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A new temperature-sensitive polymer: Poly(ethoxypropylacrylamide)
EUROPEAN POLYMER JOURNAL
European Polymer Journal 41 (2005) 2142–2149
www.elsevier.com/locate/europolj
A new temperature-sensitive polymer: Poly(ethoxypropylacrylamide) ¨ ztu¨rk b, E. Ug˘uzdog˘an a, T. C ¸ amlı b, O.S. Kabasakal c, S. Patır d, E. O b e,* E.B. Denkbasß , A. Tuncel a
e
Pamukkale University, Chemical Engineering Department, Denizli, Turkey b Hacettepe University, Chemistry Department, Ankara, Turkey c Osmangazi University, Chemical Engineering Department, Eskisßehir, Turkey d Hacettepe University, Department of Science Education, Ankara, Turkey Hacettepe University, Chemical Engineering Department, 06532 Beytepe, Ankara, Turkey
Received 18 October 2004; received in revised form 21 March 2005; accepted 5 April 2005 Available online 13 June 2005
Abstract In this study, a new temperature sensitive polymer was obtained by the solution polymerization of ethoxypropylacrylamide. The monomer, N-(3-ethoxypropyl)-acrylamide was synthesized by the nucleophilic substitution reaction of 3-ethoxy-propylamine and acryloyl chloride. The solution polymerization was performed in ethanol at 70 C, by using azobisizobutyronitrile as the initiator. Poly(N-(3-ethoxypropyl)acrylamide), PEPA, exhibited a reversible phase transition by the temperature. The effects of polymer and salt concentrations on the lower critical solution temperature, (LCST) behaviour were investigated. LCST was found to be strongly dependent on the polymer concentration. The dynamic light scattering (DLS) measurements confirmed the formation of aggregates by the association of nucleated polymer chains at the temperatures higher than LCST. However an unusual behaviour, a marked decrease in the hydrodynamic diameter by the increasing PEPA concentration was observed below the LCST. The effect of salt concentration on the critical flocculation temperature of PEPA was reasonably similar to poly(isopropylacrylamide), PNIPA. In the ethanol–water media, the reversible phase transition behaviour was observed up the ethanol concentration of 30% v/v. This study indicated that PEPA was a new alternative thermally reversible material for PNIPA. With respect to the well-defined temperature-sensitive polymers like PNIPA, polymer concentration dependent LCST of PEPA can provide significant advantages in the applications like drug targeting, affinity separation and immobilization of bioactive agents. 2005 Elsevier Ltd. All rights reserved. Keywords: Temperature-sensitive polymer; Alkylacrylamide; Isopropylacrylamide; Ethoxypropylacrylamide; LCST; Solution polymerization
1. Introduction
*
Corresponding author. Fax: +90 312 299 21 24. E-mail address: [email protected] (A. Tuncel).
Temperature-sensitive polymers have been usually obtained by the polymerization of N-alkylacrylamide monomers. These monomers are synthesized by nucleophilic substitution reaction of acryloyl chloride
0014-3057/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2005.04.007
E. Ug˘uzdog˘an et al. / European Polymer Journal 41 (2005) 2142–2149
with the appropriate amine. The most commonly used monomers for the synthesis of temperature-sensitive polymeric forms are N-isopropylacrylamide (NIPA), N-isopropylmethacrylamide and diethylacrylamide [1– 12]. Lower critical solution temperature (LCST) values of polymers are found to be controlled by the alkyl group bound to the nitrogen atom. The polymers prepared by the polymerization of NIPA generally exhibit LCST values in the range of 30–32 C depending upon the synthesis method [1–10]. In the biotechnological applications, some chemical modifications can be necessary in the structure of poly(N-isopropylacrylamide), PNIPA [3,4]. These modifications are usually made for introducing some functional groups or improving the mechanical properties of the gel matrices. In our previous studies, NIPA based, thermally sensitive linear polymers with different functionalities were synthesized and used in the temperature controlled isolation of various biomolecules like proteins, nucleotides and RNA [13– 18]. NIPA also was co-polymerized with some monomers in order to change the LCST. However, these efforts may cause a decrease in the thermosensitivity of resulting material [5–9]. The synthesis of alternative thermosensitive polymeric materials can trigger the developments in the various applications of stimuliresponsive polymers. Various n-alkylacrylamide based monomers (i.e., isopropylmethacrylamide, diethylacrylamide, methylpropylacrylamide, cyclopropylacrylamide, propylacrylamide, ethylpropylacrylamide, n- and tert-butylacrylamide and ethoxyethylacrylamide, etc.) were used for the synthesis of thermally sensitive polymers [19–29]. In some of these studies, thermoresponsive behaviour of poly(alkylacrylamide)s were comparatively investigated [19,21,26,28,29]. However no attempt was made for the preparation of ethoxypropylacrylamide based thermosensitive polymers. In our study, N-(3-ethoxypropyl)acrylamide (EPA), was synthesized by the nucleophilic substitution of acryloyl chloride with 3-ethoxypropylamine. The new thermally sensitive polymer, poly(N-(3-ethoxypropyl)acrylamide), PEPA was obtained by the solution polymerization. The thermoresponsive behaviour of PEPA homopolymer was investigated in the aqueous media.
2. Experimental 2.1. Materials The monomer, N-(3-ethoxypropyl)acrylamide (EPA) was synthesized by using acryloyl chloride (Aldrich Chemicals Co., Milwaukee, WI, USA) and 3-ethoxypropylamine (Aldrich Chemicals Co.). In the polymerizations, azobisizobutyronitrile recrystallized from methanol (AIBN) (BDH Chemicals Ltd., Poole, UK)
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and absolute ethanol (Merck A.G., Darmstad, Germany) were used as the initiator and the solvent, respectively. 2.2. Monomer synthesis A modified form of the synthesis protocol proposed for the preparation of different alkylacrylamide monomers was also used for the synthesis of N-(3-ethoxypropyl)-acrylamide (EPA) [28,29]. Typically, 3-ethoxypropylamine (0.11 mol) and p-benzoquinone (10 mg) were added to a solution of triethylamine (0.12 mol) in dichloromethane (200 ml) cooled to 0 C in an ice bath. Acryloyl chloride (0.10 mol)-dichloromethane (40 ml) mixture was dropped into the solution at approximately 0 C, in a duration of 2 h. The resulting solution was then stirred magnetically for 24 h at +4 C. The organic phase extracted with cold water and dried over MgSO4 was evaporated in vacuo for the isolation of EPA. The monomer was characterized by H NMR and FTIR spectroscopy. H NMR characteristics: (CDCl3, 400 MHz): d = 1.19 (t, 3H, CH3, J = 7.01 Hz), 1.78–1.84 (m, 2H, CH2), 3.40–3.53 (m, 6H, CH2) 5.57–5.60 (m, 1H, vinylH), 6.13–6.25 (m, 2H, vinyl-H), 6.83 (bs, 1H, NH). FTIR characteristics: 3283 (N–H, amide), 3078-2802 (alkyl), 1659 (C@C), 1552 (amide II). 2.3. Polymerization For the solution polymerization, EPA (1.2 g) and AIBN (0.035 g) were dissolved in ethanol (12 ml) in a pyrex glass polymerization reactor. Prior to the polymerization, the solution was purged with bubbling nitrogen for 5 min. The sealed reactor was placed in a shaken water bath at room temperature and the reactor was heated to the polymerization temperature (70 C) in the presence of shaking at 120 cpm. The polymerization was performed at 70 C for 12 h. After partial removal of ethanol in vacuo, the polymer was precipitated by the addition of excess petroleum ether. The polymer was redissolved in distilled water (10 ml) and the solution was reheated to the lower critical solution temperature (LCST) of PEPA. Hence the dissolved polymer was reprecipitated. The polymer was purified by successive dissolution and precipitation and dried for 2 days at 50 C in vacuo. The polymerization yield (CT% w/w) was determined according to Eq. (1), where the Mp and Mm are the weight of the polymer isolated and the weight of EPA charged into the reactor, respectively. C T ð%w=wÞ ¼ ðM p =M m Þ 100
ð1Þ
2.4. Temperature sensitivity of PEPA LCST of PEPA was determined by a UV–Vis spectrophotomer (Shimadzu, Kyoto, Japan) equipped with
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a flow-cell at which the absorbance of the polymer solution (pH 7, polymer concentration: variable between 0.1% and 5.0% w/w) was continuously measured at 500 nm with the increasing temperature. For this purpose, the polymer solution kept in a reservoir was circulated through the flow cell with a high flow rate (10 ml/ min). During the circulation, the temperature of the solution in the reservoir was increased with a heating rate of 1 C/min in a temperature range of 15–50 C. The temperature was also measured on the flow-cell with a home-made equipment. In the absorbance–temperature plot, the temperature corresponding to 10% of the total absorbance increase was evaluated as LCST. The critical flocculation temperature, CFT was also determined by the evaluation of absorbance vs. temperature curves of the aqueous polymer solutions containing NaCl with different concentrations ranging between 0.1 and 1.0 M. For this purpose, the protocol given above was used in the presence of salt (i.e., NaCl). In order to investigate the effect of alcohol concentration on the temperature sensitivity of PEPA, LCST values of PEPA in the aqueous media containing ethanol (variable between 0% and 100% v/v) were also determined by following the protocol given above. The thermosensitive behaviour of PEPA was also investigated by dynamic light scattering (DLS, Malvern, Zetasizer Nano S, UK). The DLS measurements were performed with an angle of 170 by using He–Ne laser (4 mW) operated at 633 nm. In these measurements, PEPA concentration was changed between 101 and 106 g/ml in an aqueous medium having an ionic strength of 103. The hydrodynamic size and the polydispersity index (PDI) giving the relative standard deviation for the size distribution of PEPA chains were determined by DLS. These values were measured for both polymer chains in the soluble form and the aggregates formed by the precipitated polymer chains.
high initiator concentrations. The production rate of initiator radicals increases with the increasing initiator concentration. This makes possible to initiate the polymerization via more monomer molecules and provides higher propagation rate. The increase in the propagation rate probably leads to an increase in the polymerization yield (i.e., higher monomer conversion). In order to determine the LCST of PEPA, the absorbance of aqueous PEPA solution was measured at 500 nm against temperature. For the PEPA solutions with different polymer concentrations, the variation of visible region absorbance with the temperature is given in Fig. 1. As seen here, the absorbance started to increase at lower temperature with increasing PEPA concentration. This finding indicated that the phase transition occurred at lower temperature with increasing PEPA concentration. In other words, LCST of PEPA was dependent on the polymer concentration. For the lowest PEPA concentration (i.e., 0.1% w/w), the absorbance continuously increased with the increasing temperature and reached a plateau. However, the absorbance–temperature curves obtained with higher PEPA concentrations exhibited a maximum and they were different than that observed with the lowest PEPA concentration. As described in the literature, the phase separation between polymer and water occurs at a certain temperature and the nucleated polymer chains form a stable colloidal suspension [21,23–26]. In such a case, the absorbance shows a continuous increase by the increasing concentration of nucleated polymer chains (i.e., the curve observed with the PEPA concentration of 0.1% in Fig. 1). In the presence of high polymer concentration, the small flocks formed by phase separation associate and larger aggregates are probably formed.
2.5
3. Results and discussion Thermosensitive poly(N-(3-ethoxypropyl)acrylamide), PEPA was obtained by the solution polymerization of EPA. The variation of polymerization yield with the initiator concentration is shown in Table 1. As seen here, higher polymerization yields were obtained with
Absorbance
2.0
PEPA concentration (% w/w) 0.1 0.5 1.0 5.0
1.5 1.0 0.5
Table 1 The effect of initiator concentration on the polymerization yield AIBN concentration (mg/ml)
CT (% w/w)
0.8 1.4 5.8 11.7
74.2 79.2 86.7 85.8
0.0 20
30
40
50
60
70
80
90
Temperature (°C) Fig. 1. The variation of absorbance at 500 nm with the temperature for different PEPA concentrations.
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These larger aggregates begin to precipitate when polymeric dispersion becomes unstable. The occurrence of this precipitation phenomenon is determined by a decrease in the absorbance value of the solution. In the case of high polymer concentration, the absorbance– temperature relation of the polymer solution is represented by a bell-shaped curve (i.e., for the PEPA concentrations higher than 0.5%) while it usually gives an S shaped curve with low polymer concentration (PEPA concentration of 0.1%). For each PEPA concentration, LCST was determined as the temperature corresponding to 10% of the total absorbance change observed in Fig. 1. The variation of LCST with PEPA concentration is given in Fig. 2. As seen here, LCST decreased significantly with increasing PEPA concentration. This behaviour was particularly observed in the concentration range of 0.1–1.0% PEPA. There was no significant change in LCST for the PEPA concentrations higher than 1.0%. For PNIPA homopolymer, different tendencies were reported for the variation of LCST with the homopolymer concentration. Fujishige et al. [30] reported that the phase transition of PNIPA took place at 31 C independently of either the molecular weight of the polymer or its concentration. A similar result was also reported for the variation of cloud point of PNIPA with the polymer concentration [26]. However, according to the study performed by Meyer et al. [27] LCST decreased from 41.5 to 37 C by increasing PNIPA concentration approximately 0.02–2.0% w/w . In another study performed by Tong et al. [31] LCST of NIPA decreased from approximately 33–30 C by increasing the PNIPA homopolymer concentration from 0.58 up to 70%
(w/w). The effect of polymer concentration on the LCST seemed stronger for PEPA with respect to well-known thermosensitive polymer, PNIPA. Polymer concentration dependent LCST of PEPA can provide significant advantages in the applications like drug targeting, affinity separation and immobilization of bioactive agents [3,8,13,16,27]. The aggregation behaviour of PEPA in the aqueous medium was also examined by DLS. The hydrodynamic size measurements were performed in the aqueous medium at pH 7, having an ionic strength of 1 · 103. The variation of hydrodynamic diameter with the PEPA concentration is shown in Fig. 3 at both 20 and 40 C. The lower temperature (i.e., 20 C) was the one below the LCST and the higher temperature (i.e., 40 C) was the one close to or higher than LCST in the selected PEPA concentration range. As expected the hydrodynamic radius increased with increasing PEPA concentration at a temperature close to the LCST of PEPA (i.e., 40 C). The formation of larger aggregates (i.e., an increase in the hydrodynamic radius) with increasing homopolymer concentration was also reported for PNIPA at 33 C [32]. This increase should originate from the interchain association of nucleated polymer chains with the increasing polymer concentration at a temperature close to the LCST [32]. Note that a similar increase with the polymer concentration was also observed for poly(ethylene oxide) grafted PNIPA and PNIPAblock-poly(ethylene oxide) [33,34]. At the temperatures causing the precipitation of polymer chains, the LCST decrease is attributed to the formation of larger aggregates with increasing homopolymer concentration [32,34].
103 Hydrodynamic diameter (nm)
42
40
LCST (°C)
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38
36
34
32
102
101 Temperature (°C) 20 40 100 10-6
0
1
2
3
4
5
1x10-5
1x10-4
10-3
10-2
10-1
PEPA concentration (g/mL)
PEPA concentration (% w/w) Fig. 2. The variation of LCST with PEPA concentration.
Fig. 3. The variation of hydrodynamic diameter with the PEPA concentration.
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1
1000
Polydispersity Index (PDI)
However, an unusual behaviour was obtained for the variation of hydrodynamic size with the PEPA concentration at 20 C. As seen here, the hydrodynamic size markedly decreased with the increasing PEPA concentration and the lowest hydrodynamic size values were obtained with the PEPA concentrations higher than 0.01 g/ml. By considering this behaviour, the effect of temperature on the hydrodynamic diameter and the size distribution of PEPA was investigated with a sufficiently high PEPA concentration (i.e., 0.05 g/ml). The diagrams showing the size distribution of PEPA homopolymer at different temperatures are exemplified in Fig. 4. The variation of z-average diameter by the temperature is shown in Fig. 5. The polydispersity index (PDI) values obtained at different temperatures are also shown on the same figure. As seen here, PDI exhibited a minimum at 30 C (i.e., near the LCST of PEPA) and increased with the temperature after the LCST. The relatively high PDI at 50 C should be explained by the formation of large aggregates with relatively wide size distribution. In Fig. 5, the hydrodynamic diameter showed a sharp increase at LCST. This tendency was very similar to that observed for the diblock co-polymer of poly(Nisopropylacrylamide) and poly(N-(2hydroxypropyl)methacrylamide), PNIPA-block-PHPMA [35]. In the referred study, the size increase with the increasing temperature was explained by the association of precipitated polymer chains [35]. Note that the order of magnitude of the hydrodynamic diameters measured below the LCST of PEPA were also very close to that of PNIPA-blockPHPMA below its LCST [35]. For PEPA concentrations of 0.1% and 1.0%, the plots showing the variation of absorbance with the temperature at different NaCl concentrations are given in Fig. 6a and b, respectively. As seen here, the thermal flocculation was observed with all salt concentrations at both PEPA concentrations. In the presence of salt, the temperature corresponding to the maximum point of the absorbance–temperature curve is described as the ‘‘critical flocculation temperature’’, (CFT). For
Hydrodynamic size (nm)
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100
10 Hydrodynamic diameter PDI
1
0.1 10
20
30
40
50
Temperature (°C) Fig. 5. The variation of hydrodynamic diameter and polydispersity index by the temperature.
PEPA homopolymer, the variation of CFT with the NaCl concentration is shown in Fig. 7. As seen here, CFT exhibited a linear decrease with the increasing salt concentration for PEPA homopolymer. The slopes of the straight lines in Fig. 7 were determined as 10.1 and 7.1 C/M for the PEPA concentrations of 0.1% and 1.0%, respectively. Here the slope of the linear relationship between CFT and salt concentration is a measure of the salt concentration effect on the flocculation behaviour of the thermosensitive polymer. Similar tendencies with the slope values very close to those found in our study were previously obtained by Schild and Tirrell for the effect of NaBr concentration on the LCST of PNIPA [20]. Recently, the variation of CFT with the NaCl concentration was investigated for PNIPA microgel particles in sub-micron size range [36]. For this variation, a linear tendency with a slope of 12.2 C/M was obtained with a particle concentration of 0.06% for the NaCl concentration range of 0.1–0.8 M [36]. A similar
Fig. 4. The diagrams showing the size distribution of PEPA homopolymer at different temperatures.
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a
1.0
b
1.0
0.8 Absorbance
0.8 Absorbance
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0.6
0.4
0.6
0.4
NaCl concentration (M)
NaCl concentration (M)
0.0
0.2
0.2
0.1
0.0 0.1
0.5
0.5
1.0
0.0 20
30
40
50
60
1.0
0.0 70
80
90
20
30
Temperature (°C)
40
50
60
70
Temperature (°C)
Fig. 6. The plots showing the effect of salt concentration on the variation of absorbance with temperature. (k = 500 nm), PEPA concentration: (a) 0.1% and (b) 1% w/w.
54
34
PEPA concentration (% w/w) 0.1 1.0
52 50
33
48 32 LCST (°C)
CFT (°C)
46 44 42 40
31
No phase separation
30
38 29
36 34
28
32 0.0
0.2
0.4
0.6
0.8
1.0
NaCl concentration (M)
0
10
20
30
40
50
60
70
80
90 100
Ethanol concentration (% v/v)
Fig. 7. The effect of NaCl concentration on the CFT of PEPA.
Fig. 8. The variation of LCST with the ethanol concentration in the aqueous medium. PEPA concentration: 1% w/w.
relation with approximately the same slope was also obtained by Daly and Saunders [37]. By comparing the slopes obtained for PNIPA and PEPA, one concludes that salt concentration is less effective on the temperature induced flocculation of PEPA chains with respect to PNIPA. The effect of ethanol concentration on the LCST of PEPA is shown in Fig. 8. As seen here, when the ethanol concentration was kept in the range of 0–30% (v/v), LCST significantly decreased with increasing alcohol
concentration. However, in the aqueous media with the ethanol concentration higher than 40% (v/v), the polymer was soluble in the temperature range of 20– 70 C. Thus, LCST could not be detected in this range since no chain precipitation occurred with increasing temperature. For PNIPA homopolymer, the co-nonsolvency in alcohol/water mixtures was extensively investigated by different researchers [38–41]. These results indicated that phase transition of PNIPA was observed in the methanol–water mixtures containing methanol
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European Polymer Journal 41 (2005) 2142–2149
www.elsevier.com/locate/europolj
A new temperature-sensitive polymer: Poly(ethoxypropylacrylamide) ¨ ztu¨rk b, E. Ug˘uzdog˘an a, T. C ¸ amlı b, O.S. Kabasakal c, S. Patır d, E. O b e,* E.B. Denkbasß , A. Tuncel a
e
Pamukkale University, Chemical Engineering Department, Denizli, Turkey b Hacettepe University, Chemistry Department, Ankara, Turkey c Osmangazi University, Chemical Engineering Department, Eskisßehir, Turkey d Hacettepe University, Department of Science Education, Ankara, Turkey Hacettepe University, Chemical Engineering Department, 06532 Beytepe, Ankara, Turkey
Received 18 October 2004; received in revised form 21 March 2005; accepted 5 April 2005 Available online 13 June 2005
Abstract In this study, a new temperature sensitive polymer was obtained by the solution polymerization of ethoxypropylacrylamide. The monomer, N-(3-ethoxypropyl)-acrylamide was synthesized by the nucleophilic substitution reaction of 3-ethoxy-propylamine and acryloyl chloride. The solution polymerization was performed in ethanol at 70 C, by using azobisizobutyronitrile as the initiator. Poly(N-(3-ethoxypropyl)acrylamide), PEPA, exhibited a reversible phase transition by the temperature. The effects of polymer and salt concentrations on the lower critical solution temperature, (LCST) behaviour were investigated. LCST was found to be strongly dependent on the polymer concentration. The dynamic light scattering (DLS) measurements confirmed the formation of aggregates by the association of nucleated polymer chains at the temperatures higher than LCST. However an unusual behaviour, a marked decrease in the hydrodynamic diameter by the increasing PEPA concentration was observed below the LCST. The effect of salt concentration on the critical flocculation temperature of PEPA was reasonably similar to poly(isopropylacrylamide), PNIPA. In the ethanol–water media, the reversible phase transition behaviour was observed up the ethanol concentration of 30% v/v. This study indicated that PEPA was a new alternative thermally reversible material for PNIPA. With respect to the well-defined temperature-sensitive polymers like PNIPA, polymer concentration dependent LCST of PEPA can provide significant advantages in the applications like drug targeting, affinity separation and immobilization of bioactive agents. 2005 Elsevier Ltd. All rights reserved. Keywords: Temperature-sensitive polymer; Alkylacrylamide; Isopropylacrylamide; Ethoxypropylacrylamide; LCST; Solution polymerization
1. Introduction
*
Corresponding author. Fax: +90 312 299 21 24. E-mail address: [email protected] (A. Tuncel).
Temperature-sensitive polymers have been usually obtained by the polymerization of N-alkylacrylamide monomers. These monomers are synthesized by nucleophilic substitution reaction of acryloyl chloride
0014-3057/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2005.04.007
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with the appropriate amine. The most commonly used monomers for the synthesis of temperature-sensitive polymeric forms are N-isopropylacrylamide (NIPA), N-isopropylmethacrylamide and diethylacrylamide [1– 12]. Lower critical solution temperature (LCST) values of polymers are found to be controlled by the alkyl group bound to the nitrogen atom. The polymers prepared by the polymerization of NIPA generally exhibit LCST values in the range of 30–32 C depending upon the synthesis method [1–10]. In the biotechnological applications, some chemical modifications can be necessary in the structure of poly(N-isopropylacrylamide), PNIPA [3,4]. These modifications are usually made for introducing some functional groups or improving the mechanical properties of the gel matrices. In our previous studies, NIPA based, thermally sensitive linear polymers with different functionalities were synthesized and used in the temperature controlled isolation of various biomolecules like proteins, nucleotides and RNA [13– 18]. NIPA also was co-polymerized with some monomers in order to change the LCST. However, these efforts may cause a decrease in the thermosensitivity of resulting material [5–9]. The synthesis of alternative thermosensitive polymeric materials can trigger the developments in the various applications of stimuliresponsive polymers. Various n-alkylacrylamide based monomers (i.e., isopropylmethacrylamide, diethylacrylamide, methylpropylacrylamide, cyclopropylacrylamide, propylacrylamide, ethylpropylacrylamide, n- and tert-butylacrylamide and ethoxyethylacrylamide, etc.) were used for the synthesis of thermally sensitive polymers [19–29]. In some of these studies, thermoresponsive behaviour of poly(alkylacrylamide)s were comparatively investigated [19,21,26,28,29]. However no attempt was made for the preparation of ethoxypropylacrylamide based thermosensitive polymers. In our study, N-(3-ethoxypropyl)acrylamide (EPA), was synthesized by the nucleophilic substitution of acryloyl chloride with 3-ethoxypropylamine. The new thermally sensitive polymer, poly(N-(3-ethoxypropyl)acrylamide), PEPA was obtained by the solution polymerization. The thermoresponsive behaviour of PEPA homopolymer was investigated in the aqueous media.
2. Experimental 2.1. Materials The monomer, N-(3-ethoxypropyl)acrylamide (EPA) was synthesized by using acryloyl chloride (Aldrich Chemicals Co., Milwaukee, WI, USA) and 3-ethoxypropylamine (Aldrich Chemicals Co.). In the polymerizations, azobisizobutyronitrile recrystallized from methanol (AIBN) (BDH Chemicals Ltd., Poole, UK)
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and absolute ethanol (Merck A.G., Darmstad, Germany) were used as the initiator and the solvent, respectively. 2.2. Monomer synthesis A modified form of the synthesis protocol proposed for the preparation of different alkylacrylamide monomers was also used for the synthesis of N-(3-ethoxypropyl)-acrylamide (EPA) [28,29]. Typically, 3-ethoxypropylamine (0.11 mol) and p-benzoquinone (10 mg) were added to a solution of triethylamine (0.12 mol) in dichloromethane (200 ml) cooled to 0 C in an ice bath. Acryloyl chloride (0.10 mol)-dichloromethane (40 ml) mixture was dropped into the solution at approximately 0 C, in a duration of 2 h. The resulting solution was then stirred magnetically for 24 h at +4 C. The organic phase extracted with cold water and dried over MgSO4 was evaporated in vacuo for the isolation of EPA. The monomer was characterized by H NMR and FTIR spectroscopy. H NMR characteristics: (CDCl3, 400 MHz): d = 1.19 (t, 3H, CH3, J = 7.01 Hz), 1.78–1.84 (m, 2H, CH2), 3.40–3.53 (m, 6H, CH2) 5.57–5.60 (m, 1H, vinylH), 6.13–6.25 (m, 2H, vinyl-H), 6.83 (bs, 1H, NH). FTIR characteristics: 3283 (N–H, amide), 3078-2802 (alkyl), 1659 (C@C), 1552 (amide II). 2.3. Polymerization For the solution polymerization, EPA (1.2 g) and AIBN (0.035 g) were dissolved in ethanol (12 ml) in a pyrex glass polymerization reactor. Prior to the polymerization, the solution was purged with bubbling nitrogen for 5 min. The sealed reactor was placed in a shaken water bath at room temperature and the reactor was heated to the polymerization temperature (70 C) in the presence of shaking at 120 cpm. The polymerization was performed at 70 C for 12 h. After partial removal of ethanol in vacuo, the polymer was precipitated by the addition of excess petroleum ether. The polymer was redissolved in distilled water (10 ml) and the solution was reheated to the lower critical solution temperature (LCST) of PEPA. Hence the dissolved polymer was reprecipitated. The polymer was purified by successive dissolution and precipitation and dried for 2 days at 50 C in vacuo. The polymerization yield (CT% w/w) was determined according to Eq. (1), where the Mp and Mm are the weight of the polymer isolated and the weight of EPA charged into the reactor, respectively. C T ð%w=wÞ ¼ ðM p =M m Þ 100
ð1Þ
2.4. Temperature sensitivity of PEPA LCST of PEPA was determined by a UV–Vis spectrophotomer (Shimadzu, Kyoto, Japan) equipped with
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a flow-cell at which the absorbance of the polymer solution (pH 7, polymer concentration: variable between 0.1% and 5.0% w/w) was continuously measured at 500 nm with the increasing temperature. For this purpose, the polymer solution kept in a reservoir was circulated through the flow cell with a high flow rate (10 ml/ min). During the circulation, the temperature of the solution in the reservoir was increased with a heating rate of 1 C/min in a temperature range of 15–50 C. The temperature was also measured on the flow-cell with a home-made equipment. In the absorbance–temperature plot, the temperature corresponding to 10% of the total absorbance increase was evaluated as LCST. The critical flocculation temperature, CFT was also determined by the evaluation of absorbance vs. temperature curves of the aqueous polymer solutions containing NaCl with different concentrations ranging between 0.1 and 1.0 M. For this purpose, the protocol given above was used in the presence of salt (i.e., NaCl). In order to investigate the effect of alcohol concentration on the temperature sensitivity of PEPA, LCST values of PEPA in the aqueous media containing ethanol (variable between 0% and 100% v/v) were also determined by following the protocol given above. The thermosensitive behaviour of PEPA was also investigated by dynamic light scattering (DLS, Malvern, Zetasizer Nano S, UK). The DLS measurements were performed with an angle of 170 by using He–Ne laser (4 mW) operated at 633 nm. In these measurements, PEPA concentration was changed between 101 and 106 g/ml in an aqueous medium having an ionic strength of 103. The hydrodynamic size and the polydispersity index (PDI) giving the relative standard deviation for the size distribution of PEPA chains were determined by DLS. These values were measured for both polymer chains in the soluble form and the aggregates formed by the precipitated polymer chains.
high initiator concentrations. The production rate of initiator radicals increases with the increasing initiator concentration. This makes possible to initiate the polymerization via more monomer molecules and provides higher propagation rate. The increase in the propagation rate probably leads to an increase in the polymerization yield (i.e., higher monomer conversion). In order to determine the LCST of PEPA, the absorbance of aqueous PEPA solution was measured at 500 nm against temperature. For the PEPA solutions with different polymer concentrations, the variation of visible region absorbance with the temperature is given in Fig. 1. As seen here, the absorbance started to increase at lower temperature with increasing PEPA concentration. This finding indicated that the phase transition occurred at lower temperature with increasing PEPA concentration. In other words, LCST of PEPA was dependent on the polymer concentration. For the lowest PEPA concentration (i.e., 0.1% w/w), the absorbance continuously increased with the increasing temperature and reached a plateau. However, the absorbance–temperature curves obtained with higher PEPA concentrations exhibited a maximum and they were different than that observed with the lowest PEPA concentration. As described in the literature, the phase separation between polymer and water occurs at a certain temperature and the nucleated polymer chains form a stable colloidal suspension [21,23–26]. In such a case, the absorbance shows a continuous increase by the increasing concentration of nucleated polymer chains (i.e., the curve observed with the PEPA concentration of 0.1% in Fig. 1). In the presence of high polymer concentration, the small flocks formed by phase separation associate and larger aggregates are probably formed.
2.5
3. Results and discussion Thermosensitive poly(N-(3-ethoxypropyl)acrylamide), PEPA was obtained by the solution polymerization of EPA. The variation of polymerization yield with the initiator concentration is shown in Table 1. As seen here, higher polymerization yields were obtained with
Absorbance
2.0
PEPA concentration (% w/w) 0.1 0.5 1.0 5.0
1.5 1.0 0.5
Table 1 The effect of initiator concentration on the polymerization yield AIBN concentration (mg/ml)
CT (% w/w)
0.8 1.4 5.8 11.7
74.2 79.2 86.7 85.8
0.0 20
30
40
50
60
70
80
90
Temperature (°C) Fig. 1. The variation of absorbance at 500 nm with the temperature for different PEPA concentrations.
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These larger aggregates begin to precipitate when polymeric dispersion becomes unstable. The occurrence of this precipitation phenomenon is determined by a decrease in the absorbance value of the solution. In the case of high polymer concentration, the absorbance– temperature relation of the polymer solution is represented by a bell-shaped curve (i.e., for the PEPA concentrations higher than 0.5%) while it usually gives an S shaped curve with low polymer concentration (PEPA concentration of 0.1%). For each PEPA concentration, LCST was determined as the temperature corresponding to 10% of the total absorbance change observed in Fig. 1. The variation of LCST with PEPA concentration is given in Fig. 2. As seen here, LCST decreased significantly with increasing PEPA concentration. This behaviour was particularly observed in the concentration range of 0.1–1.0% PEPA. There was no significant change in LCST for the PEPA concentrations higher than 1.0%. For PNIPA homopolymer, different tendencies were reported for the variation of LCST with the homopolymer concentration. Fujishige et al. [30] reported that the phase transition of PNIPA took place at 31 C independently of either the molecular weight of the polymer or its concentration. A similar result was also reported for the variation of cloud point of PNIPA with the polymer concentration [26]. However, according to the study performed by Meyer et al. [27] LCST decreased from 41.5 to 37 C by increasing PNIPA concentration approximately 0.02–2.0% w/w . In another study performed by Tong et al. [31] LCST of NIPA decreased from approximately 33–30 C by increasing the PNIPA homopolymer concentration from 0.58 up to 70%
(w/w). The effect of polymer concentration on the LCST seemed stronger for PEPA with respect to well-known thermosensitive polymer, PNIPA. Polymer concentration dependent LCST of PEPA can provide significant advantages in the applications like drug targeting, affinity separation and immobilization of bioactive agents [3,8,13,16,27]. The aggregation behaviour of PEPA in the aqueous medium was also examined by DLS. The hydrodynamic size measurements were performed in the aqueous medium at pH 7, having an ionic strength of 1 · 103. The variation of hydrodynamic diameter with the PEPA concentration is shown in Fig. 3 at both 20 and 40 C. The lower temperature (i.e., 20 C) was the one below the LCST and the higher temperature (i.e., 40 C) was the one close to or higher than LCST in the selected PEPA concentration range. As expected the hydrodynamic radius increased with increasing PEPA concentration at a temperature close to the LCST of PEPA (i.e., 40 C). The formation of larger aggregates (i.e., an increase in the hydrodynamic radius) with increasing homopolymer concentration was also reported for PNIPA at 33 C [32]. This increase should originate from the interchain association of nucleated polymer chains with the increasing polymer concentration at a temperature close to the LCST [32]. Note that a similar increase with the polymer concentration was also observed for poly(ethylene oxide) grafted PNIPA and PNIPAblock-poly(ethylene oxide) [33,34]. At the temperatures causing the precipitation of polymer chains, the LCST decrease is attributed to the formation of larger aggregates with increasing homopolymer concentration [32,34].
103 Hydrodynamic diameter (nm)
42
40
LCST (°C)
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38
36
34
32
102
101 Temperature (°C) 20 40 100 10-6
0
1
2
3
4
5
1x10-5
1x10-4
10-3
10-2
10-1
PEPA concentration (g/mL)
PEPA concentration (% w/w) Fig. 2. The variation of LCST with PEPA concentration.
Fig. 3. The variation of hydrodynamic diameter with the PEPA concentration.
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1
1000
Polydispersity Index (PDI)
However, an unusual behaviour was obtained for the variation of hydrodynamic size with the PEPA concentration at 20 C. As seen here, the hydrodynamic size markedly decreased with the increasing PEPA concentration and the lowest hydrodynamic size values were obtained with the PEPA concentrations higher than 0.01 g/ml. By considering this behaviour, the effect of temperature on the hydrodynamic diameter and the size distribution of PEPA was investigated with a sufficiently high PEPA concentration (i.e., 0.05 g/ml). The diagrams showing the size distribution of PEPA homopolymer at different temperatures are exemplified in Fig. 4. The variation of z-average diameter by the temperature is shown in Fig. 5. The polydispersity index (PDI) values obtained at different temperatures are also shown on the same figure. As seen here, PDI exhibited a minimum at 30 C (i.e., near the LCST of PEPA) and increased with the temperature after the LCST. The relatively high PDI at 50 C should be explained by the formation of large aggregates with relatively wide size distribution. In Fig. 5, the hydrodynamic diameter showed a sharp increase at LCST. This tendency was very similar to that observed for the diblock co-polymer of poly(Nisopropylacrylamide) and poly(N-(2hydroxypropyl)methacrylamide), PNIPA-block-PHPMA [35]. In the referred study, the size increase with the increasing temperature was explained by the association of precipitated polymer chains [35]. Note that the order of magnitude of the hydrodynamic diameters measured below the LCST of PEPA were also very close to that of PNIPA-blockPHPMA below its LCST [35]. For PEPA concentrations of 0.1% and 1.0%, the plots showing the variation of absorbance with the temperature at different NaCl concentrations are given in Fig. 6a and b, respectively. As seen here, the thermal flocculation was observed with all salt concentrations at both PEPA concentrations. In the presence of salt, the temperature corresponding to the maximum point of the absorbance–temperature curve is described as the ‘‘critical flocculation temperature’’, (CFT). For
Hydrodynamic size (nm)
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100
10 Hydrodynamic diameter PDI
1
0.1 10
20
30
40
50
Temperature (°C) Fig. 5. The variation of hydrodynamic diameter and polydispersity index by the temperature.
PEPA homopolymer, the variation of CFT with the NaCl concentration is shown in Fig. 7. As seen here, CFT exhibited a linear decrease with the increasing salt concentration for PEPA homopolymer. The slopes of the straight lines in Fig. 7 were determined as 10.1 and 7.1 C/M for the PEPA concentrations of 0.1% and 1.0%, respectively. Here the slope of the linear relationship between CFT and salt concentration is a measure of the salt concentration effect on the flocculation behaviour of the thermosensitive polymer. Similar tendencies with the slope values very close to those found in our study were previously obtained by Schild and Tirrell for the effect of NaBr concentration on the LCST of PNIPA [20]. Recently, the variation of CFT with the NaCl concentration was investigated for PNIPA microgel particles in sub-micron size range [36]. For this variation, a linear tendency with a slope of 12.2 C/M was obtained with a particle concentration of 0.06% for the NaCl concentration range of 0.1–0.8 M [36]. A similar
Fig. 4. The diagrams showing the size distribution of PEPA homopolymer at different temperatures.
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a
1.0
b
1.0
0.8 Absorbance
0.8 Absorbance
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0.6
0.4
0.6
0.4
NaCl concentration (M)
NaCl concentration (M)
0.0
0.2
0.2
0.1
0.0 0.1
0.5
0.5
1.0
0.0 20
30
40
50
60
1.0
0.0 70
80
90
20
30
Temperature (°C)
40
50
60
70
Temperature (°C)
Fig. 6. The plots showing the effect of salt concentration on the variation of absorbance with temperature. (k = 500 nm), PEPA concentration: (a) 0.1% and (b) 1% w/w.
54
34
PEPA concentration (% w/w) 0.1 1.0
52 50
33
48 32 LCST (°C)
CFT (°C)
46 44 42 40
31
No phase separation
30
38 29
36 34
28
32 0.0
0.2
0.4
0.6
0.8
1.0
NaCl concentration (M)
0
10
20
30
40
50
60
70
80
90 100
Ethanol concentration (% v/v)
Fig. 7. The effect of NaCl concentration on the CFT of PEPA.
Fig. 8. The variation of LCST with the ethanol concentration in the aqueous medium. PEPA concentration: 1% w/w.
relation with approximately the same slope was also obtained by Daly and Saunders [37]. By comparing the slopes obtained for PNIPA and PEPA, one concludes that salt concentration is less effective on the temperature induced flocculation of PEPA chains with respect to PNIPA. The effect of ethanol concentration on the LCST of PEPA is shown in Fig. 8. As seen here, when the ethanol concentration was kept in the range of 0–30% (v/v), LCST significantly decreased with increasing alcohol
concentration. However, in the aqueous media with the ethanol concentration higher than 40% (v/v), the polymer was soluble in the temperature range of 20– 70 C. Thus, LCST could not be detected in this range since no chain precipitation occurred with increasing temperature. For PNIPA homopolymer, the co-nonsolvency in alcohol/water mixtures was extensively investigated by different researchers [38–41]. These results indicated that phase transition of PNIPA was observed in the methanol–water mixtures containing methanol
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