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

Abstract

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.

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1. Introduction

As a kind of important natural product, ortho-hydroxyl compounds (such as polysaccharide, anthocyanin, procyanidine, etc.) have attracted much attention because of their good antioxidant, anti-cancer and anti-aging properties, and they have been widely applied in the field of medical care. At present, the common method of extracting natural ortho-hydroxyl compounds is the solvent extraction method. However, some impurities such as pigments and proteins would also dissolve in the 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, time-consuming and high energy consumption. Meanwhile, natural ortho-hydroxyl 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 ortho-hydroxyl compounds is highly desirable.

Over the past decade, stimuli-responsive polymers have been intensively investigated due to their specific property. These polymers undergo dramatic and reversible microstructure transformation when exposed to variations in local environment (temperature, pH, magnetic property, etc.).¹ According to this characteristic, a variety of applications based on stimuli- responsive polymers have been reported, such as drug delivery, biosensor and separation engineering.^2–4 Among various stimuli-responsive polymers, boronic acids-functionalized stimuli-responsive polymers are famous for their specific interaction with vicinal-diol-containing compounds.

Boronic acid group consists of one trivalent boron atom, one alkyl or aryl substituent, and two hydroxyl groups.⁵ As a non-natural organic acid with strong stability and many characteristic reactions, boronic acid has been first studied since 1866.⁶ According to these researches, boronic acid and its derivatives have been successfully applied for C–C bond synthesis, acid catalysis, asymmetric synthesis, carbohydrates detection, enzyme inhibition, molecular sensing, and drug release.⁷ For boronic acid and its derivatives, the most important property is their characteristic reaction with ortho-hydroxyl compounds to form boronic acid ester,⁸ which causes their obvious changes of steric configuration and physicochemical property.^9,10 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 stimuli-responsive polymer to allow subsequent reversible reaction with ortho-hydroxyl compounds. In recent years, the stimuli-responsive polymer containing boronic acid group has been successfully applied in the preparation of carbohydrates' probes^11–13 and carbohydrates' responsive micelles.^14,15 However, there are few researches concentrating on the application of boronic acids-functionalized stimuli-responsive polymer in the separation and purification of ortho-hydroxyl compounds.

In this work, thermo-responsive block copolymer PEO₂₀PPO₆₀PEO₂₀ (L64) was taken as the template and 3-carboxyphenylboronic acid groups was directly incorporated into it through chloroformylation reaction to obtain a new phenylboronic acid-functionalized 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 two-phase flotation (ATPF) system were constructed on the basis of the synthesized phenylboronic acid-functionalized thermosensitive block copolymer to extract a model vicinal-diol-containing compound (alizarin red), aiming at demonstrating the potential application of this polymer in downstream separation and purification of vicinal-diol-containing compound.

2. Materials and methods

2.1 Materials

3-Carboxyphenylboronic acid was purchased from Aladdin reagent Co., Ltd. (Shanghai, China, 99%). Oxalyl chloride (98%), dichloromethane (99.5%), N,N-dimethylformamide (99.5%), triethylamine (99%), alizarin red (99%), D-sorbitol (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 CaCl₂ and filtered.

2.2 Preparation of boronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀

Phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀ (L64-B(OH)₂) was prepared referring to the method previously reported¹⁶ and the synthesis process was shown in Scheme 1. The detailed steps are as follows.

Scheme 1 Synthesis of L64-B(OH)₂.

2.2.1 Synthesis of 3-chlorocarbonylphenylboronic acid. 0.12 g of 3-carboxyphenylboronic acid was added into 17.50 mL of dichloromethane and 124 μL of oxalyl chloride was added after 3-carboxyphenylboronic 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.

2.2.2 Synthesis of phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀. 0.50 g of L64 was dissolved in 35.00 mL of dichloromethane and 150 μL 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 ice-water bath cooling. The ice-water 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 system was achieved by heating. Finally, the bottom phase was collected and dried to gain the product. Fig. 1 shows the ¹H NMR of L64-B(OH)₂: (400 MHz, CDCl₃) δ = 6.98–7.47 (ArH), 5.99 (s, 2H), 3.05–3.76 (EO, –CH₂–; PO, –CH₂–, –RCH–), 1.06–1.08 (PO, –CH₃).

Fig. 1 ¹H NMR of L64-B(OH)₂. The illustration is enlarged diagram of the 6–9 ppm part.

2.3 Study on performance of phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀

2.3.1 Samples preparation. 0.10 g of polymer and a certain amount of D-sorbitol 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.

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.

On the other hand, the cloud point of L64-B(OH)₂ was also determined by UV-VIS spectrophotometry method.¹⁷ An appropriate amount of sample was added to the colorimetric tube and placed in a temperature-controlled 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.

Fig. 2 Cloud point determination of L64-B(OH)₂.

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 K₂HPO₄ was added and completely dissolved in the system. The salting out effect was observed after 15 min.

2.4 Application of phenylboronic acid-functionalized thermosensitive block copolymer in the separation and purification of vicinal-diol-containing compounds

In this work, alizarin red, which bears the vicinal-diol group, was taken as the model compound. The separation of alizarin red was conducted by affinity adsorption and aqueous two-phase flotation techniques based on L64-B(OH)₂.

2.4.1 Separation of alizarin red by affinity adsorption. A certain amount of L64-B(OH)₂, alizarin red, and water were added to a plastic centrifuge tube to make the total quantity of the system reach 2.00 g. After thorough mixing, a certain mass of K₂HPO₄ 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 L64-B(OH)₂, added quantity of alizarin red, concentration of K₂HPO₄, and adsorption time were discussed.

The affinity adsorption rate of alizarin red (E₁) was calculated by the eqn (1):

E₁ = M_a/M_b (1)

where M_a is the content of alizarin red in the polymer solution after affinity adsorption, M_b is the quantity of alizarin red added to the system.

2.4.2 Separation of alizarin red by aqueous two-phase flotation. An appropriate amount of K₂HPO₄, alizarin red, and L64-B(OH)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 n-propanol 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 K₂HPO₄, quantity of alizarin red, mass of collector, flotation time, and gas flow rate influencing the recovery of alizarin red were investigated.

Fig. 3 Aqueous two-phase flotation device.

The flotation recovery of alizarin red (E₂) was calculated using the eqn (2):

E₂ = C_TV_T/M_b (2)

where V_T is the volume of the top phase in the ATPF system. C_T represents the concentration of alizarin red in the top phase. M_b is the quantity of alizarin red added to the ATPF system.

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. Eqn (3) is the linear regression equation. The linear range and correlation coefficient are 0–0.1 mg mL⁻¹ and 0.9991, respectively.

A = 13.55507C + 0.02465 (3)

3. Result and discussion

3.1 Reaction mechanism between phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀ and ortho-hydroxyl compounds

To better understand the binding process between L64-B(OH)₂ and ortho-hydroxyl 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 vicinal-diol-containing compounds to generate boronic acid esters. Due to this reaction, the steric configuration of boronic acids changes,⁸ 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 physicochemical property of L64-B(OH)₂ present specific response to ortho-hydroxyl compounds. As shown in Fig. 4, L64-B(OH)₂ mainly exsits in two forms in water solution: (1) non-dissociative neutral triangular structure; (2) dissociative anion tetrahedral structure.¹⁸ The two forms have significant difference in hydrophilic properties because of their different structures. Compared with the non-dissociative neutral triangular structure, the dissociative anion tetrahedral structure shows stronger hydrophilia.¹⁹ In aqueous solution, the above two forms keep a dynamic equilibrium and the variation of this equilibrium will lead to the change in the hydrophilic–hydrophobic property of L64-B(OH)₂. According to some related literatures,⁵ the change of the system pH value and the addition of ortho-hydroxyl compounds will alter this equilibrium effectively.

Fig. 4 Equilibria of L64-B(OH)₂ in aqueous solution.

Fig. 4 shows the effect of the system pH value on the equilibrium. As the hydrogen 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 L64-B(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, L64-B(OH)₂ will mainly exist in the form of hydrophobic structure 1 and the overall hydrophilia is reduced.²⁰

Boronic acid has a higher affinity with ortho-hydroxyl compounds. When ortho-hydroxyl 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 that it is easy to be hydrolyzed.¹⁰ On the other hand, L64-B(OH)₂ with electronegativity tetrahedral structure 2 can reversibly bind with ortho-hydroxyl 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 hydrophilic–hydrophobic property of boronic acids-functionalized polymers can be adjusted by altering their aqueous environment. Adding ortho-hydroxyl compounds into L64-B(OH)₂ solution or increasing the solution pH value will lead to the right shift of the reaction balance, so that the overall hydrophilia of L64-B(OH)₂ is enhanced. Conversely, if the added amount of ortho-hydroxyl compounds or the solution pH value is reduced, more L64-B(OH)₂ will exist in the form of structure 1, thus reducing the overall hydrophilia of the modified L64.

3.2 Performance of phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀

3.2.1 Cloud point of L64 and L64-B(OH)₂. The cloud point of thermo-responsive block copolymers can effectively reflect the strength of their hydrophilia. For L64-B(OH)₂, the incorporation of boronic acid group in to the molecular structure can alter its hydrophile–lipophile balance (HLB), which will make the change in cloud point of the polymer. As described above, altering the pH value or adding ortho-hydroxyl compounds to the system will change the hydrophilic–hydrophobic property of L64-B(OH)₂. Leading to the change in cloud point of L64-B(OH)₂.²¹ Therefore, besides the variation of cloud point before and after the modification of L64, two factors affecting the cloud point of L64 and L64-B(OH)₂ including pH and added amount of ortho-hydroxyl compounds were also discussed.

As shown in Table 1, compared with the cloud point of 0.5% L64 (57.9 °C), the cloud point of 0.5% L64-B(OH)₂ (17.9 °C) decreases distinctly, which also proves that the boronic acid group is successfully introduced to the structure of L64. In order to study the influence of adding ortho-hydroxyl compounds on the cloud point of L64-B(OH)₂, sorbitol with different quantities was added into the system. It can be 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 L64-B(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 hydrophilia of L64-B(OH)₂ was greatly enhanced and its cloud point was thus improved.

Table 1 Influence of the added amount of sorbic alcohol on the cloud point of L64 and L64-B(OH)₂

Sorbic alcohol (mg)	L64 (°C)	L64-B(OH)2 (°C)
a Turbidity measurement method.b Ultraviolet spectrophotometry method.
0	57.9	17.2^a	17.0^b
5	58.1	22.0^a	22.1^b
10	58.1	26.2^a	24.9^b
20	58.0	31.7^a	31.9^b

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Fig. 5 Cloud point of L64-B(OH)₂ with different amounts of sorbic alcohol. (a) 0 mg sorbic alcohol; (b) 5 mg sorbic alcohol; (c) 10 mg sorbic alcohol; (d) 20 mg sorbic alcohol.

The above variation of cloud point is in keeping with the aforementioned mechanism, which reflects the effective combination between L64-B(OH)₂ and ortho-hydroxyl compounds and further confirms the successful preparation of L64-B(OH)₂.

The pH value of the system also has a significant impact on the cloud point of L64-B(OH)₂. As shown in Table 2, the cloud point of L64-B(OH)₂ increases with the increase of the pH value. The cloud point value determined by turbidity measurement method is in agreement with that determined by ultraviolet spectrophotometry method, which can be seen in Fig. 6. As discussed above, more L64-B(OH)₂ 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 modification of L64-B(OH)₂.

Table 2 Influence of pH on the cloud point of L64 and L64-B(OH)₂

pH	L64 (°C)	L64-B(OH)2 (°C)
a Turbidity measurement method.b Ultraviolet spectrophotometry method.
2	57.8	17.4^a
3	58.0	17.2^a
4	58.2	23.4^a	24.1^b
6	58.1	33.8^a	33.4^b
8	58.2	38.4^a	37.9^b
10	58.0	44.1^a	43.4^b
12	58.2	44.2^a	44.1^b

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Fig. 6 Cloud point of L64-B(OH)₂ solution at different pH values. (a) pH 4; (b) pH 6; (c) pH 8; (d) pH 10; (e) pH 12.

3.2.2 Salting-out experiment. The salting-out ability of a substance is closely related to the strength of its hydrophilia. In this section, the salting-out experiment was conducted by adding different amounts of K₂HPO₄ into the L64 solution (1%) and the L64-B(OH)₂ solution (1%) to evaluate the hydrophobicity–hydrophilicity strength of the polymer. As shown in Fig. 7a, there is no polymer being separated out from the L64 solution. In contrast, the precipitation of L64-B(OH)₂ can be observed obviously in Fig. 7b. In addition, with the increase of salt concentration, the precipitation amount of L64-B(OH)₂ tends to increase firstly and then decrease. During the first stage, the increase in the salting-out ability of K₂HPO₄ solution plays a leading role. With the addition of K₂HPO₄, the salting-out ability of K₂HPO₄ solution is enhanced, so more water molecules in the system would be captured by salt ions, leading to the increase in the precipitation amount of L64-B(OH)₂. However, in the second stage, as the adding amount of K₂HPO₄ further increases, the pH value of the system will increase and play the dominant role. The increase of pH value can make improvement in hydrophilicity of L64-B(OH)₂, so the precipitation amount of L64-B(OH)₂ decreases. The combined action of the above two factors cause the tendency that the precipitation amount of L64-B(OH)₂ increases firstly and then reduces with the increase of the salt concentration.

Fig. 7 Photographs of salting-out phenomenon of polymer at different K₂HPO₄ concentrations: (a) L64; (b) L64-B(OH)₂ (concentration of K₂HPO₄ is 10%, 20%, 30%, 40%, 50% from left to right, respectively.)

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 L64-B(OH)₂ shows a favorable absorption capacity to alizarin red, indicating a tight association of L64-B(OH)₂ with ortho-hydroxyl compounds. Meanwhile, it provides an effective method for the extraction of ortho-hydroxyl compounds affinity adsorption method.

Fig. 8 Photographs of alizarin red absorption: (a) alizarin red; (b) alizarin red + L64; (c) alizarin red + L64-B(OH)₂.

3.3 Application of phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀ in separation and purification of vicinal-diol-containing compounds

Alizarin red was ulitilized as the model compound to study the application of L64-B(OH)₂ for separation and purification of ortho-hydroxyl compounds. The reaction between L64-B(OH)₂ and alizarin red was verified according to the method in the literature.²² 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 L64-B(OH)₂ with the same amount. The three sample solutions were shown in Fig. 9. As an acid–base 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 3.7–5.2 and purple when the pH value is higher than 5.2. As shown in Fig. 9, it is obvious that the color of the L64-B(OH)₂ 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 L64-B(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 the result in the relevant literature.²² Hence, the effective combination between phenylboronic acid-functionalized PEO₂₀PPO₆₀PEO₂₀ and alizarin red was further confirmed.

Fig. 9 Affinity interaction between L64-B(OH)₂ and alizarin red. (a) Alizarin red; (b) alizarin red + L64; (c) alizarin red + L64-B(OH)₂.

3.3.1 Separation and purification of alizarin red by affinity adsorption system. Firstly, the influences of the concentration of L64-B(OH)₂ and the quantity of alizarin red on the affinity adsorption rate (E₁) of alizarin red were investigated while keeping the K₂HPO₄ 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 content of L64-B(OH)₂. As we know, alizarin red has only one vicinal-diol 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 L64-B(OH)₂, more alizarin red is adsorbed by the polymer. When the L64-B(OH)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 consideration, 2% of L64-B(OH)₂ concentration was selected for the following discussion.

Fig. 10 Influence of (a) concentration of L64-B(OH)₂ and quantity of alizarin red, (b) K₂HPO₄ concentration and (c) adsorption time on the affinity adsorption rate (E₁).

In addition, under different concentrations of L64-B(OH)₂, all the adsorption rate of 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 solution is quite low compared with that adsorbed by L64-B(OH)₂. As a result, the 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 L64-B(OH)₂ is 2% and the adding quantity of alizarin red is 0.3 mg, the adsorption rate of alizarin red reaches the maximum 98%.

Then the influence of K₂HPO₄ concentration on E₁, was investigated while keeping the adsorption time at 5 h and the result was revealed in Fig. 10b. With the gradual increase of K₂HPO₄ 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 K₂HPO₄, the pH value of the system rises. And so the balance of affinity reaction between alizarin red and L64-B(OH)₂ moves towards the right (Fig. 4), which makes more alizarin red be adsorbed. Meanwhile, higher salt concentration will cause stronger salting-out effect, making more modified polymer-alizarin red complex precipitate out. Therefore, the adsorption rate of alizarin red increases and reaches its maximum (98%) at 40% of salt concentration. Because the further increasing concentration of K₂HPO₄ 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 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 L64-B(OH)₂, and thus the adsorption rate would be improved. However, when the adsorption time extends to a certain value, the affinity reaction between L64-B(OH)₂ 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. Aqueous two-phase flotation is a new separation and purification technology, which is developed on the basis of aqueous two-phase extraction and solvent flotation. Compared with organic solvent extraction, aqueous two-phase extraction and solvent flotation, aqueous two-phase flotation has many advantages such as low solvent consumption, large enrichment factors, and high separation efficiency, which exhibits a promising application in the field of separation and purification.²³ Currently, there are many literatures about aqueous two-phase extraction,^24,25 but the reports on ATPF are few.^26,27 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.

Separation of water-soluble natural products from its extract liquor is always the technological difficulty for aqueous two-phase flotation, which limits its application in separation and purification of water-soluble 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 n-propanol salt ATPF system, alizarin red is more likely to distribute in aqueous phase, causing the poor flotation effect. In this section, L64-B(OH)₂ was used as the collector for the following two reasons: on the one hand, boronic acid group in L64-B(OH)₂ can 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 adsorbed on the interface of hydrophobic bubbles. The aqueous two-phase flotation schematic is shown in Fig. 11. Alizarin red can react with L64-B(OH)₂ to form a complex with surface activity, which contributes to the flotation of water-soluble alizarin red. Several single factors affecting the flotation recovery of alizarin red were discussed.

Fig. 11 Schematic diagram of aqueous two-phase flotation.

The impact of the added amount of collector (L64-B(OH)₂) on the flotation recovery of alizarin red (E₂) is shown in Fig. 12a. The flotation recovery of alizarin 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 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 E₂. From Fig. 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 E₂ (70%) was obtained. Therefore, 0.3 g of L64-B(OH)₂ was selected for the following discussion.

Fig. 12 Influence of (a) collector amount, (b) K₂HPO₄ amount, (c) alizarin red amount, (d) flow rate and (e) time on the flotation recovery (E₂).

Then, the impact of K₂HPO₄ concentration on E₂ is studied and revealed in Fig. 12b. With the increase of K₂HPO₄ concentration, the recovery of alizarin red tends to increase firstly and then decline and the maximum (87%) is obtained when the added quantity of K₂HPO₄ is 12 g. According to previous literatures,²⁸ the two incompatible phases in the flotation system were maintained by the salting-out effect. Thus, when the amount of salt is low at first, the salting-out effect is weak, which leads to the inadequate phase separation and the small volume of the top phase. Meanwhile, the collector–alizarin red complex has high solubility at the low salt concentration, so the recovery of alizarin red is low. With the increase in the K₂HPO₄ 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 K₂HPO₄ leads to the increase of pH value. Consequently, the collector prefers to exist in the form of electronegative tetrahedral structure, which is beneficial for the generation of collector–alizarin red complex. Therefore, the recovery of alizarin red increases from 36% to 87%. Whereas, the further increase in the added amount of K₂HPO₄ 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 K₂HPO₄ 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 floated 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.

Herein, the influence of nitrogen flow rate (5–25 mL min⁻¹) 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 recovery and shorten the time.²⁹ When the recovery reaches the maximum, the further increase in the nitrogen flow rate will lead to the adverse effect, because the too high nitrogen flow rate can result in the turbulent mixing in the interface.^30,31 Then many bubbles will gather in the top phase of the system³² and the phase equilibrium is broken. Consequently, alizarin red, which has been dissolved in the top phase, will re-dissolve in the bottom phase and the recovery of alizarin red declines accordingly. According to Fig. 12d, when the nitrogen flow rate is 15 mL min⁻¹, the recovery of alizarin red reaches the maximum (95%). Hence, 15 mL min⁻¹ of nitrogen flow rate was selected for the following discussion.

As described in the previous literatures, the flotation effect can rise with the extending of flotation time.^33,34 Herein, the influence of the flotation time on the effect of ATPF of alizarin red was investigated and the result is revealed in Fig. 12e. At first, the recovery rate rises with the extending of the flotation time. However, when the flotation system reaches the balance, further extending the flotation time cannot improve the flotation effect.³⁵ As shown in Fig. 12e, when the recovery of alizarin red reaches 97%, it almost keeps constant although the flotation time continues extending. Taking recovery, time and cost into consideration, 40 min was selected as the flotation time.

In addition, to verify the important function of L64-B(OH)₂ which was used as a collector during the flotation process, the following flotation comparison experiments were conducted. Under the optimal flotation condition (12 g of K₂HPO₄, 0.6 mg of alizarin red, 15 mL min⁻¹ of nitrogen flow rate, and 40 min of flotation time), ATPF was respectively carried out in three systems (with no collector, with 0.3 g L64, and with 0.3 g L64-B(OH)₂) and the flotation effects were compared. The result is revealed in Fig. 13. The recovery of alizarin red increases from 20% to 97% obviously, which indicates the significant effect of the collector (L64-B(OH)₂) for the separation of vicinal-diol-containing compounds in the ATPF system.

Fig. 13 Flotation recovery (E₂) of alizarin red under different ATPF systems.

4. Conclusions

In this work, a kind of stimuli-responsive polymer, L64-B(OH)₂, was successfully prepared. Based on the amphiphilicity and stimuli-responsive performance of PEO₂₀PPO₆₀PEO₂₀ as well as the affinity interaction of phenylboronic acid with vicinal-diol-containing compounds, this modified polymer was applied in the affinity adsorption system and aqueous two-phase flotation system for the first time. Under the optimized conditions, the recovery of the model compound (alizarin red) by affinity adsorption system and aqueous two-phase flotation system based on L64-B(OH)₂ achieved 98% and 97%, respectively. This work provides a new method to the separation and purification of vicinal-diol-containing compounds with strong hydrophilia.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31470434, 21406090, and 21576124), the Special Financial Grant from the China Postdoctoral Science Foundation (No. 2015T80510), and the Project of Science and Technology Development plan of Taicang (No. TC2015NY05).

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