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Focusing Review

Development and Pharmaceutical Applications of Functional Stationary Phases
for Capillary Electrochromatography and Chiral Separation
Kaname OHYAMA*
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, Nagasaki 852-8521, Japan

Abstract
We synthesized 3-(4-sulfo-1,8-naphthalimido)propyl-modified silica (SNAIP) for capillary electrochromatography (CEC).
The unique structure of SNAIP contributed to the retention by three interactions including hydrophobic, electrostatic and S-Sinteractions and the acceleration of electroosmotic flow at an acidic condition. The CEC employing SNAIP was successively
applied to the rapid separations of several drugs, peptides and polar compounds. Also, several examples for applying CEC toreal sample analyses were demonstrated. Furthermore, with the idea to use adamantane as a shield to reduce the peak tailingof charged solutes in CEC, adamantyl (ADM)-functionalized polymer monolith by a single-step copolymerization with themonomer containing ADM structure and a cross-linker was presented. Three chiral stationary phases with different
phenylalanine (Phe) peptide lengths, Phe4, Phe8, Phe12, were prepared to study the effect of peptide length on enantioseparationin reversed-phase HPLC. The highest resolution was observed for the selector with intermediate peptide length (i.e., Phe8).
The side chain of amino acids was also found to play a role for the separation performance of the chiral stationary phases(CSPs). The IR spectra suggested that the Phe peptides immobilized on the CSPs were assumed to be mainly in the D-helicalstate. Also, it was found that the conformation strongly contributed to the chiral recognition of the CSPs by thermodynamic
study.
Keywords: Capillary electrochromatography; Chiral separation; Stationary phase

1. Introduction
Capillary electrochromatography (CEC), which
combines the features of capillary zone electrophoresis(CZE) and high performance liquid chromatography(HPLC), is a powerful separation technique with highefficiency, high resolution, and low consumption of mobile
phase and sample. The most commonly used stationary
phase in CEC has been octadecyl silica (ODS), but in viewof the need to maintain a high enough electroosmotic flow
(EOF), it can only work in the mobile phase with relatively
high pH. When mobile phases are used with low pH, theEOF decreases tremendously owing to the protonation ofsilanol groups on the surface of ODS and the inner wall of
capillary. One way to overcome this problem is to use
silica based mixed-mode stationary phase (C18/ SCX) [1,2].
The benefit of using this type phase in CEC is the*Corresponding author: Kaname OHYAMA
Tel & Fax: +81-95-819-2446
E-mail: k-ohyama@nagasaki-u.ac.jp

contribution of a high EOF due to the ionized sulfonic acidgroup even at low pH. El Rassi and co-workers pioneered
the specially designed CEC column packed with mixed
mode stationary phase which was composed of ahydrophilic, negatively charged sublayer (sulfonic acid) anda nonpolar top layer contained octadecyl ligands andpresented its application for nucleic acids [3]. However,the synthesis procedure was multi-step and complex.
Since the numbers of the stationary phases designed for
CEC are still limited, such stationary phases are essential toshow the potential of CEC and expand its applicability.
Enantiomers of a chirally active drug often have adramatically different pharmacological or toxicologicaleffect; therefore, the separation of enantiomers is very
important. HPLC with a chiral stationary phase (CSP) is a
popular method in the field of chiral separation because of

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its good reproducibility and preparative capability. The
design and development of CSPs has attracted muchattention for a number of years. To date, CSPs withseveral chiral selectors, such as macrocyclic antibiotics [4],polysaccharide derivatives [5], modified amino acid [6],protein [7] and cinchona alkaloids [8], have been reported
and some of them are commercially available. However,these CSPs do not always give good enantioseparation oftarget analytes; therefore, successive efforts to develop a
new type of CSP are required.
In this review, the developments of novel CEC
stationary phase and CSPs with peptide chiral selectors aredescribed.
2. Functional stationary phase for CEC
In the initially described CEC, the major focus was on
the separation behavior of neutral pharmaceuticals andaromatic hydrocarbons that were well studied in reversed
phase (RP)-HPLC.
This was because these CEC
employed HPLC stationary phases, e.g. ODS. In order toexpand the applicability of CEC, the stationary phases
which produce a stable EOF at wide pH range and relyother retention mechanism different from hydrophobic
interaction. In practical, several workers employed ion
exchangers as a stationary phase or mixed-mode phase andfound them a viable alternative [9]. The S-S interaction isa type of electron donor-electron acceptor interaction,
originating from S-electron systems in two unsaturatedfunctional groups through intermolecular or intramolecularinteraction [10]. The S-S interaction can successfully beutilized in the separation of closely related compounds likemetabolites or degradation products [11]. Therefore, theretention based on S-S interaction should be included into
the retention mechanism on CEC packing materials.
In this work, 3-(4-sulfo-1,8-naphthalimido) propylmodified silyl silica gel (SNAIP, Fig. 1) was newly
synthesized with the idea to use the fixed charge of sulfonicacid group as both the EOF generator and chromatographic
retentive sites. It was easily prepared by a single reactionof aminopropyl silica (APS) with 4-sulfo-1,8-naphthalicanhydride at 150 °C for 5 h. The structure of SNAIP
could contribute to the retention by three interactions
including hydrophobic, electrostatic, and S-S interactions[12-14]. The influence of buffer pH on the EOF mobility(PEOF) was studied in the pH range of 2.5-7.5 using themobile phase composed of 40% methanol in 5 mM
phosphate (Fig. 2). By using SNAIP column, PEOF
became 1.19 and 1.55 x 10-8 m2V-1s-1 at pH 2.5 and 3.0,
respectively. These corresponded to 1.29 and 1.43-foldwhencomparedwithincreaseof
PEOF
3-(1,8-naphthalimido)propyl-modified silyl silica gel(NAIP) column. This increase was attributed to theintroduction of sulfonic acid groups, which was imperative

for a fast separation at low pH. As shown in Fig. 3, a
rapid baseline separation of five barbiturates was achieved
within 3 min by an applied voltage of 20 kV and a mobile
phase of 40% methanol in 5 mM phosphate (pH 3.8). The
prepared SNAIP column was applied for the separation of
benzodiazepines at low pH and the separation mechanismof the basic compounds on SNAIP column was studied.
The retention of each benzodiazepine decreased withincreasing the buffer concentration. The decrease in theretention was presumably a result of weakening
electrostatic binding of positively charged benzodiazepines
to the negatively charged sulfonic acid groups. This
explanation was supported by a linear relationship between
the logarithmic values of k and phosphate concentrations,which is a typical behavior in ion-exchange
chromatography [15].
On the other hand, in CEC withODS (Hypersil C18), Euerby et al. demonstrated that theelution order of studied barbiturates was completely
consistent with their hydrophobicity and phenobarbital
eluted before secobarbital even using a phenyl-bonded
stationary phase with the retention by S-S interaction [16].
In CEC with NAIP, the elution order of four barbiturates(barbital, phenobarbital, secobarbital, and thiopental) was
according to their hydrophobicity (Fig. 4). Although theretention of phenobarbital on NAIP increased comparedwith ODS due to S-S interaction between phenobarbital and
NAIP.

Fig. 1. Structure of SNAIP.

Fig. 2. Dependence of EOF mobility with SNAIP on the pH ofmobilephase. Column, total length 370 mm (packed length 90
x75
Pm
i.d.;
mobilephase,
5mM
mm)
phosphate-methanol(60:40, v/v) at different pH values; applied
voltage, 20 kV;temperature, 18 °C. Reproduced from ref. [12]with permission.

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Contrastingly, in CEC with SNAIP, a different elution
order was observed only for phenobarbital possessing an
aromatic moiety that eluted last in spite of its lower log P
value, while the order of the others was according to theirlog P values. In order to confirm the presence of S-Sinteraction, plots of log k against log P values for tested
barbiturates were drawn.
A linear relationship was
obtained for tested barbiturates, except for phenobarbital.
From these results, the difference in selectivity came from
S-S interaction between SNAIP and the aromatic moiety ofphenobarbital.
Naphthalyl group modified withelectron-acceptor group (i.e. sulfonic acid group) became
S-acidic and thus the impact of the S-S interaction could be
pronounced.
In RP-CEC, the separation of polar compounds (e.g.
carbohydrates, nuleosides) requires the use of plain aqueous
electrolytes solutions as mobile phases to obtain sufficient
retention for polar compounds [17]. Unfortunately, like in
HPLC, the highly polar mobile phase is unsuitable for a
non-wetted non-polar stationary phase, which usually leads
to high current and bubble formation within CEC column.
In addition, when the buffer concentration modulating
electrostatic interaction is too high, the bubbles can begenerated within a capillary column by Joule heating,which hinders the possibility of gradient of bufferconcentration. This bubble formation limits the use ofwide variety of mobile phases, resulting in reducing theapplicability of CEC. In this context, SNAIP is arelatively polar RP phase and therefore, using SNAIP
column, an aqueous mobile phase would be available for
CEC without bubble formation. In actual, no bubble wasobserved within SNAIP column even when the current
reached 40 PA.
This remarkable characteristic will
expand the range of aqueous mobile phase composition andbuffer concentration. At first, the effect of methanol
content in the range from 5 to 40% (v/v) on the separationof nucleosides and nucleic acid bases was studied while
keeping the buffer concentration at 10 mM and the pH of
5.0. An increase in EOF with a decrease in methanol
content was obtained; the EOF mobility increased about
65% when the methanol content decreased from 40 to 5%.
This tendency provides a significant advantage ofsuppressing the longer separation time with lowering
methanol content that is generally used for better resolution
in RP chromatographic separation. In fact, as shown in
Fig. 5, the separation of early eluting peaks, namely uridine,uracil and thymidine, was improved by the decrease of
methanol content; besides, the separation time for all
analytes, except for adenine, was hardly prolonged.
Furthermore, bubble formation was never experienced evenunder highly aqueous condition (~95% aqueous solution) of
mobile phase. Next, the effect of buffer concentration on
the CEC separations of peptides as well as protein digests

Fig. 3. Electrochromatogram of barbiturates with SNAIP.
Mobilephase: 5 mM phosphate (pH 3.8)-methanol (60:40,
v/v %). Other conditions as in Fig. 2. Peaks: 1, thiourea; 2,
barbital; 3, amobarbital; 4, secobarbital; 5, thiopental; 6,
phenobarbital. Reproduced from ref. [12] with permission.

Fig. 4. Electrochromatographic separation of barbiturates with
SNAIP and NAIP. Stationary phase, SNAIP; mobilephase, 5mM phosphate(pH 3.8)-methanol (60:40, v/v %); applied
voltage, 10 kV. Stationary phase, NAIP; mobile phase, 1 mM
citrate (pH5.0)-methanol (60:40, v/v %); applied voltage, 20 kV.
Other conditions as in Fig. 2. Peaks: 1, thiourea; 2,
phenobarbital; 3, secobarbital; 4, thiopental. Reproduced fromref. [12] with permission.

Fig. 5. Electrochromatographic separation of nucleosides andnucleic acid bases on SNAIP. Mobile phase, 10 mM phosphatebuffer (pH 5.0)-methanol (90:10,v/v %); applied voltage, 15 kV.
Other conditions as in Fig. 2. Peaks: 1, uridine; 2, uracil; 3,
thymidine; 4, thymine; 5, inosine; 6, guanosine; 7, adenosine; 8,cytidine; 9, guanine; 10, cytosine; 11, adenine. Reproduced fromref. [14] with permission.

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was investigated [18]. Fig. 6 illustrates the effect of bufferconcentration on the separation of the tryptic digests ofcytochrome c with the SNAIP column. Both mobile
phases provided a sufficient separation and, as expected,
increasing buffer concentration reduced the separation timefrom 130 min (20 mM, Fig. 6a) to 22 min (50 mM, Fig. 6b)
and sharpened all peaks, especially later peaks. Using themobile phase with 50 mM phosphate buffer, the resolutionof tryptic digests on SNAIP column was superior orcomparable to the pressurized gradient CEC in previous
work [19]. This was because basic digests could be eluted
by high buffer concentration decreasing electrostaticinteraction between sulfonic acid groups on SNAIP and thedigests. Based on these results obtained by the separationof various compounds, SNAIP can be a promisingstationary phase for applying CEC to many separation
scenes.
In order to gain a high reputation as practical technique,
CEC must demonstrate an excellent performance, withrespect to the separation speed, reproducibility anddurability, in a number of real sample analyses.
Barbiturates are widely used as sedatives, hypnotics andantiepileptics.
Since barbiturates have a narrow
therapeutic index, their improper use sometimes results inaccidental death and also barbiturates are implicated inmurder or suicide. For these reasons, the analysis for theirpresence in biological materials is frequently required.
Here, CEC with NAIP was employed for the analysis ofbarbiturates extracted from human serum by liquid-liquid
extraction, as shown in Fig. 7 [20]. The analysis ofbarbiturates in serum was successfully performed, withgood repeatability of peak response (relative standard
deviation with 11.1% for within-day and between-day) andshort separation time in less than 4.5 min. Microdialysis
is extensively used for continuous monitoring of substancesin the extracellular space of tissues and body fluid. The
advantage over other sampling methods is thatmicrodialysates are protein-free, allowing direct analysis.
The rapid separation obtained by CEC has the biggest
advantage in such direct analysis; therefore, CEC with
NAIP was applied for the analysis of caffeine and its
metabolites (1-methylxanthine and 1,7-dimethylxanthine)
in microdialysate [21]. A typical electrochromatogram ofthe microdialysates are shown in Fig. 8. The separation ofcaffeine and its two metabolites was achieved in less than
3.5 min by CEC with NAIP stationary phase, which wasapproximately 3-times less than that in HPLC with NAIP.
The within-day repeatability as well as the between-day
repeatability of the retention time and peak response wereless than 5.9% (relative standard deviation).
These
examples support the good applicability of CEC for real
sample analyses.
Organic polymer-based monolithic columns have beensuccessfully used in CEC. Polymer-based monoliths are
attractive because there are a wide variety of different
monomers available and can be used within a broad
pH-rage where they are chemically stable. They areprepared by a single-step copolymerization with monomer,
cross-linker,
pore-forming
solvent
and initiator.
Moderately orstrongly ionicmonomers,
i.e.
2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS),[2-(methacryloyloxy)ethyl]-trimethylammonium chloride,are extensively utilized in the preparation of polymer

Fig. 6. Effect of buffer concentration on the separation of
tryptic digests of cytochrome c. Mobile phase, phosphate buffer(pH 3.8)-methanol (60:40, v/v %) at different bufferconcentrations: (a) 20 mM (27 PA); (b) 50 mM (95 PA); applied
voltage, 20 kV. Other conditions as in Fig. 2. Reproduced fromref. [18] with permission.

210 nm
290 nmFig. 7. Electrochromatogram of the serum sample spiked withbarbiturates standards. Stationary phase, NAIP; column, 340
mm (packed length 90 mm) x 75 Pm i.d.; mobile phase: 1.0 mM
citrate buffer (pH5.0)-methanol (60:40, v/v %). Other conditions
Peaks: 1, barbital; 2, phenobarbital; 3,
as in Fig. 2.
secobarbital; 4, thiopental; *, Peaks for intrinsic components inserum. Reproduced from ref. [20] with permission.

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monoliths for CEC [22,23]. In the CEC separation of
charged solutes, these monoliths work as a mixed-mode
stationary phase that combines two retentive interaction (i.e.
hydrophobic and cationic-exchange or anion-exchange).
However, CEC of charged solutes on such stationary phases
often suffers from severe peak tailing due to Coulombic
attraction between solutes and monolith surface of opposite
charges. In order to juggle production of stable EOF andprevention of peak tailing in CEC, new type of capillarymonolithic columns should be developed. HPLC using
the columns packed with silica-gel particles also suffersfrom peak tailing of basic solutes due to the interaction
between solutes and silanols of packing material. In thiscontext, some groups proposed the steric exclusion of thesolute from the surface of silica-gel particles to reduce theinteraction. Hemetsberger et al. observed that longer alkyl
chains partially prevent solutes from reaching theunderlying surface and play an important role in blocking
the silanols [24]. Gilpin et al. and Guiochon et al.
demonstrated improved peak symmetry for basic solutes by
the bulky rigid structure of adamantane shielding the

Fig. 8. Electrochromatogram of microdialysate sample-spiked
with 25 Pg/ml of caffeine and its two metabolites. Conditions:mobile phase, 4.0 mM citrate buffer-methanol (20:80, v/v %).
Other conditions as in Fig. 7. Peaks: 1, 1-methylxanthine; 2,
1,7-dimethylxanthine; 3, pentoxifylline (E.S.); 4, caffeine.
Reproduced from ref. [21] with permission.

unreacted silanols from even small solutes [25,26]. With
the idea to use adamantane as a shield to reduce the peaktailing of charged solutes in CEC, we newly synthesizedadamantyl (ADM)-functionalized polymer monolith by asingle-step copolymerization with the monomer containing
ADM structure and a cross-linker [27]. Using a mobile
phase of 5 mM acetate buffer (pH 4.0) and ACN mixture,
the test solutes (aniline, N-methylaniline andN,N-dimethylaniline)
were
analyzedon
ADM-functionalized and butyl methacrylate (BMA)-basedmonolithic columns made from the polymerization mixture
consisting 23.9% 1-adamantyl-(D-trifluoromethyl) acrylate(MAF-ADE) or BMA, 15.9% ethylene dimethacrylate(EDMA), 0.4% AMPS, 6.0% water, 35.0% 1-propanol and
18.0% 1,4-butanediol. Table 1 shows retention factor andsymmetry factor for the test solutes on both columns. The
peak shapes were obviously better on ADM-functionalized
monolithic column.
The better peak shapes on
ADM-functionalized monolithic column should be due tothe reduction of the accessibility of basic solutes to sulfonicacid groups. This shielding effect of adamantane observedin polymer-based monolith conforms with that in the
ADM-modified silica used for HPLC [25,26]. However,as shown in Table 1, the retention factor for basic solutes on
ADM-functionalized monolithic column were lower thanthose on BMA-based one under the mobile phaseconditions. This also supports the existence of theshielding effects which prevents the polymerization
reaction and decreases the surface area of monolith.
In another approach to juggle production of stable EOF
and prevention of peak tailing in CEC, methacrylic acid
(MAA), a weakly ionic monomer, was employed in the
preparation of BMA-based monolith for CEC in place of
AMPS [28,29].
A weakly ionic monomer is analternative; but the use of it is limited [30,31] because it is aweak EOF generator. Therefore, compared to AMPS, highcontent of MAA was employed in order to increase theionizable functional groups (i.e. carboxylic acid groups)
and then, strengthen EOF. Because polymer monolithic
column containing high content of an ionic monomer is
supposed to decrease its rigidity, less than 1.0% of ionic

Table 1. Peak shapes of basic solutes on ADM-functionalized and BMA-based monolithic columnsc).

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monomers were generally used [32,33]. Here, the effectof MAA content (0.05, 0.5 and 2.5%) on the CEC
performance was studied with varying the composition of
pore-forming solvents. It was noted that the higher MAA
content provided the higher EOF mobility and the better
column efficiency. In this study, the high MAA content isthought to swell the monolith than the low one does.
Because of the high efficiency, the baseline separation of
six test solutes was achieved in less than 100 seconds whenusing an applied voltage of 25 kV and the short-end
injection method (9.0 cm capillary packed length).
3.
Preparation
andCharacterizationofpoly(L-phenylalanine) chiral stationary phase
Since CSPs with a protein chiral selector have excellent
enantioselectivity, a peptide chiral selector would seem tooffer promise. Indeed, research on peptide chiral selectors
has progressed. Li et al. developed polyproline-based
CSPs and demonstrated their broad enantioselectivity innormal-phase mode [34].
Also, Chen usedpoly(N-benzyl-L-glutamine)-coated silica for the chiralseparation of hydantoins [35]. We developed a novel CSP
with phenylalanine (Phe) peptide as chiral selector [36-38].
The CSP was prepared on APS by solid-phase synthesis
with tert-butoxycarbonyl (Boc) amino acids. N-terminusof all the CSPs were end-capped by benzoyl chloride (BZ).
For a peptide chiral selector, the secondary structure
introduces more conformational rigidity into the selector
and can influence its enantioselectivity. The secondary
structure of a peptide varies by its length [39], thus in thisstudy CSPs with different peptide lengths were also

individually prepared. At first, we studied peptide chiralselectors made from Boc-phenylglycine (Phg), Boc-valine(Val) and Boc-proline (Pro) in order to find out the kind of
amino acid that is favorable for this class of CSPs.
Warfarin enantiomers were used as a model chiral analyte
in order to compare the newly synthesized CSPs, i.e.
APS-(Phg)8-BZ, APS-(Val)8-BZ and APS-(Pro)8-BZ, with
APS-(Phe)8-BZ under RP-mode. Enantioseparation wasachieved on APS-(Val)8-BZ (kA, 19.2; kB, 20.1; D, 1.05; Rs,
1.26) and APS-(Phe)8-BZ (kA, 27.3; kB, 29.2; D, 1.07; Rs,
1.60) while no chiral resolution was observed on
APS-(Phg)8-BZ and APS-(Pro)8-BZ. This result suggeststhat the side chain of amino acids plays a role for the
separation performance of the CSPs. Phg and Phe differ
only by the presence of the methylene group between
D-carbon and phenyl group; this methylene group probably
gives a high steric flexibility to the interactive site (i.e.
phenyl group) with analytes. In this context, the side
chain of Val is also sterically flexible. Pro does not have aflexible interaction site and thus, may not be suitable forour proposed CSPs. Besides warfarin enantiomers, theCSPs were used for the enantioseparation oftrihexylphenidyl and benzoin which were partially
However, no chiralseparated on APS-(Phe)8-BZ.
separation was observed on the three CSPs. This can bebecause the side chain of Phe peptide works as a flexible
and strong interactive site in the chiral recognition. Next,
we investigated the effect of Phe peptide length (CSP1,
Phe4; CSP2, Phe8; CSP3, Phe12) on chiral separation in
HPLC. Investigation of the chemical structure of the Phe
peptide reveals that a combination of hydrophobic

Table 2. Chiral separation of R/S-wafarin on the CSP1-3.

Fig. 9. Typical chromatographic separations of R/S-warfarin with CSP1-3. Mobile phase: 0.5M sodium perchlorate (pH 2.0)-acetonitrile(77:23, v/v %). Reproduced from ref. [36] with permission.

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Fig. 10. a) Effect of column temperature on resolution factor and b) van’t Hoff plot of warfarin enantiomers. Mobile phase: 0.5 M
sodium perchlorate (pH 2.0)-acetonitrile (77:23, v/v %). Reproduced from ref. [38] with permission.

interaction, S-S interaction and hydrogen bonding would
seem to contribute to the retention mechanism.
Accordingly, a longer peptide chiral selector was expected
to be more effective on the chiral separation because ofincreased hydrophobicity as the Phe peptide length
increases and in general, the chiral recognition based on thesecondary structure of peptide becomes more pronounced.
The parameters (k, D, Rs) and the representative
chromatograms in the separation of R/S-warfarin on
CSP1-3, using a mobile phase consisting of 0.5M aqueous
sodium perchlorate (pH 2.0) and acetonitrile (77/23, v/v),
are shown in Table 2 and Fig. 9, respectively. Surprisingly,
best resolution was achieved by CSP2, which had anintermediate peptide length. The same tendency wasobserved for the aminobutyl silica-based CSP having thesame chiral selectors as CSP1-3. By making the Phe
peptide selectors longer, hydrophobic interaction and S-Sinteraction are enhanced. The hydrogen bonding may bedecreased, but its influence on the chiral recognition isrelatively weak. Therefore, the lower enantioselectivity of
CSP3 than that of CSP2 could be attributed to thesecondary structure of Phe12. The secondary structure of a
peptide depends on its chain length [39] and variation insecondary structure could impact on enantioselectivity.
An IR spectra is widely used to estimate the conformationof peptides and proteins from their amide I and IIabsorption bands [40,41]. IR measurement on CSP1-3,
initially dispersed in the mobile phase for 12 h and thendried in vacuo at ambient temperature, was carried out.
The resultant IR spectra provided an amide I absorption
band at 1645-44 cm-1 and an amide II absorption band at
1541-35 cm-1. Absorption bands at 1650-40 and 1550-20

are characteristic of D-helical structure [40,41] and thus thePhe peptides immobilized on CSP1-3 were assumed to bemainly in the D-helical state. Therefore, the lower
enantioselectivity of CSP3 compared to CSP2 could not be
explained by differences in secondary structure.
Inaqueous solution, the major environmental factor that could
affect the energetics of the helix-coil transition of peptides
is temperature. Therefore, the contribution of column
temperature to the chiral recognition of the poly( L-Phe)
CSP was investigated using warfarin enantiomers as amodel. Fig. 10a shows dependence of the resolution
factor of warfarin on column temperature. Resolution
factor decreased as temperature increased. Especially, it
dramatically decreased when temperature increased from30 to 50 °C. Furthermore, van’t Hoff plot, which is thethermodynamic relationship between retention factor andtemperature in Kelvin, was not linear (Fig. 10b).
Non-linear van’t Hoff plots may reflect a change in therelative contributions by enthalpy and entropy of transfer ofthe solute from the mobile phase to the stationary phase[42]. The changes in these relative contributions andpossible changes in the retention mechanism are often
thought to be related to conformation changes in thestationary phase (e.g. C18 stationary phases) [43]. Ma etal. reported that the D-helical conformations of polycationicpeptide melt into extended ones as the temperature
increased (from 1 to 60 °C) [44]. Similarly, Muñoz andSerrano reported a decrease of helical content as increasing
temperature using polyalanine peptide [45]. From theseobservations, the chiral recognition of the poly( L-Phe)peptide selector was sensitive to the column temperatureprobably due to change of its secondary structure. We

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further evaluated the enantioselectivity of the CSPs innormal phase (NP)-LC using four chiral analytes. The
best resolution was obtained for benzoin enantiomers andas n-hexane content increased, the resolution factor
increased due to the increase in NP mode retention. In RP
mode, enantioseparation could not be achieved for
mandelic and tropic acids, however partial separation was
possible in NP mode, reciprocally warfarin which could befully separated in RP mode [36] could not be separated in
NP mode. The separable chiral analytes on this class of
CSPs were changeable by the separation mode. In
contrast to RP-LC, the current study showed that only the
CSP with the longest peptide selector (Phe12) provided anenantioselectivity in NP-LC. This was mainly due to its
relatively high hydrophilicity; additionally, the peptide
secondary structure, which was pronounced by thehydrogen bonds facilitated in organic mobile phase, might
contribute to the chiral recognition. The optimum peptide
length of our proposed CSPs for enantioseparation should
be varied according to the separation mode.
4. Conclusion
The experimental data have confirmed that the
separation mechanism in CEC with SNAIP stationary phase
was a hybrid of electrophoretic migration andchromatographic
retention
involvinghydrophobic,
electrostatic as well as S-S interactions. Using low pH
mobile phase, the baseline separation of five barbiturates inless than 3 min was observed due to the higher EOF.
SNAIP is easily prepared on a single reaction and shows adifferent selectivity than conventional RP stationary phases.
The application of the SNAIP stationary phase for the CECof a wide range of analytes including pharmaceuticals,amino acids, peptides, protein digests, nucleosides andnucleic acid bases. Furthermore, it is demonstrated that
ADM-functionalized monolithic stationary phase canprovide good peak shape of basic solutes because of its
shielding effect.
A new class of CSPs with a different number of Pheunits immobilized on APS have been prepared andevaluated. The longest Phe peptide selector did notdemonstrate the best enantioselectivity as expected, but thehighest resolution was achieved by the CSP having
intermediate peptide length. This indicated that enhanced
enantioselectivity is not always obtained by a longer
peptide chiral selector. Therefore, when a different aminoacid is used to develop a peptide chiral selector,
optimizationofpeptidelengthcanenhanceenantioselectivity. The side chain of amino acids was also
found to play a role for the separation performance of the
CSPs. The IR spectra suggested that the Phe peptides
immobilized on the CSPs were assumed to be mainly in the
D-helical state. Also, it was found that the conformationstrongly contributed to the chiral recognition of the CSPs
by thermodynamic study.
Acknowledgements
I would like to sincerely thank Drs. Naotaka Kuroda,
Mitsuhiro Wada and Naoya Kishikawa (Nagasaki
University); Drs. Kenichiro Nakashima and Yoshihito Ohba
(Nagasaki International University); Drs. Chang-Kee Lim
and Gwyn A. Lord (London University).
The author is grateful to the support provided by theSpecial Coordination Funds for Promoting Science andTechnology of the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), a Grant-in-Aid forYoung Scientist from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, a grant from
Mitsui Sumitomo Insurance Welfare Foundation, The
Shimabara Science Promotion Foundation, a Yamada Bee
Farm Grant, and a Grant-Aid for Scientific Research from
Nagasaki University.
Finally, the author gratefully thanks the award
committee and all the members of the Japan Society for
Chromatographic Sciences.
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