Hemin associated to cetyltrimethylammonium broide micelles: A biomimetic catalyst for 2,4,6-trichlorophenol degradation

Article abstract of DOI:10.1007/s11426-014-5238-0

For the first time, an efficient, green, economical biomimetic catalyst (hemin-cetyltrimethylammonium bromide micelles) was discovered to degrade 2,4,6-trichlorophenol (TCP). The degradation experiments indicate that pH, temperature, the addition of 2-methylimidazole, and the amount of hydrogen peroxide influence the degradation process. Test of reusability revealed that CTAB micelles can protect hemin from destruction by H₂O₂ and that the materials can be recycled. This material can be of great use for waste-water treatment.

Full text of DOI:10.1007/s11426-014-5238-0

SCIENCE CHINA
Chemistry
January 2015 Vol.58 No.1: 1–7
doi: 10.1007/s11426-014-5238-0
• ARTICLES •
doi: 10.1007/s11426-014-5238-0
Hemin associated to cetyltrimethylammonium broide micelles: a
biomimetic catalyst for 2,4,6-trichlorophenol degradation
Lihui Zhang1,2, Cheng Gu1*, Ran Hong1 & Haiping Zhang1
1State Key Laboratory of Pollution Control & Resource Reuse; School of the Environment, Nanjing University, Nanjing 210023, China
2School of Chemistry and Chemical Engineering, Pingdingshan University, Pingdingshan 467000, China
Received August 29, 2014; accepted October 15, 2014
For the first time, an efficient, green, economical biomimetic catalyst (hemin-cetyltrimethylammonium bromide micelles) was
discovered to degrade 2,4,6-trichlorophenol (TCP). The degradation experiments indicate that pH, temperature, the addition of
2-methylimidazole, and the amount of hydrogen peroxide influence the degradation process. Test of reusability revealed that
CTAB micelles can protect hemin from destruction by H2O2 and that the materials can be recycled. This material can be of
great use for waste-water treatment.
hemin-CTAB micelles, biomimetic catalyst, degrade, TCP, recycle
environmentally friendly. Second, FPs can be conducted
under mild conditions [12–14].
1 Introduction
H2O2 does not oxidize TCP in the absence of catalysts,
however. Previous studies have shown that, using hydrogen
peroxide as the oxidant, horseradish peroxidase (HRP)
[15–17], the extracellular lignin peroxidases of phanero-
chaete chrysosporium [18], myoglobin (Mb) under condi-
tions of oxidative stress [19], and chloroperoxidase (CPO)
from caldariomyces fumago [20,21] could mediate the oxi-
dation of chlorinated phenols. The iron complexes of por-
phyrins (e.g. hemin) are known to form the active sites of a
variety of systems such as HRP, Mb and CPO. Studies have
shown that hemin can be used to mimic the functions of
natural enzyme [22–33]. However, a hemin molecule in the
absence of a protein matrix easily undergoes dimerization in
aqueous solution, which has limited its utility for the deg-
radation of contaminants in waste-water.
Polychlorinated phenols such as 2,4,6-trichlorophenol (TCP)
have been widely used as herbicides, fungicides, pesticides,
insecticides, pharmaceuticals and dyes. Due to their high
toxicity, carcinogenic properties, and persistence in the en-
vironment, they have been listed as a group of priority pol-
lutants by the US Environmental Protection Agency [1].
Therefore, an efficient chemical treatment process is needed
in order to effectively degrade chlorophenols. Recently, an
advanced oxidation process (AOP) has been developed.
During AOP, highly reactive hydroxyl radicals are produced
that can oxidize most of the organic pollutants; therefore,
AOP technique has been widely used for degradation of
many persistent contaminants [2–5]. As one of the AOPs,
the Fenton process (FP) has been used in the degradation of
phenols and chlorophenols [6–9]. Compared to other AOPs,
FP has several advantages. First, H2O2 is used as the stoi-
chiometric oxidant, which gives water as the only
by-product [10,11]. The degradation process is therefore
To stabilize the monomeric form of hemin, it is immobi-
lized on various substrates such as methacrylamide-ethylene
glycol dimethacrylate copolymer [34], ß-cyclodextrin [35],
and the surface of a glassy carbon electrode to degrade TCP
[36]; 2,6-dichlorobenzo-quinone (DCQ) is the main product
of these approaches (Scheme 1). However, these immobili-
*Corresponding author (email: chenggu@nju.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2015
chem.scichina.com link.springer.com

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Zhang et al. Sci China Chem January (2015) Vol.58 No.1
tion of amino acid residues. In addition, we conducted a
recycling experiment to test the stability of this new materi-
al.
Scheme 1 Fenton oxidation of TCP to form quinone product by a hemin-
H2O2 system.
2 Experimental
2.1 Chemicals
zation processes are generally so complicated as to increase
the degree of difficulty in practical application. Moreover,
during these immobilization processes, only a small part of
the hemin could be adsorbed to the substrate. It is therefore
necessary to develop an efficient, environmentally friendly,
and economical biomimetic catalyst [26].
Hemin, CTAB, imidazole (Im), 2-methylimidazole (2-
MeIm), 4-methylimidazole (4-MeIm), histamine, and TCP
were purchased from Sigma-Aldrich (USA). Methanol
(MeOH), acetonitrile (CH3CN), and acetic acid (HAC) were
of HPLC grade and purchased from Tedia (USA). C6H5Na3-
O7·2H2O, Na2HPO4·7H2O, NaH2PO4·2H2O, CH3COO-
Na·3H2O, Na2B4O7·10H2O, sodium hydroxide, boric acid,
citric acid, dimethyl sulfoxide (DMSO), and H2O2 (30%)
were of all analytical grade and obtained from Nanjing
Chemical Corporation (China). All of the chemicals were
used as received.
Hemin stock solution (5 mmol/L) was prepared by dis-
solving hemin in 1 mL of DMSO and shaking until fully
dissolved. TCP stock solution (5 mmol/L) was prepared in
methanol. Im, 2-MeIm, 4-MeIm, and histamine were dis-
solved in methanol to prepare 5 mmol/L of solution. H2O2
(3%) was obtained by diluting H2O2 (30%) by ultrapure
water. Various pH buffers were made from the correspond-
ing salts, acids, and bases. Sodium citrate-sodium phosphate
was used for pH 3.0, sodium acetate buffer for pH 4.0–5.0,
and sodium phosphate buffer for pH 6.0–7.0.
The surfactant molecule is usually composed of two parts:
a long-chain hydrocarbon “tail” and a polar “head”. The two
polar head groups undergo a repulsive electrostatic interac-
tion to form micelles above a well-defined threshold con-
centration (i.e. the critical micelle concentration, CMC). At
CMC, the long-chain hydrocarbon tails meet at a common
center space and the charged polar groups form a mul-
ticharged surface. Micelles, which are usually spherical in
shape, can be cationic, anionic, or nonionic. Cetyltrimethyl-
ammonium bromide (CTAB) is one kind of cationic surfac-
tant (Sheme 2). Generally, the CMC of CTAB is 0.0009
mol/L in water, and the aggregation number of one spheri-
cal micelle is 62 at 25 °C. In a solution containing surfac-
tants, an organic molecule is usually located in the hydro-
phobic center of the micelles and could increase the con-
centration of dissolved organic matter in the solution
[37–43]. For example, the insoluble porphyrins are highly
soluble in water in the presence of detergents such as CTAB
(Figure 1).
As we know, the catalytic efficiency of hemin is low in
comparison with natural peroxidase enzyme due to the lack
of hydrophobic polypeptide and amino acid residues such as
histidine-170 and histidine-42 [44–51]. The imidazole group
in these two forms of histidine is generally considered to be
the essential functional group in the catalytic oxidation pro-
cess of HRP. The objective of this study was to form a
hemin-CTAB complex to stabilize the monomeric form of
hemin and utilize this composite as the catalyst to degrade
TCP. Some imidazole bases were used to mimic the func-
2.2 Preparation of CTAB micelles and hemin-CTAB
micelles
When the concentration is below its CMC, CTAB mole-
cules exist as the monomer in aqueous solution [52–56].
When the initial CTAB concentration is greater than its
CMC [57–59], CTAB micelles form in the solution. In our
study, 24 mg of CTAB was added to 30 mL of acetic buffer
(pH 4) to obtain CTAB micelles (the concentration of
CTAB is 2.2 mmol/L, larger than its CMC of 0.9 mmol/L).
Then, 180 μL of hemin (5 mmol/L) was added into the solu-
tion of CTAB micelles until the concentration of hemin was
30 μmol/L. The hemin-CTAB micelles were used as the
catalyst for the degradation of TCP by H2O2.
Scheme 2 Chemical structure of CTAB.
2.3 Batch degradation assays
The concentrations of TCP during the degradation process
were monitored by a high-performance liquid chromatog-
raphy (HPLC, Waters Alliance, USA) instrument equipped
with a Symmetry C18 column. The mobile phase was com-
posed of 15% acidified water (0.5% acetic acid) and ace-
tonitrile (15:85, v/v) at a flow rate of 1.0 mL/min. The UV
Figure 1 The formation of monomeric hemin intercalated in CTAB
micelles.

Zhang et al. Sci China Chem January (2015) Vol.58 No.1
3
detector was operated at a wavelength of 220 nm; the injec-
tion volume was 20 L. The column temperature was set at
30 °C. The total analysis time was 5 min; the elution time of
the TCP was 3.9 min. The standard curve of the TCP con-
centrations is provided in Figure S1 (see Supporting Infor-
mation online). Ratio of H2O2/TCP, reaction pH, molar ratio
of substrate-to-catalyst, effect of imidazole structure, and
temperature were optimized for the degradation of TCP.
Last, the degradation kinetic of TCP was carried out in
these optimum conditions and the results were compared to
those in ordinary conditions.
2.4 Test of reusability
The test for reusability of hemin-CTAB-micelle catalyst
was conducted under the optimized conditions described
above. The initial concentration of TCP was 90 μmol/L and
the radio of hydrogen peroxide to the substrate was 5:1.
This test revealed that most of the TCP was degraded in 2 h.
Therefore, we calculated the degradation efficiency by the
remaining concentration of TCP determined by HPLC after
2 h. Next, the amount of TCP was replenished to maintain
the initial concentration and the same amount of H2O2 was
added to initiate the reaction. After another 2 h, the degra-
dation efficiency was obtained for the second cycle, which
allowed us to calculate the reusability of the materials.
Figure 2 UV-Vis spectra of 30 μmol/L hemin in CTAB micelles in ace-
tate buffer (pH 4).
3.2 Optimization of the reaction conditions
3.2.1 The ratio of H2O2/TCP
The degradation of TCP in different H2O2/TCP ratios is
presented in Figure 3. It may be observed that doses of
H2O2 significantly influenced the degradation of TCP. At
the low ratio of H2O2/TCP (1:1), more than 50% of the ini-
tial TCP was not degraded. When we increased the ratio of
H2O2/TCP to 2:1, about 93% of the TCP was degraded. At a
5:1 ratio of H2O2/TCP, about 97% of the TCP was degraded.
When we continued to increase the ratio of H2O2/TCP to
10:1 and 15:1, there was no obvious change in the degrada-
tion of TCP. However, we must consider the destruction of
the catalyst-hemin in the presence of large amounts of H2O2
in the solution. To achieve the efficient removal of TCP and
also recycle the catalyst, we set the optimum ratio of
H2O2/TCP at 5:1.
3 Results and discussion
3.1 UV-Vis scan of the hemin-CTAB micelles
Metal porphyrins are not soluble in neutral and acidic
aqueous solution. They also have a marked tendency to ag-
gregation to dimers and oligomers in alkaline solution,
which has little or no activity. However, they are highly
solubilized as the active monomer in the presence of deter-
gents such CTAB (see Introduction). The UV-Vis absorp-
tion of hemin in CTAB micelles solution has a Soret peak at
396 nm, which indicates that monomer is the predominant
form (Figure 2). The molar extinction coefficient for the
hemin-CTAB micelles system is 0.42×105 L/(mol cm).
The color of the hemin-CTAB micelles solution is red-
dish. Simplicio et al. [41] discovered that intercalated hemin
is associated with an acid-base equilibrium between a red
diaquo form (M(H2O)2·Micelles) and a green monoaquom-
onohydroxy form (Me(OH)·Micelles):
3.2.2 pH dependence
The degradation of TCP by Fenton reaction is greatly in-
fluenced by the pH of the system. In the literature, many
researchers studied the Fenton reaction for treating waste-
water and concluded that the optimal pH range of the Fen-
ton process was between 2 and 4 [60–63]. Figure 4 shows
the pH dependence of TCP oxidation by hemin-CTAB mi-
celles solution. At pH 3–5, higher degradation of TCP was
observed (>70%). However, there was little degradation at
pH 6–7. At pH 4, almost all of the TCP could be degraded.
Given these results above, the remainder of our experiment
was carried out at pH 4.
M(H2O2 )Micelles  Me(OH)Micelles+H
3.2.3 The substrate-to-catalyst molar ratio
The pKa of this equilibrium is 5.5; the green (Me (OH)·
Micelles) changes to a reddish M(H2O)2·Micelles below
pH 5 [41]. Therefore, at pH 4, hemin mainly takes a red
diaquo form (M(H2O)2·Micelles).
The substrate-to-catalyst (S/C) molar ratio usually reveals
catalytic efficiency. It is a great challenge to decrease the
catalyst amount to obtain a high catalytic efficiency. The
degradation of hemin-CTAB micelles toward TCP oxida-

4
Zhang et al. Sci China Chem January (2015) Vol.58 No.1
tion at different S/C molar ratios is listed in Table 1. It
could be concluded that TCP could be fully degraded by
hemin-CTAB micelles solution at the S/C ratio of 3–14.
The monomeric CTAB molecules could form micelles
through a self-assembly process as the concentration reach-
es its CMC. Then, the CTAB micelles would encapsulate
the catalyst-hemin in the center and isolate it from each
other to retain the monomeric form. However, because there
is interspace in the CTAB micelles, the H2O2 could disperse
into the hydrophobic region of the micelles to react with
hemin to form a ferryl porphyrin radical cation species
(Compound I). The substrate “TCP” in the micelles would
then be degraded in the interspace. Such a reaction is ho-
mogenous, which is quite different from the hemin immobi-
lized on the substrate. However, the CTAB-micelles could
act as the hydrophobic center to protect the hemin from de-
struction by H2O2 and radical-induced damage, and could
concentrate the substrate around the catalyst to facilitate the
reaction.
3.2.4 Effect of imidazole bases
The distal histidine (His-42) of HRP can promote the for-
mation of Compound I and plays an important role in pe-
roxidase catalysis [64–66]. Basically, it is the imidazole
group in histidine that is generally considered to be as the
essential functional group in the catalytic oxidation process
of HRP [44–51]. We therefore investigated the effects of
different imidazole bases on the activity of hemin-CTAB
micelles (Figure S2). The activity for hemin-CTAB micelles
without the addition of imidazole base was defined as 100%.
At pH 4, the reaction was enhanced by the presence of im-
idazole bases at the low ratio of imidazole bases/hemin
(1:1). For the most effective imidazole base 2-MeIm, the
relative activity could increase to 150%. Similar results
have appeared in the literature. Newmyer et al. [51] com-
pared the effectiveness of different imidazoles in stimulat-
ing catalysis of an H42A mutant of HRP. They concluded
that 2-substituted imidazoles such as 2-MeIm were the most
effective imidazoles because the 2-substituted imidazoles
did not coordinate to the iron. Im and 1-MeIm, which both
coordinated to the heme iron atom, were found to be less
effective. Therefore, in the literature, 2-substituted imidaz-
oles are generally considered to be the sterically hindered
imidazole bases [67]. Similarly, Uno et al. [68] have dis-
covered that at acidic pH, the bis-coordination of the imid-
azole base to the iron atom of hemin is enhanced, and that
the unhindered bases are less effective activators. Sterically
hindered 2-MeIm, which could effectively lower the for-
mation of bis-ligating hemin, resulted in higher activity.
Therefore, due to the stereo-hindrance effect, 2-MeIm can
effectively inhibit the bis-ligating hemin and has the best
effect on the degradation of TCP.
Figure 3 The ratio of H2O2/TCP on the degradation of TCP. Reaction
conditions: 30 μmol/L hemin-CTAB micelles, 100 μmol/L TCP at H2O2/
TCP ratios of 1:1, 2:1, 5:1, 10:1, and 15:1.
Figure 4 pH dependence for degradation of TCP by hemin-CTAB mi-
celles solution. Reaction conditions: 30 μmol/L hemin-CTAB micelles,
100 μmol/L TCP, 5:1 H2O2/TCP ratio.
We changed the ratio of 2-MeIm/hemin to find the opti-
mum ratio for the degradation of TCP (Figure 5). At the low
ratio of 2-MeIm/hemin (1:1), the relative activity could in-
crease to 150%. However, as more 2-MeIm was used
(2-MeIm/hemin ratios of 2:1, 3:1, and 4:1), the relative ac-
tivity decreased to about 110%. This result was probably
because that part of the excessive imidazole bases coordi-
nated with iron atoms to form bis-ligating hemin, even in
the sterically hindered imidazole bases “2-MeIm”. As a
result, both the fifth and sixth sites were occupied by
2-MeIm, and there was no blank site for H2O2 to oxidize the
Table 1 Hydrogen peroxide oxidation of TCP catalyzed by hemin-CTAB
micelles
Run
1
Catalyst
Hemin
Hemin
Hemin
Hemin
Hemin
C/S (mol%)
pH
4
Conversion (%)
3
4
100
98.9
98.8
98.0
97.0
2
4
3
6
4
4
8
4
5
14
4
a) Reaction conditions: 30 μmol/L hemin in the CTAB micelles, and
the ratio of H2O2:TCP is 5:1.

Zhang et al. Sci China Chem January (2015) Vol.58 No.1
5
of 60 °C). The degradation kinetic results of TCP are shown
in Figure 7. Without 2-MeIm at room temperature, the deg-
radation curve was nearly linear over a total degradation
time of 12 h (Figure 7(a)). However, with the addition of
2-MeIm at 60 °C, 90% of the TCP was degraded in 2 h
(Figure 7(b)). We can thus optimize conditions and obtain a
high degradation rate for TCP, which may have applications
in waste-water treatment. Today, the degradation of poly-
chlorinated phenols is carried out by photocatalytic method
and electrochemical method. Compared to the Fenton reac-
tion, these two methods have higher mineralization ratios
and faster degradation rates [69,70]. However, the Fenton
reaction is comparably simple and does not require either
without the visible-light irradiation or the preparation of
electrodes.
Figure 5 The ratio of 2-MeIm/hemin for degradation of TCP by hemin-
CTAB micelles (comparing the degradation percentage of TCP in the first
30 min). Reaction conditions: 30 μmol/L hemin-CTAB micelles, 230
μmol/L TCP, 5:1 H2O2/TCP ratio.
3.3 Test of reusability
In the successive TCP oxidations with hemin-CTAB
ferric hemin(III) to form Compound I to degrade the TCP.
Therefore, in the following experiment, we set the ratio of
2-MeIm/hemin to 1:1.
3.2.5 Effect of temperature
The effect of temperature on the degradation rate of TCP
was studied at 25, 40 and 60 °C (Figure 6). With the in-
crease of temperature, the degradation percentage of the
TCP increased. The activity for hemin-CTAB micelles at
25 °C was defined as 100%. The relative activity was re-
spectively enhanced to 160% at 40 °C and 300% at 60 °C.
Because high temperature results in high-energy collisions
among the substrate, catalyst, and H2O2, and also increases
the substrate degradation. We could increase the removal
rate of TCP by elevating the reaction temperature. However,
the enzyme-catalyzed reaction rate initially rises as the
temperature increases. The rate is then negatively affected
at the higher temperatures; most of the enzymes will be de-
activated at 40 °C. In addition, the enzyme will be stored at
4 °C or lower. These, harsh conditions for the storage and
usage of the enzyme as well as the high price of the enzyme
have greatly limited its use in practical application. Howev-
er, the newly formed hemin-CTAB micelles show better
thermostability at high temperatures. Moreover, hemin-
CTAB micelles show extremely better activity (three fold)
for the degradation of TCP at high temperatures than at
room temperature. For all of these reasons, the easily ob-
tained hemin-CTAB-micelles catalyst is promising for the
treatment of waste water containing TCP, instead of using
HRP and other enzymes.
Figure 6 Effects of temperature on the relative activity of hemin-CTAB
micelles as indicated by the removal of TCP (comparing the degradation
percentage of TCP in the first 30 min). Reaction conditions: 30 μmol/L
hemin-CTAB micelles, 250 μmol/L TCP, 5:1 H2O2/TCP ratio.
Above all, the degradation experiments indicate that pH,
temperature, the addition of 2-MeIm and the amount of hy-
drogen peroxide influence the degradation process. We car-
ried out two reactions at the optimum ratio of H2O2/TCP
(5:1) in an acetate buffer (pH 4), but only one was success-
ful (with the addition of 2-MeIm and at a high temperature
Figure 7 The degradation kinetic of TCP. Reaction conditions: (a) 30
μmol/L hemin-CTAB micelles, 5:1 H2O2/TCP ratio, temperature 25 °C; (b)
30 μmol/L hemin-CTAB micelles, 5:1 H2O2/TCP ratio, 30 μmol/L 2-MeIm,
temperature 60 °C.

6
Zhang et al. Sci China Chem January (2015) Vol.58 No.1
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cycle was 93% (100% for the first run), which confirmed
that CTAB micelles can protect hemin from destruction by
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Supporting information
The supporting information is available online at chem.scichina.com and
link.springer.com/journal/11426. The supporting materials are published as
submitted, without typesetting or editing. The responsibility for scientific
accuracy and content remains entirely with the authors.
This work was financially supported by the National Natural Science
Foundation of China (21222704, 21237002), the National Key Basic Re-
search Program of China (2014CB441102), and the Collaborative Innova-
tion Center for Regional Environmental Quality. We thank the Analytical
Center and High Performance Computing Center of Nanjing University for
the characterization of the samples and for computational study.
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