Showcasing research from Laboratories of Analytical Chemistry,
and Materials Chemistry, Department of Applied Chemistry,
Faculty of Science and Technology, Keio University,
Yokohama, Japan.

As featured in:Volume 7 Number 31 21 August 2019 Pages 4727–4868

Journal of

Materials Chemistry B

A biomimetic hybrid material consisting of CaCO3 mesoporous
microspheres and an alternating copolymer for reversed-phase
HPLC
Inspired by CaCO3 based biominerals like eggshells, alternating
amphiphilic copolymer-modified monodisperse mesoporous
CaCO3 microspheres were applied as a HPLC stationary phase
for analyses of basic drugs requiring alkaline mobile phases.

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Wenjun Zhang et al.
Harnessing combinational phototherapy via post-synthetic
PpIX conjugation on nanoscale metal–organic frameworks

See Yuki Hiruta et al.,J. Mater. Chem. B, 2019, 7, 4771.

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A biomimetic hybrid material consisting of CaCO3
mesoporous microspheres and an alternating
copolymer for reversed-phase HPLC†
Mai Mochida, Yuta Nagai, Hiroto Kumagai, Hiroaki Imai,
Yuki Hiruta *Daniel CitterioandWe developed a biomineral-inspired hybrid material composed of CaCO3 and an organic polymer as acolumn packing material for HPLC. This material combines a hierarchical mesoporous structure and thefunctionality of the polymer. The surface of monodispersed mesoporous CaCO3 microspheres was
modified with poly(maleic acid-alt-1-octadecene) (PMAcO) comprising hydrophobic alkyl chains andanionic carboxylate groups. PMAcO adsorbed onto the surface of CaCO3 through electrostatic interaction
between Ca2+ sites and carboxylate groups, resulting in an octadecene coated microsphere interface.
These microspheres were applied as a HPLC column and exhibited reversed-phase retention behavior in
Received 20th May 2019,
Accepted 8th July 2019the separation of alkylbenzenes. This column showed high alkaline mobile phase resistance compared with

DOI: 10.1039/c9tb01014a

clomipramine and imipramine spiked into whole blood was achieved with an alkaline mobile phase,rsc.li/materials-b

demonstrating the potential of the biomineral-inspired material as a HPLC stationary phase for practicalapplications in routine analyses of basic drugs requiring alkaline mobile phases.

the conventionally applied ODS column packing material. Quantitative analysis of the basic antidepressantsIntroduction
Biominerals are inorganic–organic hybrid materials with hierarchically organized structures from nanoscopic to macroscopic
scales.1 For example, eggshells, seashells and corals have mesostructures of CaCO3 nanocrystals, which are elaborately controlled
by biopolymers.2,3 Focusing on eggshells as an example, theirstructure has pores that are necessary for gas exchange allowing
respiration, and the presence of biopolymers prevents virus
invasion. Furthermore, they provide relatively high mechanicalstrength to protect the inner shell.4 These characteristics ofhierarchical structures combining CaCO3 nanocrystals and biopolymers enable the control of substance distribution and provide
mechanical strength. Therefore, material scientists have been
attracted by the elaborate architecture and formation processesof these biominerals, attempting to develop new fabrication
technologies and to obtain novel functionalities through imitation of these features.5,6Paying attention to the hierarchical structure of biominerals
to control the distribution of substances, a normal phase chromatography stationary phase based on biopolymer-removed crushed

Department of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,
223-8522 Yokohama, Japan. E-mail: hiruta@applc.keio.ac.jp† Electronic supplementary information (ESI) available: Additional material
characterizations, chromatographic results. See DOI: 10.1039/c9tb01014a

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seashells has been developed.7 Although it was shown that thehierarchical structure of biominerals can be applied to achromatographic separation material, the functionality of the
biopolymer of biominerals like egg shells in terms of controlling substance distribution was not fully used for separation
purposes.
In order to control the morphology of CaCO3, crystallization
methods involving anionic small molecules8,9 or anionicpolymers10–12 were studied by mimicking the formation process of biominerals. We have also reported that monodispersed
mesoporous CaCO3 microspheres with 20 nm mesopores andcontrollable particle size can be obtained by a biomimetic
process.13 In this method, an anionic polymer, poly(sodium
4-sulfonate) (PSS), controls crystal growth (i.e. assemblage of
20 nm CaCO3 nanocrystals) by electrostatic adsorption, resulting
in the formation of a metastable phase vaterite crystal. In addition,
we have modified these microspheres with thermo-responsivepoly(N-isopropylacrylamide) (PNIPAAm) to transport chemical
substances from an organic to an aqueous phase by temperatureswitching.14 However, after modification with PNIPAAm, the interparticular space was fully filled with the thermo-responsive
polymer, resulting in the loss of the hierarchical pore structurethat is important in controlling substance distribution.
Cooperative interaction between the hierarchical structure
and organic molecules is important to obtain the functions of
biominerals and the high mechanical strength as in the case of

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Fig. 1

Journal of Materials Chemistry B

Concept image.

eggshells.15 To the best of our knowledge, a biomimetic material
that synergistically combines the hierarchical structure and thefunction of organic molecules to enable substance distribution
control has not been reported. Herein, we have developed
biomineral-inspired amphiphilic polymer-modified monodisperse
CaCO3 microspheres having a hierarchical pore structure and thechemical functionality of the polymer used for modification. In
addition, due to the ordered assembled structures composed ofpolymers and abundant inorganic CaCO3 (Fig. 1), excellent physicalproperties are achieved. We thought that these properties of thebiomineral-inspired hybrid material are suitable for controlling
substance distribution similarly to egg shells, and we applied it
as a column packing material for high performance liquid
chromatography (HPLC).
Column chromatography was originally developed by the
botanist Mikhail Tswett, using liquid-adsorption columns containing CaCO3 in the form of a powder of ground chalk (i.e.
calcite crystals). This implies that synthetic CaCO3 mesoporous
microspheres are applicable as a stationary phase packing
material for HPLC.16 In current HPLC columns, octadecyl silica(ODS) is the most widely used stationary phase for reversed-phase
chromatography. It consists of mesoporous inorganic silica gel
modified with organic hydrophobic octadecyl moieties. Because
biomineral-inspired materials also possess a hierarchical mesoporous structure, their application as a reversed-phase HPLC
column packing material similar to ODS is possible by hydrophobic alkyl polymer modification of their surface.
As with CaCO3-based biominerals wherein hierarchicalstructures are controlled by electrostatic adsorption between
CaCO3 crystals and biopolymers while stably maintaining themorphology,2 the imitation of their formation process allows
the modification of synthetic CaCO3 mesoporous microsphereswith an anionic polymer by electrostatic adsorption. By thismethod, mesoporous CaCO3 particles can be stably modified,due to polymer adsorption at multiple sites on the surface of
CaCO3, while eﬀectively retaining the mesopores.17
In addition, CaCO3 is stable against dissolution in alkaline
solutions, since it is a basic salt. On the other hand, the use of
ODS is limited due to poor hydrolytic stability against alkaline
mobile phases. The fact that alkaline mobile phases have to be
avoided when using low alkaline resistant silica gel column
materials is a disadvantage leading to low separation performancefor basic analytes.18 By replacing ODS with the polymer-modified

4772 | J. Mater. Chem. B, 2019, 7, 4771--4777

CaCO3 particles, it is possible to realize an alkaline stablepacking material overcoming the disadvantages of conventional
stationary phases.
We have developed a biomineral-inspired inorganic–organic
hybrid material by modifying the surface of monodisperse
mesoporous CaCO3 microspheres through electrostatic adsorption of a regularly alternating copolymer having both anionicgroups and functional groups, while maintaining the mesoporous structure. Whereas the anionic groups serve the purposeof adsorbing the polymer to the crystal surface, the alternating
functional groups impart their functionality. Thus, efficientmodification and functionalization of the surface of the monodisperse mesoporous CaCO3 microspheres were simultaneously
achieved. Especially, maleic anhydride based alternating copolymers can obtain various functionality by radical copolymerization of maleic anhydride with styrene, a-olefins, or vinyl ethers.19
In this work, poly(maleic acid-alt-1-octadecene) (PMAcO), consisting of regularly alternating hydrophobic alkyl chains and two
carboxylate units, was used (Fig. 1). The presence of the octadecylgroups on the surface of the monodisperse mesoporous CaCO3
microspheres imparts a strong hydrophobic character, similar tothe octadecyl groups of ODS, and enables the application as aversatile packing material in reversed-phase HPLC.

Results and discussion
Preparation and characterization of amphiphilic polymer
modified monodisperse CaCO3 microspheres
Bare monodisperse mesoporous CaCO3 microspheres wereprepared by a simple mixing method of two solutions at room
temperature according to the literature.13 CaCO3 crystal growthduring microsphere synthesis was controlled with PSS, followedby the decomposition of PSS using aqueous NaClO solution andcalcination. PMAcO was obtained by the hydrolysis of repeating
maleic anhydride units in poly(maleic anhydride-alt-1-octadecene)(PMAO), as shown in Scheme 1.20 Hydrolysis was confirmed by
FT-IR spectra (Fig. S1, ESI†), in which the peak at 1780 cm 1
derived from maleic anhydride decreased, while the peak at
1729 cm 1 derived from maleic acid was enhanced. The surfaceof the bare CaCO3 microspheres was modified with PMAcO by
simply dispersing the microspheres in an organic solution of
PMAcO. PMAcO adsorbed onto the surface of CaCO3 throughelectrostatic interaction between cationic calcium sites andanionic carboxylate groups, resulting in an octadecyl-coatedmicrosphere interface referred to as ‘‘CaCO3–PMAcO’’.
Particle morphologies of bare CaCO3 and CaCO3–PMAcO
particles were observed by scanning electron microscopy (SEM),

Scheme 1

Hydrolysis reaction of PMAO to form PMAcO.

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Fig. 2Paper

SEM images of (A–D): bare CaCO3 particles and (E–H): polymer-modified CaCO3–PMAcO particles.

as shown in Fig. 2. Both before and after modification with
PMAcO, monodisperse spherical particles with identical average particle diameters of 3.5  0.4 mm and 3.5  0.6 mm,
respectively, were observed (n = 400, measured from Fig. 2D and H).
The particle size distributions are shown in Fig. S2 (ESI†).
CaCO3 microspheres obtained by the applied synthesis method
have been reported to consist of aggregates of 20 nm-sized
CaCO3 crystals,21 which was also observed for the modified
CaCO3–PMAcO particles (Fig. S3, ESI†), confirming that themorphology of the mesoporous microspheres was retained
after modification with PMAcO. According to the XRD patterns(Fig. 3A), the bare CaCO3 particles were assigned to be purevaterite, with the crystal structure unchanged by modification
with PMAcO. FT-IR spectra (Fig. 3B) showed the appearance ofpeaks at 2924 and 2858 cm 1 derived from –CH2– moieties after
modification with PMAcO. In addition, the mass reduction in

the thermogravimetric (TG) curves in the 240–540 1C range(Fig. 3C) indicated the presence of 5 wt% PMAcO on the surfaceof CaCO3 microspheres, which was comparable to oleic acid for
CaCO3 surface modification prepared in analogy to the CaCO3–
PMAcO particles (Fig. 3D). These results demonstrate that
PMAcO was successfully modified onto the surface of CaCO3
microspheres while maintaining their properties. The pore size
distributions of CaCO3 microspheres before and after PMAcO
modification were obtained by nitrogen adsorption–desorption
isotherms (Fig. 3E). While the pore size distributions hardly
changed upon modification with the polymer, the pore diameter decreased from 22 to 16 nm (Fig. 3F). The thickness of an
octadecanoic acid monolayer on CaCO3 has been reported tobe E2.6 nm.22 Therefore, these results infer that a uniform
monolayer of repeating octadecene units was formed on thesurface of the pores by PMAcO modification. Since ODS

Fig. 3 Physical properties of bare CaCO3 and CaCO3–PMAcO microspheres: (A) XRD spectra, (B) FT-IR spectra, (C) TG curves, (D) TG curves of CaCO3–
PMAcO and CaCO3–oleic acid before and after washing; ‘‘-W’’ indicates samples after washing, (E) N2 adsorption–desorption isotherms, and (F) poresize distributions.

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particles used in HPLC column packings have a uniform
particle size of 2–5 mm with 6–30 nm pores, the current
CaCO3–PMAcO microspheres with comparable uniform particle
size and pore size fulfil the basic requirements to be applied asa HPLC column packing material. For successful application as a
packing material for long-term reproducible HPLC analysis, durability against the mobile phase solvent is required. Since the
PMAcO polymer has a plurality of adsorption sites, it is expected to
be stably adsorbed onto the surface of CaCO3. The durability of the
PMAcO modification was experimentally evaluated by comparing
the amount of organic components by means of the TG curves in
the 240–540 1C regime recorded before and after washing with awater/methanol mixture (50/50, v/v) representing a common
mobile phase. The results shown in Fig. 3D indicate that theamount of PMAcO did not change during the washing process. On
the other hand, in a comparative experiment using oleic acid for
CaCO3 surface modification (prepared in analogy to the CaCO3–
PMAcO particles), a drastic decrease of the organic compoundfrom 5 to 1 wt% upon washing was observed, corresponding to an
80% reduction. This significant difference is attributed to the factthat oleic acid only has a single carboxylate residue to interact with
the CaCO3 surface, in contrast to PMAcO with multiple bindingsites, leading to the higher durability of the latter. Therefore, thebiomineral inspired surface modification with the polymer byinteraction between polymeric functional groups and calcium sites
seems to contribute to the long-term durability of CaCO3–PMAcO
and, hence, its applicability as a packing material for HPLC.
Evaluation of CaCO3–PMAcO microspheres as a column
packing material for HPLC
CaCO3–PMAcO microspheres were packed into a column (100 
2.1 mm I.D.) and their performance as a stationary phase for
HPLC was evaluated. Fig. S4A (ESI†) shows the relationshipbetween the mobile phase mixing ratio and the back pressure
for methanol/water mixtures. At a flow rate of 0.4 mL min 1, theback pressure of the column was below 15 MPa (Fig. S4B, ESI†).
Furthermore, the back pressure was found to be stable and,

Journal of Materials Chemistry B

therefore, suitable for use with a conventional HPLC system. The
separation mode of this column was evaluated through theelution behavior of naphthalene and alkylbenzenes (C0–C10)
depending on the mixing ratio of methanol/water as the mobile
phase. The retention factors of naphthalene and alkylbenzenes
increased with decreasing proportions of methanol (Fig. S5,
ESI,† and Fig. 4A) and longer alkyl chains resulted in higherhydrophobicity (Fig. 4A). These results indicate that CaCO3–
PMAcO behaved as a reversed-phase mode stationary phase forthe separation of naphthalene and alkylbenzenes, while pure
CaCO3 generally works as normal-phase mode stationary phase.7
The octadecene groups of the CaCO3–PMAcO microspheres atthe stationary phase to mobile phase interface performed a role
identical to the octadecyl groups of ODS, resulting in reversedphase characteristics. During the separation of a mixture of
11 alkylbenzenes, all peaks could be clearly observed with abaseline resolution (peak resolution Rs = 1.8–4.2 4 1.5) in thechromatogram (Fig. 4B).
The durability of the CaCO3–PMAcO column against an alkaline
mobile phase (mixture of pH 10.8 aqueous Na2B4O7 buffer andmethanol) was evaluated by monitoring the elution behavior ofnaphthalene. The deterioration of the column was estimated by the
relative change of the retention time during the continuous flow of
the mobile phase. A comparative study was carried out using an
ODS column prepared from a commercially available 3 mm ODS
silica packing material through the same procedure as that for the
CaCO3–PMAcO column. As shown in Fig. 4C and Fig. S6 (ESI†), theretention time and the number of theoretical plates of naphthalene
were retained over 3500 column volumes (CVs) of alkaline mobile
phase purging in the case of the CaCO3–PMAcO column (RSD ofretention time: 0.7%), while it continuously decreased when using
the ODS packed column (over 35% decrease after 3500 CV purges).
It is assumed that the amount of octadecyl groups on the silica
surface gradually decreased due to the inherent solubility of silica inalkaline media, resulting in a reduction of hydrophobic interaction
with naphthalene. In contrast, the performance of the CaCO3–
PMAcO packing in the alkaline mobile phase was preserved, sinceFig. 4 (A) Retention factors of C0–10 alkylbenzenes obtained with a CaCO3–PMAcO packed column. Each sample was eluted at each mixing ratioof the methanol/water mobile phase ranging from 10/90 (v/v) to 90/10 (v/v); error bars represent mean values 1s (n = 3); flow rate of mobile phase
0.3 mL min 1. (B) Chromatogram showing the separation of a mixture of C0–10 alkylbenzenes by the CaCO3–PMAcO column. Gradient elution wasapplied at a flow rate of 0.1 mL min 1 starting with methanol/water (50/50, v/v) for 4 min, followed by the gradient program up to 80/20 (v/v) over 36 min
and finally elution with 80/20 (v/v) for 20 min. (C) Relative change of the retention time of naphthalene upon continuous running of alkaline mobile phase(50/50, v/v methanol/pH 10.8 Na2B4O7 buffer) in the case of CaCO3–PMAcO and ODS columns; flow rate of mobile phase 0.3 mL min 1.

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Paper

Fig. 5 (A) Chemical structures and pKa of the basic antidepressants clomipramine and imipramine; chromatograms of basic antidepressants for the
CaCO3–PMAcO column flowed at 0.3 mL min 1: (B) clomipramine (0.1 mg mL 1) with neutral and alkaline mobile phases. Methanol/water (63/37, v/v)
was used as a neutral mobile phase and methanol/pH 10.8 Na2B4O7 buffer (63/37, v/v) was used as an alkaline mobile phase. (C) Mixture of two basic
antidepressants (clomipramine and imipramine) with a methanol/pH 10.8 Na2B4O7 buffer (55/45, v/v). (D) Basic antidepressants (clomipramine andimipramine) spiked into whole blood of porcine with a methanol/pH 10.8 Na2B4O7 buffer (55/45, v/v).

the solubility of CaCO3 decreases with increasing pH. These
results indicated that CaCO3–PMAcO packed columns show
excellent durability at high pH and could be used for routine
analyses requiring alkaline mobile phases.

Table 1

Quantitative analysis of basic antidepressants spiked into
whole blood using a CaCO3–PMAcO packed column

Imipramine
5.0
Clomipramine 5.0

As a proof-of-concept demonstration, we conducted the analysis
of basic compounds requiring an alkaline mobile phase forsuccessful separation. When a neutral mobile phase is used,basic compounds become positively charged, resulting inunwanted electrostatic and hydrogen bond interactions with thestationary phase in addition to hydrophobic interactions. On theother hand, these are eliminated by deprotonation of basic
compounds in alkaline mobile phases, allowing the separationof basic compounds as free bases, which is attractive for routine
analysis. Silica-based columns are reluctantly used because of
their inherent instability to alkaline mobile phases.23 Herein, we
demonstrate the analysis of the basic antidepressants clomipramine
and imipramine (pKa of 9.3 and 9.5, respectively,24 shown in
Fig. 5A), known as tricyclics. Since addiction to psychotropic
drugs is a serious problem, the routine analysis of these substances in biological fluids is of significance.25 First, the eﬀect ofthe mobile phase pH on the elution behavior of clomipramine
using a CaCO3–PMAcO packed column was investigated. Fig. 5Bshows the chromatograms of clomipramine on a CaCO3–PMAcO
stationary phase eluted with a neutral or alkaline mobile phase.
A sharper peak was obtained when using the alkaline mobile
phase compared to the neutral mobile phase (1.4-fold number oftheoretical plates; 0.7-fold peak asymmetry factor). Protonationof clomipramine in the neutral mobile phase resulted in theinteraction with anionic carboxylates or carbonates, causing
tailing and extension of the retention time. The suppression ofclomipramine protonation eliminated these unwanted interactions, leading to a sharp chromatographic peak with simple
retention behavior only governed by hydrophobic interactions

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Recovery of basic drugs from a whole blood sample

Level
Basic drug

From peak area

From peak height

Recovery RSD
Recovery RSD(ng mL 1) (%, n = 3) (%, n = 3) (%, n = 3) (%, n = 3)
96.4
97.6

0.2
0.7

95.6
96.2

0.3
0.7

between clomipramine and octadecene groups. In addition,
clomipramine and imipramine were successfully separated by
the CaCO3–PMAcO column in combination with the alkaline
mobile phase (Fig. 5C). Linear calibration curves for clomipramine
and imipramine were obtained based on the peak areas or heightsfrom chromatograms of mixed samples of the two basic antidepressants (Fig. S7A and B, ESI†). Finally, porcine whole blood
spiked with clomipramine and imipramine was analyzed using
the CaCO3–PMAcO packed column and the alkaline mobile phase.
The sample was prepared according to a general whole blood
pretreatment method26 before HPLC analysis. Fig. 5D shows thecorresponding chromatogram. Blood constituents (mainly serum
albumin) and the two basic antidepressants were successfully
separated. Furthermore, good recovery values (95.6–97.6%) andsmall relative standard deviations (RSD o 0.7%) calculated fromthe calibration curves (Fig. S7A and B, ESI†) were achieved (Table 1).
On the other hand, the two basic antidepressants were not successfully separated with the ODS packed column in a neutral mobile
phase (Fig. S8, ESI†). These results indicated that HPLC analysis with
CaCO3–PMAcO packed columns in an alkaline mobile phase ispractically applicable to the routine analysis of basic antidepressants.

Conclusions
This work is to the best of our knowledge the first example of abiomimetic material in which the hierarchical structure andthe polymer function are fused to enable eﬀective control ofsubstance distribution. By mimicking the formation process of

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Paperbiominerals, mesoporous CaCO3 microspheres were fabricatedand surface-modified with an alternating amphiphilic copolymer
consisting of hydrophobic alkyl chains and anionic carboxylategroups. Because of the monodisperse and mesoporous structure
modified with hydrophobic octadecene groups on its surface,
this biomineral-inspired hybrid material was found to be suitable for application in HPLC. When applied as a HPLC column
packing material, it showed reversed-phase retention behavior
and high resistance against alkaline mobile phases. In a proofof-concept application, whole blood spiked with basic antidepressants clomipramine and imipramine was successfully analyzed with an alkaline mobile phase, in which conventional ODS
packings have significant stability limitations. Thus, the present
work clearly demonstrates the potential of biomineral-inspired
PMAcO-modified mesoporous CaCO3 microspheres as a HPLC
column packing material. By decreasing the particle size of themesoporous CaCO3 microspheres, the pore size distribution canbe narrowed. Furthermore, since decreasing the particle diameter leads to an expansion of the specific surface area and an
improvement of packing density, it is expected that a higher
separation ability can be obtained by decreasing the particle
diameter. Furthermore, it is expected that various retention
modes can be imparted to this packing material with a variety
of maleic acid-based alternating polymers. Therefore, we expectthat this work opens a new route for biomimetic materials inseparation applications.

Experimental section
Materials and instruments
Calcium chloride (CaCl2), sodium carbonate (Na2CO3), 5 wt%
aqueous sodium hypochlorite (NaClO) solution, tetrahydrofran(THF) and HPLC-grade methanol were purchased from Kanto
Chemical (Tokyo, Japan). Toluene, acetone, potassium hydroxide(KOH), trifluoroacetic acid (TFA), oleic acid, sodium tetraborate
decahydrate (Na2B4O710H2O), sodium hydroxide (NaOH),
naphthalene and clomipramine hydrochloride were purchasedfrom Wako Pure Chemical (Osaka, Japan). Poly(sodium 4-sulfonate)(PSS, Mw: B70 000) and poly(maleic anhydride-alt-1-octadecene)(PMAO, Mn: 30 000–50 000) were purchased from Sigma-Aldrich(St. Louis, MO). Benzene, methylbenzene, ethylbenzene, amylbenzene, butylbenzene, hexylbenzene, heptylbenzene, n-octylbenzene,
nonylbenzene, decylbenzene and imipramine hydrochloride werepurchased from Tokyo Chemical Industry (Tokyo, Japan). A 3 mm
fully porous ODS silica gel (CHEMCOSORB 3-ODS-L) was purchased from Chemco Scientific (Osaka, Japan). Porcine whole
blood with 0.3 wt% citric acid was purchased from Tokyo
Shibaura Zouki CO (Tokyo, Japan). Ultrapure water (18.2 MOcm) was obtained from a PURELAB flex water purification system(ELGA, Veolia Water, Marlow, U.K.).
Fabrication of CaCO3 microspheres
128 mL of 1 M CaCl2 solution was added to 4.0 mL of 16 mM
Na2CO3 solution containing 1.0 g L 1 PSS. After mixing, thesolution was stirred at 1500 rpm for 90 s with a discoid shape4776 | J. Mater. Chem. B, 2019, 7, 4771--4777

Journal of Materials Chemistry B

magnetic stirrer tip (diameter: 30.5 mm, and height: 12 mm)
and then left for 24 h. After filtration, the organic components
included in the products were extracted by immersion in 5 wt%
NaClO aqueous solution for 48 h. The resultant microspheres
were washed extensively with purified water and annealed at
400 1C, leading to bare CaCO3 microspheres.
Preparation of PMAcO by hydrolysis of PMAO
315 mg of solid KOH was added into 10 mL of a solution of 1.0 g
of PMAO in THF, followed by vigorous stirring for 2 hours atroom temperature. After evaporating the solvent, the residue
was dissolved in a small amount of acetone, and an excess
amount of TFA was added. Aggregates were re-dissolved in 5 mLof acetone and slowly poured into 100 mL of water to precipitate the polymer. This purified polymer was collected by suction
filtration with water washing and dried in a vacuum.
Modification of PMAcO onto the surface of CaCO3
microspheres
100 mg of PMAcO was dissolved in 20 mL of THF and 30 mL of
toluene. After addition of 1.0 g of bare CaCO3 microspheres,
stirring was vigorously continued for 2 hours at 60 1C. The
collected ‘‘CaCO3–PMAcO’’ microspheres were washed with asmall amount of THF and dried at 60 1C. Oleic acid-modified
CaCO3 microspheres were obtained by the same procedure.
100 mg of oleic acid was dissolved in 50 mL of toluene. After
addition of 1.0 g of bare CaCO3 microspheres, stirring was
vigorously continued for 2 hours at 60 1C. The collected‘‘CaCO3–oleic acid’’ microspheres were washed with a small
amount of THF and dried at 60 1C.
Characterization of PMAcO, CaCO3 particles and modified
CaCO3 particles
The morphologies of bare CaCO3 and CaCO3–PMAcO wereobserved using scanning electron microscopy (SEM, S-4700,
Hitachi, Tokyo, Japan). Before observation, samples were
coated with osmium using an osmium coater (HPC-1S, Vacuum
Device, Ibaraki, Japan) for 15 s at 10 mA. For the observation of
fractured CaCO3–PMAcO, the sample was prepared by a thickcoating with osmium using an osmium coater for 60 s at 10 mA,
followed by crushing with a microspatula. The size of microspheres was measured using the image processing software
ImageJ (National Institutes of Health). The Brunauer–Emmett–Teller (BET) surface area, pore volume and pore size distributions were determined by N2 sorption (3Flex, Micromeritics,
SHIMADZU, Kyoto, Japan). Before measurements, samples
were degassed for 6 h at 160 1C. The crystal structures of thesamples were confirmed by X-ray diffraction (XRD, MiniFlex IIdiffractometer, Rigaku, Tokyo, Japan) using CuKa radiation.
Organic components of samples were also characterized using
high throughput Fourier transform infrared spectroscopy (FTIR, model-Alpha, Bruker, Germany). The contents of organic
components of microspheres were measured by thermogravimetric analysis (TG, TG/DTA7200, SII, Chiba, Japan). Durabilityof PMAcO modification was evaluated by comparing the amountsof organic components in the 240–540 1C regime of the TG

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Journal of Materials Chemistry B

curves before and after washing with solvent. CaCO3–PMAcO waswashed 10 times with 10 mL (total 100 mL) of a mixed solvent ofwater and methanol (50/50, v/v) with suction filtration and driedin a vacuum. CaCO3–oleic acid was evaluated according to thesame procedure.

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Column packing
A slurry of CaCO3–PMAcO was packed into stainless steel
columns (100 mm  2.1 mm I.D., Chemco Scientific). The
slurry of CaCO3–PMAcO beads (0.7 g) in methanol (13 mL) was
poured into a slurry reservoir (Chemco Scientific) connected tothe stainless steel column. Methanol was flowed through theslurry reservoir using a HPLC pump (LC-6AD, SHIMADZU) for
30 min, followed by flowing methanol/water (50/50, v/v) for
60 min with a constant pressure of 35 MPa. CHEMCOSORB
3-ODS-L was packed by the same procedure.
Chromatographic analyses
Chromatographic analyses were carried out using a ProminenceI LC2030C (SHIMADZU) system equipped with a UV-Vis detector.
All measurements were conducted at 25 1C with 1 mL injection
volume. Elution behavior of all samples was monitored at
254 nm. All samples were dissolved in methanol and filteredthrough a 0.2 mm membrane filter. The pH of the 50 mM
Na2B4O7 buﬀer was adjusted to 10.8 with 100 mM NaOH aq.
Chromatographic parameters were calculated using thefollowing equations:
Retention factor: k = (tRt0)/t0, where t0 and tR are theretention times of solvent peak and the target analyte,
respectively.
Peak resolution: RS = 2(tR2 tR1)/(W1 + W2), where tR is theretention time (tR2 4 tR1), W is the peak width of the analyte,
and Rs 4 1.5 indicates complete separation.
Theoretical plate: N = 16(tR/W)2
Peak asymmetry factor: As = b/a, where a is the time from theleading edge of the peak to the peak midpoint, and b is the timefrom the peak midpoint to the tailing edge.
Preparation of whole blood samples
Porcine whole blood samples were pretreated using a protein
precipitation method.26 To a 2 mL centrifuge tube, 200 mL ofwhole blood, 100 mL of basic drug mixture (50 mg mL 1
imipramine, and 50 mg mL 1 clomipramine in methanol) or
100 mL of methanol as a blank sample, and 700 mL of acetonitrile
were added. These solutions were mixed using a vortex mixer for
10 minutes and centrifuged at 10 000 rpm for 10 minutes. The
supernatant was filtered through a 0.2 mm membrane filter.

Conflicts of interest
There are no conflicts to declare.

This journal is © The Royal Society of Chemistry 2019

Paper

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