Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids.
ABSTRACT A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-microm diol-silica particles. The CSP mixed with 5-microm amino silica particles (3:1) was packed into 75-microm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1-3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor Rs=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)-water-acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)-water-methanol-acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12-20 min).
- [show abstract] [hide abstract]
ABSTRACT: In the past few decades, macrocyclic antibiotic molecules have become among the most useful chiral selectors in analytical HPLC, thin-layer chromatography and capillary electrophoresis and also in preparative methods. The macrocyclic glycopeptides, such as teicoplanin, vancomycin and ristocetin A and its analogs, are perhaps the most useful selectors for the enantioseparation of nonprotected and N-protected peptides and amino acids, β-blockers, β-agonists, nonsteroidal anti-inflammatory drugs, antineoplastics and various other biologically important compounds. This article discusses the physicochemical properties, method developments, mechanisms and applications for separations on macrocyclic antibiotics. Since comprehensive reviews on the HPLC separation of biologically important analytes on macrocyclic antibiotic chiral stationary phases have been published up to the middle of the 2000s, the recent progress covers the period 2006–2010, focussing on the HPLC applications of antibiotic phases.Separation and Purification Reviews - SEP PURIF REV. 01/2012; 41(3):207-249.
- [show abstract] [hide abstract]
ABSTRACT: Enantiomers represent a class of compounds extensively investigated since they can show totally different behaviors when they interact with a chiral environment. Because of their identical chemical structure (they differ only in the spatial arrangement of the atoms in the molecule), the separation of optical isomers is a challenging task of analytical chemistry. So far employed methods for the separation of enantiomers are mainly based on chromatography. CE as well was considered as an analytical technique suitable for chiral separations, characterized by high efficiency and low consumption of reagent. Recently, miniaturization was introduced in LC to answer the needs to perform analyses in the minimum time, to use the smallest amount of samples and to reduce environmental pollution. Nano-LC represents nowadays a valid alternative to the abovementioned conventional analytical techniques, and can be advantageously exploited for enantiomeric separation especially because it needs minute amounts of the chiral material necessary to carry out enantiomeric separations. This review describes the development and applications of nano-LC in the field of chiral separations. The data reported in literature show its relevance for the study enantiomers-chiral selectors interaction, as well as for application in pharmaceutical and clinical research.Journal of Separation Science 11/2012; · 2.59 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The present review intends to summarize recent developments in the field of enantioselective separations and analysis by CEC. It covers studies published in English language in common peer-reviewed journals within the period between 2003 and 2006. Both, methods making use of chiral mobile phase additives as well as chiral stationary phases for electrochromatographic enantiomer separations, are reviewed. Achievements that have been made on the various column technologies, such as open-tubular, particle-packed, inorganic, organic and particle-fixed (hybrid-type) monolithic as well as molecularly imprinted polymer phases, are discussed.Electrophoresis 09/2007; 28(15):2527-65. · 3.26 Impact Factor
Journal of Chromatography A, 990 (2003) 143–151
U se of short-end injection capillary packed with a glycopeptide
antibiotic stationary phase in electrochromatography and capillary
liquid chromatography for the enantiomeric separation of
Salvatore Fanali , Paolo Catarcini , Carla Presutti , Rosanna Stancanelli
aIstituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche (CNR), Area della Ricerca di Roma, P.O. Box 10,
00016 Monterotondo Scalo, Rome, Italy
Dipartimento Farmaco Chimico, Facolta di Farmacia, Universita di Messina, Viale Annunziata, 98168 Messina, Italy
Dipartimento di Studi Farmaceutici, Universita degli Studi di Roma ‘La Sapienza’, P. le A. Moro 5, 00185 Rome, Italy
A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic
belonging to the teicoplanin family, with 5-mm diol-silica particles. The CSP mixed with 5-mm amino silica particles (3:1)
was packed into 75-mm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing
several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode
in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric
resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition
(organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on
enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the
separation of mandelic acid enantiomers in less than 72 s with resolution factor R 52.18 applying a voltage of 30 kV and
eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was
also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the
mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long
analysis times were observed (12–20 min).
2003 Elsevier Science B.V. All rights reserved.
Keywords: Injection methods; Chiral stationary phases, electrochromatography; Chiral stationary phases, LC; Electro-
chromatography; Glycopeptide; Peptides; Antibiotics; Hydroxy acids; Teicoplanin; Mandelic acid
1 . Introduction
environmental, agrochemical, pharmaceutical inter-
est, because they contain in their chemical structure
one or more asymmetric center, exist as one or more
couples of enantiomers. Very often the two enantio-
mers may exhibit different pharmacological or bio-
chemical properties  and therefore in the case of
the introduction in the market of a new drug, the
A wide number of compounds including those of
*Corresponding author. Tel.: 139-6-9067-2256; fax: 139-6-
E-mail address: email@example.com (S. Fanali).
0021-9673/03/$ – see front matter
2003 Elsevier Science B.V. All rights reserved.
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151
pharmacological effect and the study of the metabo-
lism have to be carefully studied.
In the last few years analytical methods able to
resolve chiral compounds were studied focusing
attention on the development of new chiral stationary
phases (CSPs) and/or chiral selectors achieving good
enantioresolution in short time and good efficiency.
Analytical methods so far used for the enantio-
meric separations include gas chromatography (GC),
high-performance liquid chromatography (HPLC),
supercritical fluid chromatography (SFC) and recent-
ly capillary electrophoresis (CE) [2–8]
CE is a powerful electromigration technique ex-
hibiting high efficiency and high resolution in short
analysis time towards a wide number of enantiomeric
compounds belonging to different classes such as
organic and inorganic ions/neutral molecules, pep-
tides, proteins, herbicides, drugs, etc. More recently
capillary electrochromatography (CEC) was success-
fully applied to the enantiomeric resolution of a wide
number of compounds utilizing the advantages of
both CE and HPLC (high efficiency and selectivity,
In CEC a relatively strong electroosmotic flow
(EOF) with a flat flow profile carries to the detector
both enantiomers and mobile phase, while the
stereoselectivity and the enantioresolution can be
achieved because of interactions with the chiral
selector. The former is present in the system, e.g.,
bonded to the capillary wall (open-CEC) or to the
packed stationary phase (p-CEC) or to a polymeric
material or added to the mobile phase [8,10].
Several chiral selectors were successfully em-
ployed in CEC and among them glycopeptide anti-
biotics (GAs) resulted to be powerful enantiorecogni-
tion agents towards a wide number of compounds,
e.g., amino acid derivatives, herbicides, drugs, etc.
Vancomycin and teicoplanin, belonging to this
class of chiral selectors, exhibited very high enan-
tioresolution capability mainly towards basic com-
pounds. Here the studies were carried out employing
either packed or monolithic columns containing
separately the two GAs [11–19]. In a recent work
, with the aim to find a CSP useful for the
separation of acidic enantiomeric compounds, we
studied a new silica-based CSP employing a Hepta-
Tyr antibiotic (MDL 63,246) (a modified teicop-
lanin) as the chiral selector. The same GAs was
studied by us using capillary zone electrophoresis
(CZE) for the enantiomeric resolution of acidic
compounds such as herbicides, drugs and hydroxy
Very often it is desirable to perform analyses as
fast as possible, especially when a large number of
samples have to be handled, therefore appropriate
method optimization must be carefully studied. Ex-
amples of fast chiral resolutions were already re-
ported in both CZE and CEC [23–25].
In this study part of the short-end injection (8.4
cm) was packed with the CSP containing MDL
63,246 mixed with amino propyl silica (3:1) and
used for CEC experiments in order to perform
enantiomeric resolution of selected hydroxy acids as
fast as possible.
In order to optimize the enantiomeric separation of
the selected racemic compounds we studied the
effect of the mobile phase composition modifying
the content and the type of organic solvent as well as
the pH of the buffer.
2 . Experimental
2 .1. Instrumentation
Electrochromatographic experiments were carried
out using an Agilent 3D CE instrument (Waldbronn,
Germany) equipped with a UV-diode array detector
operated at 195 nm (unless otherwise stated) and a
thermostated capillary cartridge applying different
voltages in the range 5–30 kV. Injection was done at
the short end of the capillary (cathodic polarity)
applying 12 bar, 0.2 min. During the experiments
both ends of the capillary were pressurized at 8 bar
in order to avoid bubble formation.
The fused-silica capillaries, 75-mm I.D.3375-mm
O.D., used in this work were purchased from
Composite Metal Services (Hallow, UK). Capillary
packing was done by using a LC series 10 HPLC
pump (Perkin-Elmer, Palo Alto, CA, USA).
2 .2. Materials and methods
DL-m-Hydroxymandelic acid (m-OH-MA), and DL-
3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-
MA) were purchased from Sigma (St. Louis, MO,
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151 145
USA); D-(2)- and L-(1)-mandelic acids (MA) were
from Carlo Erba (Milan, Italy) while DL-p-hydroxy-
mandelic acid (p-OH-MA) was from Ega-Chemie
PhL) and 4-chloro-DL-mandelic acid (4-Cl-MA) were
purchased from Fluka (Buchs, Switzerland). Metha-
nol (MeOH) and acetonitrile (ACN), of pure reagent
grade were obtained from BDH (Poole, UK). Am-
monium acetate, sodium cyanoborohydride, sodium
periodate, LiChrospher diol silica phase 5-mm par-
ticle diameter (pore size 100 A) were purchased
from Merck (Darmstadt, Germany). Kromasil amino
silica 5 mm was a gift of Aka Chemicals (Bohus,
Sweden), MDL 63,246 glycopeptide antibiotic was
synthesized at Lepetit Research Center (Gerenzano,
Italy)  and purified by Righetti’s group with a
preparative isoelectric focusing method (see Ref.
Aqueous mobile phases were prepared by adding
the appropriate volume of organic modifier to the
buffer solutions at a controlled pH 6.
One mg/ml analyte stock solutions were prepared
in methanol and stored at 4 8C; the solutions were
daily diluted with the mobile phase at the desired
concentrations and injected for the CEC analysis.
silica, sonicated for 60 min, centrifuged and washed
The recovered modified silica CSP was treated
with 30 ml of 50 mM phosphate buffer at pH 3.1
containing 10 mM of NaCNBH
60 min, washed with water as in point (B). The
modified silica particles were washed three times
with 20 ml each of methanol and the solvent
evaporated at room temperature under vacuum.
The following steps were used in order to prepare
the CSP packed capillaries: (i) the capillary was
connected to a mechanical temporary frit and
packed, using an LC pump, with a slurry of Li-
Chrospher diol–silica (2:1) in 10 mM NaCl solution;
the frit was prepared with a heating wire (about
350 8C, 10 s). The capillary was cut close to the frit,
connected to the pump and flushed with water in
order to eliminate the excess of silica phase, the
capillary was then connected to the pump with the
opposite side for next step: (ii) a slurry of 40 mg
MDL–amino silica (3:1, w/w) in 1.5 ml water–
acetonitrile (1:1, v/v) mixture was prepared, soni-
cated and used for packing the capillary (about 2000
p.s.i., 6.6 cm; 1 p.s.i.56894.76 Pa). (iii) The capil-
lary was then packed with diol–silica (2:1, w/w) for
4 cm and the end frit prepared close to the chiral
stationary phase (0.4 cm). The part of the capillary
where the polyimide was removed was covered with
a layer of epoxy resin. The total length of the
capillary was 34.4 cm; second frit at 7.0 cm;
effective length at 8.4 cm.
Fig. 1 shows the scheme of the separation capil-
lary used for CEC experiments where the effective
length was the short end of the tube.
DL-2-Phenyllactic acid (2-
2 .3. Synthesis of the chiral stationary phase and
packing capillary procedure
The Hepta-Tyr chiral stationary phase (MDL-
CSP) was prepared in our laboratory according to a
previously published method for the synthesis of
vancomycin CSP .
(A) Four hundred mg of 5 mm LiChrospher DIOL
silica particles were added to a 30 ml mixture of
water–methanol (4:1, v/v) containing 60 mM of
NaIO and sonicated for 60 min in order to oxidize
the diol to aldehyde groups. The mixture was
centrifuged at 4000 rpm for 5 min and the solution
eliminated; the solid material was washed for three
times with 20 ml of water.
(B) One hundred and sixty mg of Hepta-Tyr
antibiotic (MDL 63,246) were dissolved in a mixture
of 50 mM NaH PO , pH 7.04, titrated with NaOH
containing 10 mM of NaCNBH
trile (6 ml) in order to have a 3 mM solution of
MDL. The solution was added to the oxidized diol
3 . Results and discussion
3 .1. Test of the capillary packed at the short-end
The use of CEC for the separation of chiral
compounds can offer great advantages over other
established CE modes, e.g., free zone electrophoresis
(CZE). In fact in CEC the same column packed with
the CSP can be used several times, strongly reducing
the amount of expensive chiral selector. On the
contrary in CZE, besides only a few milligrams of
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151
Fig. 1. Scheme of the packed capillary used in this study.
chiral selectors (CSs) being employed, it is necessary
to change the BGE after each run. Furthermore the
CSs usually not being transparent at the UV wave-
lengths used for detection, are responsible for the
low sensitivity. In this case the partial filling method
combined with the counter-current process was
proposed analyzing a wide number of chiral com-
GAs such as vancomycin, teicoplanin and teicop-
lanin derivatives, resulted to be excellent chiral
selectors and successfully tested for the enantiomeric
resolution of a wide number of compounds, includ-
ing amino acid derivatives and drugs in both CZE
When analyzing a large number of enantiomeric
samples it is desirable to perform the work in a short
time and therefore several approaches can be consid-
ered. Among them it is noteworthy to mention (i) an
increase of the applied electric field (higher voltages
or shorter capillary) and (ii) the use of a short
effective length of the capillary. In both cases we
have some limitations due to the fixed maximum
voltage that can be applied to the minimum length
that the capillary cartridge can allow and to the fixed
effective length (8.4 cm).
In this study the capillary was packed at the
short-end injection with slurry composed by MDL
63,246 antibiotic and amino silica. The GA and
amino silica present in the packed column, posses-
sing amino groups were positively charged at the
operating experimental conditions, pH,7. Therefore
a strong EOF was expected due to the positive
charge of the stationary phase.
The packed capillary at the short-end injection was
tested analyzing racemic mandelic acid; the mobile
phase was a mixture of ammonium acetate, pH 6, in
50% (v/v) acetonitrile applying a voltage in the
range 5–30 kV. The injection was done from the
outlet side of the capillary and analytes were moving
as anions through the short end (8.4 cm) to the
detector carried by a strong electroosmotic flow and
the self electrophoretic mobility of the analyte. The
experimental set-up resulted to be quite stable, but
some instability can be expected due to the fact that
two different EOF are involved in the separation
process: the first (towards the anode) due to the CSP
(positively charged) and the second (towards the
cathode) originating from the capillary wall (nega-
tively charged). The measured EOF was in the range
5.8–0.35 min (2.5–30 kV), clearly showing that the
influence of the capillary wall was negligible; this
was also shown by Dittmann and Rozing using two
different reversed-phase columns  where the
RP18 silica particles were packed into two different
capillaries (coated and uncoated), obtaining the same
The plot of reduced plate heights versus linear
velocity of the EOF (see Fig. 2) showed an increase
of efficiency by decreasing the velocity of the EOF.
However 20 kV was the selected voltage for further
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151147
namely mandelic acid, m- and p-hydroxymandelic
acid, 3-hydroxy-4-methoxymandelic acid, 4-chloro-
mandelic acid and 2-phenyllactic acid was studied by
CEC using the mobile phase containing ammonium
acetate at pH 6 and 30–60% of ACN. The content of
the organic modifier was limited at the studied range
because current instability and too long analysis time
were observed at concentrations higher than 60% and
lower than 30%, respectively. All studied compounds
were baseline resolved at any concentration of ACN
except 2-PhL which showed R 50.8–1.0. The re-
corded enantioresolution factors resulted 10–50%
lower than those previously observed  employing
the same stationary phase with a longer effective
length (26 cm, with chiral length of 24 cm).
The enantioresolution did not change remarkably
by increasing the MeCN concentration while higher
efficiencies were observed at 50 and 60%. Therefore
we studied the effect of MeOH (0–50%) added to
the mobile phase containing ammonium acetate at
pH 6 and acetonitrile (50–0%) on enantioresolution.
The trend of R
versus organic modifier con-
centration ratio was quite similar to our previous
results achieved using the longer effective capillary
length, R increased by increasing the MeOH con-
centration with maximum value at 50% of MeOH.
However the lowest efficiencies were observed at
this MeOH concentration. This is documented in Fig.
3 where mobile phases with different concentration
ratio of MeOH–ACN were employed for the enan-
tioresolution of 2-PhL.
Fig. 4a,b shows the effect of organic modifier
concentration ratio on efficiency of the first eluting
enantiomeric hydroxy acids. As can be observed, the
efficiency increased by increasing the MeOH con-
centration (decreasing ACN%) up to 20% MeOH
and than decreased for MA, m-OH-MA, 2-PhL, 4-
Cl-MA and 3-OH-4-MeO-MA. In the case of p-OH-
MA a similar trend was observed but with a maxi-
mum efficiency at 30% MeOH while for 3,4-di-OH-
MA the addition of MeOH caused a reduction of
efficiency. Twenty or 30% MeOH added to the
acetonitrile–water–buffer system seems to offer the
highest efficiency with good resolution and reason-
ably short analysis time. This is shown in Fig. 5a–d
where the enantiomeric separation of m-OH-MA,
p-OH-MA, 3-OH-4-MeO-MA, 4-Cl-MA, 2-PhL are
Fig. 2. Plot of reduced plate heights versus linear velocity of
electroosmotic flow. Sample L-(1)-mandelic acid. Capillary, 34.4
cm (effective length 8.4 cm, stationary phase, 6.6 cm, frit at 7.0
cm)375 mm I.D. Mobile phase 50 mM ammonium acetate (pH
6)–water–ACN (1:4:5, v/v). Applied voltage, 2.5–30 kV(0.9–9.9
mA); injection at short-end side (cathode) 12 bar, 0.2 min of 0.2
mg/ml of racemic MA followed by a mobile phase plug at 12 bar,
0.15 min. The capillary was pressurized at both ends at 8 bar.
Capillary temperature, 20 8C.
experiments achieving good efficiencies and satisfac-
tory analysis time.
The repeatability was calculated by analyzing the
racemic mandelic acid mixture (n57), eluting with
the mobile phase employed in the above-described
experiments and measuring the RSD% of the re-
tention time of the two enantiomers (t
resolution factor (R ) and enantioselectivity (a)
(RSD51.2, 1.3, 0.7 and 0.7%, respectively). These
results were comparable with those previously
achieved using a capillary packed with the same
stationary phase but with a longer effective length
(26 cm) .
A sample mixture containing L-(1)- and D-(2)-
mandelic acid (3:1, v/v) was analyzed in order to
verify the affinity of the two enantiomers towards the
chiral selector and we found that the D-(2) enantio-
mer was the most retained compound.
3 .2. Effect of organic modifier on
enantioresolution of hydroxy acid compounds
The effect of acetonitrile concentration on enantio-
separation of selected hydroxy acid enantiomers,
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151
Fig. 3. Effect of organic modifier concentration ratio on enantiomeric separation of 2-phenyllactic acid. Applied voltage, 20 kV; sample
concentration, 0.2 mg/ml; mobile phase, 50 mM ammonium acetate (pH 6)–water–organic modifier mixture (1:4:5, v/v); sample
concentration, 0.2 mg/ml except 3,4-di-OH-MA that was 0.4 mg/ml. MeCN stands for ACN. For other experimental conditions see Fig. 2.
3 .3. Use of short-end injection packed capillary in
the CSP. Efficiencies measured in nano-HPLC analy-
sis were generally lower than those achieved using
the same mobile phase in CEC, e.g., N m
delic acid: CEC583 250; nano-HPLC543 357.
Table 1 shows the values of retention time,
retention factor, enantioselectivity and resolution
factor of the studied hydroxy acid enantiomers
separated by nano-HPLC. The repeatability was
studied analyzing a racemic mixture of m-hydroxy-
mandelic acid recording satisfactory results. The
RSDs were: 1.4, 1.5 and 1.5% for void volume and
retention times of the first and second eluting
enantiomer, respectively; 0.4, 1 and 0.1% for re-
tention factors, enantioresolution and enantioselec-
tivity, respectively. From the above-discussed results
we can remark that the CSP based on Hepta-Tyr
antibiotic can also be used in nano-HPLC; however,
an applied pressure higher than 12 bar is necessary in
order to reduce the analysis time to the level
achieved in CEC.
The capillary packed at the short-end injection was
also evaluated for nano-HPLC enantiomeric sepa-
ration of selected studied hydroxy acid derivatives
running the experiments with the same instrument
used for CEC studies. The mobile phase selected was
that containing 50 mM ammonium acetate (pH 6)–
water–MeOH–ACN (1:4:2:3, v/v) which allowed to
achieve baseline resolution of all selected hydroxy
acid enantiomers by CEC. All studied compounds
were baseline resolved by nano-HPLC applying a
relatively low pressure: 12 bar (the maximum per-
mitted by the commercial instrumentation). Besides,
higher enantioresolution was achieved in nano-HPLC
than in CEC, retention times were higher due to the
relatively low pressure applied. The higher resolution
observed in nano-HPLC can be probably explained
by the longer time spent by analytes in contact with
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151149
Fig. 4. Effect of organic modifier concentration ratio on efficiency of the first eluting enantiomeric hydroxy acids. (a) MA, m-OH-MA and
p-OH-MA; (b) 3,4-di-OH-MA, 3-H-4-MeO-MA, 4-Cl-MA and 2-PhL. MeCN stands for ACN.
4 . Conclusions
lution factor; however, decreased efficiencies were
observed at MeOH concentrations higher than 20–
30%. The shortest analysis time was obtained using
50% of ACN, which was also a compromise for
good efficiency and enantioresolution. A further
increase of applied voltage (30 kVwas the maximum
allowed by the instrumentation) allowed the enantio-
meric resolution of mandelic acid in less than 72 s as
is illustrated in Fig. 6.
The CSP was also tested in the nano-HPLC mode
achieving interesting results concerning the re-
peatability data (RSD 1–1.5% for retention time).
Hepta-Tyr antibiotic CSP packed in the short-end
injection of the capillary was successfully used for
the separation of hydroxy acid enantiomeric com-
pounds by CEC. A mobile phase containing am-
monium acetate, pH 6, and ACN or ACN–MeOH,
allowed the enantioresolution of all studied com-
pounds. Retention time, resolution and efficiency
were strongly influenced by the mobile phase com-
position. In fact the addition of MeOH was a
predominant factor for achieving higher enantioreso-
S. Fanali et al. / J. Chromatogr. A 990 (2003) 143–151
Fig. 6. Fast enantiomeric separation of racemic mandelic acid by
CEC. Applied voltage, 30 kV; injection at the short-end of the
capillary; mobile phase 50 mM ammonium acetate (pH 6)–water–
ACN (1:4:5, v/v). For other experimental conditions see Fig. 2.
Thanks are due to Professor P.G. Righetti, Depart-
ment of Agricultural and Industrial Biotechnology,
University of Verona (Italy) for the kind donation of
the Hepta-Tyr glycopeptide antibiotic and to Aka
Chemicals AB (Bohus, Sweden), for the gift of
Kromasil amino silica 5 mm. Furthermore we are
grateful to ‘Ministero dell’Universita e della Ricerca
Scientifica, MURST’, Italy (grant cofin 40%) for
supporting P.C. with a grant.
Fig. 5. Electrochromatographic enantiomeric separation of 2-PhL,
4-Cl-MA, 3-OH-4-MeO-MA, m-OH-MA and p-OH-MA. Mobile
phase, 50 mM ammonium acetate (pH 6)–water–ACN–MeOH
(1:4.30:20, v/v). For other experimental conditions see Fig. 2.
Further study is being carried out in our laboratory in
order to use pressures higher than 12 bar to find
experimental parameters useful for decreasing analy-
sis time at the level observed in CEC. It is not clear
whether the Hepta-Tyr antibiotic is bonded or ad-
sorbed on the stationary phase, therefore additional
investigations (e.g., NMR) are needed in order to
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Retention time (t
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tography (CLC) for the enantiomeric separation of hydroxy acids
), retention factor (k), enantioresolution (R )
The capillary was the same used for CEC experiments; mobile
phase 50 mM ammonium acetate (pH 6)–water–methanol–ace-
tonitrile (1:4:2:3, v/v); applied pressure, 12 bar; capillary tem-
perature 20 8C.
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