Chiral ligand-exchange resolution of underivatized amino acids on a dynamically modified stationary phase for RP-HPTLC

Article abstract of DOI:10.1002/chir.22324

The synthesis of Spi(τ-dec), derived from the selective alkylation of L-spinacine (4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) at the τ-nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ-dec) was successfully employed in the past to prepare home-made chiral columns for chiral ligand-exchange high-performance liquid chromatography. In the present article a new method is described, using Spi(τ-dec) as a chiral selector in high-performance thin-layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ-dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive. Chirality 26:313-318, 2014. 2014 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22324

CHIRALITY 26:313–318 (2014)
Chiral Ligand-Exchange Resolution of Underivatized Amino Acids on a
Dynamically Modified Stationary Phase for RP-HPTLC
*
MAURIZIO REMELLI, STEFANIA FACCINI, AND CHIARA CONATO
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Ferrara, Italia
ABSTRACT
The synthesis of Spi(τ-dec), derived from the selective alkylation of L-spinacine
(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) at the τ-nitrogen of its heteroaromatic
ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time.
Spi(τ-dec) was successfully employed in the past to prepare home-made chiral columns for
chiral ligand-exchange high-performance liquid chromatography. In the present article a new
method is described, using Spi(τ-dec) as a chiral selector in high-performance thin-layer
chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ-dec)
and then treated with copper sulfate. The performance of this new chiral stationary phase
was tested against racemic mixtures of aromatic amino acids, after appropriate optimization
of both the conditions of preparation of the plates and the mobile phase composition.
The enantioselectivity values obtained for the studied compounds were higher than
those reported in the literature for similar systems. The method employed here for
the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive.
Chirality 26:313–318, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: HPTLC; chiral separation; amino acid enantiomers; ligand-exchange chromatography;
spinacine; chiral coated stationary phase
INTRODUCTION
reagent with the support; (ii) immersion of the plates into a
solution of the impregnating reagent; (iii) developing the plate
with a solution of the impregnating material; (iv) exposing the
layers to the vapors of the impregnating reagent; (v) spraying
the impregnating reagent on to the plate; (vi) chemically modi-
fying the inert support with a suitable reagent. Starting from the
pioneering works of the 1980s,10–19 most of these different
approaches have been applied to chiral separation of amino
acids.6 Chiral thin layer chromatographic techniques have
been recently reviewed.6,7
One of the best methods to separate racemic mixtures of
underivatized amino acids is chiral ligand-exchange chroma-
tography (CLEC), introduced by Rogozhin and Davankov,20
which is based on the stereoselectivity of homochiral and
heterochiral metal complexes of the same ligands, as in the
case of aliphatic and aromatic amino acids.21 The metal most
often used as complexing agent is Cu(II). In the past, some
CCSPs for CLEC have been successfully prepared and
employed for the enantioseparation of amino acids11,12,16,22–26
and other chelating compounds.27–29 Based on the ligand-
exchange mechanism, TLC plates Chiralplate (Macherey-
Nagel, Duren, Germany) and HPTLC Chir (Merck, Darmstadt,
Germany) were commercially developed. In this context, sev-
eral years ago our research group prepared and studied new
CCSPs for the chiral separation of amino acids and dipeptides,
for both high-performance liquid chromatography (HPLC) and
HPTLC. The chiral selector used was Nτ-n-decyl-L-histidine
(His(τ-dec)) a derivative of L-His, selectively alkylated at the
The resolution of enantiomeric mixtures is one of the most
challenging and fascinating topics in chromatography. In fact,
most of the compounds of biological interest are chiral, in-
cluding simple amino acids. Since the biological environment
is intrinsically chiral, it follows that the different enantiomers
of chiral substances, used, e.g., as drugs, can have very dif-
ferent pharmacological effects. Unfortunately, this was
tragically experienced at the end of 1950s, with the case of
thalidomide1; at that time it was not possible, or very complex
and expensive, to obtain an optically pure drug and the race-
mate was commercialized, containing both the “eutomer”
(the enantiomer having the desired pharmacological activity)
and the “distomer” (the other enantiomer, often nonactive but
teratogen, in the case of thalidomide). Enantiomers have
identical chemical and physical behavior in an achiral environ-
ment; therefore, in order to be separated by chromatography,
a chiral stationary or mobile phase is required; alternatively, a
precolumn derivatization, to obtain the corresponding diaste-
reomers, should be performed.2 All chromatographic tech-
niques have been used for chiral separations.3–6 Among them
a special place is occupied by thin-layer chromatography
(TLC), for its characteristics of simplicity, convenience, and
affordability.7 Although different chiral phases (actually quite
expensive) for TLC are commercially available, the preparation
of home-made plates is possible, with the advantages of
modulating the properties of the stationary phase, e.g., its
enantioselectivity, purity, or capacity, at relatively low cost. In
this context, the so-called chiral coated stationary phases
(CCSPs) can be prepared by covering a nonchiral commercial
plate with a suitable chiral selector, which almost entirely
changes the properties of the plate.8 In order to prepare these
“impregnated” TLC plates, several strategies may be used.
The most important of them have been classified by Bhushan
and Martens8,9 as follows: (i) mixing of the impregnating
© 2014 Wiley Periodicals, Inc.
*Correspondence to: M. Remelli, Dipartimento di Scienze Chimiche
e
Farmaceutiche, Università di Ferrara, via Fossato di Mortara 17, 44121
Ferrara, Italia. E-mail: rmm@unife.it
Received for publication 24 January 2014; Accepted 3 March 2014
DOI: 10.1002/chir.22324
Published online 28 April 2014 in Wiley Online Library
(wileyonlinelibrary.com).

314
REMELLI ET AL.
pyrrole-like nitrogen of its imidazole ring.30–32 In this molecule,
the long alkyl chain acts as an anchor for the RP-18 silica
support, while the His residue can form stationary ternary
copper complexes with the enantiomers moving in the mobile
phase. More recently,33 the use in HPLC-CLEC of a new chiral
selector, Spi(τ-dec), has been described; here the starting
amino acid (and the chiral center) was L-spinacine ((4,5,6,7-
tetrahydro-1H-imidazo[4,5 -c] pyridine -6 -carboxylic acid),
Spi). For both those chiral selectors the chromatographic
results were encouraging.
225°C. FTIR (potassium bromide): 3374, 3273, 2958, 2922, 2854,
2671, 2617, 1645, 1597, 1410, 1321 cm-1.
The instruments used for the L-Spi characterization were the following:
Buchi capillary melting point apparatus; polarimeter Perkin-Elmer
(Norwalk, CT) 241; MALDI-TOF spectrometer HP model G2025 ALD-
TOF; elemental analyzer Carlo Erba mod. 1106; FTIR Bruker (Billerica,
MA) IFS 88 (resolution 0.6 cm-1); NMR Varian (Palo Alto, CA) 300 MHz;
DSC/TGA NETZSCH.
Methods
Chiral plate preparation. Merck HPTLC plates (highly hydrophobic
RP-18 or partially hydrophilic RP-18 WF254S) were stabilized in an oven
at 120°C for 15 min and then left to cool in air. The mechanical stability
of the stationary phase was then checked by dipping the plate in pure
methanol or hydro-alcoholic solutions (30%, 50%, 70%, or 90% of water)
for 15 min. After drying in air for several hours the plate coverage proved
compact in all the cases. The plate was first immersed in a hydro-
alcoholic solution of the chiral selector and, after room-temperature
drying, in a second solution containing a Cu(II) salt (details are reported
below). The use of dipping solutions in this order allows the complete
recovery of the clean chiral selector in excess.
The first dipping procedure was optimized considering that the most
uniform coverage and the highest chiral selector loading were desirable.
The homogeneity of the coverage was judged both visually and under the
Wood lamp, where the presence of empty spots due to bubbles was evi-
dent. The chiral selector loading was evaluated measuring the absor-
bance of Spi(τ-dec) adsorbed on the plate at the TLC-Scanner (Camag,
254 and 365 nm, reflectance mode).
In this article the synthesis of Spi(τ-dec) and its application
for the preparation of a CCSP for HPTLC is described for the
first time. The main operating parameters, for both the
preparation of the plates and their development, have been
studied in detail and optimized. The separation of enantio-
meric mixtures of aromatic amino acids is shown.
MATERIALS AND METHODS
Materials
Methanol (MeOH), ethanol (EtOH), isopropanol (iso-PrOH), acetoni-
trile (ACN), and tetrahydrofuran (THF) were HPLC-grade solvents
(Sigma-Aldrich, St. Louis, MO); water was tetra-distilled. Copper(II)
acetate, sulfate, perchlorate, nitrate, and chloride (Riedel-de Haën,
Aldrich, Sigma), acetic acid (Carlo Erba, Rodano, Italy) were used to
prepare mobile phases and amino acids (Aldrich and Sigma) were high-
purity products, employed without further purification. Mobile phase
pH was adjusted by addition of suitable amounts of standard NaOH or
HCl solutions, under potentiometric control.
The copper ion loading procedure was optimized as well on the basis of
the retention and enantioselectivity of selected samples (see below).
Synthesis and characterization of the chiral selector Spi(τ-dec). The
chiral selector Spi(τ-dec) (see Scheme 1) was synthesized by condensation
of Nτ-n-decyl-L-histidine (His(τ-dec)) with formaldehyde (FA), using a
method derived from that previously described for the synthesis of Spi,
described below.34
Chromatographic conditions. Amino acids, in
a water/methanol
solution (0.1% w/v), were applied to the plates either through a glass
capillary or with a Linomat IV (Camag, Mutenz) equipped with a 100 μl
microsyringe (Hamilton, Reno, NV); sample volume: 1÷3 μl; delivery rate:
4 sec/μl. The samples were developed in a presaturated chamber (1 h),
allowing the plate to equilibrate with eluent vapors before starting the
development. After drying at room temperature, the plates were dipped
for a few seconds in a 0.15% w/v ninhydrin solution in acetone and then
oven dried at 110°C for about 3 min. Intense red or violet spots developed
on a yellow field. Throughout, solute retention is given as Rm = log(1/Rf –1),
where Rf is the retention factor, and enantioselectivity is computed as
α = Rf(L)/Rf(D).
His(τ-dec) was synthesized as previously described.32 His(τ-dec)
(1.25 g = 4.23 mmol) was dissolved in
a mixture of FA (37% w/v,
0.32 cm3, 4.23 mmol) and HCl (37% w/v, 10 cm3); the solution was then
stirred for 24 h at room temperature. Additional 0.32 cm-3 of FA was then
added and the mixture was refluxed and stirred for 3 h. The resulting
solution was then concentrated by evaporation at reduced pressure and
nearly neutralized (pH = 4) with NaOH 1 M under potentiometric control.
The white precipitate was collected by filtration and dried. The remaining
solution was concentrated again and stored at 4°C for 1 night. The new
precipitate thus formed was collected by filtration and dried. The two
portions of the product proved identical at the analytical tests and suffi-
ciently pure to be mixed and used without further purification. Yield
RESULTS AND DISCUSSION
The most important factors affecting retention and selectivity
of the chiral plates are the following: characteristics of the plate
employed as a chiral-selector support; stationary-phase coating
procedure; and solvent mixture used as mobile phase. These
three factors were investigated and optimized.
25
78%; mp 229°C dec; [α]D -34.8° (c, 2.5 methanol). MALDI-TOF spec-
trum: one single peak at m/z = 308.9 Da, corresponding to the protonated
sample. Anal.: Calcd. for C17H29N3O2 · 1.5 H2O (FW = 334.52): C, 61.1%; H,
9.6%; N, 12.6%. Found: C, 61.8%; H, 9.4%; N, 12.6. 1H-NMR (CD3OD): δ
(ppm) 0.9 (3H, t, 18CH3), 1.3 (14H, m, 11-17CH2), 1.75 (2H, m, 10CH2),
2.95 (1H, dd, 7CH2), 3.24 (1H, dd, 7CH2), 3.93 (2H, m, 9CH2), 4.00
6
4
4
2
Optimization of the Chiral-Plate Preparation
(1H, m, CH), 4.27 (1H, d, CH2), 4.40 (1H, d, CH2), 7.75 (1H, s, CH).
13C-NMR (CD3OD): δ (ppm) 14.4 (1C-18), 23.7 (1C-17), 26.7 (1C-7), 27.5
(1C-16), 30.2 (1C-11), 30.4 (1C-12), 30.6 (2C-13,14), 31.7 (1C-10), 33.0
(1C-15), 40.0 (1C-4), 46.0 (1C-9), 58.6 (1C-6), 120.2 (1C-3a), 134.1 (1C-7a),
139.3 (1C-2), 173.1 (1C-8). TGA: thermally stable up to 200°C, with a
progressive weight loss (from 90 to 160°C) of 2.6%, corresponding to
1.5 water molecules. DSC: exothermic peak (in air) starting from
Plate type. Different TLC plates could be used as solid
support for modification with Spi(τ-dec). However, a basic
property should be their hydrophobicity, required to strongly
fix the chiral selector through its long hydrocarbon chain and
thus leaving the chiral head free. Two Merck plates, normally
employed for reversed phase HPTLC, were tested in the
present study: the classical RP-18 (end-capped and highly
hydrophobic) and the RP-18 W type, which is only partially
covered with C18 chains and is moderately hydrophilic. The
presence of a certain amount of free silanols allows the latter
to be wet by aqueous solvents employed either as mobile
phase, for the dipping solutions used in the chiral stationary
phase preparation, or for sample solutions. Both the plates
Scheme 1. The chiral selector Nτ-n-decyl-L-spinacine (Spi(τ-dec)).
Chirality DOI 10.1002/chir

CHIRAL HPTLC WITH A DYNAMICALLY COATED STATIONARY PHASE
315
proved able to strongly absorb the chiral selector, but the
coverage homogeneity was generally better for the W type.
0.2% ( 8 · 10-3 mol dm-3) in H2O/MeOH 90:10; after drying at
room temperature, the plates were ready for use or could be
stored for later use. By following this protocol, the reproduc-
ibility of the plate preparation was excellent, being the varia-
bility of the retention factor of L-Phe, used as reference,
within the range of 5%. The chiral plate, conserved in a desic-
cator, remained unchanged for several weeks.
Chiral-selector coating procedure. The good solubility of Spi
(τ-dec) in MeOH, even at room temperature, makes it a good
solvent to prepare the chiral selector solution employed in
the first dipping step (see Materials and Methods). Both pure
MeOH and a 1:1 H2O/MeOH mixture were tested and the
former gave the most uniform stationary phase coverage. Spi
(τ-dec) concentrations ranging from 0.1 to 1.0% w/v were
employed: under 0.4% a nonuniform coverage was obtained,
while increasing the concentration above 0.4% did not yield a
higher surface coating, as deduced from the scanner signal.
Dipping times ranging from 1 to 10 min were then tested: the
optimal dipping time was 5 min, since shorter times gave a
nonuniform coverage and longer times did not give any im-
provement in the obtained chiral layer.
Optimization of the Mobile Phase
The solvent mixture employed as a mobile phase should be
able to dissolve the racemic amino acids investigated as
samples, without perturbing the chiral-plate dynamic coating.
A hydro-organic solvent, with a high percentage of water,
fulfils these requirements, provided the aqueous portion
contains the amount of copper necessary to maintain the
copper layer on the stationary phase. Thus, the experimental
parameters examined are the following: type and percentage
of copper salt, type and percentage of organic modifier,
aqueous-solution pH, and the corresponding buffer concentration.
Copper loading. The copper solution used for the second
dipping step must be able to wet the chiral layer, allowing
the formation of stationary Cu(II) complexes with Spi(τ-dec),
without detaching the chiral selector already adsorbed on
the hydrophobic support. Previous experience32 suggested
the use of an H2O/MeOH 90:10 solvent. In fact, Spi(τ-dec) is
practically insoluble in water but a certain amount of organic
solvent is required for good contact between the dipping
solution and the stationary phase. Variable dipping times
(1–10 min) and copper concentrations (0.1–1.0% w/v) were
tested: increasing each parameter, higher and more uniform
copper loadings were obtained, as could be inferred by an
inspection under the Wood lamp. However, it is worth noting
that copper ions can also bind the free silanols of the hydro-
phobic support, thus giving nonchiral stationary complexing
sites which increase the solute retention but do not contribute
to enantioselectivity. In order to judge the best copper loading
conditions, some chromatographic tests were performed on
plates prepared by using different copper-solution concentra-
tions and dipping times: DL-Phe and DL-Trp racemic samples
were run and the separation and shape of the spots were
examined. The best results corresponded to a dipping time
of 10 min into a 0.2% copper solution. Similar tests were also
performed to check the role played by the copper counter-
ion: copper acetate, sulfate, nitrate, chloride, and perchlorate
were examined and copper sulfate was chosen.
Copper salt. The data reported in Table 1 reveal that the
anion of the copper salt used in the eluent mixture has
moderate influence on the Rm values and on the selectivity
of the aromatic amino acids. Higher selectivity values were
obtained using copper acetate, analogously with the results
previously reported for an HPLC study with His(τ-dec);31 this
result can be attributed to the higher pH value, due to hydro-
lysis of the acetate (the eluent used for these tests was not
buffered).
Copper concentration. The Cu(II) ion concentration in the
aqueous portion of the eluent was varied of two orders of
magnitude (from 10-2 to10-4 mol · dm-3); the effect on the re-
tention of Phe enantiomers was negligible (see Table 2),
while, in the case of Trp, the Rm values show some decrease,
resulting in a maximum of selectivity when copper acetate is
1 · 10-3 mol · dm-3. This concentration was chosen as the opti-
mum value for further investigation. It is worth noting that
this effect is lower than what is expected on the basis of that
reported in the literature for similar systems.14 In fact, copper
concentration affects the “solvent strength” as it participates
in the ligand-exchange retention mechanism: higher metal
ion concentrations are expected to shift the complex-formation
equilibrium in favor of the mobile phase, thus reducing the
sojourn time in the stationary phase. On the other hand, the
amount of copper bound to the stationary chiral selector is in
equilibrium with the copper concentration in the eluent: when
the latter is reduced also the former decreases and, in turn,
this leads to lower retention.
In view of above results, the following standard procedure
for the chiral coating preparation was then followed: Merck
RP-18 WF254S plates were immersed in 0.4% w/v solution
of Spi(τ-dec) in pure MeOH for 5 min and then left to dry
before the following treatment with copper. The plates were
then immersed for 10 min in a copper sulfate solution,
TABLE 1. Retention parameters for the separation of racemic mixtures of Phe and Trp on the home-made chiral plates, using dif-
ferent copper salts in the aqueous portion of the eluent (70% MeOH)
Trp
Phe
Copper salt
pH
Rm(L)
Rm(D)
α
Rm(L)
Rm(D)
α
Cu(CH3COO)2ÁH2O
CuSO4Á5H2O
6.0
5.5
5.5
5.4
5.4
0.82
0.82
0.79
0.87
0.87
1.19
1.06
1.06
1.12
1.12
2.2
1.9
2.1
1.7
2.1
0.10
0.12
0.16
0.18
0.19
0.33
0.31
0.33
0.37
0.37
1.4
1.3
1.3
1.3
1.3
Cu(NO3)2Á3H2O
Cu(ClO4)2Á6H2O
CuCl2
The measured pH of the aqueous copper solution is also reported.
Chirality DOI 10.1002/chir

316
REMELLI ET AL.
TABLE 2. Retention parameters for the separation of racemic
mixtures of Phe and Trp on the home-made chiral plates, using
different copper acetate concentrations in the aqueous portion
of the eluent (MeOH 30%)
Organic modifier. The main roles played by the organic
solvent present in the mobile phase are: (i) to assure that
the plate is effectively wetted by the eluent and (ii) to mediate
the interaction between the solutes and the hydrophobic
support. The former mainly affects the eluent velocity and
the run time. The latter influences both the solute retention
and selectivity in different ways, especially when the solute
bears hydrophobic or aromatic side chains. In fact, retention
partially depends on the direct hydrophobic interactions
between the solute and the RP support and this retention
mechanism is not enantioselective. In addition, the polarity
of the mobile phase affects the strength of the coordinative
(mainly electrostatic) interactions, and thus the stability, of
the ternary copper complexes formed between the stationary
chiral selector and the sample; also, this effect can be consid-
ered not enantioselective. Finally, the enantioselection
requires an additional interaction of the sample with the
stationary phase, besides the two-point chelation,35,36 and this
is clearly affected by the polarity of the mobile phase.
Copper
Trp
Phe
concentration
(mol · dm-3)
Rm(L)
Rm(D)
α
Rm(L)
Rm(D)
α
1 · 10-2
5 · 10-3
1 · 10-3
5 · 10-4
1 · 10-4
0.86
0.87
0.82
0.75
0.79
1.12
1.06
1.19
1.00
0.90
1.7
1.5
2.2
1.7
1.3
0.16
0.14
0.10
0.16
0.18
0.35
0.37
0.33
0.33
0.35
1.3
1.4
1.4
1.3
1.3
The formation of ternary Cu(II) complexes of L-Spi and L-
or D-amino acids, in aqueous solution, has been studied in
the past by our research group.34 In equimolar solutions,
the formation of mixed species is favored over that of binary
complexes. The hypothesized structure is that of a distorted
octahedron with the Cu(II) ion in the center. The two amino
acids are linked to the metal in the so-called “glycine-like”
mode, i.e., by means of their α-amino nitrogen and carboxylic
oxygen. The donor atoms are located in the equatorial plane
Scheme 2. Structural hypothesis of the stationary ternary complex of Cu(II)
with the chiral selector, Spi(τ-dec), and L/D-Phe, showing the possible interaction
with the hydrophobic support as the probable source of enantioselectivity.
Fig. 1. Chromatographic parameters concerning the separation of tryptophan enantiomers on the chiral plates, using as mobile phase different binary water
(copper acetate 1 · 10-3 mol dm-3)/organic solvent mixtures, at variable ratios.
Chirality DOI 10.1002/chir

CHIRAL HPTLC WITH A DYNAMICALLY COATED STATIONARY PHASE
317
of the complex, most likely in a trans arrangement. The rigid
structure of L-Spi hinders, in solution, additional intramolecu-
lar interactions between the two ligands, and thus the forma-
tion of such ternary species is not stereoselective. In the
chromatographic medium, where the structure of the mixed
complexes can be assumed very similar to that found in solu-
tion, Spi(τ-dec) is adsorbed on the hydrophobic support and
the interaction between the side chain of the L- or D-amino
acid and the octadecyl silica is possible (Scheme 2). This is
most likely the origin of the observed enantioselectivity.
In order to shed light on the specific action of several
organic solvents, also as a function of their percentage (i.e.,
the eluent polarity), the chromatographic behavior of two
sample racemates (L/D-Phe and L/D-Trp) was investigated.
The results for L/D-Trp are graphically shown in Figure 1;
the data are reported in the Supplementary Material.
The best separation of aromatic amino acids is always
achieved with the lowest percentages of organic modifier.
Among the organic solvents here employed, only methanol
shows the “typical” RP behavior: the corresponding values
of Rm steadily and roughly linearly decrease with increasing
of the solvent strength. In the case of the other organic
solvents, Rm decreases when their percentage increases up
to about 50% (60% in the case of THF) to increase again at
higher percentages. As far as the corresponding values of α
are concerned, they always decrease with the organic
modifier percentage. This means that the new effect respon-
sible for retention at high organic solvent amounts is not
enantioselective. The simplest explanation for this behavior
is the reduced amino acid solubility when the eluent becomes
less and less polar.
TABLE 3. Retention parameters for the separation of racemic
mixtures of Phe and Trp on the home-made chiral plates, at
different pH values of the aqueous portion of the eluent
(acetate buffer 1 · 10-2 mol dm-3, Cu(ClO4)2 1 · 10-3 mol dm-3/
MeOH 70:30)
Trp
Phe
pH
Rm(L)
Rm(D)
α
Rm(L)
Rm(D)
α
3.5
4.1
4.5
5.0
5.5
6.0
0.80
0.90
0.95
0.87
0.82
0.81
1.14
1.29
1.14
1.14
1.05
1.04
2.0
2.2
1.4
1.7
1.6
1.6
0.16
0.17
0.23
0.23
0.19
0.24
0.36
0.39
0.39
0.43
0.38
0.40
1.3
1.4
1.3
1.4
1.3
1.3
Eluent pH. In order to evaluate the effect of mobile phase pH
on retention and selectivity, a mixture of MeOH / acetate buffer
solution 1 · 10-2 mol dm-3 (adjusted at different pH values, in the
range of 3.5–6.0) and copper perchlorate 1 · 10-3 mol dm-3 30:70
was employed as eluent. The effect of pH was reported to be
very important in the corresponding HPLC system,33 where it
is one of the most important parameters ruling the amino acid
retention. Data reported in Table 3 show that, in the present
system, the effect of pH on retention is rather low; in the case
of Trp, the best enantioselectivity is observed at the lowest
pH value, in agreement with the literature.24
Optimization of the eluent composition. In order to obtain the
best separation of aromatic amino acids, the composition of
the eluent was optimized through the method “PRISMA,”
developed by Nyiredy et al.37,38 This method relies on the
preparation of a multicomponent eluent mixture, optimized
through a guided trial-and-error sequence. Once the station-
ary phase has been chosen, the set of solvents forming the
mobile phase must be selected: in the present work, MeOH,
ACN, and THF (to be mixed with H2O) were taken. The
second step is to define the appropriate “strength” of the
eluent, in order to obtain suitable Rf values (optimal Rf values
range from 0.2 to 0.8). This can be achieved by varying the
Fig. 2. Scanned chromatogram concerning the separation of a mixture of
racemic Trp, Phe, and 3,4-dihydroxyphenylalanine (DOPA). Eluent: MeOH/
ACN/THF:aqueous copper acetate 10-3 mol · dm-3 8:1:10:81.
TABLE 4. Retention parameters for the separation of racemic mixtures of some aromatic amino acids on the home-made chiral
plates
Sample
Rm(1)
Rm(2)
α (this work)
α (Ref. 32)
α (Ref. 16)
α (Ref. 24)
Trp
Phe
DOPA
Tyr
α-methyl-Trp
α-methyl-Phe
α-methyl-Tyr
5-methyl-Trp
0.82a
0.10a
-0.27a
-0.10b
0.09c
0.48a
-0.16b
0.52d
1.19a
0.33a
-0.12a
0.10b
0.33c
0.66a
0.10b
0.83d
2.17a
1.38a
1.14a
1.18b
1.77c
1.40a
1.34b
1.45d
1.33
1.28
1.24
1.21
1.38
1.43
1.27
1.33
1.20
1.20
1.23
1.14
—
1.27
1.19
1.19
1.00
—
1.25
1.11
—
—
—
—
aAqueous copper acetate 1 · 10-3 mol dm-3 /MeOH 70:30.
bAqueous copper acetate 1 · 10-3 mol dm-3 /EtOH 80:20.
cAqueous copper acetate 1 · 10-3 mol dm-3 /ACN 80:20.
dAqueous copper acetate 1 · 10-3 mol dm-3 /MeOH 60:40.
Chirality DOI 10.1002/chir

318
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ACKNOWLEDGMENTS
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experiments and fruitful discussion. The financial contribution
of University of Ferrara (FAR) is gratefully acknowledged.
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SUPPORTING INFORMATION
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