Synthesis, self-assembling properties, and atom transfer radical polymerization of an alkylated L-phenylalanine-derived monomeric organogel from silica: A new approach to prepare packing materials for high-performance liquid chromatography

Article abstract of DOI:10.1002/chem.200701302

The monomer N′-octadecylN^α-(4-vinyl)-benzoyl-L- phenylalanineamide (4) based on L-phenylalanine has been simply but effectively synthesized, and its self-assembling properties have been investigated. FTIR and a variable-temperature ¹H NMR spectroscopic investigation demonstrated that the aggregation of compound 4 in various organic solvents is due to the formation of intermolecular hydrogen bonds among the amide moieties. UV/Vis measurements indicated that the multiple π-π interactions of the phenyl groups also contribute to the self-as-sembly. As was observed by ¹³C cross-polarization magic-angle spinning (CP/MAS) NMR and variable-temperature ¹H NMR measurements, the ordered alkyl chains also played an important role in the molecular aggregation by van der Waals interactions. Compound 4 was polymerized by surface-initiated atom transfer radical polymerization from porous silica gel to prepare a packing material for HPLC. The results of thermogravimetric analysis showed that a relatively large amount of polymer was grafted onto the silica surface. The organic phase on silica was in a noncrystalline solid form in which the long alkyl chain exists in a less-ordered gauche conformation. Analysis of chromatographic performance for polyaromatic hydrocarbon samples showed higher selectivity than conventional reversed-phase HPLC packing materials.

Full text of DOI:10.1002/chem.200701302

DOI: 10.1002/chem.200701302
Synthesis, Self-Assembling Properties, and Atom Transfer Radical
Polymerization of an Alkylated l-Phenylalanine-Derived Monomeric
Organogel from Silica: A New Approach To Prepare Packing Materials for
High-Performance Liquid Chromatography
M. Mizanur Rahman, Miklós Czaun, Makoto Takafuji, and Hirotaka Ihara*[a]
Abstract: The monomer N’-octadecyl-
Na-(4-vinyl)-benzoyl-l-phenylalaninea-
mide (4)based on l-phenylalanine has
been simply but effectively synthesized,
and its self-assembling properties have
been investigated. FTIR and a varia-
ble-temperature 1H NMR spectroscopic
investigation demonstrated that the ag-
gregation of compound 4 in various or-
ganic solvents is due to the formation
of intermolecular hydrogen bonds
among the amide moieties. UV/Vis
measurements indicated that the multi-
ple p–p interactions of the phenyl
groups also contribute to the self-as-
sembly. As was observed by 13C cross-
polarization magic-angle spinning (CP/
MAS)NMR and variable-temperature
1H NMR measurements, the ordered
alkyl chains also played an important
role in the molecular aggregation by
van der Waals interactions. Compound
4 was polymerized by surface-initiated
atom transfer radical polymerization
from porous silica gel to prepare a
packing material for HPLC. The results
of thermogravimetric analysis showed
that a relatively large amount of poly-
mer was grafted onto the silica surface.
The organic phase on silica was in a
noncrystalline solid form in which the
long alkyl chain exists in a less-ordered
gauche conformation. Analysis of chro-
matographic performance for polyaro-
matic hydrocarbon samples showed
higher selectivity than conventional re-
versed-phase HPLC packing materials.
Keywords: gels · liquid chromatog-
raphy · polyaromatic hydrocarbons ·
polymerization · self-assembly
Introduction
Such characteristic self-assembled structures and the poten-
tial applications of polymers derived from amino acids have
attracted researchers to develop new synthetic routes to pre-
pare a wide variety of amino acid based polymers by using
various polymerization techniques.[8]
In recent years, there has been renewed interest in synthetic
polypeptides because of their potential application as biode-
gradable and biomedical polymers,[1] as well as their ability
to form highly ordered hierarchical structures through non-
covalent forces such as hydrogen bonding.[2] Incorporation
of a high degree of amino acid functionality and chirality in
polymer chains can enhance the potential to form secondary
structures (a helix and b sheet)and higher-ordered struc-
tures.[3] These synthetic polymers can be useful as chiral rec-
ognition stationary phases for HPLC,[4] metal-ion absorb-
ents,[5] drug-delivery agents,[6] and biocompatible materials.[7]
In principle, the formation of polymer-grafted inorganic
particles can be approached in two ways. 1)The “grafting
to” technique[9] consists of the synthesis of end-functional-
ized polymers followed by the immobilization of these poly-
mers onto the surface through anchoring groups. The “graft-
ing to” method is experimentally simple but it has a limita-
tion, namely the difficulty in achieving high grafting density
because of the steric crowding of the surface by the already
grafted polymers.[10] 2)On the contrary, in surface-initiated
polymerization (“grafting from”)polymer chains grow in
situ from initiator molecules that have been pregrafted to
the surface of inorganic particles.[11] The “grafting from” ap-
proach is considered to give higher densities because only
monomer molecules have to diffuse to the active species.
“Living”/controlled radical polymerization (CRP)meth-
ods, such as nitroxide-mediated polymerization (NMP),
atom transfer radical polymerization (ATRP), reversible ad-
[a] Dr. M. M. Rahman, Dr. M. Czaun, Dr. M. Takafuji, Prof. H. Ihara
Applied Chemistry and Biochemistry
Kumamoto University
2-39-1 Kurokami, Kumamoto 860-8555 (Japan)
Fax : (+81)96-342-3662
E-mail: ihara@kumamoto-u.ac.jp
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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FULL PAPER
dition–fragmentation chain transfer (RAFT)polymerization,
living anionic polymerization (LAP), and living cationic
polymerization (LCP), provide an ability to produce particu-
lar polymer architectures with controlled molecular weight
and molecular weight distributions.[12] The major difference
between the conventional radical polymerization techniques
and CRP is the lifetime of the propagating radicals. While
radicals derived from the thermal decomposition of conven-
tional radical polymerization initiators (azobisisobutyroni-
trile type)may undergo termination reactions within a few
seconds, the lifetime of the propagating radicals can be ex-
tended to several hours in controlled processes.[13] Among
the CRP processes, increasing attention has been paid to
ATRP since its discovery by Matyjaszewski and Sawamo-
to,[14] because this method does not require rigorous experi-
mental conditions like LAP or LCP.
The reaction between the activator complex (often CuBr-
chelated by N-donor ligands)and the dormant initiator re-
sults in the formation of a propagating radical and deactiva-
tor complex by a reversible halogen-atom transfer reaction
(Scheme 1). A low stationary concentration of propagating
Scheme 2. Synthesis of compound 4 from l-phenylalanine (l-Phe). Z-Cl:
carbobenzoxy chloride; DEPC: diethylphosphorocyanidate; TEA: trie-
thylamine.
lanine and Z-Cl, was reacted with stearylamine in THF in
the presence of a peptide synthesis coupling agent DEPC,
affording N’-octadecyl-Na-carbobenzoyl-l-phenylalaninea-
mide (2). Deprotection of the a-amino group of 2 by hydro-
genation (catalyzed by Pd/carbon black)resulted in N’-octa-
decyl-l-phenylalanineamide (3). The acylation of the a-
amino group was carried out with 4-vinylbenzoic acid in al-
kaline medium in the presence of DEPC in THF at 08C, to
give the octadecylated l-phenylalanine-derived monomer 4
as a white powder. The characterization of 1–4 was carried
out by elemental analysis and different spectroscopic meth-
ods (see Figures S1 and S2 in the Supporting Information).
Compound 4 was then used for surface-initiated living radi-
cal polymerization from a silica surface to produce a high-
density chromatographic stationary phase.
Scheme 1. Mechanism of ATRP.
radical (<10À8 m)is maintained by the dynamic equilibrium
which is established after a short period of time (in a few
seconds).[13] Recent advances in controlled/living polymeri-
zation processes have encouraged the preparation of a mul-
titude of macromolecules with controllable architecture,
functionality, composition, and topology.[15]
The use of amino acid based assemblies as delivery vehi-
cles is induced principally by their ability to incorporate and
release poorly water-soluble, hydrophobic, and/or highly
toxic compounds, while also minimizing drug degradation
and wastage, and hence increasing bioavailability.[16] To date
there is no report on the synthesis, self-assembling proper-
ties, and surface-initiated ATRP of alkyl l-phenylalanine-de-
rived monomers. Herein, we present the synthesis, charac-
terization, and subsequent polymerization of an alkyl l-phe-
nylalanine monomer from ATRP initiator-grafted silica par-
ticles. Furthermore, an application of these polymer-coated
silica particles as a stationary phase for HPLC is introduced.
Self-assembling properties of compound 4: It is well known
that hydrogen bonding is one of the driving forces for self-
assembly of organogelators in organic solvents.[17] IR and
1H NMR spectroscopies are powerful tools to study hydro-
gen-bonding interactions. Extensive precedent indicates that
secondary amide groups (NH)engaged in the standard
À
amide–amide hydrogen bonds (C=O···H N)display stretch-
ing bands in the range 3370–3250 cmÀ1, while stretching
bands in the range 3500–3400 cmÀ1 are attributed to “free”
secondary amide groups that are not involved in hydrogen
bonding.[18] The FTIR spectrum of compound 4 in CHCl3, in
which no self-assembly occurs, showed an absorption band
[19]
Results and Discussion
at 3434 cmÀ1 attributed to free N H groups
(Figure S3,
À
Supporting Information). On the other hand, the FTIR
spectrum of 4 in a cyclohexane gel ([4]=30 mm)showed ab-
sorption bands at 3302 cmÀ1 as well as 1658 and 1631 cmÀ1,
arising from the intermolecular hydrogen-bonded amide
moieties (Figure S4, Supporting Information). The FTIR
measurements also provide information on the alkyl groups.
The absorption bands of the antisymmetric (nas)and sym-
Synthesis of N’-octadecyl-Na-(4-vinyl)-benzoyl-l-phenylala-
nineamide: We selected l-phenylalanine as chiral origin due
to its neutral character to avoid synthetic difficulty and be-
cause of its high hydrophobicity. Scheme 2 illustrates the
synthetic process of the l-phenylalanine derivatives. N-Ben-
zyloxycarbonyl-l-phenylalanine (1), derived from l-phenyla-
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H. Ihara et al.
metric (ns)CH stretching vibrations of 4 appeared at 2927
2
À1
(nas, CH2)and 2855 cm
(ns, CH2)in CHCl , while in the
3
benzene gel they shifted to 2919 and 2850 cmÀ1, respectively
(Figure S5, Supporting Information). Such a frequency shift
to lower wavenumbers is induced by the restricted mobility
of the alkyl chains in 4, thus indicating that van der Waals
interactions among the alkyl chains also plays an important
role in the self-assembly of 4 molecules.[20]
Figure 1 shows variable-temperature 1H NMR measure-
ments of compound 4 in [D12]cyclohexane gel from 30 to
Figure 2. Partial 13C CP/MAS NMR spectrum of compound 4 at room
temperature.
evidence that the aromatic moieties also aggregate through
p–p interactions (Figure S7, Supporting Information). The
FTIR, NMR, and UV/Vis measurements demonstrated that
intermolecular hydrogen bonding among the amide moiet-
ies, van der Waals interaction between the alkyl chains, and
p–p interactions of the aromatic groups played the most im-
portant roles in the self-assembly of compound 4. The mo-
lecular packing model of compound 4 was estimated by Hy-
perChem version 5.1 with molecular mechanics by using the
semiempirical AM1 method (Figure 3). Calculations were
1
Figure 1. Partial H NMR spectra of compound 4 in [D12]cyclohexane gel,
indicating the shift of amide protons.
708C with the assignments of key amide protons: N-alkyla-
mide (H1)and the a-amide (H2). As the temperature in-
creased, the peaks for amide protons were consistently visi-
ble as representative of the associated amide function. Upon
increasing the temperature, the intensity of the two bands
increased and shifted upfield, demonstrating the presence of
one-mode association alternation between the free and the
associated species. The intensity of the peaks ascribed to the
aliphatic moieties at d=1.2 ppm also increased within the
temperature range of 30 to 708C and became sharper gradu-
ally, which suggests that the mobility of the alkyl chain in-
creases as a function of temperature (Figure S6, Supporting
Information). The ordered structural conformation of the
alkyl chain of 4 was evaluated by 13C cross-polarization
magic-angle spinning (CP/MAS)NMR measurements
(Figure 2). The higher intensity of the peak at d=33.4 ppm
attributed to the trans conformation indicates that most of
the alkyl chains are in an ordered structure at ambient tem-
perature.[21]
To monitor the interaction between the phenyl groups of
4, UV/Vis spectra were collected in cyclohexane at variable
temperature (80–08C). The intensity of the absorption band
at 262 nm decreased as the temperature fell, providing clear
Figure 3. Molecular packing model of compound 4.
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Packing Materials for HPLC
FULL PAPER
stopped when the difference in the energy level after two
consecutive iterations was less than 0.001 kcalmolÀ1. The
computer simulation confirmed the experimental results,
namely the driving force for the self-assembly of 4 is partly
based on the intermolecular hydrogen bonding between the
oxygen atom derived from the carbonyl group and the nitro-
gen atom of the amide moiety. Indeed, it can be described
by the two symmetrical hydrogen bonds between the amide
nitrogen and carbonyl oxygen atoms of the l-phenylalanine
moiety, and furthermore between the a-amide nitrogen and
the carbonyl oxygen atoms of the 4-vinylbenzoyl moiety.
Chromatographic application of molecular gels: We have re-
ported the development of a poly(octadecyl acrylate)deriv-
ative as a lipid membrane analogue and its successful appli-
cation as a HPLC stationary phase after immobilization
onto silica. Our detailed investigations revealed that the
highly ordered structure induced the orientation of the car-
bonyl groups that work as a source of p–p interaction be-
tween the stationary phase and the solute molecules. The
aligned carbonyl groups provide effective recognition of the
molecular planarity and linearity of polyaromatic hydrocar-
bons (PAHs)through multiple p–p interactions.[22] We have
also reported that dialkyl l-glutamide-derived amphiphilic
lipids form nanotubes, nanohelices, and nanofibers induced
by the bilayer structures in water or organic solvents, and by
intermolecular hydrogen bonds among the amide moieties
that also contribute to self-assembly.[23] Successful applica-
tion of a dialkyl l-glutamide-derived organogel as a HPLC
stationary phase revealed that the highly ordered molecules
form a condensed thin layer over the silica surface by inter-
molecular hydrogen bonding among the amide moieties,
which keeps the carbonyl groups in an ordered form favora-
ble for multiple p–p interactions with the guest molecule.
Enhanced molecular shape selectivity was observed through
p–p interactions between the carbonyl groups and the delo-
calized electrons of PAHs for the glutamide-derived station-
ary phase.[24] As similar noncovalent interactions, namely hy-
drogen bonding among the amide moieties and aromatic p–
p interactions, were found to contribute to the self-assembly
of compound 4, Sil-poly4 (Scheme 3)could be considered as
a novel stationary phase for HPLC applications.
Scheme 3. Synthetic steps for the preparation of poly4-grafted silica parti-
cles (Sil-poly4). PMDETA: 1,1,4,7,7-pentamethyldiethylenetriamine.
nitiator suspended in the mixture of monomer and toluene
in the presence of CuBr and PMDETA as catalyst precur-
sors (see the Supporting Information). After the ATRP pro-
cess, Sil-poly4 was purified by repeated washing in different
solvents to remove the nongrafted polymers from the sur-
face. Structural characterization of Sil-5 and Sil-poly4 was
carried out by different spectroscopic (diffuse reflectance in-
frared Fourier transform (DRIFT), NMR) and thermoana-
lytic methods (thermogravimetric analysis (TGA)).
Thermogravimetric analysis of Sil-poly4: To assess the or-
ganic content of the initiator-functionalized and polymer-
modified silica, repeated TGA runs were conducted and
almost identical curves were obtained. As the good reprodu-
cibility does not prove that significant errors are not incur-
red, control TGA experiments with nongrafted poly4 were
needed. Before measurements, each sample was first kept
under vacuum at 358C for 5 h to remove solvent traces, then
TGA measurements were run at a constant heating rate of
108CminÀ1 in air by using an empty crucible as a reference.
The heating process was carried out up to 8008C, which has
been demonstrated to be sufficiently high to degrade all sur-
face-bonded organosilanes.[26] Typical TGA curves for the
bare silica, Sil-5, and Sil-poly4 are depicted in Figure 4.
The weight retention profile of Sil-5 reached a plateau at
1108C (drying period), indicating the removal of surface
water. After the thermal degradation of the initiator the
weight of the sample was constant from 650 to 8008C.
Polymerization of 4 from silica by surface-initiated ATRP:
To synthesize polymer-grafted silica particles we broke the
process down into two steps (Scheme 3): 1) immobilization
of ATRP initiator on silica particles and 2)polymerization
from initiator-grafted silica. Radical polymerization initiator
[11-(2-bromo-2-methyl)propionyloxy]undecyltrichlorosilane
(5)was synthesized according to a method reported in the
literature.[25] Initiator 5 was grafted onto silica in dry toluene
in the presence of TEA under a nitrogen atmosphere (see
Experimental Section). The reaction between the surface-ac-
cessible OH group of silica and the anchoring group of the
A plateau in the weight retention curve of Sil-poly4 was
also observed as the temperature reached 6008C, confirming
that there is no polymer material remaining on the silica at
8008C. Considering the TGA curve of Sil-5 as reference, the
weight of the immobilized initiator can be calculated as
9.6 wt% of the total mass. Similarly, TGA revealed that
À
initiator ( SiCl3)resulted in the formation of a chemical
bond between the silica surface and initiator 5 (Sil-5).
ATRP processes were carried out by using Sil-5 as a macroi-
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H. Ihara et al.
1500 cmÀ1 are shown in Figure 5. In spectrum b, the ester
group introduced by initiator 5 is clearly noticeable, as indi-
cated by the C=O bond stretching at 1717 cmÀ1. A group of
peaks at 2931 and 2859 cmÀ1 is attributed to the CH bond
stretching of the long alkyl chain in initiator 5. The FTIR
spectrum of 4 (Figure 5, spectrum d)shows intense bands at
1659 and 1630 cmÀ1 corresponding to the carbonyl stretching
of the two amide bonds, respectively (the latter overlapped
with the C=C stretching band of the vinyl moiety). A broad
signal (Figure 5, spectrum c)could be observed at
1634 cmÀ1, indicating the presence of grafted polymer. The
spectrum of Sil-poly4 also displays a peak at 1719 cmÀ1 due
to the carbonyl stretching of initiator 5, but the intensity in
this case is not as strong as that detected in the spectrum of
À
Sil-5. Equally important is the appearance of N H stretch-
ing (3293 cmÀ1)in spectrum c derived from poly 4, providing
further evidence that monomer 4 was successfully polymer-
Figure 4. TGA curves for a)bare silica particles, b)Sil- 5, and c)Sil-poly 4.
À
ized from the silica surface. The Si OH bonds of the pure
silica show an absorption band at 3652 cmÀ1, indicating that
some surface OH groups remained unfunctionalized (Fig-
ure 5a and c). These results clearly proved that a considera-
bly large amount of poly4 could be immobilized on the
silica surface.
25.5 wt% poly4 is grafted on the silica surface if the weight
retention of Sil-5 was considered as reference at 8008C. The
former weight difference (9.6%)was translated into an
average grafting density of 0.61 initiator molecules per nm2.
However, this value is one order of magnitude lower than
the generally accepted silica surface hydroxyl group density
(five OH groups per nm2)[27] but slightly higher than that
demonstrated in other reports.[28] From the results of TGA
measurements it can be concluded that a fairly large amount
of polymer was grafted onto the silica surface by the ATRP
process, and Sil-poly4 can be considered as a high-density
organic phase for HPLC.
NMR studies of Sil-poly4: In liquid- or suspended-state
NMR spectroscopy, only those molecules or parts of mole-
cules are detectable that have very fast rotational mo-
tions.[29] The motion must be in such a fast range that it can
average out dipolar coupling and chemical shift anisotropy
until the related NMR peaks become narrow enough to be
1
detected. The suspension-state H NMR spectroscopy of Sil-
poly4 was measured from 25 to 508C. Neither the half-
height width (line width)of methylene groups nor the spin–
spin relaxation time (T2)showed any significant change with
temperature (20–508C). We observed that the intensity of
the NMR peaks representing terminal methyl and methyl-
ene groups of octadecyl moieties increased slightly and were
detectable when a very high vertical scale was used for
graphical presentation. These results indicate that the organ-
ic phase on the silica surface is in the solid state at room
temperature.
IR analysis: Immobilization of ATRP initiator 5 and sur-
face-initiated polymerization of 4 were also confirmed by
DRIFT spectroscopy. The absorbance spectra for bare silica
particles, Sil-5, and Sil-poly4 in the region from 3700 to
Solid-state 13C CP/MAS NMR spectroscopy is a powerful
tool for evaluation of the chemical composition of modified
surfaces and for confirming the integrity of the immobilized
alkyl groups. 13C CP/MAS NMR spectra of Sil-poly4 were
acquired at variable temperature (25–508C). It is well
known that the 13C NMR signals for (CH2)n carbon atoms
appear at two resonances, one at d=32.2 ppm due to the
trans conformation, indicating the presence of rigid and or-
dered chains, and the other at d=30.0 ppm due to the
gauche conformation.[21] The intense signal at d=29.9 ppm
in the 13C CP/MAS NMR spectra, attributed to the methyl-
ene carbon atoms of the octadecyl groups, indicates that the
N-alkyl chains of Sil-poly4 are arranged in a less ordered
gauche conformational form, and no alteration to the or-
dered trans conformation could be observed in the tempera-
ture range 25–508C (Figure S8, Supporting Information).
Figure 5. DRIFT spectra of a)bare silica particles, b)Sil- 5, and c)Sil-
poly4; d)FTIR spectrum of 4.
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Packing Materials for HPLC
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29Si CP/MAS NMR investigation shows the differentiation
between geminal silanol groups (Q2)and free silanol groups
Figure 6, this phase showed a much higher retention for
both alkylbenzenes and PAHs. It was also observed that
logk and logP plots of alkylbenzenes and PAHs in ODS
were parallel and almost coincided with each other, provid-
ing evidence that ODS can recognize only the hydrophobici-
ty of analytes.
It was found that Sil-poly4 showed a higher retention for
PAHs compared to its values for alkylbenzenes. For in-
stance, the logP of naphthacene (5.71)is smaller than that
of octylbenzene (6.30), while the logk value of naphthacene
(2.95)is higher than that of octylbenzene (2.09.) The in-
crease of logk for PAHs was accompanied by selectivity en-
hancement, which provides specific interactive sites for
PAHs that can recognize aromaticity as well as molecular
hydrophobicity.
4
(Q3)besides the siloxane groups (Q ), which are indicated
by signals at d=À92, À102, and À111 ppm, respectively. In
initiator- and polymer-grafted silica the signal corresponding
to residual geminal silanols is not seen. When the initiator
was reacted with the silica surface, a large amount of cross-
linked T2-type silicon species (d=À57 ppm)was observed
while polymer grafting increased the cross-linked surface, as
indicated by the appearance of T3 signals (d=À65 ppm)and
the absence of a signal for T1[30] (29Si CP/MAS NMR spectra
are given in Figure S9 in the Supporting Information).
To demonstrate an application of the new amino acid de-
rived monomer, Sil-poly4 was suspended in chloroform/hex-
anol and packed in a stainless-steel column of length 25 cm
and internal diameter (i.d.)4.6 mm by using methanol as
packing solvent. The packed column was used for chromato-
graphic analysis and the results are described in the follow-
ing sections.
Chromatographic performance of Sil-poly4: The first chro-
matographic evaluation was performed by using the Tanaka
test mixture containing hydrophobic probes.[32] Figure 7
shows the chromatogram obtained, in which it can be ob-
Retention mode: It is known that conventional octadecylsi-
lane (ODS)or alkyl phases can recognize the hydrophobici-
ty of analytes in HPLC, and this hydrophobicity is measured
by the methylene activity of the stationary phases. This ac-
tivity reflects the possibility of the phase being able to sepa-
rate two molecules that differ only in methylene groups, for
example, amylbenzene and butylbenzene or ethylbenzene
and toluene. The retention mode, as well as the extent of hy-
drophobic interaction among the analytes and the packing
materials in HPLC, can be determined by retention studies
of alkylbenzenes.[31]
To evaluate the retention mode and chromatographic per-
formance, Sil-poly4 was packed in a stainless-steel column
and separation experiments were carried out. Figure 6
shows the correlation between logk and logP for Sil-poly4
and a conventional ODS phase. Notably, the retention mode
of Sil-poly4 showed a reversed-phase mode compared to
that of the conventional ODS phase. As indicated in
Figure 7. Chromatogram for Tanaka test mixture with Sil-poly4. Mobile
phase: MeOH/H2O (90:10), flow rate: 1 mLminÀ1, column temperature:
308C, UV detection (254 nm).
served that all compounds were separated with good effi-
ciencies and good peak shapes. This characterization proto-
col is a well-developed approach that is recommended to
obtain information about the functionality of the silyl re-
agent and the methylene selectivity, as well as to establish
the repeatability and reproducibility of the separation be-
havior of commercially available reversed phases.[32] The
most relevant properties, which are measured by the chro-
matographic parameters for the separation of seven com-
pounds, are shape and methylene selectivities, hydrogen
bonding, and ion-exchange capacities in neutral media.
The chromatogram (Figure 7)shows the separation of two
homologous alkylbenzenes and nonplanar PAHs, and it was
observed that all four compounds are well resolved. The re-
tention of some PAH isomers and aromatic positional iso-
mers was examined to assess the shape selectivity by Sil-
Figure 6. Logk versus logP plots for ODS and Sil-poly4 stationary
phases; a: benzene, b: toluene, c: ethylbenzene, d: butylbenzene, e: hex-
ylbenzene, f: octylbenzene, g: decylbenzene, h: dodecylbenzene, i: naph-
thalene, j: anthracene, k: naphthacene.
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H. Ihara et al.
poly4, and the retention data were compared with those for
the conventional ODS phase. Several size and shape param-
eters for PAHs were introduced for systematic investigations
of retention behavior. The retention data for PAH isomers
on Sil-poly4 and their comparison with ODS are given in
Table 1.
Table 1. Retention and separation factors of PAHs for Sil-poly4 and
ODS stationary phases.
Analyte[a]
Sil-poly4
Sil-ODS
k
a
k
a
benzene
0.974
0.74
2.36
6.30
1.78
3.55
naphthalene
anthracene
pyrene
2.30
1.32
2.63
3.76
4.57
4.87
4.89
5.60
2.08
2.20
3.07
4.45
4.45
6.141
10.28
14.42
12.64
14.70
19.10
3.22
Figure 8. Chromatogram of
a
mixture of naphthalene, anthracene,
1.41
1.23
1.43
1.90
1.21
1.29
1.30
1.49
pyrene, and triphenylene with Sil-poly4. Mobile phase: MeOH/H2O
(90:10), flow rate: 1 mLminÀ1, column temperature: 308C, UV detection
(254 nm).
triphenylene
benzo[a]anthracene
chrysene
defined silica–polymer hybrid materials were prepared by
initiator immobilization on a silica surface and subsequent
surface-initiated ATRP by using initiator-modified particles
as macroinitiators.
naphthacene
cis-stilbene
trans-stilbene
o-terphenyl
m-terphenyl
p-terphenyl
1.44
1.05
4.63
The octadecyl chains on the polymer-grafted silica parti-
cles were a disordered, solid, and noncrystalline form that
did not show any phase-transition behavior. Sil-poly4 was
used as a HPLC packing material and showed a reversed-
phase retention mode. The chromatographic results for
PAH isomers showed better separation behavior than with a
conventional ODS phase. This new polymer-grafted silica
could be efficiently used for the separation of PAHs in re-
versed-phase HPLC. Furthermore, compound 4 may also be
applied towards the functionalization of magnetic particles,
and the magneto-responsive composite materials ultimately
could be used for magnetic hyperthermia. By introducing
cationic functional groups, the 4 molecule could be a poten-
tial candidate for the preparation of carriers that can deliver
nucleic acids into cells to investigate the properties of gene
sequences.
4.468
8.71
1.95
3.10
1.44
1.44
13.81
[a] Mobile phase: methanol/water (90:10); column temperature: 30 8C;
flow rate: 1.00 mLminÀ1; UV detection (254 nm).
To evaluate the planarity recognition capability of ODS
phases, Tanaka et al.[33] introduced the selectivity for two
solutes, o-terphenyl (F=9, L/B=1.11)and triphenylene
(F=9, L/B=1.12). (Definitions of F and L/B are given in
the Supporting Information.)We observed that Sil-poly 4
(atriphenylene/o-terphenyl =3.23)showed extremely enhanced mo-
lecular planarity recognition compared to ODS (atriphenylene/o-
terphenyl =1.4). A typical example for selectivity towards
PAHs on Sil-poly4 is shown in Figure 8. The retention data
show that Sil-poly4 yielded higher separation for all sets of
PAHs and geometrical isomers than the conventional re-
versed-phase HPLC stationary phase, regardless of the fact
that the alkyl chain in Sil-poly4 yielded a disordered gauche
transformation and no inversion to ordered transformation
was seen up to a temperature of 508C.
Experimental Section
General methods and materials: TEA (Wako, 99+ %)was distilled over
potassium hydroxide. Trichlorosilane (TCA, 97%), 2-bromoisobutyryl
bromide (Aldrich, 98%), 10-undecene-1-ol (Aldrich, 98%), platinum(0)-
1,3-divinyl-1,1,3,3-tetramethyldisiloxane (Karstedt catalyst; Aldrich, 0.1m
in xylenes), PMDETA (Wako, 98.0%), and copper(I) bromide (Aldrich,
99.999%)were used as received. Toluene (Wako, 99 + %)and diethyl
ether (Wako, 99.5+ %)were distilled from sodium/benzophenone and
stored under argon when not used. Porous silica particles (YMC-GEL)
were purchased from YMC (Kyoto, Japan); their average diameter, pore
size, and surface area were 5 mm, 12 nm, and 300 m2 gÀ1, respectively.
Conclusion
A new polymerizable N’-octadecyl-l-phenylalanine-derived
monomer has been synthesized and found to be self-assem-
bled by intermolecular hydrogen bonding. Structurally well-
HPLC-grade methanol and PAH samples were obtained from Nacalai
Tesque (Japan). Analytical thin-layer chromatography was performed on
0.25 mm silica gel plates, and silica gel column chromatography was car-
1318
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 1312 – 1321

Packing Materials for HPLC
FULL PAPER
ried out with silica gel 60 (Wakogel C-300). IR measurements were con-
ducted on a Jasco (Japan)FTIR-4100 Plus instrument in KBr. For
DRIFT measurements, the accessory DR PRO410-M (Jasco, Japan)was
used. TGA was performed on a Seiko EXSTAR 6000 TG/DTA 6300 ther-
addition of NaOH (2m, 20 mL). The addition of Z-Cl and NaOH was re-
peated four more times at 15 min intervals. After completion of Z-Cl ad-
dition the mixture was stirred for 1 h at 08C and 5 h at room tempera-
ture. The reaction mixture was extracted with diethyl ether (3”50 mL)to
remove unreacted Z-Cl and the aqueous layer was separated. HCl (6m)
was added to the aqueous layer until the pH reached 2.00. The mixture
was extracted with ethyl acetate (5”50 mL); the organic layer was then
washed with distilled water (5”50 mL), dried over Na2SO4, and concen-
trated under reduced pressure. n-Hexane (300 mL)was added and the
mixture was stirred with a glass rod until white crystals appeared, then
kept in a refrigerator for 24 h. The white solid obtained was isolated by
filtration and dried in vacuo to give 1 (83.2 g, yield: 85.6%). M.p. 79–
mobalance in static air from 30 to 8008C at a heating rate of 108CminÀ1
.
1
For characterization of synthesis, H and 13C NMR spectra were recorded
on a JEOL JNM-LA400 (Japan)instrument. Chemical shifts ( d)of 1H
and 13C were expressed in parts per million (ppm)with use of the inter-
nal standard Me4Si (d=0.00 ppm). Coupling constants (J)are reported in
Hertz. Elemental analyses were carried out on a Perkin–Elmer CHNS/
O 2400 apparatus. UV/Vis spectra were measured on a Jasco V-560 spec-
trophotometer by using a quartz cell of width 1 cm.
1
Solid- (13C CP/MAS NMR and 29Si CP/MAS NMR) and suspended-state
1H NMR measurements: NMR spectra were measured by a Varian Uni-
tyInova AS400 instrument at a static magnetic field of 9.4 T by using a
GHX nanoprobe for suspension-state NMR and a solid probe for CP/
MAS NMR spectroscopy at a spin rate of 2000–3500 Hz for suspension-
state NMR and 4000–4500 Hz for solid-state NMR measurements. The
samples for suspension-state 1H NMR spectroscopy were made by using
Sil-poly4 (10 mg)in CD 3OD (100 mL)with tetramethylsilane (0.03%.)
1H NMR spectra were recorded at 20–508C at 58C intervals by using a
GHX Varian AS400 nanoprobe. The parameters used for measurement
were delay time 1.5 s, pulse width 2.2 ms, transient number 32, and spec-
tral width 6000 Hz. Water was suppressed by using a presaturation pulse
sequence with a saturation delay of 1.5 s and a saturation power of 2 dB.
For assigning peaks, after determination of a pulse width of 908, simple
RELAY COSY (correlation spectroscopy test)was carried out and the
chemical shifts of the terminal methyl and methylene proton of the alkyl
chain were determined. For solid-state 13C CP/MAS, the NMR measuring
parameters were: spectral width 50000 Hz, proton pulse width 90=
11.6 ms, contact time for cross polarization 5 ms, and delay before acquisi-
tion 2 s. High-power proton decoupling of 63 dB with fine attenuation of
dipole r=2500 was used only during detection periods. 29Si CP/MAS
NMR spectra were collected with the same instrument. Representative
samples (200–250 mg)were spun at 3500 Hz by using 7 mm double-bear-
ing ZrO2 rotors. The spectra were obtained with a cross-polarization con-
tact time of 5 ms. The pulse interval time was 1.5 s. The transmitter fre-
808C; H NMR (400 MHz, CDCl3): d=7.33 (m, 5H; C6H5), 7.25 (m, 5H;
C6H5), 5.13 (d, J=8.76 Hz, 1H; *CHNHC(O)), 5.10 (s, 2H;
C(O)CH2C6H5), 4.70 (m, 1H; *CH)3.18 (dd, J=8.76 Hz, 1H; *CHCH),
3.08 ppm (dd, J=8.76 Hz, 1H; *CHCH); 13C NMR (100 MHz, CDCl3):
d=176.26, 155.83, 136.02, 135.41, 129.30, 128.65, 128.51, 128.22, 128.08,
127.21, 127.04, 67.14, 54.55, 37.68 ppm; IR (KBr): n˜ =3329, 3150, 3087,
3063, 3033, 1718, 1698, 1531, 1496, 1454 cmÀ1; elemental analysis calcd
(%)for C 17H17NO4: C 68.21, H 5.73, N 4.68; found C 67.93, H 5.81, N
4.58.
N’-Octadecyl-Na-carbobenzoyl-l-phenylalanineamide (2): N-Carbobenzo-
yl-l-phenylalanine (1)(30.0 g, 100.23 mmol)and stearylamine (29.65 g,
110.25 mmol)were dissolved in dry THF (300 mL)by stirring. Anhy-
drous TEA (25.30 g, 250.58 mmol)was added to the mixture followed by
DEPC (17.98 g, 110.25 mmol)and stirring was continued for 1 h at 0 8C.
The ice bath was removed and the mixture was stirred overnight at room
temperature. The mixture was concentrated under reduced pressure and
the residue was dissolved in CHCl3 (250 mL). The chloroform solution
was washed with 10% NaHCO3 solution, HCl (0.2m), and distilled water.
The solution was dried over Na2SO4, concentrated under reduced pres-
sure, recrystallized from methanol, and dried in vacuo to give a white
powder (40.1 g, yield: 72.5%). M.p. 122–1238C; 1H NMR (400 MHz,
CDCl3): d=7.33 (m, 5H; C6H5), 7.25 (m, 5H; C6H5), 5.46 (s;
NHC(O)*CH), 5.35 (s, 1H; *CHNHC(O)), 5.09 (s, 2H; C(O)CH2C6H5)),
4.28 (t, J=13.6 Hz; *CH), 2.95 (m, 2H; *CHCH2C6H5), 3.10 (m, 2H;
CH2NHC(O)*CH), 1.55 (m, 2H; CH2CH2CH2NHC(O)*CH), 1.25 (m,
30H; CH3CH2 ”15), 0.86 ppm (t, J=12.0 Hz, 3H; CH3); 13C NMR
(100 MHz, CDCl3): d=170.41, 155.83, 136.82, 136.14, 129.29, 128.70,
128.54, 128.21, 128.02, 127.03, 67.03, 56.53, 39.70, 39.53, 31.91, 29.68,
29.64, 29.57, 29.53, 29.48, 29.34, 29.26, 29.20, 26.91, 26.74, 22.68,
14.10 ppm; IR (KBr): n˜ =3302, 3150, 3087, 3063, 3033, 2919, 2849, 1686,
1
quencies of 29Si and H were 59.59 and 300.13 MHz, respectively. Typical-
ly, 1.5-k FIDs with an acquisition time of 30 ms were accumulated in
1 kilobyte (kb)data points and zero filling to 8 kb prior to Fourier trans-
formation. The line broadening used was 30 Hz and the spectral width
for all spectra was about 25 kHz.
1654, 1534, 1467, 1439 cmÀ1
; elemental analysis calcd (%)for
HPLC measurement: The chromatographic system consisted of a Gulliv-
er PU-980 intelligent HPLC pump, a Rheodyne sample injector with a
20 mL loop, and a Jasco multiwavelength UV detector MD 2010 plus. The
column temperature was maintained by using a column jacket that had a
circulator with a heating and a cooling system. A personal computer con-
nected to the detector with Jasco–Borwin (Ver 1.5)software was used for
system control and data analysis. As the sensitivity of the UV detector
was high, 5 mL of sample solution was used for each injection. To avoid
overloading effects, special attention was given to the selection of opti-
mum experimental conditions. Separations were performed with HPLC-
C35H54N2O3: C 76.30, H 9.88, N 5.09; found C 76.30, H 9.80, N 5.13.
N’-Octadecyl-l-phenylalanineamide (3): N’-Octadecyl-Na-carbobenzoyl-
l-phenylalanineamide (2)(14.0 g, 25.41 mmol)was dissolved in ethanol
(300 mL)with heating and Pd carbon black (1.4 g)was added to the solu-
tion. H2 gas was bubbled slowly into the solution for 4 h at 608C. The Pd
carbon black was removed by filtration, then the solution was concentrat-
ed under reduced pressure, recrystallized from methanol, and dried in
vacuo to give a white powder (7.8 g, yield: 73.65%). M.p. 78–798C;
1H NMR (400 MHz, CDCl3): d=7.21 (m, 5H; C6H5), 3.57 (q, J=16 Hz;
*CH), 3.21–3.29 (m, 3H; CH2NHC(O)*CH, *CHCHC6H5), 2.66 (q, J=
grade methanol/water (90:10)as mobile phase at
a flow rate of
1.00 mLminÀ1. Measurement of the retention factor (k)was carried out
under isocratic elution conditions. The separation factor (a)is the ratio
of the retention factor of two solutes that are being analyzed. The reten-
tion time of D2O was used as the void volume (t0)marker (the absorption
of D2O was measured at 400 nm, which is actually considered as injection
shock). All data points were derived from at least triplicate measure-
ments, with the value of retention time (tR)varying by Æ1%. The water/
1-octanol partition coefficient (P)was measured by retention studies
with ODS (monomeric; Inertsil ODS, 250”4.6 mm i.d., GL Science,
Tokyo, Japan): logP=3.579+4.207 logk(r)0.999997). [34]
23.4 Hz,
1H;
*CHCHC6H5),
1.47
(d,
J=6.8 Hz,
2H;
CH2CH2CH2NHC(O)*CH), 1.25 (m, 30H; CH3CH2 ”15), 0.86 ppm (t,
J=13.4 Hz, 3H; CH3); 13C NMR (100 MHz, CDCl3): d=173.9, 137.98,
129.29, 128.66, 126.75, 56.46, 41.05, 39.09, 31.90, 29.67, 29.63, 29.57, 29.56,
29.53, 29.34, 29.29, 26.92, 22.66, 14.09 ppm; IR (KBr): n˜ =3366, 3295,
3087, 3063, 3030, 2956, 2918, 2849, 1635, 1550, 1471 cmÀ1; elemental anal-
ysis calcd (%)for C 27H48N2O: C 77.82, H 11.61, N 6.73; found: C 77.53,
H 11.38, N 6.75.
N’-Octadecyl-Na-(4-vinyl)-benzoyl-l-phenylalanineamide (4): N’-Octadec-
yl-l-phenylalanineamide (3)(5.0 g, 12.00 mmol)and 4-vinylbenzoic acid
(2.0 g, 13.2 mmol)were dissolved in dry THF (200 mL)and stirred. An-
hydrous TEA (3.03 g, 30.2 mmol)was added followed by DEPC (2.2 g,
13.2 mmol)and stirring was continued for 1 h at 0 8C in an ice bath. The
mixture was stirred at room temperature overnight, then concentrated
under reduced pressure and the residue was dissolved in CHCl3
Synthesis of l-phenylalanine-derived self-assembling monomeric organo-
gelator
N-Carbobenzoyl-l-phenylalanine (1): l-Phenylalanine (45 g, 272.4 mmol)
was dissolved in NaOH solution (2m, 250 mL)and stirred in an ice bath
at 08C. Z-Cl (10.5 mL, 61.5 mmol)was added dropwise followed by the
Chem. Eur. J. 2008, 14, 1312 – 1321
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1319

H. Ihara et al.
(150 mL). The chloroform solution was washed with 10% NaHCO3, HCl
(0.2m)and distilled water (3”50 mL)and then the organic layer was
dried over Na2SO4. The mixture was concentrated and recrystallized
from ethanol and dried in vacuo to give a white powder (6.0 g, yield:
91.5%). M.p. 137–1388C; 1H NMR (400 MHz, CDCl3): d=7.70 (d, J=
8.76 Hz, 2H; C6H5), 7.4 (d, J=8.76 Hz, 2H; C6H5), 7.3 (m, 5H; C6H5),
7.23 (d, J=5.88 Hz, 1H; *CHNHC(O)), 6.70 (q, J=28.32 Hz, 1H;
C6H5CHCH2), 5.8 (d, J=17.56 Hz, 1H; C6H5CHCH-H), 5.5 (s, 1H; CH2-
NH-C(O)*CH), 5.34 (d, J=10.8 Hz, 1H; C6H5CHCH-H), 4.73 (t, J=
9.6 Hz; *CH), 3.25 (m, 1H; CH2NHC(O)*CH), 3.03 (m, 2H;
*CHCH2C6H5), 1.30 (m, 32H; CH3CH2 ”16), 0.86 ppm (t, J=12.0 Hz,
3H; CH3); 13C NMR (100 MHz, CDCl3): d=170.60, 166.70, 140.97,
136.92, 135.93, 132.90, 129.39, 128.71, 127.40, 127.05, 126.33, 116.08, 55.21,
39.62, 38.91, 31.94, 29.72, 29.67, 29.61, 29.52, 29.37, 29.30, 29.25, 26.81,
22.70, 14.11 ppm; IR (KBr): n˜ =3306, 3087, 3063, 3033, 2919, 2850, 1658,
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C36H54N2O2: C 79.06, H 9.96, N 5.12; found C 78.65, H 9.83, N 5.02.
Immobilization of initiator 5 on silica particles: Silica (5 g)was suspend-
ed in toluene (30 mL)in a round-bottomed flask, [11-(2-bromo-2-meth-
yl)propionyloxy]undecyltrichlorosilane (5; 2.150 g, 4.72 mmol)was added
and the suspension was rotated for 5 min. Then Et3N (1.44 g, 14.2 mmol)
was added and the rotation was continued under an inert atmosphere for
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Surface-initiated ATRP from Sil-5: Initiator-grafted silica (Sil-5; 4.1 g),
compound 4 (3.94 g, 7.2 mmol,)and PMDETA (0.536 g, 3.1 mmol)were
suspended in dry toluene (17 mL)and the suspension was purged with ni-
trogen. CuBr (0.085 g, 2.02 mmol)was added and the mixture was de-
gassed by three freeze–pump–thaw cycles. The flask was then placed in
an oil bath with a preset temperature of 908C and rotation at a slow ve-
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Packing Materials for HPLC
FULL PAPER
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Received: August 22, 2007
Published online: November 21, 2007
Chem. Eur. J. 2008, 14, 1312 – 1321
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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