Preparation of MIP composite membrane for effective separation of phenylalanine racemate solution : 패닐알라닌 라세믹 용액의 효과적 분해를 위한 분자각인 고분자 복합 막 제조

다국어 초록 (Multilingual Abstract)

L-Phe and D-Phe imprinted P(MAA-co-EGDMA) submicron/nanoscale beads (LIBs and DIBs) were prepared by modified suspension polymerization.
The percentages of template removal from LIBs and DIBs were 95.5% and 92.5% respectively. Adsorption equilibrium time was determined as 6 h.The adsorption capacity of the prepared submicron/nanoscale beads increased continuously with increase in concentration of racemate solution. The adsorption capacity of beads increased continuously with increase in concentration of racemate solution. Maximum adsorption selectivity was obtained at 100 mg L-1 of Phenylalanine racemate solution. The trend of adsorption capacity and selectivity at different pH was pH 2 ≥ 6 > 4. LIBs prepared for the first time by modified suspension polymerization showed the highest adsorption capacity 0.35 mg/g and selectivity 1.62 at STP. LIBs and DIBs maintained their higher adsorption capacity and selectivity up to 5 successive batches. The trend of adsorption capacity and selectivity was LIBs ≥ DIBs > non-imprinted beads (NIBs).
FE-SEM analysis showed that the presence of template had caused greater effect on the beads sizes. The size of LIBs and DIBs was slightly larger (100nm~1.5µm) than NIBs (100nm~800nm). Similarly, FT-IR study revealed higher number of binding sites in LIBs than that of DIBs and NIBs. 13C CP-MAS NMR spectroscopy was used to evaluate the basic structure of the prepared beads. It also helped in correlated the beads sizes with the extent of polymerization. This whole study revealed that the newly prepared Phe imprinted submicron/nanoscale beads showed higher adsorption capacity and selectivity compared to the previously reported Phe imprinted microbeads.
D-Phe and L-Phe imprinted P(AA-co-AN) membranes (DIM and LIM) were prepared by phase inversion method. The average thickness of DIMs, LIMs and non-imprinted P(AA-co-AN) membrane (NIM) was in the range of 40 µm - 50 µm. A total of 83.75% and 84.29% of template was removed during complete washing of DIMs and LIMs respectively. The trend of improved physical properties, adsorption capacity and selectivity was LIM ≥ DIMs > NIMs. FT-IR analysis supported the higher number of binding sites in LIMs compared to DIMs and NIMs.
Four types of composite membranes i.e, composite membrane composed of LIBs and LIM (CMLIBLIM), composite membrane composed of DIBs and DIM (CMDIBDIM), composite membrane composed of LIBs and DIM (CMLIBDIM) and composite membrane composed of DIBs and LIM (CMDIBLIM) were prepared for chiral resolution of Phe racemate solution. These composite membranes were prepared by phase inversion technique, after a uniform dispersion of 3 % (w/w) of the prepared submicron/nanoscale beads into Phe incorporated P(AN-co-AA) solution using a simple physico-mechanical process (vertexing and sonication).
In all types of composite membranes the extraction of template (73% – 75%) during coagulation step and after complete washing (88% - 90%) was higher compared to Phe imprinted membranes (control membranes).
The average thickness of the resultant Phe imprinted composite membranes was 70 µm ~ 80 µm. The higher thickness, large surface area and large number of ionic sites in the composite membranes caused improvement in physical properties of the composite membranes compared to control. The maximum percent swelling ratio (387% - 426%) and water holding capacity (0.16 g/g - 0.17 g/g) of Phe imprinted composite membranes were higher than that of control membranes. The wet weight decrease rate was 1.5 to 2 times lower than that of control membranes.
FE-SEM analyses clarified that all types of composite membranes were composed of three portions i.e. upper and lower dense layers and a middle broad layer. The thickness of the two cover layers was in the range of 6 µm ~7 µm. The average size of the nano-pores observed in the prepared composite membranes was in the range of 6 nm ~16 nm.
The structure characterization of composite membranes was done by FT-IR study. In case of composite membranes the shifting of OH peaks to lower wave numbers indicated the interaction of incorporated beads with the composite membranes matrix. FT-IR inferred that the Phe imprinted submicron/nanoscale beads maintained the active functional groups after incorporation in the Phe imprinted composite membranes matrix.
Assemblies of the prepared composite membranes were employed separately in ultrafiltration for chiral separation of D,L-Phe racemate solution.
CMLIBLIM and CMDIBDIM followed the selective adsorption phenomenon. CMDIBDIM and CMLIBLIM showed 1.35 - 1.50 times higher adsorption capacity and 1.5 times higher adsorption selectivity compared to the control membranes. The selective rejection by MIP composite membranes was reported for the first time. Percent rejection of Phe imprinted composite membranes was lower compared to control membranes. The permselectivity value of these composite membranes was less than 1 (~0.43).
CMLIBDIM and CMDIBLIM followed the facilitated permeation mechanism. These membranes showed 1.5 - 1.6 times higher separation factor (permselectivity) than that of control membranes. Adsorption capacity of CMLIBDIM and CMDIBLIM was 1.35 - 1.45 times higher than that of control membranes while it was much higher than that of Phe imprinted beads. CMLIBDIM and CMDIBLIM also showed a selective but lower rejection of the enantiomers compared to control membranes.
The adsorption capacity and separation factor of these newly prepared composite membranes are higher compared to previously reported MIP composite membranes used for enantioseparations. The selective rejection phenomenon by the MIP composite membranes was observed for the first time.
It was concluded that these composite systems, where two different MIP formats i.e., Phe imprinted submicron/nanoscale beads and Phe imprinted membranes were combined and both were functionally active, will be useful in future for several industrial and biomedical applications. The enhanced adsorption ability and improved separation properties of these newly developed composite MIP membranes, provide a good basis for use in the practical applications such as in the faster and effective resolution of enantiomers, in the pre-concentration processes as well as in blood filtration, drug delivery, food processing and in scaffold preparation etc.

국문 초록 (Abstract)

L-Phe 과 D-Phe 각인 P(MAA-co-EGDMA) 초미세/나노 beads (LIBs and DIBs) 가 개량된 고분자 합성법에 의하여 제조 되었다.
LIBs와 DIBs로 부터의 template 제거률은 각 95.5%와 92.5%이다. 흡착 평형 시간은 6 h시간으로 측정 되었다. bead의 흡착률(adsorption capacity)은 라세믹 용액(racemate solution)의 농도가 높아짐에따라 지속적으로 높아졌다. 최대 흡착도(adsorption selectivity)는 100 mg L-1 의 phenyalanine 라세믹 용액에서 얻을 수 있었다. 다양한 경향성의 흡착률과
다양한 pH (pH 2≧6＞4)에서의 흡착도를 얻을 수 잇었다. 초기 LIBs는
서스펜션(suspension) 중합으로 제작되었고 STP에서 가장 높은 흡착률은
0.35 mg/g 과 흡착도 1.62으로 측정 되었다. LIBs와 DIBs는 5 batch까지 높은 흡착률과 흡착도를 보였다. 흡착률과 흡착도의 크기는 LIBs ≧ DIBs ＞ non imprinted beads (NIBs) 순으로 나타났다.
FE-SEM 분석을 통하여 template의 존재가 beads의 크기에 큰 영향을 미침을 확인하였다. LIBs 와 DIBs의 크기는 (100nm ~ 1.5µm) NIBs (100nm~800nm) 보다 약간 큰 것으로 관찰 되었다. 유사하게 FT-IR 분석에서는 LIBs가 DIBs와 NIBs보다 더 많은 결합부위를 가지는 것으로 분석되었다. 13C CP-MAS NMR 분광분석를 통해 beads의 기본 구조를 알고자 하였다. 이러한 일련의 연구를 통하여 새롭게 제작한 Phe 각인 미세/나노 고분자 beads가 Phe imprinted microbeads보다 더 높은 흡착률과 흡착도를 보임을 알 수 있었다.
D-Phe 와 L-Phe 각인 P(AA-co-AN) 막 (DIM 과 LIM) 은 상 전환 법으로 제조 되었다. DIMs, LIMs 그리고 non-imprinted P(AA-co-AN) 막 (NIM) 의 평균 두께는 40µm - 50µm 였다. 각 83.75% 와 84.29% 의 template이 DIMs와 LIMs의 완전 세척 공정에서 씻겨 나갔다. 물리적 성질과 흡착률 그리고 흡착도는 LIBs ≧ DiBs ＞ non imprinted beads (NIBs) 순으로 나타났다. 이 결과는 FT-IR 분석에서 LIM이 DIMs와 NIMs보다 많은 결합 부위를 가진다는 사실을 뒷받침 해준다.
네 종류의 합성 막 [DIBs와 DIM (CMDIBDIM), LIBs 와 LIM (CMLIBLIM), LIBs와 DIM (CMLIBDIM), DIBs와 LIM (CMDIBLIM)] 이 chiral resolution Phe 라세믹 용액의 처리에 사용되었다. 이 네가지 종류의 막은 상 전환 법으로 제조되었고 3 % 의 미세/나노 beads를 pHe incorporated P(AN-co-AA) 용액을 사용하여 확산시켜 제조 하였으며 간단한 기계-물리적 공정 (vortexing and sonication)방법을 사용하였다.
모든 종류의 합성 막에서 template의 추출(73% ~ 75%)은 응고과정에에서 일어났고 완전 세척 (88% ~ 90%)은 Phe 각인 막(비교군)에서 더 높았다.
평균 두께는 Phe 각인 중합막이 70~80㎛로 나타났다. 중합 막에서의 더 넓은 두께, 큰 표면적, 더 많은 량의 이온결합이 비교군 보다 더 향상된 물리적 성질을 띄게 한 것으로 판단 되었다. Phe imprinted composite막의 최대 팽윤(swelling) 률과 (387% ~ 476%)와 용수량 (0.16 g/g ~ 0.17 g/g)역시 비교군 보다 컸다. 수분 감소량은 비교군 보다 1.5~2배 가량 늦었다.
FE-SEM 분석을 통하여 모든 종류의 중합막이 세가지 부분으로 구성되어 있음을—아래 위의 얇고 밀집된 층과 가운데 부분에 넓게 퍼진 막으로 구성되었다. 두 장의 덮게 막은 6~7 ㎛ 이다확인 할 수 있었다. 중학 막에서 관찰된 nano-기공은 6 nm ~ 16 nm 범위였다.
중합막의 구조 분석은 FT-IR을 통하여 이루어 졌다. 중합막의 경우에서 OH peak의 더 낮은 파장으로의 이동이 의미하는 바는 bead와 중합 막간의 상호작용에 의한 결과로 설명 할 수 있다. FT-IR 분석을 통하여 미세/나노 beads가 Phe 각인 미세/나노 막 상태에서도 작용기가 제 역할을 할 수 있음을 확인 할 수 있었다. 중합 막의 분류는 D,L-Phe 라세믹 용액의 ultrafilteration chiral 분리과정에서 개별적으로 이루어 졌다.
CMLIBLIM과 CMDIBIM은 선택적 흡착 현상을 보였다. CMDIBDIM과 DMLIBLIM은 비교군 보다 1.35~1.50배 높은 흡착률과 1.5배 높은 흡착도를 보였다. MIP 중합 막에서 선택적 rejection 현상이 처음으로 연구 되었다. Phe 각인 중합막의 rejection 현상이 비교군 보다 낮았다. Perm selectivity 값은 모든 중합막에서 1 이하(~0.43)으로 측정되었다.
CMLIBDIM 과 CMDIBLIM은 중계 삼투 기작을 따른다. 이러한 막들은 비교군 보다 1.5~1.6배 가량 높은 값을 가진다. CMLIBDIM 과 CMDIBLIM의 흡착도는 비교군보다 1.35 ~ 1.45배 높으며 Phe 각인 beads 보다는 훨씬 크다. CMLIBDIM 과 CMDIBLIM은 좋은 흡착도를 보이지만 구조 이성질체의 rejection은 비교군 보다 낮았다.
이러한 중합 막은 흡착률과 분리정도는 기존의 MIP 중합 막보다 높았다. 선택적 rejection 현상은 학계에서 처음으로 보고되었다.
본 연구를 통하여 Phe 각인 미세/나노 beads 와 Phe 각인 막이 결합되었으며 두 가지 모두 제 기능을 잃지 않았다. 이러한 결과를 통하여 미래 많은 산업, 의료 분양에의 적용이 가능할 것이라 예상된다. 본 연구를 통한 MIP 중합막의 흡착률과 분리 정도의 향상은 더 빠르고 효과적인 구조 이성질체 분리의 기반을 공고히 하였고, 앞으로의 연구를 통해 혈액 여과, 약물 투입, 식품제조 등의 기술 적용방법 모색의 필요성이 있다.

목차 (Table of Contents)

• 1.Introduction
• 1.1.Molecularly imprinted polymers 2
• 1.2. Molecular imprinting mechanism 5
• 1.2.1. Pre-organization of functional groups 5
• 1.2.2. Shape specificity of the binding sites 7
• 1.3. Types of imprinting 8
• 1.3.1. Covalent imprinting 8
• 1.3.2. Non-covalent imprinting 9
• 1.3.3. Semi-covalent or Hybrid imprinting 10
• 1.3.4. Ion imprinting 12
• 1.4. Origin and history of molecular imprinting technology 13
• 1.5. Applications of molecularly imprinted polymers 15
• 1.5.1 Separation 15
• 1.5.2. Assays and sensors 18
• 1.5.3. Catalysis 20
• 1.5.4. Drug delivery 21
• 1.5.5. In biotechnology 22
• 1.6. MIP formats and polymerization processes 25
• 1.6.1 MIP particles 25
• 1.6.2 MIP monoliths 26
• 1.6.3 MIP beads 28
• 1.6.4 MIP membranes 28
• 1.6.5 MIP composite membranes 29
• 1.7. Chirality and its significance 31
• 1.8. Phenylalanine 34
• 1.9. CONCLUSIONS 41
• 1.10. AIM OF THIS THESIS 42
• 1.11. REFERENCES 45
• 2. Preparation, characterization and application of Phenylalanine imprinted membranes
• 2.1. INTRODUCTION 62
• 2.2. EXPERIMENTAL 66
• 2.2.1. Materials 66
• 2.2.2. Preparation of D-Phe, L-Phe imprinted and non-imprinted membranes 66
• 2.2.3. Removal of template during coagulation 71
• 2.2.4. Extraction of template from DIMs and LIMs 73
• 2.2.5. Physical characterization 73
• 2.2.6. FE-SEM analysis 74
• 2.2.7. Fourier transform infrared spectroscopy (FT-IR) 75
• 2.2.8. Ultrafiltration (UF) experiments 75
• 2.2.9. High performance liquid chromatography (HPLC) 76
• 2.3. RESULTS AND DISCUSSIONS 80
• 2.3.1. Extraction of template 80
• 2.3.2. Physical characterization 84
• 2.3.3. FE-SEM study 88
• 2.3.4. FT-IR analyses 96
• 2.3.5. Adsorption and adsorption selectivity study 100
• 2.3.6. Permeation and permeation selectivity study 111
• 2.3.7. Rejection and rejection selectivity study 118
• 2.4. CONCLUSIONS 125
• 2.5. REFERENCES 126
• 3. Preparation, characterization and application of Phenylalanine imprinted submicron/nanoscale beads
• 3.1. INTRODUCTION 133
• 3.2. EXPERIMENTAL 144
• 3.2.1. Materials 144
• 3.2.2. Preparation of molecularly imprinted submicron/nanoscale beads 144
• 3.2.3. Preparation of non-imprinted submicron/nanoscale beads 146
• 3.2.4. Washing of the prepared beads 149
• 3.2.5. FE-SEM analysis 150
• 3.2.6. = Fourier transform infrared spectroscopy (FT-IR) 150
• 3.2.7. Solid state 13C CP-MAS NMR 150
• 3.2.8. Batch experiments 151
• 3.2.9. High performance liquid chromatography (HPLC) 152
• 3.3. RESULTS AND DISCUSSIONS 154
• 3.3.1. Extraction of template from the polymer matrix of DIBs and LIBs 154
• 3.3.2. FE-SEM study and beads size distribution 158
• 3.3.3. FT-IR and 13C CP-MAS NMR spectroscopy for structural characterization 165
• 3.3.4. Adsorption capacity and selectivity 177
• 3.3.5. Effect of pH and template 180
• 3.3.6. Effect of concentration of racemate solution 183
• 3.3.7. Equilibrium adsorption time 187
• 3.3.8. Desorption and reusability 190
• 3.4. CONCLUSIONS 193
• 3.5. REFERENCES 194
• 4. Preparation, characterization and application of Phenylalanine imprinted composite membranes
• 4.1. INTRODUCTION 209
• 4.2. EXPERIMENTAL 217
• 4.2.1. Materials 217
• 4.2.2. Preparation of submicron/nanoscale beads and polymeric solutions for making the composite membranes 217
• 4.2.3. Preparation of composite membranes 218
• 4.2.4. Washing of the prepared composite membranes 221
• 4.2.5. Physical characterization of composite membranes 221
• 4.2.6. FE-SEM analysis 222
• 4.2.7. Fourier transform infrared spectroscopy (FT-IR) 223
• 4.2.8. Ultrafiltration (UF) experiments 223
• 4.2.9. High performance liquid chromatography (HPLC) 224
• 4.3. RESULTS AND DISCUSSIONS 230
• 4.3.1. Extraction of template 230
• 4.3.2. Physical characterization 235
• 4.3.3. Composite membrane morphology and ultrafiltration 240
• 4.3.4. FT-IR study 251
• 4.3.5. Permeability coefficient and permselectivity 264
• 4.3.6. Permeability coefficient and permselectivity of CMLIBDIM and CMDIBLIM 264
• 4.3.7. Permeability coefficient and permselectivity of CMDIBDIM and CMLIBLIM 271
• 4.3.8. Adsorption and adsorption selectivity 277
• 4.3.9. Adsorption and adsorption selectivity of CMDIBDIM and CMLIBLIM 277
• 4.3.10. Adsorption and adsorption selectivity of CMLIBDIM and CMDIBLIM 284
• 4.3.11. Rejection and rejection selectivity 290
• 4.3.12. Rejection and rejection selectivity of CMDIBDIM and CMLIBLIM 290
• 4.3.13. Rejection and rejection selectivity of CMLIBDIM and CMDIBLIM 297
• 4.3.14. Comparison with Phe imprinted membranes and beads 302
• 4.3.15. Comparison with previously reported MIP composite membranes 304
• 4.4. CONCLUSIONS 311
• 4.5. REFERENCES 313
• THESIS ABSTRACT (English) 323
• THESIS ABSTRACT (Korean) 328