip

heny

l

ADM

PC(d

iffer

entl

oadi

ng)c

oate

dsi

lica

mon

olith

;100

�m

id×

20.0

cmUV

(254

nm)

n-He

xane

/i-Pr

OH(9

0:10

,v/v

)[1

13]

Four

ster

eois

omer

sof

the

N-b

enzy

loxy

-ca

rbon

ylph

osph

inic

pseu

dodi

pept

ide

met

hyle

ster

benz

ylox

ycar

bony

l-ho

mop

heny

lala

nine

Z-hP

he�

(PO 2

HCH 2

)Ph

e-OC

H 3an

dof

the

corr

espo

ndin

gN

-2,4

-di

nitro

phen

yl-h

Phe�

(PO 2

HCH 2

)Phe

-OH

tBuC

QDim

mob

ilize

don

silic

am

onol

ith;1

00�

mid

×25

.0cm

UV(2

16or

360

nm)

ACN

/MeO

H(5

0:50

v/v)

,com

pris

ing

200

mM

acet

icac

id,2

00m

Mfo

rmic

acid

and

4m

Mtri

ethy

lam

ine

CEC,

CE,H

PLC

[119

]

Clen

bute

rol,

sota

lol,

pron

etha

lol,

mefl

oqui

ne,

mefl

oqui

ne-t-

buty

lcar

bam

ate,

rimite

rol,

salb

utam

ol,t

alin

olol

trans

-(1S,

2S)-2

-(N-4

-Ally

loxy

-3,5

-di

chlo

robe

nzoy

l)am

ino

cycl

ohex

anes

ulfo

nic

acid

mod

ified

silic

am

onol

ith;1

00�

mid

×25

.0or

8.0

(CEC

)and

33.5

cm(n

ano-

LC)

UV(2

16nm

)Aq

ueou

san

dno

naq

ueou

sm

obile

phas

esCE

C[1

20]

Mefl

oqui

ne,m

efloq

uine

-O-te

rt-bu

tyl-

carb

amat

e,an

dpr

onet

halo

ltra

ns-(1

S,2S

)-2-(N

-4-A

llylo

xy-3

,5-

dich

loro

benz

oyl)a

min

ocy-

cloh

exan

esul

foni

cac

idm

odifi

edsi

lica

mon

olith

;100

�m

id×

25.0

cm

UV(2

16nm

)AC

N/M

eOH

(80:

20v/

v)co

ntai

ning

(R,S

)-2-a

min

o-1-

buta

nola

ndfo

rmic

acid

CEC

[121

]

Flav

anon

eM

ono-

(6-a

zido-

6-de

oxy)

-�-C

Dgr

afte

don

orga

nic

poly

mer

(3st

eps

synt

hesi

s-cl

ick

chem

istry

);75

�m

id×

31.0

cm

UV(2

14)

MEO

H/5

mM

bora

tebu

ffer,

pH8.

2(4

0:60

,v/v

)CE

C[1

24]

1-In

dano

l,1-

phen

ylet

hyla

min

e,�

-phe

nyl

glyc

inol

,1-(4

-bro

mop

heny

l)et

hano

l,1-

(2-c

hlor

ophe

nyl)

etha

nol

(R)-A

cryl

oylo

xy-�

-�-d

imet

hyl-�

-but

yrol

acto

nem

odifi

edsi

lica

mon

olith

;100

�m

×50

.0cm

UV(2

54nm

)n-

Hexa

ne/i-

PrOH

(98:

2,v/

v)[1

22]



Ta

ble

1.

Co

nti

nu

ed

Anal

ytes

Colu

mn

Dete

ctio

nM

obile

phas

eCo

mpa

rativ

eRe

fere

nce

sepa

ratio

nm

ode

13ra

cem

icco

mpo

unds

(stru

ctur

eon

lyre

porte

d)Ph

-�-C

D-si

lica

hybr

idm

onol

ith;7

5�

mid

×30

.0cm

UV(2

14nm

)He

xane

/i-Pr

OH(9

0:10

v/v)

orM

eOH/

triet

hyla

mm

oniu

mac

etat

e,pH

4.2

(60:

40v/

v)

[112

]

Keto

prof

en,f

enop

rofe

n,flu

rbip

rofe

n,su

prof

enPG

Aim

mob

ilize

don

achi

rale

poxy

-sili

cam

onol

ith;1

00�

mid

×7.

0or

14.0

cmUV

(200

nm)

50m

Mph

osph

ate

buffe

r,pH

7.0

CEC

[65]

Mol

ecul

arim

prin

ted

poly

mer

icco

lum

nsDN

S-ph

enyl

alan

ine

DNS-

L-ph

enyl

alan

ine

mol

ecul

arim

prin

tpol

ymer

;25

�m

id×

85.0

cm,o

pen

tubu

lar

UV(2

80nm

)M

eCN

/ace

ticac

id(9

9.5:

0.5,

v/v)

CEC

[125

]

Bupi

vaca

ine,

mep

ivac

aine

,and

ropi

vaca

ine

Bupi

vaca

ine,

mep

ivac

aine

,orS

-rop

ivac

aine

tem

plat

efo

rgra

fting

poly

mer

izatio

non

orga

nic

poly

mer

icm

onol

ith;1

00�

mid

×5.

0cm

UV(2

15nm

)AC

N[1

28]

THP,

Trog

er’s

base

L-TH

Pan

d(5

S,11

S)-(-

)-Tro

ger’s

base

impr

inte

dsi

lica

base

dm

onol

ith;7

5�

mid

×8.

5,20

.0,a

nd24

.5cm

UV(2

14nm

)AC

N/5

mM

acet

ate

buffe

r,pH

6.0

(80:

20,v

/v)

CEC

[129

]

Keto

prof

en,n

apro

xen,

ibup

rofe

n,fe

nopr

ofen

S-Ke

topr

ofen

mol

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derivative has been demonstrated as highly effective for enan-tiomer separations. In addition, the selectivity of this class ofCS can be further modulated by the introduction in the phenylring of electron-donating and electron-withdrawing groups,suggesting that the most important adsorbing sites for chi-ral discrimination on phenylcarbamate derivatives may bethe polar carbamate groups. Initially, their performance wasoptimized in normal phase conditions, even due to the sol-ubility of polysaccharides derivatives in some solvent. How-ever, later a relatively high enantiorecognition capability wasshown in aqueous organic mobile phases and recently, theiremployment with polar organic mobile phases was demon-strated to be successful [50–53]. Polysaccharides-based CSswere deeply investigated in miniaturized techniques as well,namely nano-LC and CEC, as demonstrated by the large num-ber of works reported in Table 1, and very interesting resultswere achieved (see below).

Even if present in a lower number of research papers,macrocyclic antibiotics (MAs) assume important implica-tions for nano-LC enantioseparations, too. MAs possess manystereogenic centres and functional groups, which are respon-sible of multiple interactions, i.e. hydrogen bonds, ionic-,�–� interactions. Furthermore, glycopeptides antibiotics,such as vancomycin, teicoplanin, and ristocetin A, are char-acterized by an aglycon portion with basket shape made byfused macrocyclic rings that allows the formation of inclu-sion complexes [54–56]. All these features are responsible oftheir high enantioselectivity toward several chiral compoundsand it is noteworthy mentioning, among all the applicationsof enantioseparative nano-LC to real samples, the most arerelated with the use of MAs (see Section 5.2).

Proteins and glycoproteins result instead less employedin nano-LC, with only five works present in literature (see Ta-ble 1). They are well known to bind stereoselectively drugs andcan be considered as polymers composed of several chiral sub-units (amino acids), characterized by a 3D structure, wherebyhydrophobic, electrostatic interactions, and hydrogen bondsguarantee chiral recognition. The high specificity of some pro-tein reduces their spectrum of selectivity and, further, whena protein has to be incorporated into a matrix to create a CSP,special attention has to be paid during immobilization in or-der to maintain natural conformation of the protein, essentialfor chiral discrimination. [57–60]. These drawbacks can be re-sponsible of their limited use in nano-LC. When they wereused in OT-LC, low retention factors, efficiency and resolutionwere observed [61,62]. However, proteins immobilization onsilica monoliths seems to be promising since higher coverageof proteins, and consequently higher resolution factors, thanthose obtained with silica particles or the organic polymer-based monolith were achieved [63, 64]. The work proposedby Gotti et al. [65], and discussed later on, go as well in thisdirection, showing high stability of the protein-based chiralselector and improved enantioselectivity and efficiency, dueto the covalent immobilization on monolithic silica capillarycolumn.

Finally, a quite large number of works is related withthe use of low molecular mass selectors (mainly with ligand

exchange properties), especially in combination with mono-lithic matrix, as described in the following sections.

5 Enantioseparations by means

of nano-LC

Considering the aforementioned advantages of nano-LC, theemploy of this miniaturized technique for enantiomeric as-sessment is furthermore gainful, since a minute amount ofexpensive CSP or CMPA is needed. Most of applications ofchiral nano-LC relate with the pharmaceutical field. In thisreview, works will be classified considering the type of chiralcolumn used (OT, monolithic, particulate packed or molecu-lar imprinted polymer, MIP), the employ of a chiral selectoras mobile phase additive and eventually the use of indirectmethod for enantioseparation. In Table 1 all the researchpapers concerning chiral separation by means of nano-LCare reported. Since many works used the same column (i.e.CEC), the same CSP (i.e. HPLC), or the same CS (i.e. CZE)for comparative studies, the table column entitled “Compar-ative separation mode” takes into account this evaluation.Furthermore, when reviewed research papers gave detailedinformation concerning, e.g. particles and pore size, amountof coating, etc., we reported those data in the Table 1, sincewe consider them useful for a critical estimation of the works.

5.1 OT chiral separation

The first chiral separation in nano-LC was obtained bySchurig et al. [66] employing an OT column (50 �m id) coatedwith ChirasilDEX, a permethylated-�-CD chemically linked todimethylpolysiloxane. Considering hexobarbital as test com-pounds, it was demonstrated the versatility of the same col-umn toward different techniques, as GC, supercritical fluidchromatography, and CEC (see Fig. 1). The same type of col-umn was evaluated in OT-LC and OT-CEC, coupled with MS,for a series of chiral drugs and alcohols [67]. In this regards,it should be underlined that several works are based on com-parative studies between nano-LC and CEC, where the samecolumn and, quite often, the same instrumentation was usedfor both techniques.

Francotte et al. separated both acidic and basiccompounds of pharmaceutical interest employing cap-illaries coated with cellulose derivative, 3,5-dimethyl-phenylcarbamoyl cellulose or p-methylbenzoyl cellulose [68].Even if both CSPs showed high enantioselectivity, they werecharacterized by short lifetime. To increase the stability of thecoating of the column, Wakita et al. [69] proposed a copoly-merization reaction of another cellulose derivative, cellulosetris(3,5-dichlorophenylcarbamate) (CDCPC) and carried outthe separation of several racemates.

Recently, Zou et al. proposed the use of nanoparti-cles to improve phase ratio and consequently chiral reso-lution in OT-LC [70]. Mobile crystalline material (MCM-41)type silica nano-particles were immobilized on a bare fused



Figure 1. Enantiomer separation of hexo-barbital on a 50-�m id × 1 m fused sil-ica column coated with Chirasil-DEX byGC, SFC, nano-LC, and CEC. Effective col-umn length in nano-LC and CEC, 85.0 cm.Buffer, borate-phosphate, pH 7 (the arrowindicates the dead volume). From [66] withpermission.

silica capillary and successively coated with cellulose tris(3,5-dimethylphenyl carbamate) (CDMPC) at different concen-trations. The capillary column obtained with this procedureshowed higher enantioselectivity than a bare-fused silica cap-illary coated with the same amount of CS (0.3% w/v CDMPC).

In addition to polysaccharides-based CSP, proteins aswell were used as CS in OT-LC. BSA, chemically boundto the capillary wall, was used for the enantioseparation ofdinitrophenyl-amino acids, oxazepam, and lorazepam [61],while with avidin (a glycoprotein), physically adsorbed on thecapillary wall, the enantioseparation of dansyl-amino acidsand some drugs was achieved [62].

In an interesting work, OT-LC was also proposed to per-form enantioselective studies by frontal affinity chromatog-raphy. The gamma isoform ligand-binding domain of perox-isome proliferator-activated receptor (PPAR�), immobilizedinto a capillary column, was used to characterize, in combi-nation with MS, the enantioselective interaction of some an-tagonist for this biological target. Different binding affinitiesof enantiomers to PPAR� resulted in a characteristic biphasicfrontal profile as the result of the two saturation events [71].Despite of the promising result showed, the number of re-search papers related to OT-LC chiral separations is quitemodest mainly due to their poor selectivity arising from thelow phase ratio [23].

5.2 Particulate packed chiral capillary columns

A large number of works is present in literature whereenantioseparations were carried out with packed columns.Permethylated-�-CD-modified silica was used to performenantiomeric separation and to compare the perfor-mance of CEC and nano-LC [72, 73]. As expected, effi-ciency in CEC was higher than nano-LC. Gong et al.tested two bonded CSPs, 8-aminoquinoline-2-ylmethyl- and8-aminoquinoline-7-ylmethyl-diaza-18-crown-6-capped [3-(2-O-�-CD)-2-hydroxypropoxy] propylsilyl silica particles (non-porous, 1.5 �m), where the combination of crown ether with�-CD was exploited to obtain better selectivity toward po-sitional isomers and chiral compounds [74]. Analyses were

performed in a capillary column (75 �m id), working at highpressure (>8000 p.s.i.) and high column efficiencies wereachieved. Three other different �-CD-based CSP, containingpernaphthylcarbamoylated �-CD, peracetylated �-CD, andpermethylated �-CD, respectively, already used in HPLC,were also studied in nano-LC and CEC. It was noted thatthe presence of carbonyl groups as well as aromatic ringincreased the enantioseparation capability. Concerning thefirst examined CSP, it showed better enantioseparation of �-blockers than HPLC, and this phenomenon was attributed toimprovement of separation efficiency due to different columnfabrications [75].

A CSP based on �-CD was also employed by Si-Ahmedet al. in nano-LC to perform the enantioseparation of severalflavanoids and diastereoisomeric separation of two flavanoneglycosides [76]. Good eanantioresolutions were achieved, inshorter analysis time compared to the results reported in lit-erature, for the same compounds, with conventional HPLC.The same CSP, phenyl-carbamate-propyl-�-CD stationaryphase, was used to perform analysis of hesperetin, a chiral fla-vanone abundant in citrus fruits with antioxidant, anticancer,anti-inflammatory, and analgesic activities, in human urineafter ingestion of blood orange juice [77]. Precisely, after thevalidation of the methods, in terms of sensitivity (LOD andLOQ, values 0.1 and 0.5 �g/mL, respectively), linearity, pre-cision, accuracy, and recovery of the extraction procedure,the excretion of R- and S-hesperitin in urine was monitoredwithin 24 h and it was observed that S-hesperetin was ex-creted at higher concentrations than R-hesperetin, while theflavanoid was almost completely eliminated in about 12 h.

CSPs widely employed are based on polysaccharidederivatives. In particular, those derived from cellulose andamylose, were extensively investigated and compared innano-LC as well as in CEC by Chankvetadze’s group. Ina preliminary study, polyacrylamide, and polysaccharidederivatives, namely Chiraspher R© (prepared by radical copoly-merisation of N-acryloyl-L-phenyl-alanineethylester with sil-ica gel) and CDMPC, were tested as CSP for the separa-tion of several racemic drugs, working in both normal andreversed phase conditions [78]. While the polyacrilamide-based SP was covalently bonded to silica particles, cellulose



derivative was coated. The feasibility of miniaturizationfrom HPLC to nano-LC and CEC for chiral separation wasdemonstrated, underlining the importance to avoid columnoverloading in downscaled separation in order to not fade theperformance.

In a following study, CDMPC (also called ChiracelOD) enantio-resolving capability was compared withthose ones of Chiralpak AD, consisting of amylose-tris(3,5-dimethylphenylcarbamate) and cellulose-tris(4-methylbenzoate) (Chiralcel OJ) for the separation ofthalidomide and its hydroxilated metabolites in convectionalHPLC column, nano-LC, and CEC [79]. In nano-LC, evenif Chiralpak AD showed higher enantioselectivity, thebaseline resolution of all racemic compounds in one run wasobtained only with a CSP composed by a combination of twopolysaccharide derivatives (16% AD plus 14% OD) coatedonto aminopropylsilica.

Later on, the same CPSs were evaluated considering theloading of the chiral selector on aminopropyl derivatized silicagel, the pore, and particle size of silica gels, temperature and,obviously, the mobile phase composition [80–84].

A CSP based on CDCPC was also studied in terms of load-ing of the chiral selector, particle, pore size, and type of the sil-ica support, and optimization of separative medium [85–88].Employing capillary columns packed with CDCPC, nano-LC,and CEC techniques were compared for the determination ofenantiomeric purity of the contraceptive drug levonorgestrelin a commercial sample [89]. Despite of higher efficiencyoffered by the electrochromatographic method, the minorenantiomeric impurity ((+)-norgestrel) of a drug formulationwas better detected with nano-LC, due to its minor baselinenoise. Even if the method did not allow to clearly detect anyenantiomeric impurity of (+)-norgestrel in the samples of lev-onorgestrel used in the study, the standard addition methodclearly indicated that the impurity on the level below 0.1%w/w might be present. Consequently, 0.1% w/w was consid-ered as the apparent LOD of detection by the authors, beingthe levonorgestrel sample at the concentration of 5 mg/mL.

The effect of the presence of different electron-donatingand electron-withdrawing groups on polysaccharide deriva-tives was investigated introducing cellulose tris(3-chloro-4-methylphenylcarbamate) (Sepapak-2) [90], which was laterproposed for the enantiomeric separation of fluorenylmethy-loxycarbonyl derivatives of amino acids in nano-LC andCEC, and the determination of the content and the enan-tiomeric purity of the non-protein amino acid citrullinein food supplements by CEC [91]. Sepapak-2 has been re-cently compared with Sepapak-4 ((cellulose tris(4-chloro-3-methylphenylcarbamate)) for the enantioseparation of 16 pes-ticides [92]. Sepapak-4 provided better resolution toward highnumber of compounds and was selected for further studiesin CEC.

Amylose tris(5-chloro-2-methylphenylcarbamate)-basedCSP was also subject of an accurate testing in both nano-LCand CEC, and showed good enantio-resolving capability for aseries of racemic drugs [93]. Recently, cellulose tris(4-chloro-3-methylphenylcarbamate) was also coated on core-shell silica

particles and slightly better results, in terms of chiral selec-tivity and resolution, were obtained compared with totallyporous material [94].

A bonded type of cellulose derivative CSP, cellulosetrisphenylcarbamate, was also proposed by Chen et al. [95] forenantioseparations in nano-LC and CEC and good stability ofthe column was demonstrated. The same group proposedafter a positively charged cellulose derivative to be used inCEC and nano-LC [96]. Working in normal phase conditions,the enantiosepareation of some drug candidates was achievedwith a capillary column packed for 8 cm only.

Another class of chiral selectors widely employed in nano-LC are MAs. Fanali et al. obtained the enantioseparation ofseveral hydroxy acids employing a capillary column packedfor only 7 cm with silica stationary phase modified with MDL63 246 (Hepta-Tyr), a glycopeptide antibiotic from the te-icoplanin family [97]. The same type of column was selectedfor a study of enantiopurity of a pharmaceutical formula-tion of a new chiral drug (loxiglumide) under investigationfor the treatment of gastrointestinal diseases [98]. The assayof the pharmaceutical sample (ampoule) declared to containonly D-loxiglumide (0.5%, w/v) gave a recovery of the drugin agreement with the labeled content (recovery = 99.88 ±2.16%), while L-loxiglumide was not detected because absentor at concentration lower than the LOD value (0.5 �g/mL forL-loxiglumide).

Several works are related with the use of a stationaryphase derivatized with vancomycin. Enantioseparation ofacid and basic drugs, such as no steroidal anti-inflammatorydrugs, �-blockers, and clofibric acid [99, 100], chlorophenoxyacid herbicides [101] were obtained in nano-LC. Validatedmethods were applied for the determination of metopro-lol in pharmaceutical formulation [99] and to detect simul-taneously mirtazapine, a second-generation antidepressantdrug, and its metabolites in serum samples as shown inFig. 2 [102]. In this work, coupling the nano-LC system withan ESI-MS interface, it was possible to obtain about oneorder lower LOD and LOQ than those obtained with UV.LOD and LOQ values for mirtazapine, 8-hydroxymirtazapine

Figure 2. Separation of mirtazapine, 8-hydroxymirtazapine, andN-desmethylmirtazapine with (R)-1-(2-naphthyl)ethylamine as in-ternal standard. Chromatographic conditions: capillary column,75 �m id × 23.5 cm packed length; mobile phase, ammoniumacetate 500 mM pH 4.5/H2O/MeOH/ACN (1:14:40:45, v/v/v/v), flowrate: 200 nL/min; injected volume, about 60 nL. From [101] withpermission.



and N-desmethylmirtazapine, with UV detection were in therange 5–15 and 10–40 �g/mL, respectively, while with MSdetection were 0.3–5 and 0.6–35 �g/mL, in that order. Forsome �-blockers, a study concerning on-column focusingwas carried out in order to improve the sensitivity and thedeveloped method was applied to determination of alprenololin human plasma [103]. Dissolving the analyte in methanol,which demonstrated a lower eluting capability than that of themobile phase used in the experiments, it was possible to in-ject a sample volume such high as 1500 nL, resulting in LODand LOQ values for both alprenolol enantiomers of 9.0 and15.6 ng/mL, respectively.

A capillary column packed with vancomycin-modified sil-ica particles was selected to carry out enantiomeric separationof several D- and L-amino acids derivatized with FITC, bymeans of nano-LC coupled with MS. The method was ap-plied to verify the quality of orange juice samples, since thepresence of D-amino acids can be attributed to, e.g. micro-biological contamination and adulteration [104]. In order toachieve detection limits suitable for this kind of analysis, an-alytes were focused on a C18 cartridge, employing a column-switching system. This apparatus allowed the injection of5-�L sample volume. Further, the chromatographic separa-tion system was coupled with an ion-trap mass spectrometerthrough a nano spray interface and finally it provided LODvalues as low as 8 ng/mL. Figure 3 shows nano-LC–MS chro-matograms of the analysis of a fresh (a) and a commercial (b)orange juice, respectively.

A CSP based on teicoplanin, another glicopeptidic antibi-otic, was also used for the determination of racemic atenololin human urine by nano-LC hyphenated with MS [105]. Aftercomparing the sensitivity of the nano-LC method obtainedby using a conventional on-column UV detector for CE, or azeta cell (3 nL volume), the ion-trap MS detection was chosen

since offered the highest sensitivity (LOD, 50 ng/mL, andLOQ, 400 ng/mL for each atenolol enantiomer).

Less common CSPs to carry out enantioseparations bymeans of nano-LC are also reported in literature. Widepore aminopropyl silica gel coated with helically chiralpoly(diphenyl-2-pyridylmethyl methacrylate) was used as CSPfor a comparative study among conventional HPLC, nano-LC, pressure-assisted CEC, and CEC. Enantioseparations ob-tained in nano-LC showed higher peak efficiency, and con-sequently higher resolutions, than those achieved with acommon-size HPLC column. Comparing with CEC, in nano-LC similar efficiencies were unexpectedly observed [106].Fanali et al. investigated the use of tert-butylbenzoylatedtartardiamide chiral stationary phase for the separation ofracemic acidic compounds of pharmaceutical and environ-mental interest, working in normal phase mode [107]. Tert-butylbenzoylated tartardiamide chiral stationary phase be-longs to a series of CSP based on optically active �-amino acidsor tartaric acid diamides able to form weak diasteromericcomplexes with analytes based on hydrogen bonding. A studyconcerning the influence of capillary temperature on enan-tioresolution was also carried out demonstrating that reten-tion of analytes was an exothermic process. It should be un-derlined that this kind of study can be easily performed withcapillary columns, since they are rapidly conditioned at thedesired temperature.

In normal phase, chiral separations of amino acid amideswere also obtained using (S)-N-(3,5-dinitrobenzoyl)leucine-N-phenyl-N-propylamide-bonded silica as CSP. The chiralrecognition mechanism of this CSP utilizes the �−� donor–acceptor and hydrogen-bonding interactions. The data ob-tained in nano-LC were compared with those from CEC andit was concluded that enantioselectivities were similar, butresolution was better in CEC. However, the authors stated

Figure 3. Comparison of two differ-ent nano-LC-MS profiles from orangejuice: fresh squeezed and commercialjuice. Capillary chiral column, 75 �mid × 25.0 cm packed length; mobilephase, 500 mM ammonium formatepH 3.5/water/MeOH, 4:11:95 v/v/v; MSconditions: positive ion mode; capil-lary voltage: 30 V; ion-spray voltage:2.0 kV; capillary temperature: 170C.From [104] with permission.



that nano-LC separation was not optimized for injection vol-ume and extra-column band broadening contributions [108].Another comparison between nano-LC and CEC, was re-cently reported by Lee et al. [109]. (−)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid-bonded silica as CSP was employed forenantioseparation of some �-amino acids. Chiral recognitionmechanism involves the tripodal complexation of the proto-nated primary amino group (R-NH3

+) inside the cavity ofthe 18-crown-ether ring via three +N–H···O hydrogen bondsand additional roles are played by the two free carboxylic acidgroups acting as chiral barriers, enantioselective hydrogen-bonding sites, or ionic interaction sites. Generally, CEC gavebetter enantioselectivity and resolutions than nano-LC, as aconsequence of higher efficiency due to the flat EOF profilepresent CEC.

5.3 Chiral monolithic columns

Monolithic media are widely used for capillary columns sincethey can be easily prepared in situ and do not require frits toretain the stationary phase. The latter aspect is particularly ad-vantageous when CSPs have to be employed, because whensilica particles have been derivatized with a CS, quite oftenthey are no more suitable for frit preparation or new appro-priate conditions of sintering have to be found. Two maincategories of polymeric material can be distinguished: thoseobtained by polymerization of organic monomers and thoseconsisting of an inorganic silica network. The polymeriza-tion of silica monoliths can be performed by sol-gel processor by fusion of porous silica packing material in a capillaryvia sintering, entrapping in a silica network or by sol-gel pro-cess. In the last case, a sort of hybrid between monolithicand packed bed is obtained. Wistuba et al. prepared a chi-ral silica monolith, by sintering a bare silica bed followed byon-column modification with permethylated �-CD (Chirasil-Dex) [110]. The column was successfully tested in nano-LC,pressure assisted–CEC (p-CEC) and CEC for the enantiomerseparation of several chiral compounds. Later, the same groupentrapped silica particles into a silica matrix by sol-gel pro-cess, creating a so-called particle-glued monolithic columnby means of immobilization of Chira-Dex-silica (permethyl-�-CD-silica) [111]. Also in this work, the same column wasutilized for chiral separation in nano-LC, p-CEC, and CEC aswell. Considering hexobarbital as test compound and apply-ing a pressure of only 10 bar in nano-LC and 10 bar plus 20 kVin p-CEC, retention time in nano-LC was four time longer,which demonstrated that in p-CEC the EOF is the main driv-ing force. When the applied pressure was 150 bar for bothtechniques (carprofen was used as test compound), reten-tion time was slightly higher in CEC, since electrophoreticmobility of carprofen (negatively charged) was in the oppo-site direction of EOF. A small improvement of enantiores-olution and efficiency was also observed. When the polarityof the applied voltage was inverted, a reduction of analysistime was observed, but resolution decreased as well. Em-ploying another CD derivative, perphenylcarbamoylated �-

CD (Ph-�-CD), as chiral selector, a silica hybrid monolith wasproposed by Zou et al. [112]. The capillary column was pre-pared by a “one-pot” approach. First, mono (6A-N-allylamino-6A-deoxy)-Ph-�-CD was synthesized starting from mono-6A-deoxy-6A-(p-tolylsulfonyl) �-CD. After, the polycondensationof alkoxysilanes and in situ copolymerization of mono (6A-N-allylamino-6A-deoxy)-Ph-�-CD and vinyl group on the pre-condensed siloxanes took place in a pretreated fused silicacapillary. The influence of several parameters on the synthe-sis of the chiral monolith were evaluated, such as polyconden-sation temperature, amount of chiral monomer, the ratio ofN,N-dimethylformamide/methanol, and the content of waterin the polymeric mixture. Finally, the capability of the col-umn for enantioseparation was demonstrated resolving 13racemates in normal- and reversed phase modes.

Monolithic silica columns were derivatized with polysac-charide derivatives, exploiting the high permeability and ef-ficiency offered by monolithic silica materials as well asavoiding frits fabrication. In a preliminary study, nativesilica-based monolith was modified in situ with CDMPC[13]. Working with mobile phases composed by n-hexane/2-propanol mixtures, enantioseparation of analytes as 2,2,2-trifluoro-1-(9-anthryl)ethanol, benzoin, 2,2’-dihydroxy-6,6’-dimethylbiphenyl, flavanone, and trans-stilbene oxide, wereobtained in less than 4 min, while for several �-blockers enan-tioresolution was achieved at most within 12 min (see Fig. 4).The same column showed enantiorecognition capability alsooperating with reversed phase conditions. Moreover, a studywas carried out in order to further decrease analysis time and,as an example, it was reported the chiral separation of 2,2,2-trifluoro-1-(9-anthryl)-ethanol obtained in less than 30 s, witha column of 12 cm packed length. However, for this columnthe coating procedure with the cellulose derivative was re-peated three times. The obtained positive results promptedthe authors to modify silica monolith with amylose tris(3,5-dimethylphenylcarbamate) [113]. The effect of the amountof amylose tris(3,5-dimethylphenylcarbamate) coated ontomonolithic silica on separation characteristics was also stud-ied and it was noted that, even if increasing the concentrationof the CS during the coating procedure brought to higherresolutions, it negatively affected efficiency. In situ modifica-tion of monolithic-fused silica capillary columns was realizedcovalently bonding 3,5-disubstituted phenylcarbamate deriva-tives of cellulose and amylase as well, to increase the stabilityof these CSP toward different solvents [114]. Although, as ex-pected, polysaccharide derivatives coated onto silica supportshowed fairly higher enantiorecognition compared to the ma-terials covalently attached onto the surface, covalent immobi-lization of polysaccharide derivatives allowed multiple coat-ing and immobilization steps with a consequent increasedretention and separation factors, with few exceptions.

A monolithic silica capillary column was used also for thephysical adsorption of avidin and tested for nano-LC and CECchiral separation [115]. The effect of type and concentrationof activating capillary reagent (sodium hydroxide solutionsor ammonia solutions) was investigated and optimized in or-der to increase the adsorbed amount of avidin. Reducing the



Figure 4. Enantioseparation of 2,2,2-trifluoro-1-(9-anthryl)ethanol (A), benzo-in (B), 2,2’-dihydroxy-6,6’-dimethyl-biphenyl (C), trans-cyclopropanedi-carboxylic acid dianilide (D), flavanone(E), and trans-stilbene oxide (F) onmonolithic silica capillary column(100 �m id × 20.0 cm) modified with50 mg/mL solution of CDCPC inacetone. Mobile phase was n-hexane/i-PrOH (90:10, v/v). Applied pressurewas varied in the range 1.0–2.0 MPadepending on the analyte. From [13]with permission.

column length from 27 to 6.5 cm some tested racemic ana-lytes were baseline resolved within 4 min in both CEC andnano-LC. Finally, to enhance the sensitivity, field-enhancedsample injection was successfully coupled to the techniques.Recently, penicillin G acylase, an enzyme, was instead co-valently immobilized on a monolithic epoxy silica capillarycolumn [65]. It resulted in good stability of the chiral selec-tor and satisfactory enantioselectivity and efficiency towardarylpropionic acids. Employing a capillary column packed for7 cm only, the simultaneous enantioseparation of racemicketoprofen, suprofen, and flurbiprofen was obtained within5 min, as reported in Fig. 5. The method was finally vali-dated for the quantitation of the distomer R-ketoprofen inconcentrations within the range 0.25–2.0% w/w with respectof S-ketoprofen (1 mg/mL) in an artificial solution, and afterin a pharmaceutical sample (tablets), being 0.25% w/w theLOQ value.

Other chiral selectors were employed in combinationwith silica monoliths. Dansyl amino acids and hydroxylacids were enantioseparated by CEC and nano-LC, usingL-prolinamide as CS based on a ligand exchange mecha-nism [116]. Cu(II) was grafted on the surface of the CSPconditioning with aqueous solution. With this kind of CS,the chiral discrimination is due to the exchange of one lig-and in the Cu(II) complex with an analyte ligand, whichform ternary-mixed copper complexes with different stabil-

Figure 5. Simultaneous enantioseparation of ketoprofen, supro-fen, and flurbiprofen in nano-LC using a duplex column consti-tuted of a separation penicillin G acylase (PGA)-monolithic sec-tion (100 �m id × 7.0 cm) and an open fused-silica capillarysection (50 �m id × 26.0 cm). The mobile phase was 50 mMsodium phosphate buffer, pH 7.0. The applied pressure was 12bar. From [65] with permission.

ity. The CSP offered higher efficiency in CEC. Subsequently,the same procedure was adopted to realize CSP containingL-phenylalaninamide or L-alaninamide. These CSP, togetherwith that based on L-prolinamide were evaluated for nano-LCapplications [117], and further investigated also in CE andCEC [118] to better explain chiral recognition mechanism. In



nano-LC, among all the studied CSP, L-phenylalaninamide-modified monolithic column showed higher selectivity thanalaninamide-modified one, while L-prolinamide- and L-phenylalaninamide-modified CSPs showed a different enan-tioselectivity. Preinerstorfer et al. modified a monolithicsilica column with O-9-(tert-butylcarbamoyl)quinidine, cin-chona alkaloid-derived anion-exchange-type chiral selector,for the stereoselective analysis of some phosphinic acid pseu-dodipeptides in CEC. The same column was tested in nano-LC as well, but the lower efficiency gave a worse separation.However, the same negative outcome was observed in con-ventional HPLC, too [119]. A silica monolith functionalizedby a cation exchange type chiral selector, trans-(1S,2S)-2-(N-4-allyloxy-3,5-dichlorobenzoyl)aminocyclohexanesulfonic acid,was studied by the same team for the enantioseparation ofvarious chiral bases in nano-LC and CEC. The influence ofCS coverage was considered and it was concluded that nei-ther the second immobilization cycle (29 nmol/cm of CSinstead of 22 nmol/cm with first immolization) nor the dy-namic flow-through immobilization improved enantiomer-resolving ability [120]. In a following work, considering meflo-quine, mefloquine-O-tert-butylcarbamate, and pronethalol astest solutes, individual migration contributions in CEC (elec-trokinetic and chromatographic) were better investigated toexplain the behavior of the silica-based monolithic capil-lary column under CEC and nano-LC conditions and theseparation mechanism of charged solutes in stereoselec-tive ion-exchange CEC. Comparing the performance of bothtechniques in terms of kinetic plots, it was noted that theefficiency gain in CEC was mainly due to a greatly reducedA-term contribution to peak dispersion, while apparently thecolumn gave comparable efficient mass-transfer character-istics, with a C-term slightly smaller in CEC [121]. Recently,(R)-acryloyloxy-�-�-dimethyl-�-butyrolacton, a low-molecularmass CS was employed to create a brush type CSP. Afterthe derivatization of native-free OH silica monolith with 3-methacrylamidopropyl-triethoxysilane, the resulting hybridmonolith was in situ copolymerized with the CS. A set ofracemic secondary alcohols and a chiral amine were sepa-rated and the main mechanism of enantiorecognition wasascribed to the H-bond acceptor and the hydrophobic interac-tion with the gem dimethyl group of the butyrolacton [122].

Organic polymer monolith materials were also used toprepare chiral media. Generally, they offer easier and fasterprocedures of preparation than silica-based monolith, butlower efficiency and reproducibility. Schmid et al. prepared achiral ligand exchange stationary phase by a one-step in situpolymerization of acrylamide based continuous bed, utiliz-ing N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline as CS[123]. The column was projected to be used in CEC, however,for D, L-phenylalanine enantioresolution was demonstratedin nano-LC, too.

Guerrouache et al. synthesized an organic polymer basedon N-acryloxysuccinimide and ethylene dimethacrylate, byUV-initiated free radical polymerization [124]. In a secondstep, succinimide groups were derivatized with propargy-lamine to insert alkine moieties, and finally an azido deriva-

tive of �-CD (mono-(6-azido-6-deoxy)-�-CD) was grafted onthe monolithic bed, exploiting a click reaction. As prelimi-nary result, the enantioseparation of flavanone was achievedin both CEC and nano-LC.

5.4 MIP stationary phases

Monolithic matrices give the possibility to prepare MIP, bythe addition of a template (imprint) molecule in the poly-meric mixture. Monomers arrange around the template, be-cause of secondary interactions (e.g. hydrogen bonds) or theformation of covalent bonds between functional groups ofthe template and those of the monomers. Once polymeriza-tion is completed, the imprint molecule is extracted and thepolymer network will posses cavities having shape, size, andfunctional groups complementary to those of the template.As a consequence it shows a remarkable affinity toward thetemplate. For this characteristic, MIP has been widely stud-ied for synthetic chemistry, catalysis, solid-phase extractions,sensor design, and as CSP for chromatographic separations.For conventional LC, MIPs are synthesized in bulk and af-ter ground, sieved and packed into columns. However, thisprocedure presents some drawbacks, such as wide particlesize distribution and irregular particles shape, which nega-tively affect efficiency. The introduction of capillary columnsallowed the synthesis in situ of MIPs as well [125–127].

Dansyl-L-phenylalanine was used as template by Tan andRemcho, to prepare 25-�m id capillary columns for chiral OT-LC and OT-CEC [125]. The chiral separation of a mixture of D-and L-dansyl phenylalanine was obtained applying a pressurein the range of 100–500 mbar. At 350 mbar enantiomers wereresolved in less than 8 min, with a column with an effectivelength of 85 cm.

A limitation when using one-step polymerizations to pre-pare MIPs is the need to optimize the conditions to obtainthe desired porosity for each used polymeric mixture and tem-plate. A way to manage surface chemistry without stronglychange the permeability of a given material, consist of graft-ing procedure. If a porogenic agent is added to the graftingmixture, it is possible to graft a mesoporous layer with aconsequent increase of the surface area and the number offunctional groups. Courtois et al. proposed this expedientto prepare capillary columns for low abundance sample en-richment [128]. Mepivacaine, bupivacaine, and enantiopureS-ropivacaine were alternatively used as imprint molecules.During their evaluation, authors observed that the columnprepared with S-ropivacaine showed high selectivity for thesame enantiomer, and a split peak for mepivacaine, charac-teristic of racemic compounds. This phenomenon could befurther investigated to prepare analytical column by meansof the same procedure. A similar approach was developed byOu et al. to anchor MIP onto the surface of a monolithic sil-ica capillary column [129]. L-Tetrahydropalmatine (THP) and(5S,11S)-(–)Troger’s base were used as template, respectively.L-THP was also used as imprint molecule to prepare an or-ganic monolithic capillary column in a one-step procedure.



Both L-THP-based columns, composite monolith and organicmonolith, were tested in nano-LC and it was noted that theretention of THP enantiomers was stronger on the organiccolumn, as a consequence of large number of recognitionsites on its surface, but concerning column efficiency andseparation time the composite monolith gave better perfor-mance.

Ziadi et al. reported in situ preparation of an OT MIPmonolithic column where S-ketoprofen was used as tem-plate [130]. To secure formation of the MIP layer into a silicacapillary of 100-�m id, the reaction mixture was rinsed intothe capillary and let to react in a water bath at 50C for 3 h.Then the capillary was flushed with a 0.5 MPa nitrogen flowfor 5 min, and was again placed in the water bath for 2 h tocomplete MIP formation. The column showed high enantios-electivity not only toward ketoprofen, but for ibuprofen andnaproxen as well, demonstrating high cross-selectivity.

5.5 CMPA

As already reported in this manuscript, miniaturized tech-niques offer the advantages to use the chiral selector dissolvedin the mobile phase in its free form, since very few amountof it is needed to perform analysis. The use of a CMPA can beconsidered convenient compared to a stationary phase con-taining it, when CMPA results less expensive, the synthesisof the CSP is difficult, accessibility of the CS present in freesolution for interaction with analytes is easier. Furthermore,the combination of achiral stationary phases with CMPAs orof different chiral additives can provide higher flexibility forthe selection of experimental conditions or improve chiralrecognition.

Healy et al. employed methyl-�-CD as CMPA in combina-tion with a C18-RP capillary column for the enantioseparationof the anti-inflammatory drug naproxen. They demonstratedthat, working with the same experimental conditions used inconventional HPLC, in nano-LC the consumption of mobilephase was reduced more than 1000 folds [131].

Later on, Rocco and Fanali carried out a study onthe enantioseparation of several racemic nonsteroidal anti-inflammatory drugs employing different achiral stationaryphases in combination with heptakis (2,3,6-tri-O-methyl)-�-CD (TM-�-CD) [132]. Among all investigated stationaryphases, i.e. cyano, C8 and different types of C18 (5- and3-�m particle size, bidentate 4.2-�m particle size), C18, 3 �m,reversed phase gave best results in terms of achieved enan-tioresolution. The chromatographic method was also appliedfor the determination of the association constant betweenracemic analyte and CD. An analogous work about the com-bination of CD with an appropriate stationary phase, regardedthe development and validation of an analytical methodfor the determination of a chiral fluoroquinolone antibiotic,ofloxacin. In this case, enantioresolution was achieved em-ploying heptakis-(2,3-diacetyl-6-sulfo)-�-CD as CMPA and anachiral reversed phase capillary column packed with Pinna-cle II Phenyl 3 �m [133]. The method performance was eval-

uated thorough the following analytical parameters: LOD,LOQ, linear range, accuracy, and precision. Satisfactory datawere achieved such as LOD values of 1.94 and 3.75 �g/mLfor the R-(+) and S-(–) enantiomer, respectively, and intradayand interday precision of peak areas, expressed as RSD, aslow as 4.74%.

The same group accomplished a research about the com-parison of the performance of a RP-C18 monolithic columnand a RP-C18-packed column of the same dimensions, usedin combination with TM-�-CD or hydroxypropyl-�-CD (HP-�-CD) [49]. As expected, the monolithic column showed highpermeability but lower selectivity compared to the packedone. For this reason, in case of TM-�-CD, only when enan-tioresolutions were high enough with the packed column,compounds could be still baseline resolved with the mono-lithic column and in reduced analysis time. The monolithiccolumn resulted particularly advantageous when HP-�-CDwas selected as CMPA. In fact with this CS, enantiodiscrimi-nation occurs with very low percentage of organic modifier oreven in a completely aqueous mobile phase. With the packedcolumn in those conditions (0–10% organic modifier), mostof studied compounds not eluted in reasonable time, whilewith the monolithc column they were resolved within 30 minat most.

6 Conclusions

The downscaling of chromatographic technique, up tonanoscale level, aroused lively interest among scientists in-volved in separation science, since this miniaturization in-troduced advantages as low consumption of reagent, smallamount of sample needed, reduced analysis time, good effi-ciency, and easy coupling with MS. For these reasons, nowa-days, nano-LC represents a valid alternative to classical chro-matographic techniques in many analytical fields, includingchiral analysis.

The main characteristic of nano-LC that determined itssuccess in the field of enantioseparation is related to the lowrequirement of CSP or CMPA, since this material can be veryexpensive. Nano-LC, e.g. is suitable for the study of new CSP,especially when small amount is available.

Several chiral separations by means of nano-LC are re-ported in literature. A large number of works is related tothe study of selectand/selector interactions, however, applica-tions in the pharmaceutical, clinical, environmental, and foodareas have been carried out as well. Among chiral selectors,polysaccharides, CDs, and MAs were widely employed. Thereduced flow rate and the consequent limited consumptionof mobile phase, allows the use of chiral additives. Most ofresearch papers involve the use of particulate-packed capillarycolumns. However, as a common trend in other applicationfields, the synthesis of chiral monolithic columns is continu-ously investigated. In fact, due to the capillary format of thecolumns employed in nano-LC, they can be easily preparedin situ, offer high permeability and do not need frits to re-tain the stationary phase. Furthermore, they allow to realize



highly selective material as MIP and seem to represent a goodopportunity to realize nano-LC in microdevices.

The authors have declared no conflict of interest.

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Anna Rocco graduated in Pharmaceutical Chemistry and Technologies from the Univer-sity “La Sapienza”, Rome, in 2003. Since 2004 she is working at the Institute of ChemicalMethodologies CNR, in Rome. In 2008 she started her Ph.D. studies at the Faculty ofNatural Science, Vytautas Magnus University, Kaunas. She has experience in analysis ofsamples of interest in pharmaceutical, environmental and food fields by CE, CEC andn-LC. She has also practice in preparing packed and monolithic capillary columns.



Audrius Maruska graduated from Kaunas University of Technology (1985), obtainedthere PhD degree in synthesis and evaluation of cellulose adsorbents (1990) and Habili-tated Dr. degree at Vilnius University in the field of stationary phases and techniques formicroseparations (2002), spent long-term scientific stays at Mainz, Marburg and UppsalaUniversities. Currently he is a head of research group at Vytautas Magnus Universityactive in the field of development of microseparations, synthesis of stationary phases,coupling of methods and phytochemical analysis.

Salvatore Fanali achieved the title of Doctor in Chemistry at the University of Rome “LaSapienza”. His research include either chromatography and electromigration techniquesin conventional and miniaturized modes. Special attention was paid in coupling nano-LC, CE and CEC with mass spectrometry. He is head of a research group at Instituteof Chemical Methodologies active in the development of miniaturized instrumentationand applications in the field of food analysis, enantiomers separation, new columntechnology, environmental analysis etc.


enantiomeric separations by means of nano-lc

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