Synthesis of a phenylboronic acid-functionalized thermosensitive block copolymer and its application in separation and purification of vicinal-diol-containing compounds

Article abstract of DOI:10.1039/c6ra16327k

A phenylboronic acid-functionalized amphiphilic thermosensitive block copolymer was prepared by taking PEO₂₀PPO₆₀PEO₂₀ as the template and a detailed study of its performance in downstream separation processes was presented. Alizarin red was used as the model compound to study the feasibility of using an affinity adsorption system and aqueous two-phase flotation system based on the phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀ for the separation and purification of ortho-hydroxyl compounds. The optimized conditions of the affinity adsorption experiment were: 2% phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀, 0.3 mg alizarin red, 40% K₂HPO₄ and 4 h adsorption time. The affinity adsorption rate of alizarin red reached 98% under such conditions. In addition, a flotation recovery of alizarin red of 97% was obtained under the optimal conditions of aqueous two-phase flotation: 0.3 g of collector, 12 g K₂HPO₄, 0.6 mg alizarin red, 15 mL min^-1 nitrogen flow rate and 40 min flotation time. Both methods are capable of separating alizarin red, suggesting their broad application prospect in downstream separation engineering of vicinal-diol-containing compounds.

Full text of DOI:10.1039/c6ra16327k

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Page 1 of 36
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DOI: 10.1039/C6RA16327K
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Synthesis of phenylboronic acid-functionalized thermosensitive
block copolymer and its application in separation and
purification of vicinal-diol-containing compounds
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a
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Yun Wang,* Yuanyuan Li, Juan Han,* Xu Tang, Liang Ni, Xiaowei Hu and Lei
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Wang
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School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang
212013, PR China
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School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013,
PR China
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* Corresponding authors
Tel: +86 158 9638 5156; Fax: +86ꢀ0511ꢀ88791800
Eꢀmail: yunwang@ujs.edu.cn
Tel: +86 137 7553 4923; Fax: +86ꢀ0511ꢀ88791800
Eꢀmail: hanjuan@ujs.edu.cn
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Abstract: Phenylboronic acidꢀfunctionalized amphiphilic thermosensitive block
copolymer was prepared by taking PEO20PPO60PEO20 as the template and a detailed
study of its performance in downstream separation processes was presented. Alizarin
red was used as the model compound to study the feasibility of using affinity
adsorption system and aqueous twoꢀphase flotation system based on the
phenylboronic acidꢀfunctionalized PEO20PPO60PEO20 for the separation and
purification of orthoꢀhydroxyl compounds. The optimized condition of affinity
adsorption experiment was: 2% of phenylboronic acidꢀfunctionalized
PEO PPO PEO , 0.3 mg of alizarin red, 40% of K HPO and 4 h of adsorption time.
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The affinity adsorption rate of alizarin red reached 98% under such condition. In
addition, the flotation recovery of alizarin red, 97% was obtained at the optimal
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condition of aqueous twoꢀphase flotation: 0.3 g of collector, 12 g of K HPO , 0.6 mg
of alizarin red, 15 mL/min of nitrogen flow rate and 40 min of flotation time. Both
methods are capable of separating alizarin red, suggesting their broad application
prospect in downstream separation engineering of vicinalꢀdiolꢀcontaining compounds.
Keywords:
phenylboronic
acidꢀfunctionalized
PEO20PPO60PEO20;
vicinalꢀdiolꢀcontaining compounds; affinity adsorption; aqueous twoꢀphase flotation
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1. Introduction
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As a kind of important natural products, orthoꢀhydroxyl compounds (such as
polysaccharide, anthocyanin, procyanidine, etc.) have attracted much attention
because of their good antioxidant, antiꢀcancer and antiꢀaging properties, and they have
been widely applied in the field of medical care. At present, the common method of
extracting natural orthoꢀhydroxyl compounds is solvent extraction method. However,
some impurities such as pigment and protein would also dissolve in extraction solvent.
Many separation techniques including precipitation, membrane separation and
chromatographic separation are employed to get rid of these impurities. The
traditional separation method adopts the strategy of removing impurities, which has
the shortcomings of process complexity, timeꢀconsuming and high energy
consumption. Meanwhile, natural orthoꢀhydroxyl compounds are easily inactivated
when undergo a long period of separation process. In view of this, a simple, rapid,
mild and efficient method for the separation of natural orthoꢀhydroxyl compounds is
highly desirable.
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Over the past decade, stimuliꢀresponsive polymers have been intensively
investigated due to their specific property. These polymers undergo dramatic and
reversible microstructure transformation when exposed to variations in local
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environment (temperature, pH, magnetic property, etc.). According to this
characteristic, a variety of applications based on stimuliꢀ responsive polymers have
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ꢀ4
been reported, such as drug delivery, biosensor and separation engineering. Among
various stimuliꢀresponsive polymers, boronic acidsꢀfunctionalized stimuliꢀresponsive
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polymers are famous for their specific interaction with vicinalꢀdiolꢀcontaining
compounds.
Boronic acid group consists of one trivalent boron atom, one alkyl or aryl
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substituent, and two hydroxyl groups. As a nonꢀnatural organic acid with strong
stability and many characteristic reactions, boronic acid has been first studied since
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1866. According to these researches, boronic acid and its derivatives have been
successfully applied for CꢀC bond synthesis, acid catalysis, asymmetric synthesis,
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carbohydrates detection, enzyme inhibition, molecular sensing, and drug release. For
boronic acid and its derivatives, the most important property is their characteristic
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reaction with orthoꢀhydroxyl compounds to form boronic acid ester, which causes
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their obvious changes of steric configuration and physicochemical property.
The
covalent interaction of boronic acid with diol is readily reversible upon the change of
local environment. It is advantageous to incorporate this distinct interaction within
stimuliꢀresponsive polymer to allow subsequent reversible reaction with
orthoꢀhydroxyl compounds. In recent years, the stimuliꢀresponsive polymer
containing boronic acid group has been successfully applied in the preparation of
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carbohydrates’ probes
and carbohydrates’ responsive micelles.
However, there
are few researches concentrating on the application of boronic acidsꢀfunctionalized
stimuliꢀresponsive polymer in the separation and purification of orthoꢀhydroxyl
compounds.
In this work, thermoꢀresponsive block copolymer PEO20PPO60PEO20 (L64) was
taken as the template and 3ꢀcarboxyphenylboronic acid groups was directly
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incorporated into it through chloroformylation reaction to obtain a new phenylboronic
acidꢀfunctionalized thermosensitive block copolymer. Besides that, the relevant
property of the novel polymer including cloud point and solubility were studied.
Herein, two kinds of separation systems―affinity adsorption system and aqueous
twoꢀphase flotation (ATPF) system were constructed on the basis of the synthesized
phenylboronic acidꢀfunctionalized thermosensitive block copolymer to extract a
model vicinalꢀdiolꢀcontaining compound (alizarin red), aiming at demonstrating the
potential application of this polymer in downstream separation and purification of
vicinalꢀdiolꢀcontaining compound.
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2.1 Materials
3ꢀcarboxyphenylboronic acid was purchased from Aladdin reagent Co., Ltd.
(Shanghai, China, 99%). Oxalyl chloride (98%), dichloromethane (99.5%),
N,Nꢀdimethylformamide (99.5%), triethylamine (99%), alizarin red (99%), Dꢀsorbitol
(98%), hydrochloric acid (36%ꢀ38%), sodium hydroxide (96%), dipotassium
phosphate (98%) were purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). PEO20PPO60PEO20 was purchased from BASF China Co., Ltd, one
end of which was hydroxide radical. All the solvents were dried over CaCl
filtered.
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and
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2.2 Preparation of boronic acid-functionalized PEO PPO PEO
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Phenylboronic acidꢀfunctionalized PEO20PPO60PEO20 (L64ꢀB(OH)
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) was prepared
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referring to the method previously reported and the synthesis process was shown in
Scheme 1. The detailed steps are as follows.
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Scheme 1 Synthesis of L64ꢀB(OH) .
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2.2.1 Synthesis of 3-chlorocarbonylphenylboronic acid
0.12 g of 3ꢀcarboxyphenylboronic acid was added into 17.50 mL of
dichloromethane and 124 ꢁL of oxalyl chloride was added after
3ꢀcarboxyphenylboronic acid was thoroughly dissolved. Then, 4 drops of DMF were
dropped to the mixture solution to trigger the reaction. The obtained solution was
stirred for 12 h at room temperature. The yellow liquid product was finally obtained
by removing the solvent through rotary evaporation. The product was applied to the
next reaction without any aftertreatment.
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2.2.2 Synthesis of phenylboronic acid-functionalized PEO20PPO60PEO20
0.50 g of L64 was dissolved in 35.00 mL of dichloromethane and 150 ꢁL of
triethylamine was subsequently added to get solution A. Afterwards, 0.7 mmol
chlorocarbonylphenylboronic acid was dissolved in 17.50 mL of dichloromethane to
obtain solution B. Next, solution B was dropped into solution A with iceꢀwater bath
cooling. The iceꢀwater bath was removed after finishing dropping and the system was
stirred for 12 h at room temperature. The solvent was removed by rotary evaporation
and a certain amount of water was added into the system. The phase separation of the
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system was achieved by heating. Finally, the bottom phase was collected and dried to
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gain the product. Fig. 1 shows the H NMR of L64ꢀB(OH)
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: (400 MHz, CDCl ) δ =
6.98ꢀ7.47 (ArH), 5.99 (s, 2H), 3.05ꢀ3.76 (EO, ꢀCH
2
ꢀ; PO, ꢀCH ꢀ, ꢀRCHꢀ), 1.06ꢀ1.08
2
3
(PO, ꢀCH ).
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Fig. 1 H NMR of L64ꢀB(OH)
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. The illustration is enlarged diagram of the 6ꢀ9 ppm
part.
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2.3 Study on performance of phenylboronic acid-functionalized
PEO PPO PEO
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2.3.1 Samples preparation
0.10 g of polymer and a certain amount of Dꢀsorbitol and water were added to a
25.00 mL colorimetric tube to make the total quantity of the system reach 20 g. The
solution pH was adjusted by HCl and NaOH solution.
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2.3.2 Determination of cloud point
On the one hand, the cloud point was measured with turbidity measurement method.
The sample solution was heated in a thermostatic bath until it turned turbid. Then the
turbid solution was removed from the bath and cooled at the room temperature. The
cloud point of polymer solution was obtained by capturing the catastrophe point at
which the turbidity disappeared. The determination of each cloud point was repeated
three times.
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On the other hand, the cloud point of L64ꢀB(OH) was also determined by UVꢀVIS
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spectrophotometry method. An appropriate amount of sample was added to the
colorimetric tube and placed in a temperatureꢀcontrolled bath at different temperatures
for 15 min. The samples with different temperatures were determined at 356 nm and
the distilled water was regarded as the reference solution. The cloud point was
determined by finding out the temperature interval where the absorbance of the
sample increased sharply, as shown in Fig. 2.
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.10
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CMT
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Temperature
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Fig. 2 Cloud point determination of L64ꢀB(OH)2.
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2.3.3 Salting-out experiment
0.20 g of polymer and an appropriate amount of water were added to a 5.00 mL
colorimetric bottle to make the total quantity of the system reach 2.00 g. Then a
certain amount of K HPO was added and completely dissolved in the system. The
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salting out effect was observed after 15 min.
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2.4 Application of phenylboronic acid-functionalized thermosensitive
block copolymer in the separation and purification of
vicinal-diol-containing compounds
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In this work, alizarin red, which bears the vicinalꢀdiol group, was taken as the
model compound. The separation of alizarin red was conducted by affinity adsorption
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and aqueous twoꢀphase flotation techniques based on L64ꢀB(OH) .
2.4.1 Separation of alizarin red by affinity adsorption
A certain amount of L64ꢀB(OH) , alizarin red, and water were added to a plastic
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centrifuge tube to make the total quantity of the system reach 2.00 g. After thorough
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mixing, a certain mass of K HPO was dissolved in the system. The polymer was
separated from the system and diluted with water after affinity adsorption to
determine the content of alizarin red in the polymer solution. Herein, several factors
affecting the affinity adsorption rate of alizarin red including the mass of L64ꢀB(OH)2,
2 4
added quantity of alizarin red, concentration of K HPO , and adsorption time were
discussed.
1
The affinity adsorption rate of alizarin red (E ) was calculated by the Equation 1:
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=
M
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(1)
where
M
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is the content of alizarin red in the polymer solution after affinity
is the quantity of alizarin red added to the system.
phase flotation
, alizarin red, and L64ꢀB(OH)
adsorption,
M
b
2.4.2 Separation of alizarin red by aqueous two
An appropriate amount of K HPO
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2
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2
were mixed and
dissolved in a 50 mL colorimetric tube. Then the mixture solution was diluted to the
tick mark with distilled water. After the mixture solution was transferred to a 50 mL
flotation column, a certain volume of nꢀpropanol was added on the top of the column.
The flotation device was illustrated in Fig. 3. Adsorbing on the surface of the nitrogen
gas bubble ascending at a certain flow rate, alizarin red along with collector was
eventually floated to the top phase after a period of time. After flotation, the volume
of the top phase was read and alizarin red was measured. All the ATPF processes were
performed at room temperature for three times. Several variables, namely
concentration of K HPO , quantity of alizarin red, mass of collector, flotation time,
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and gas flow rate influencing the recovery of alizarin red were investigated.
The flotation recovery of alizarin red ( ) was calculated using the Equation 2:
(2)
represents the
E
2
2 T T b
E =C V /M
where
T T
V is the volume of the top phase in the ATPF system. C
concentration of alizarin red in the top phase. Mb is the quantity of alizarin red added
to the ATPF system.
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Fig. 3 Aqueous twoꢀphase flotation device.
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2.5 Concentration determination of alizarin red
1.00 mL of the sample and 9.00 mL of citric acid―sodium citrate buffer solution
(pH 4) were thoroughly mixed in the 10.00 mL plastic centrifuge tube. The sample
was determined at 424 nm and the distilled water was regarded as the reference
solution. The experiment was conducted in triplicate.
Alizarin red solutions at a series of concentration were prepared. A calibration
curve of alizarin red was obtained by taking the absorbance (A) as the ordinate and
concentration (C) as the abscissa. Equation 3 is the linear regression equation. The
linear range and correlation coefficient are 0~0.1 mg/mL and 0.9991, respectively.
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A=13.55507C+0.02465
(3)
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3. Result and discussion
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3.1 Reaction mechanism between phenylboronic acid-functionalized
PEO PPO PEO and ortho-hydroxyl compounds
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To better understand the binding process between L64ꢀB(OH) and orthoꢀhydroxyl
2
compounds as well as the reasons for the physicochemical property changes of the
polymer, here their reaction mechanism was discussed. It is well known that boronic
acids can react with vicinalꢀdiolꢀcontaining compounds to generate boronic acid esters.
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Due to this reaction, the steric configuration of boronic acids changes, thus resulting
in the alteration of the physicochemical property of boronic acids. Owing to the
incorporation of phenylboronic acid into L64, the steric configuration and
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physicochemical property of L64ꢀB(OH) present specific response to orthoꢀhydroxyl
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compounds. As shown in Fig. 4, L64ꢀB(OH)
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mainly exsits in two forms in water
solution: (1) nonꢀdissociative neutral triangular structure; (2) dissociative anion
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tetrahedral structure. The two forms have significant difference in hydrophilic
properties because of their different structures. Compared with the nonꢀdissociative
neutral triangular structure, the dissociative anion tetrahedral structure shows stronger
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hydrophilia. In aqueous solution, the above two forms keep a dynamic equilibrium
and the variation of this equilibrium will lead to the change in the
hydrophilicꢀhydrophobic property of L64ꢀB(OH) . According to some related
2
5
literatures, the change of the system pH value and the addition of orthoꢀhydroxyl
compounds will alter this equilibrium effectively.
Fig. 4 shows the effect of the system pH value on the equilibrium. As the hydrogen
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ions are released during the transformation from structure 1 to structure 2 and are
neutralized when the pH value of the system increases, the reaction balance will move
toward the right side. In this way, more L64ꢀB(OH)
2
with hydrophilic
electronegativity structure 2 in the aqueous solution will exist and the overall
hydrophilia is thus increased. Otherwise, the reaction balance will move toward the
left side when the pH value of the system decreases. As a result, L64ꢀB(OH)
2
will
mainly exist in the form of hydrophobic structure 1 and the overall hydrophilia is
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reduced.
Boronic acid has a higher affinity with orthoꢀhydroxyl compounds. When
orthoꢀhydroxyl compounds are added to the system, on the one hand, boronic acid
esters with the ring structure are generated through combining boronic acid with
electroneutral triangular structure 1; however, this boronic acid ester is so unstable
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that it is easy to be hydrolyzed. On the other hand, L64ꢀB(OH)
2
with
electronegativity tetrahedral structure 2 can reversibly bind with orthoꢀhydroxyl
compounds to generate boronic acid ester 3, which makes the balance move toward
the direction of forming anionic forms 2 and 3 with higher hydrophilicity.
On the whole, the hydrophilicꢀhydrophobic property of boronic
acidsꢀfunctionalized polymers can be adjusted by altering their aqueous environment.
Adding orthoꢀhydroxyl compounds into L64ꢀB(OH) solution or increasing the
2
solution pH value will lead to the right shift of the reaction balance, so that the overall
hydrophilia of L64ꢀB(OH)
2
is enhanced. Conversely, if the added amount of
orthoꢀhydroxyl compounds or the solution pH value is reduced, more L64ꢀB(OH)
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will exist in the form of structure 1, thus reducing the overall hydrophilia of the
modified L64.
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Fig. 4 Equilibria of L64ꢀB(OH) in aqueous solution.
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3.2
Performance
of
phenylboronic
acid-functionalized
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PEO PPO PEO
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3.2.1 Cloud point of L64 and L64-B(OH)2
The cloud point of thermoꢀresponsive block copolymers can effectively reflect the
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strength of their hydrophilia. For L64ꢀB(OH) , the incorporation of boronic acid
2
group in to the molecular structure can alter its hydrophileꢀlipophile balance (HLB),
which will make the change in cloud point of the polymer. As described above,
altering the pH value or adding orthoꢀhydroxyl compounds to the system will change
2
the hydrophilicꢀhydrophobic property of L64ꢀB(OH) . Leading to the change in cloud
2
1
point of L64ꢀB(OH)2. Therefore, besides the variation of cloud point before and
after the modification of L64, two factors affecting the cloud point of L64 and
L64ꢀB(OH)
discussed.
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including pH and added amount of orthoꢀhydroxyl compounds were also
o
As shown in Table 1, compared with the cloud point of 0.5% L64 (57.9 C), the
o
cloud point of 0.5% L64ꢀB(OH) (17.9 C) decreases distinctly, which also proves
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that the boronic acid group is successfully introduced to the structure of L64. In order
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to study the influence of adding orthoꢀhydroxyl compounds on the cloud point of
L64ꢀB(OH) , sorbitol with different quantities was added into the system. It can be
2
observed from Table 1 and Fig. 5 that the addition of sorbitol with different quantities
almost has no impact on the cloud point of L64; while the cloud point of L64ꢀB(OH)2
increases significantly. As previously mentioned, boronic acid exists in the form of
electrically neutral triangular structure (1) or electronegativity tetrahedral structure (2)
in the aqueous solution. The electronegativity tetrahedral structure (2) can reversibly
react with sorbitol to produce boronic acid ester (3), which makes the balance move
toward the direction of generating hydrophilic anion structure (2) and (3). Hence, with
the rising of sorbitol concentration, the balance moves to the right. Consequently, the
1
1
1
1
1
hydrophilia of L64ꢀB(OH)
improved.
2
was greatly enhanced and its cloud point was thus
Table 1. Influence of the added amount of sorbic alcohol on the cloud point of L64
and L64ꢀB(OH)2
Sorbic alcohol
L64
L64ꢀB(OH)
2
o
o
(mg)
( C)
( C)
a
b
b
0
57.9
58.1
58.1
17.2
22.0
26.2
31.7
17.0
22.1
24.9
31.9
a
5
a
b
1
0
0
a
b
2
58.0
a
b
1
1
5
6
turbidity measurement method
;
ultraviolet spectrophotometry method.
15

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2
1
1
1
1
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
.8
.6
.4
.2
.0
2
.2
a
2.0
1.8
b
1
1
1
1
0
.6
.4
.2
.0
.8
Cloud point
Cloud point
0.6
0
0
.4
.2
0.0
0
5
10 15 20 25 30 35 40 45 50 55 60
Temperature
0
5
10 15 20 25 30 35 40 45 50 55 60
Temperature
1
1
1
1
1
1
0
0
0
0
0
.8
.6
.4
.2
.0
.8
.6
.4
.2
.0
1
1
1
.4
.2
.0
c
d
0.8
0
0
0
.6
.4
.2
Cloud point
Cloud point
0.0
10
0
5
10 15 20 25 30 35 40 45 50 55 60
Temperature
15
20
25
30
35
40
45
50
55
60
Temperature
2
3
Fig. 5 Cloud point of L64ꢀB(OH)
sorbic alcohol (b) 5 mg sorbic alcohol
alcohol.
2
with different amounts of sorbic alcohol. a) 0 mg
4
5
6
7
8
9
0
1
2
；
；
(c) 10 mg sorbic alcohol (d) 20 mg sorbic
；
The above variation of cloud point is in keeping with the aforementioned
mechanism, which reflects the effective combination between L64ꢀB(OH) and
orthoꢀhydroxyl compounds and further confirms the successful preparation of
L64ꢀB(OH)
2
1
1
1
2
.
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1
Table 2. Influence of pH on the cloud point of L64 and L64ꢀB(OH)
2
pH
L64
L64ꢀB(OH)
2
o
o
(
C)
( C)
a
a
a
a
a
2
3
4
6
8
57.8
58.0
58.2
58.1
58.2
58.0
58.2
17.4
17.2
23.4
33.8
38.4
b
24.1
33.4
37.9
43.4
44.1
b
b
a
b
1
1
0
2
44.1
44.2
a
b
a
b
2
3
turbidity measurement method; ultraviolet spectrophotometry method.
4
5
6
7
8
9
0
1
2
The pH value of the system also has a significant impact on the cloud point of
L64ꢀB(OH) . As shown in Table 2, the cloud point of L64ꢀB(OH) increases with the
2
2
increase of the pH value. The cloud point value determined by turbidity measurement
method is in agreement with that determined by ultraviolet spectrophotometry method,
2
which can be seen in Fig. 6. As discussed above, more L64ꢀB(OH) will exist in the
form of hydrophilic electronegativity structure (2) with the increase in the pH value of
solution, which results in the rise of its cloud point. The experimental results are in
accordance with the above discussed mechanism, which confirms the successful
1
1
1
2
modification of L64ꢀB(OH) .
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0
0
0
0
0
0
0
.7
.6
.5
.4
.3
.2
.1
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
a
b
Cloud point
Cloud point
0.0
0
5
10 15 20 25 30 35 40 45 50 55 60
Temperature
0
5
10 15 20 25 30 35 40 45 50 55 60 65
Temperature
1
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
0
0
0
0
0
0
0
0
.40
.35
.30
.25
.20
.15
.10
.05
c
d
Cloud point
Cloud point
0.00
0
5
10 15 20 25 30 35 40 45 50 55
Temperature
0
5
10 15 20 25 30 35 40 45 50 55
Temperature
2
0
0
0
0
0
0
0
0
0
.40
.35
.30
.25
.20
.15
.10
.05
.00
e
Cloud point
0
5
10 15 20 25 30 35 40 45 50 55
Temperature
3
4
Fig. 6 Cloud point of L64ꢀB(OH) solution at different pH values. (a) pH 4; (b) pH 6;
2
5
6
7
(c) pH 8; (d) pH 10; (e) pH 12.
18

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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3.2.2 Salting
-
out experiment
out ability of a substance is closely related to the strength of its
The salting
ꢀ
hydrophilia. In this section, the saltingꢀout experiment was conducted by adding
different amounts of K HPO into the L64 solution (1%) and the L64ꢀB(OH)
2
4
2
solution(1%) to evaluate the hydrophobicityꢀhydrophilicity strength of the polymer.
As shown in Fig. 7a, there is no polymer being separated out from the L64 solution.
2
In contrast, the precipitation of L64ꢀB(OH) can be observed obviously in Fig. 7b. In
addition, with the increase of salt concentration, the precipitation amount of
L64ꢀB(OH) tends to increase firstly and then decrease. During the first stage, the
2
1
1
1
1
1
1
1
1
1
1
2
2
2
increase in the saltingꢀout ability of K
addition of K HPO , the saltingꢀout ability of K
water molecules in the system would be captured by salt ions, leading to the increase
in the precipitation amount of L64ꢀB(OH) . However, in the second stage, as the
adding amount of K HPO further increases, the pH value of the system will increase
2
HPO
4
solution plays a leading role. With the
2
4
2
HPO solution is enhanced, so more
4
2
2
4
and play the dominant role. The increase of pH value can make improvement in
hydrophilicity of L64ꢀB(OH) , so the precipitation amount of L64ꢀB(OH) decreases.
The combined action of the above two factors cause the tendency that the
2
2
precipitation amount of L64ꢀB(OH)
increase of the salt concentration.
2
increases firstly and then reduces with the
Moreover, a certain amount of alizarin red was added to three different systems and
the results were shown in Fig. 8. From Fig. 8, we could easily see that L64ꢀB(OH)
shows a favorable absorption capacity to alizarin red, indicating a tight association of
2
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1
2
L64ꢀB(OH)
2
with orthoꢀhydroxyl compounds. Meanwhile, it provides an effective
method for the extraction of orthoꢀhydroxyl compounds―affinity adsorption method.
3
4
5
Fig. 7 Photographs of saltingꢀout phenomenon of polymer at different K
concentrations: (a) L64; (b) L64ꢀB(OH) (Concentration of K HPO is 10%, 20%,
30%, 40%, 50% from left to right, respectively.).
2 4
HPO
6
7
8
2
2
4
9
0
1
1
1
1
Fig. 8 Photographs of alizarin red absorption: (a) alizarin red; (b) alizarin red + L64;
(c) alizarin red + L64ꢀB(OH)
1
2
3
2
.
20

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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.3
Application
of
in
vicinal-diol-containing compounds
phenylboronic
acid-functionalized
purification of
PEO PPO PEO
separation
and
20
60
20
Alizarin red was ulitilized as the model compound to study the application of
L64ꢀB(OH) for separation and purification of orthoꢀhydroxyl compounds. The
reaction between L64ꢀB(OH) and alizarin red was verified according to the method
2
2
2
2
in the literature. A certain amount of alizarin red was firstly dissolved in the PBS
(pH 7). After that, the alizarin red solution was divided into triplicate and two of them
were respectively added into L64 and L64ꢀB(OH) with the same amount. The three
2
1
1
1
1
1
1
1
1
1
1
2
sample solutions were shown in Fig. 9. As an acidꢀbase indicator, the color of alizarin
red solution will change with the variation of pH value. Alizarin red solution is yellow
when the pH value is between 3.7ꢀ5.2 and purple when the pH value is higher than 5.2.
2
As shown in Fig. 9, it is obvious that the color of the L64ꢀB(OH) solution is partial
yellow, which proves that its pH value is lower than that of the other two sample
solutions. This result reveals that a large number of hydrogen ions are released due to
the combination of L64ꢀB(OH)
2
with alizarin red and the pH value of the solution is
thus reduced. Moreover, the color changes in this work are nearly in accordance with
22
the result in the relevant literature. Hence, the effective combination between
phenylboronic acidꢀfunctionalized PEO PPO PEO and alizarin red was further
2
0
60
20
confirmed.
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1
2
Fig. 9 Affinity interaction between L64ꢀB(OH) and alizarin red. (a) alizarin red; (b)
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
2
alizarin red + L64; (c) alizarin red + L64ꢀB(OH) .
3.3.1 Separation and purification of alizarin red by affinity adsorption system
Firstly, the influences of the concentration of L64ꢀB(OH) and the quantity of
alizarin red on the affinity adsorption rate (E ) of alizarin red were investigated while
2
1
2 4
keeping the K HPO concentration 40% and adsorption time 5 h. The results are
shown in Fig. 10a. Under different adding quantities of alizarin red, the adsorption
rate of alizarin red increases firstly and then keeps almost constant with the increasing
1
1
1
1
1
1
1
1
1
1
2
content of L64ꢀB(OH) . As we know, alizarin red has only one vicinalꢀdiol group, so
the boronic acid group can react with alizarin red at the ratio of 1:1. Because of this,
most of alizarin red could not be adsorbed if the concentration of the polymer was low,
leading to the low adsorption rate of alizarin red. With the increasing concentration of
L64ꢀB(OH) , more alizarin red is adsorbed by the polymer. When the L64ꢀB(OH)2
2
concentration increases to a certain degree, the modified polymer is able to adsorb
almost all of alizarin red. At this time, no more alizarin red can be adsorbed if the
polymer is added continuously and the adsorption rate of alizarin red keeps nearly
constant. Taking the reagents consumption and separation efficiency into
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1
2
2
consideration, 2% of L64ꢀB(OH) concentration was selected for the following
discussion.
a 100%
0
0
.05mg alizarin red
.1mg alizarin red
90%
80%
70%
60%
50%
0.2mg alizarin red
0.3mg alizarin red
.4mg alizarin red
0
0.5
1
1.5
2
2.5
Concentration of L64ꢀB(OH) (w/w)
2
3
1
20%
00%
100%
b
c
1
80%
60%
40%
20%
0%
80%
60%
40%
20%
0%
10% 20% 30% 40% 50%
0.5
1
2
3
4
5
Concentration of K HPO (w/w)
Time (h)
4
5
2
4
Fig. 10 Influence of (a) concentration of L64ꢀB(OH)
2
and quantity of alizarin red, (b)
).
6
7
8
9
0
1
2
3
4
K
2
HPO concentration and (c) adsorption time on the affinity adsorption rate (E
4
1
In addition, under different concentrations of L64ꢀB(OH) , all the adsorption rate of
2
alizarin red with 0.05, 0.1, 0.2, 0.3, 0.4 mg tend to increase firstly and then decrease.
As a hydrophilic substance, alizarin red possesses an excellent solubility in aqueous
solutions. Hence, a small amount of alizarin red is hard to be adsorbed by the polymer,
causing the low adsorption rate of alizarin red at its low concentration. As the alizarin
red concentration increases, the amount of alizarin red remaining in the aqueous
1
1
1
1
1
2
solution is quite low compared with that adsorbed by L64ꢀB(OH) . As a result, the
23

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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
adsorption rate of alizarin red increases gradually with the addition of alizarin red and
reaches the maximum when its adding quantity is 0.3 mg. At this point, affinity
adsorption of alizarin red reaches its saturation and the adsorption rate reduces with
the further increase of the alizarin red concentration. Therefore, 0.3 mg of alizarin red
was selected for the following discussion.
To sum up, when the concentration of L64ꢀB(OH)
alizarin red is 0.3 mg, the adsorption rate of alizarin red reaches the maximum 98%.
Then the influence of K HPO concentration on E , was investigated while keeping
the adsorption time at 5 h and the result was revealed in Fig. 10b. With the gradual
increase of K HPO concentration, the adsorption rate of alizarin red tends to increase
firstly and then keep nearly constant. The possible causes for the above tendency are
as follows. At first, with the increase in the concentration of basic salt K HPO , the
2
is 2% and the adding quantity of
2
4
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
4
2
4
pH value of the system rises. And so the balance of affinity reaction between alizarin
red and L64ꢀB(OH) moves towards the right (Fig. 4), which makes more alizarin red
2
be adsorbed. Meanwhile, higher salt concentration will cause stronger saltingꢀout
effect, making more modified polymerꢀalizarin red complex precipitate out. Therefore,
the adsorption rate of alizarin red increases and reaches its maximum (98%) at 40% of
2 4
salt concentration. Because the further increasing concentration of K HPO almost
has no influences on the adsorption effect, 40% of salt concentration was selected for
the following discussion.
Finally, the impact of adsorption time on the adsorption effect of alizarin red was
revealed in Fig. 10c. With the increase of adsorption time, the adsorption rate of
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
alizarin red increases firstly and then keeps almost constant. This is because it takes a
certain time to reach the balance of the affinity reaction between boronic acid and
alizarin red. With the increase of adsorption time at the beginning, more alizarin red
reacts with L64ꢀB(OH) , and thus the adsorption rate would be improved. However,
2
when the adsorption time extends to a certain value, the affinity reaction between
2
L64ꢀB(OH) and alizarin red reaches the balance, and then the adsorption rate of
alizarin red cannot be effectively improved with the further increase of the adsorption
time. Therefore, taking time cost and adsorption efficiency both into consideration, 4
h of adsorption time was selected for the affinity separation of alizarin red.
3.3.2 Aqueous two-phase flotation of alizarin red
1
1
1
1
1
1
1
1
1
1
2
2
2
Aqueous twoꢀphase flotation is a new separation and purification technology,
which is developed on the basis of aqueous twoꢀphase extraction and solvent flotation.
Compared with organic solvent extraction, aqueous twoꢀphase extraction and solvent
flotation, aqueous twoꢀphase flotation has many advantages such as low solvent
consumption, large enrichment factors, and high separation efficiency, which exhibits
2
3
a promising application in the field of separation and purification. Currently, there
24,25
are many literatures about aqueous twoꢀphase extraction,
but the reports on ATPF
2
6,27
are few.
According to the existing reports, ATPF systems were mainly established
to separate the target compounds from the fermentation broth or extract liquor based
on the hydrophobicity or surface activity of target molecules, which shows the
drawbacks of narrow application and poor selectivity.
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1
2
Fig. 11 Schematic diagram of aqueous twoꢀphase flotation.
3
4
5
6
7
8
9
0
1
2
3
Separation of waterꢀsoluble natural products from its extract liquor is always the
technological difficulty for aqueous twoꢀphase flotation, which limits its application
in separation and purification of waterꢀsoluble natural products. The use of collector is
the critical factor for solving this difficulty. As a dye with very strong hydrophilia,
alizarin red has an excellent solubility in water. Thus, in nꢀpropanol―salt ATPF
system, alizarin red is more likely to distribute in aqueous phase, causing the poor
1
1
1
1
flotation effect. In this section, L64ꢀB(OH)
2
was used as the collector for the
can
following two reasons: on the one hand, boronic acid group in L64ꢀB(OH)
2
effectively interact with alizarin red; on the other hand, as a surface active agent, the
hydrophobic block PPO of modified PEO20PPO60PEO20 is able to be effectively
26

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1
2
3
4
5
adsorbed on the interface of hydrophobic bubbles. The aqueous twoꢀphase flotation
schematic is shown in Fig. 11. Alizarin red can react with L64ꢀB(OH) to form a
2
complex with surface activity, which contributes to the flotation of waterꢀsoluble
alizarin red. Several single factors affecting the flotation recovery of alizarin red were
discussed.
a 100%
6
5
4
3
2
1
100%
80%
60%
40%
5.5
5
E2
Volume of top phase
b
E2
Volume of top phase
80%
60%
40%
20%
4.5
4
3.5
3
20%
2.5
0%
0%
2
0.05
0.1
Added amount of collector (g)
00%
0.3
0.3
0.4
8
10
12
14
16
K
HPO (g)
4
2
6
1
120%
96%
92%
88%
84%
80%
76%
c
d
e 1
00%
80%
60%
40%
20%
0%
8
6
4
2
0%
0%
0%
0%
0%
0
.1 0.2 0.4 0.6 0.8
1
5
10 15 20 25
10 20 30 40 50
Time (min)
Alizarin red (mg)
Flow rate (mL/min)
7
8
Fig. 12 Influence of (a) collector amount, (b) K
2
HPO
4
amount, (c) alizarin red amount,
).
9
0
(d) flow rate and (e) time on the flotation recovery (E
2
1
1
1
1
1
1
1
1
2
3
4
5
6
The impact of the added amount of collector (L64ꢀB(OH)
2
) on the flotation
recovery of alizarin red (E ) is shown in Fig. 12a. The flotation recovery of alizarin
2
red tends to increase firstly from 34% to 70% and then decrease with the continuous
addition of collector. The reversible binding between alizarin red and boronic acid
group of the collector is the key factor for the flotation of alizarin red to the top phase.
A small amount of collector is insufficient for trapping alizarin red, leading to the low
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
flotation recovery. With the increase in the added amount of collector, more alizarin
red was captured and floated to the top phase, causing the increase of E . From Fig.
2
12a, the volume of the top phase decreases with the increase of the added amount of
collector. Therefore, as the content of the collector further increases, the recovery of
alizarin red drops. When 0.3 g of the collector was added to the system, the maximum
2 2
E (70%) was obtained. Therefore, 0.3 g of L64ꢀB(OH) was selected for the
following discussion.
Then, the impact of K
2
4 2
HPO concentration on E is studied and revealed in Fig. 12b.
With the increase of K HPO concentration, the recovery of alizarin red tends to
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
increase firstly and then decline and the maximum (87%) is obtained when the added
2
8
quantity of K
phases in the flotation system were maintained by the salting
the amount of salt is low at first, the salting out effect is weak, which leads to the
2
HPO
4
is 12 g. According to previous literatures, the two incompatible
ꢀ
out effect. Thus, when
ꢀ
inadequate phase separation and the small volume of the top phase. Meanwhile, the
collectorꢀalizarin red complex has high solubility at the low salt concentration, so the
2 4
recovery of alizarin red is low. With the increase in the K HPO amount, the
salting out effect is enhanced and large volume of the top phase is obtained due to the
ꢀ
complete phase separation. Furthermore, as stated previously, the increase in the
amount of K HPO leads to the increase of pH value. Consequently, the collector
2
4
prefers to exist in the form of electronegative tetrahedral structure, which is beneficial
for the generation of collectorꢀalizarin red complex. Therefore, the recovery of
alizarin red increases from 36% to 87%. Whereas, the further increase in the added
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
amount of K
2 4
HPO will make the viscosity of flotation system too high, which affects
the mass transfer of bubbles in the system. As a result, the flotation recovery of
alizarin red tends to decrease with high salt concentration. Therefore, 12 g of K
2 4
HPO
was selected for the following discussion.
In this section, the influence of the added amount of alizarin red was investigated.
As shown in Fig. 12c, the recovery of alizarin red increases firstly and then reduces
with the increase of the added amount of alizarin red. As a hydrophilic dye, alizarin
red has high solubility in water and the successful flotation of alizarin red mainly
depends on the addition of the collector. When an excessive amount of alizarin red is
added, they cannot be completely flotated to the top phase because the capture ability
of the collector is limited. Thus, the residual amount of alizarin red in the bottom
phase increases and the recovery declines. From Fig. 12c, the maximum recovery of
alizarin red (92%) is obtained when 0.6 mg of alizarin red is selected. Eventually, 0.6
mg of alizarin red was selected for the following discussion.
1
1
1
1
1
1
1
1
1
1
2
2
2
Herein, the influence of nitrogen flow rate (5ꢀ25 mL/min) on the flotation recovery
was investigated, as shown in Fig. 12d. With the increase of nitrogen flow rate, the
recovery rate tends to rise firstly and then decrease. As stated in the previous literature,
increasing the nitrogen flow rate within limits can effectively improve the flotation
29
recovery and shorten the time. When the recovery reaches the maximum, the further
increase in the nitrogen flow rate will lead to the adverse effect, because the too high
30,31
nitrogen flow rate can result in the turbulent mixing in the interface.
Then many
3
2
bubbles will gather in the top phase of the system and the phase equilibrium is
29