A bio-based benzoxazine surfactant from amino acids

Article abstract of DOI:10.1039/d0gc00218f

A novel bio-based benzoxazine (Ca-g) surfactant was synthesizedviaa Mannich reaction using renewable glycine and cardanol as raw materials. The structure of Ca-g was characterized by NMR, FT-IR, and elemental analysis. Styrene containing emulsions and polystyrene (PS) latex were prepared based on the Ca-g surfactant. These results showed that the Ca-g surfactant exhibited high efficiency for stabilizing styrene emulsions. An addition of only 1.7 (w/v)% stabilized the high internal phase emulsion (HIPE) with a styrene volume fraction up to 90%. More interestingly, HIPE exhibited pH-sensitivity and could be formed at pH values from 9.0 to 12.0. Furthermore, PS particles were obtainedviaHIPE polymerization. In addition, thein vitrocytotoxicity of Ca-g and its triglyceride emulsion was also investigated. Ca-g showed lower cytotoxicity against the HeLa cell line. A stable triglyceride-in-water emulsion stabilized by Ca-g was prepared. The bio-based Ca-g surfactant may have potential use in cosmetics and daily cleaning products.

Full text of DOI:10.1039/d0gc00218f

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Page 1 of 22
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DOI: 10.1039/D0GC00218F
Bio-based benzoxazine surfactant from amino acid
Zheng Wang,a Shu Yao,b Kun Song,b Xinjian Gong,a Sujuan Zhang,a Sheng Gaoa and
Zaijun Lu*a
a
Key Laboratory for Special Functional Aggregated Materials of Ministry of
Education, School of Chemistry and Chemical Engineering, Shandong University,
Jinan, 250100, P. R. China
b
Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University,
Jinan, 250100, P. R. China
*Corresponding author
E-mail: z.lu@sdu.edu.cn; Fax: +86 531 88564464; Tel: +86 531 88361599
Abstracts
A novel bio-based benzoxazine (Ca-g) surfactant was synthesized via a Mannich
reaction using renewable glycine and cardanol as raw materials. The structure of Ca-g
was characterized by NMR, FT-IR, and elemental analysis. Styrene containing
emulsions and polystyrene (PS) latex were prepared based on Ca-g surfactant. These
results showed that Ca-g surfactant exhibited high efficiency for stabilizing styrene
emulsions. An addition of only 1.7 (w/v)% stabilized the high internal phase emulsion
(HIPE) with styrene volume fraction up to 90 %. More interestingly, HIPE exhibited a
pH-sensitive property and could be formed at pH values from 9.0 to 12.0.
Furthermore, polystyrene (PS) particles were obtained via HIPE polymerization. In
addition, the in vitro cytotoxicity of Ca-g and its triglceride emulsion were also
investigated. Ca-g showed lower cytotoxicity against the Hela cell line. A stable
triglceride-in-water emulsion stabilized by Ca-g was prepared. The bio-based Ca-g
surfactant may have potential use in cosmetics and daily cleaning products.
Keywords: Bio-based surfactant Benzoxazine chemistry Amino acid Emulsion

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DOI: 10.1039/D0GC00218F
Introduction
Compared with petrochemical-based surfactants, bio-based surfactants have
several significant advantages: (1) Their raw materials are renewable biomass such as
vegetable oils,1-4 carbohydrates,5-7 and amino acids.8-10 They are not only abundant in
source, but also have seasonally harvestable sustainability.11 (2) They are naturally
biodegradable which confers a significant environmental friendliness.7,8 Therefore,
bio-based surfactants have attracted more and more attention from both academia and
industry.12
Recently, the developing benzoxazine chemistry can easily connect various
phenol derivatives and amine derivatives in one pot.13-18 It provides a facile method to
synthesize bio-based benzoxazine surfactants. Moreover, not only does bio-based
benzoxazine surfactant display emulsification properties comparable to traditional
surfactant,13,19 but also the benzoxazine ring can undergo polymerization to become a
polymerized surfactant.20 At present, only bio-based benzoxazine surfactants derived
from phenol biomass have been reported by a few research groups. For example,
Ishida et al. synthesized a benzoxazine monomer using vanillin and aniline as starting
materials, and then the aldehyde group of the benzoxazine monomer reacted with
amine-terminated poly(ethylene oxide)19-co-poly(propylene oxide)3 to form the
nonionic surfactant.19 Krajnc et al. synthesized a nonionic benzoxazine surfactant
using natural cardanol, amine-terminated poly(ethylene oxide)19-co-poly(propylene
oxide)3, and paraformaldehyde.20 However, the synthesis of bio-based benzoxazine
surfactant derived from amine biomass has not been reported so far.
In this article, we report the first synthesis of a bio-based benzoxazine surfactant
using amino acids as amine biomass. Amino acids are very rich in species and
quantities in nature. Their amino groups can be used as amine source to synthesize
benzoxazine monomer. Their carboxyl groups can be served as the hydrophilic part of
surfactants. Therefore, amino acids are especially suitable for the synthesis of
benzoxazine surfactants. Cardanol is easily available in large quantities from the
cashew nut shell, an agricultural waste product.21-23 Its long aliphatic side chain can

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DOI: 10.1039/D0GC00218F
be served as the hydrophobic part of a surfactant. Therefore, cardanol is selected as
the phenol source. The paraformaldehyde can be attained by catalytic oxidation of
bio-methanol.24 The above-mentioned renewable feedstocks were combined to
synthesize a bio-based benzoxazine surfactant via the Mannich reaction. Using the
benzoxazine surfactant as an emulsifier, styrene-in-water high internal phase emulsion
and polystyrene (PS) particles were successfully prepared. Furthermore, the in vitro
cytotoxicity of Ca-g and its triglceride emulsion were also investigated.
Experimental
Materials
Glycine (99.5 %), azobisisobutyronitrile (AIBN, 98 %), and paraformaldehyde
(analytically pure) were purchased from Shanghai Macklin Biochemical Co., Ltd.
3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazoliumbromide (MTT) was obtained
from Fule Biological Technology Co., Ltd. 4,7-Di(2-thienyl)-2,1,3-benzothiadiazole
(DBT, 99 %) and 3-pentadecylphenol (95 %) were supplied by TCI (Shanghai)
Development Co., Ltd. Hela cell line was provided by Qilu Hospital, Shandong
University. Cardanol (98 %) and capryli/capric triglceride were purchased from
Jinquan Chemical Co., Ltd. Deionized water was purified using an Ultrapure water
purifier. Methanol, ethanol, ethyl acetate, dimethyl sulfoxide (DMSO), sodium
hydroxide and hydrochloric acid were purchased from Fuyu Fine Chemical Co., Ltd.
Styrene (99 %, Shanghai Macklin Biochemical Co., Ltd.) was purified by filtering
through aluminium oxide. All the other reagents and solvents were used as received.
Synthesis of benzoxazine monomers of Ca-g and Pe-g
To a 50 ml three-neck round flask, 0.75 g (0.01 mol) of glycine, 0.40 g (0.01 mol) of
sodium hydroxide, and 10 ml water were added and stirred at 25 °C for 12 hours. The
solvent was distilled and dried under vacuum to yield a white solid.
To the flask containing sodium glycinate, 3.0 g (10 mmol) of cardanol or 3.04 g (10
mmol) 3-pentadecylphenol, 0.60 g (20 mmol) of paraformaldehyde, and 30 ml
methanol were added. The reaction mixtures were stirred and heated at 65 °C for 6

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DOI: 10.1039/D0GC00218F
hours. The crude product was dropped into ethyl acetate for precipitation. The filter
cake was collected for vacuum drying to give Ca-g (yield 70 %) or Pe-g (yield 55 %).
Ca-g, white powder. Elemental analysis: Found: C, 72.1, H, 8.85, N, 3.3%. δH (300
MHz, CD3OD, ppm): 6.94-6.56 (3H, m, ArH), 5.48-5.30 (3H, m, -CH=CH-), 4.87
(2H, s, O-CH2-N), 4.00 (2H, s, Ar-CH2-N), 3.39 (2H, s, N-CH2-COONa), 2.9-2.75
(2H, m, CH2-(CH=)2), 2.53 (2H, t, benzylic CH2-), 2.15-2.0 (3H, m, -CH2-CH=),
1.7-1.25 (15H, m, -CH2-), 0.95 (2H, q, -CH3). δC (75MHz, CD3OD, ppm): 176.68
(N-CH2-C=O), 156.90, 153.73, 143.63, 142.23, 136.56, 129.85, 129.78, 129.53,
129.44, 129.26, 128.86, 127.87, 127.65, 127.31, 127.04, 126.39, 120.43, 118.42,
117.20, 115.73, 115.48, 113.62, 82.51 (O-CH2-N), 56.32, 55.19 (N-CH2-C=O), 51.08,
50.11 (Ar-CH2-N), 35.19, 31.49, 31.10, 31.06, 29.40, 29.36, 29.25, 29.00, 28.89,
28.82, 28.60, 26.72, 26.70, 26.67, 25.13, 25.03, 22.43, 22.27, 12.98, 12.68. FT-IR
(KBr, cm-1): 3010 (C-H of benzene ring), 2926 and 2853 (CH2 of oxazine ring as well
as alkyl side chain of cardanol), 1507 (C=C of benzene ring), 1410 (carboxyl group
stretching of the sodium carboxylate), 1245 and 1035 (C-O-C of oxazine ring), 934
(C-O-C stretching of oxazine ring and C-H vibration of aromatic ring).
Pe-g, white powder. δH (300 MHz, CD3OD, ppm): 6.94-6.56 (3H, m, ArH), 4.84 (2H,
s, O-CH2-N), 4.00 (2H, s, Ar-CH2-N), 3.37 (2H, s, N-CH2-COONa), 2.53 (2H, t,
benzylic CH2-), 1.4-1.25 (26 H, m, -CH2-), 0.91 (3H, t, -CH3). FT-IR (KBr, cm-1):
2925 and 2848 (CH2 of oxazine ring as well as alkyl side chain of
3-pentadecylphenol), 1507 (C=C of benzene ring), 1410 (carboxyl group stretching of
the sodium carboxylate), 1239 (C-O-C of oxazine ring), 933 (the benzene ring
attached to oxazine ring).
Preparation of styrene-water emulsions
To a 25 ml vial, the styrene (at an volume fraction of 50 %, 60 %, 70 %, 75 %, 80 %,
90 %) and aqueous solution of 1.7 (w/v)% Ca-g or Pe-g at pH = 12.0 were added.
Emulsions were obtained by homogenizing the mixture with a homogenizer (ANGNI,
18 mm head) operating at 10,000 rpm for 2 min at 25 °C. The pH values of emulsions
were adjusted by addition of HCl or NaOH solution to cover the pH range from 5.5 to
12.0.

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Preparation of triglceride emulsion
To a 25 ml vial, the capryli/capric triglceride at an volume fraction of 85 % and
aqueous solution of 1.7 (w/v)% Ca-g were added. A triglceride emulsion was obtained
by homogenizing the mixture with a homogenizer (ANGNI, 18 mm head) operating at
10,000 rpm for 2 min.
Preparation of PS particles
HIPEs containing 22.5 ml styrene, 7.5 ml aqueous solution of 1.7 (w/v)% Ca-g at pH
= 12.0, and 0.11 g AIBN were prepared using a homogenizer operating at 10,000 rpm
for 2 min. The prepared HIPEs were transferred into a 50 ml three-neck round flask
and stirred mechanically under nitrogen at 60 °C for 24 hours. After polymerization,
the resulting latex was diluted by adding water, and then poured into excess ethanol.
The product was obtained by filtration, and then dried in a vacuum oven to afford the
white powder 15.4 g (yield 75 %).
In vitro cytotoxicity of Ca-g
The in vitro cytotoxicity of Ca-g against Hela cell line was determined by the MTT
method. Hela cells were planted into 96-well culture plates at a density of 1×104 cells
per well and incubated for 24 hours at 37 °C. The cells were treated with different
concentrations of Ca-g for a specific time. Then 20 μl of MTT (5 mg/ml) solution was
added to each well and incubated for another 4 hours. After the cell culture medium
was removed, DMSO (200 μl) was added to each well. The absorbance at a
wavelength of 570 nm was measured. Results were presented as average ± standard
deviation.
Measurements
13
1H and C NMR spectra were recorded on a Avance-300 spectrometer (Bruker, US)
with CD3OD as deuterated solvent.
FT-IR spectroscopy was performed using a Tensor 27 FTIR spectrometer (Bruker, US)
with a resolution of 4 cm-1 in the absorbance mode. Samples were pressed into KBr
pellets for analysis.

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Surface tension of aqueous solutions of Ca-g or Pe-g at pH = 12.0 were measured on
JYW-200B (Dahua, China) automatic surface tension instrument by Du Noüy ring
method at 45 ± 0.1 °C.
The conductivity of emulsion was measured using a DDS-307 digital conductivity
meter (Leici, China). The conductivity of the emulsion above 10 μS cm-1 suggested
that it was an O/W emulsion. On the other hand, the conductivity of the emulsion
below 10 μS cm-1 suggested that it was a W/O emulsion.
Drop test was used to confirm the emulsion type suggested by the conductivity
measurement. An emulsion droplet was added to deionized water or oil. An O/W
emulsion droplet dispersed quickly in water and remained as a drop in oil. On the
other hand, a W/O emulsion droplet dispersed quickly in oil and remained as a drop in
water.
The droplet diameter of the emulsion was measured using a Zetasizer Nano ZS
instrument (Malvern, UK). The test for each emulsion was repeated at least three
times at 25 °C.
Optical microscope images of emulsions and the polystyrene microspheres were
recorded with a SMZ-168 optical microscope (Motic, China).
Confocal images of emulsions were viewed by a Fluoview 500 confocal microscopy
(Olympus, Japan). The styrene phase was marked with DBT fluorescent dye. When
excited by a 488 nm laser, DBT had a yellow emission indicating the styrene phase.
The surface morphology of the polystyrene microsphere was characterized with G300
FE-SEM (Carl Zeiss, UK). Before test, the sample was coated by a layer of gold.
Results and discussion
Synthesis and characterization of Ca-g
When commercial amino acids exist as the zwitterions, the amino groups are
protonated, reducing their nucleophilicity. It is very difficult to carry out Mannich
reaction for the preparation of benzoxazine monomer. The reported solutions in
literature include deprotonating the amino groups by converting amino acids into their

Page 7 of 22
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corresponding quaternary ammonium salts25 or esters.26,27 Our modifier solution is to
convert amino acid into its sodium salt using a stoichiometric amount of sodium
hydroxide. In this way, the abundant amino acids can be used as the amino source for
benzoxazine synthesis. In addition, the carboxylate group can act as the hydrophilic
part of the surfactant.
DOI: 10.1039/D0GC00218F
Cardanol is a mixture of naturally occurring phenolic sources. In fact, the
bio-based benzoxazine (abbreviated as Ca-g) surfactant derived from cardanol is also
a mixture. We synthesized a single benzoxazine (abbreviated as Pe-g) surfactant
derived from 3-pentadecylphenol and glycine as a model compound for comparison.
OH
O
N
COONa
reflux
+
COONa
2 HCHO
+
NH2
R
R
or
R =
single
(a)
mixed
(b)
Scheme 1 Synthetic route to mixed benzoxazine surfactant of Ca-g (a) and single
benzoxazine surfactant of Pe-g (b).
Scheme 1 illustrates the synthetic route to mixed benzoxazine surfactant of Ca-g
(a) and single benzoxazine surfactant of Pe-g (b). The glycine initially reacted with
sodium hydroxide to form its sodium salt. Subsequentially, the cardanol reacted with
sodium glycinate and paraformaldehyde at a mole ratio of 1: 1: 2 to produce Ca-g.
The reaction system was homogeneous at early stage. As the reaction continued, the
powdered product constantly precipitated from solution. After the reaction finished,
the Ca-g was isolated by filtration.

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Fig. 1 1H NMR spectrum of Ca-g.
1
Fig. 1 shows the H NMR spectrum of Ca-g. The two resonances centered at
4.87 ppm and 4.00 ppm are attributed to the protons of O-CH2-N (a) and Ar-CH2-N
(b) in the benzoxazine ring structure, respectively. The resonance at 3.39 ppm is
assigned to the protons of the N-CH2-COONa (c). The resonance signal at 2.52 ppm
is assigned to the protons of the Ar-CH2- (g) in long alkyl side chain. In addition, the
relative integration ratios of those resonance signals at δ 4.00, 3.39, and 2.52 ppm are
2.04: 2.02: 2.04, respectively. These agree well with the theoretical value of 2.00:
2.00: 2.00.
Fig. 2 13C NMR spectrum of Ca-g.
13
Fig. 2 shows the C NMR spectrum of Ca-g. The two resonances at 82.51 ppm
and 50.11 ppm are assigned to the methylene carbon resonances of O-CH2-N (a) and

Page 9 of 22
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Ar-CH2-N(b), respectively. The resonances at 176.68 ppm and 55.19 ppm are
assigned to the carbonyl carbon resonance of N-CH2-C=O (n) and methylene carbon
resonance of N-CH2-C=O (c), respectively.
Fig. 3 FT-IR spectrum of Ca-g.
Fig. 3 shows the FT-IR spectrum of Ca-g. The peak at 934 cm-1 corresponds to
the C-O-C stretching of oxazine ring and C-H vibration of aromatic ring.28 Besides,
the peak at 1410 cm-1 assigns to the symmetric stretching of carboxyl group.29
In addition, Pe-g was also characterized by1H NMR and FT-IR (see Fig. S1 and
Fig. S2).
Emulsifying properties of Ca-g and Pe-g
Fig. 4 shows surface tensions at different concentrations of Ca-g and Pe-g. As
the concentration of Ca-g increases, the surface tension drops sharply. When the
concentration is 0.0035 mol/L, the surface tension reaches a minimum value (γcmc) of
36.9 mN/m. Further increasing the concentration of Ca-g, the surface tension is
almost unchanged. Therefore, the critical micelle concentration (cmc) of Ca-g is
0.0035 mol/L. Similarly, the cmc and γcmc of Pe-g are 0.004 mol/L and 37.8 mN/m,
respectively.

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Fig. 4 Surface tensions at different concentrations of Ca-g and Pe-g at 45 °C and
pH= 12.0.
The effectiveness of the surface tension reduction (Пcmc), the maximum surface
excess concentration (Γmax), and minimum area occupied by one surfactant molecule
adsorbed at the air/water interface (Amin) of Ca-g and Pe-g surfactants are listed in
Table 1. The cmc, γcmc, and Пcmc values of Ca-g are closed to Pe-g. It suggests that the
surface activity of Ca-g surfactant is comparable to Pe-g surfactant. For the absorption
of Ca-g and Pe-g surfactants at the air/water interface, Ca-g has a larger Amin value
compared with Pe-g. This indicates that the Ca-g molecules have a lower packing
density than that of Pe-g at the air/water interface.
Table 1 Surface properties of benzoxazine surfactants Ca-g and Pe-g at 45 °C and
pH= 12.0.
a
b
c
Surfactants
cmc
(mol/L)
0.0035
0.004
γcmc
(mN/m)
36.9
Пcmc
Γmax
Amin
(mN/m)
31.8
(μmol/m2)
2.13
(Å2)
77.9
72.8
Ca-g
Pe-g
37.8
30.9
2.28
aПcmc = γ0 －γcmc, where γ0 was the surface tension of pure water and γcmc was the
1
dγ
surface tension at cmc. bΓmax
=
, where γ, C, R, and T represented the surface
)
(
nRT d lnC

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tension, surfactant concentration, gas constant, and absolute temperature, respectively.
1016
c
The value of n was taken as 1 in our system. Amin
=
, where NA respected the
NAΓmax
Avogadro’s number.
Fig. 5 shows photographs of emulsions with different styrene volume fractions
stabilized by Ca-g (a) and Pe-g (b) after standing for 24 hours. When the styrene
volume fraction is 50 %, the freshly prepared emulsions stabilized by Ca-g and Pe-g
undergo obvious creaming after one day of storage. These systems separate into a
cream layer in the top and a serum layer in the bottom. The mean diameters of styrene
droplets are 5.7 μm (see Table 2). Therefore, the Brownian diffusion kT of those
large-sized styrene droplets is less than the force of gravity (mass × acceleration due
to gravity g) 30,31
:
kT < (4/3) πR3Δρgh
(1)
where k, T, R, Δρ, and h represent the Boltzmann constant, the absolute temperature,
the droplet radius, the density difference between styrene droplets and water phase,
and the height of the container, respectively. In this case, the styrene droplets move to
the top due to their lower density compared with the continuous water phase, leading
to creaming of emulsions. The cream layer volume increases with increasing styrene
volume fraction from 50 % to 70 %. This is because that the apparent viscosity of the
emulsion increases with increasing styrene volume fraction due to the interdroplet
interactions and crowding effects.32 This hinders the upward movement of styrene
droplets, reducing the occurrence of creaming phenomenon.33,34

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Fig. 5 Photographs of styrene-in-water emulsions with different styrene volume
fractions stabilized by Ca-g (a) and Pe-g (b) after standing for 24 hours at room
temperature and pH = 12.0. (c), (d), and (e) Confocal images of the emulsions
stabilized by Ca-g having 50, 75, and 90 % styrene volume fraction, respectively
(Ca-g and Pe-g concentration: 0.04 mol/L).
These emulsions are O/W type determined by conductivity measurements and
drop tests. Confocal microscope images (see Fig. 5c) reveal that the yellow styrene
droplets disperse in a continuous water medium (black), further confirming the
formation of the O/W emulsions. According to Davies’ method, we calculate the
hydrophilic-lipophilic balance (HLB) value of Ca-g surfactant. HLB = 7 + Σ H + Σ L
= 7 + H-COONa × N-COONa + Htertiary amine × Ntertiary amine + H-O- × N-O- + Laromatic ring
Naromatic ring + Laliphatic -CH3, -CH2, -CH, = CH2, and = CH × Naliphatic -CH3, -CH2, -CH, = CH2, and = CH
×
=
26.6, where H, L, and N represent the contribution of each hydrophilic group, the
contribution of each lipophilic group, and numbers of each hydrophilic group or
lipophilic group, respectively. This high HLB value of 26.6 indicates that Ca-g

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surfactant has a stronger partitioning tendency in water compared with oil phase.
The high hydrophilicity is beneficial to stabilize O/W type emulsion.
When the styrene volume fraction is 75 %, the entire mixture forms a high
internal phase emulsion (HIPE). As the styrene volume fraction increases, the
viscosity of HIPE increases. When the styrene volume fraction reaches 90 %, the
HIPE does not show any fluidity in an inverted vial. The confocal microscopy image
(see Fig. 5e) shows that the yellow styrene droplets exhibit a polygonal shape, which
is the typical structure for a HIPE.
The characterization data of these emulsions are listed in Table 2. The mean
diameter of the emulsion droplet stabilized by Ca-g is close to that of the emulsion
stabilized by Pe-g, indicating the comparable emulsifying capacity.
Table 2 Mean diametera of emulsion droplets stabilized by Ca-g and Pe-g at different
styrene volume fractions.
Styrene volume
fraction
Mean diameter (μm)
Ca-g
Pe-g
50 %
60 %
70 %
75 %
80 %
90 %
5.7±2.5
6.0±2.4
6.4±2.3
6.7±2.1
7.1±2.6
7.6±3.0
5.2±2.8
5.7±2.3
6.2±2.6
6.4±2.5
6.8±2.2
7.4±3.5
a Measured with a Malvern Zetasizer instrument.
It should be noted that the low molecular weight surfactants usually need to be
added large amounts of 5-50 (w/v)% for the stabilization of HIPEs.37 The bio-based
Ca-g surfactant shows high efficiency for the stabilization of HIPEs. A low
concentration of 1.7 (w/v)% (i.e., 0.04mol/L) is enough to stabilize the
styrene-in-water HIPE with an internal phase fraction up to 90 %.

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Since the carboxylic acid group is pH-sensitive, we further explored the
pH-sensitivity of the emulsions. Fig. 6 shows photographs of styrene-in-water HIPEs
stabilized by Ca-g and Pe-g at different pH values after standing for 2 hours.
Fig. 6 Photographs of styrene-in-water HIPEs stabilized by Ca-g (a from visual
observation, b from optical microscope) and Pe-g (c from visual observation, d from
optical microscope) at different pH values after standing for 2 hours at room
temperature (Ca-g and Pe-g concentration: 1.7 (w/v)%, styrene volume fraction:
75 %).
When the pH value decreases from 12.0 to 9.0, digital photographs depict that
the HIPEs remain stable. Optical microscopy images of HIPEs reveal spherical and
dispersed styrene droplets (see Fig. 6b, d). Although the appearance of the emulsion
has not changed obviously, the diameter of styrene droplets gradually increases with
decreasing the pH value (see Table 3). Since pK1 (the dissociation constant of
carboxyl group) and pK2 (the dissociation constant of protonated amino group)38 of
Ca-g are 2.4 and 7.8 respectively, the carboxyl groups are completely negatively
charged in the pH range of 9.0 - 12.0. As the pH decreases gradually from 12.0 to 9.0,
a small amount of tertiary amino groups are protonated gradually. We think that the
positively charged tertiary amino groups interact with the negatively charged carboxyl
groups of neighbor molecules, leading to a reduced emulsifying capacity of Ca-g and
an increase in the diameter of styrene droplets.

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a
Table 3 Mean diameter of emulsion droplets stabilized by Ca-g and Pe-g at different
pH values.
Mean diameter (μm)
pH value
Ca-g
Pe-g
12.0
11.0
10.0
9.0
6.7±2.1
6.8±2.7
7.0±3.0
7.6±3.4
8.0±5.7
6.4±2.5
7.0±2.7
7.3±3.0
8.2±4.2
8.0
a Measured with a Malvern Zetasizer instrument.
When the pH value decreases to 8.0, digital photographs show the separated
yellow styrene layer containing DBT on top of the HIPE. The demulsification
phenomenon on the macroscopic level begins to occur. When the pH value decreases
to 5.5, the complete demulsification occurs. This is because that the tertiary amino
groups of Ca-g are almost completely positively charged as pH value of emulsion is
close to the isoelectric point (pI = 5.1) of Ca-g. The emulsifying capacity of Ca-g is
insufficient to stabilize styrene droplets and thus results in the demulsification of the
HIPE. The pH sensitivity of HIPEs stabilized by Ca-g is similar to the HIPE stabilized
by Pe-g.
Fabrication of PS latex
We prepared the HIPE with 75 vol% styrene using AIBN as initiator at pH =
12.0. HIPE polymerization was carried out at 60 °C for 24 hours. Figure 7 shows the
photographs of PS latex before and after demulsification. After polymerization, the
latex becomes milky white (see Fig. 7a). It was visualized by an optical microscope
(see Fig. 7b). The image shows that PS particles exhibit a spherical shape. It is
consistent with the spherical shape of styrene droplets in HIPE with 75 vol% styrene
at pH = 12.0.

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Fig. 7 Photographs of PS latex before (a from visual observation, b from optical
microscope) and after demulsification (c from visual observation, d from scanning
electronic microscope).
The latex was diluted by adding a small amount of water and then poured into
ethanol for demulsification. The precipitate was isolated by filtration (see Fig. 7c).
The surface morphology of the PS particles was determined using a scanning
electronic microscope. The image reveals spherical and polydisperse PS particles (see
Fig. 7d). The average particle size of PS particle was 5.3±3.1 μm (polydispersity
index: 0.26), which slightly smaller than the mean diameter of styrene droplets (6.7±
2.1 μm; polydispersity index: 0.20) in HIPEs.
Fabrication of triglceride emulsion
We used the MTT method to assay the cytotoxycity of Ca-g against the Hela cell
line. Figure 8 shows the in vitro cytotoxicity of Ca-g against the Hela cell line. Cell
activity decreases with increasing the concentration of Ca-g. The half maximal
inhibitory concentration (IC50) of Ca-g is 105 μg/ml. This concentration is larger than
the IC50 of SDS (31.40 μg/ml),39 which is commonly used in shampoo, toothpaste and
cosmetics. It suggests that Ca-g has lower cytotoxicity than the traditional

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petroleum-based surfactant SDS. This result is consistent with that of other reported
amino acid-based surfactants.39-41
Fig. 8 In vitro cytotoxicity of Ca-g against the Hela cell line. Data are presented as the
average ± standard deviation of three independent experiments (n = 5).
According to cosmetic ingredients, we further prepared a triglceride emulsion
using Ca-g as the surfactant. The caprylic/capric triglceride, an ingredient commonly
used in cosmetics, was selected as the oil phase. An O/W type HIPE with 85 vol%
triglceride was produced by dispersion through a high speed homogenizer (see Fig.
9a). The HIPE does not exhibit any fluidity. Optical microscope image (see Fig. 9b)
shows that the oil droplets of triglceride in HIPE have a polygonal shape. This
emulsion has good centrifugal stability and storage stability. No demulsification
occurs after centrifugation for 30 min at 360*g. This emulsion does not show any
change in appearance after room temperature storage for 7 months.

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Fig. 9 Photograph of triglceride-in-water HIPE stabilized by 1.7 (w/v)% Ca-g after
standing for 24 hours (a from visual observation, b from optical microscope).
Conclusions
In summary, through converting amino acids to their sodium salts, a bio-based
benzoxazine mixed surfactant of Ca-g has been successfully prepared using renewable
glycine and cardanol as starting materials.
The critical micelle concentration (cmc) of mixed surfactant of Ca-g was 0.0035
mol/L. By comparing the values of cmc, γcmc, Пcmc, Γmax, and Amin, the surface activity
of mixed surfactant of Ca-g was comparable to single surfactant of Pe-g. The Ca-g
molecules had a lower packing density than that of Pe-g at the air/water interface.
The Ca-g surfactant exhibited high emulsifying efficiency, and a low
concentration of 1.7 (w/v) % was enough to stabilize the O/W type HIPE with styrene
volume fraction up to 90 %. The HIPEs showed pH sensitivity and were readily
prepared at pH 9.0-12.0.
The bio-based Ca-g surfactant can be used to carry on emulsion polymerization
and prepare polystyrene particles. Additionally, the amino acid-based surfactant Ca-g
shows lower cytotoxicity against the Hela cell line. The bio-based Ca-g surfactant
may have potential use in the fields of daily cleaning products and cosmetics.
Conflicts of interest
There are no conflicts to declare.

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Acknowledgements
We would like to thank the Natural Science Foundation of Shandong Province
(ZR2018MB022), the National Nature Science Foundation of China (51973104), and
the Science and Technology Plan of Jinan city (201602102) for their financial
support.
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A bio-based benzoxazine surfactant is synthesized using amino acids as amine source,
which may have potential use in cosmetics and cleaning products.