K. Fremielle Lim,1 Jessica Scanlon,1 Jordan Velich1, Michael Bowyer2 and Clovia Holdsworth,1. 1Discipline of Chemistry, School of Environmental and Life ...
Characterisation of Molecularly Imprinted Microspheres by NMR K. Fremielle Lim,1 Jessica Scanlon,1 Jordan Velich1, Michael Bowyer2 and Clovia Holdsworth,1 1Discipline
of Chemistry, School of Environmental and Life Sciences, Callaghan, University of Newcastle, Australia 2Discipline of Applied Sciences, School of Environmental and Life Sciences, Ourimbah, University of Newcastle, Australia
Introduction Molecularly Imprinted microspheres (MIMs) are structured porous materials with molecular recognition sites of predetermined selectivity, which are produced conventionally by free radical polymerisation.1 These recognition sites are formed by co-polymerisation of the chosen cross-linker and functional monomer that form complexes with the target compound or template prior to polymerisation. Removal of the template from the material leaves a cavity, which is shape and size specific for the template. The binding efficiency of the MIPs are analysed using a blank polymer or a non-imprinted microspheres (NIMs)2 , which are prepared the same way as MIMs but without the template. This study focuses on the use of NMR for the characterisation of MIMs from copolymer composition to rebinding studies, which have not been reported before.
N
N
OH
Table 1. MIM systems analyzed using quantitative NMR.
N H
N
N
OH
H
N
H O
OH
O
O
N
N
O
(A)
H3 C
CH3
(B)
H3 C
OH
(C)
HO
(D) 4
O
(E)
1
O
3
O
2
N H
O
(F)
Cross-linker
(F) Ethylene Glycol Dimethacrylate (EGDMA)
2
O
(G)
3
(H)
Template (A) Caffeine and (B) Theophylline
O O
Functional monomer
1
N
O
N H
N
O
O
O
O
Polymerisation Feed
System
OH
H N
O
Figure 1. Overview of the Molecular Imprinting Process. (1) Assembly of the pre-polymerisation complex (2) Polymerisation process (3) Removal of the template / Rebinding process.
Methacrylic Acid (MAA)
(C) 3,5-Dimethylphenol, (D) Orcinol, (E)Phloroglucinol
(G) 2,6 -bis-(acryl)amido pyridine (BAAPy)
(H) 2,3,5-Tri-O-acetyl uridine (TOAU)
• MIMs are prepared by precipitation polymerisation using 10 mL of acetonitrile in 0.500 mmol of total monomer (EGDMA+ MAA).
Figure 2. Molecular Structures of (A) Caffeine (B) Theophylline (C) 3,5-Dimethylphenol (D) Orcinol (E) Phloroglucinol, and (F) ethylene glycol dimethacrylate (EGDMA), (G) 2,6 -bis-(acryl)amido pyridine (BAAPy) and (H) 2,3,5-Tri-O-acetyl uridine (TOAU)
2 O 120
2 O
100
1,4Dioxane
1:100 1:50 1:25 1:10 1:5
80
60 Figure 3. The NMR setup showing the coaxial insert on a 5 mm tube.
40
4
20
0 MIP
NIP
3
% EGDMA
MIP
NIP % MAA
MIP % template
Figure 4. Copolymer composition of System 2 imprinted with (E) at various initiator : total monomer ratios.
The incorporation of the templates in the polymers is influenced by the amount of the initiator. In general, higher amount of initiator results to lower amount of template incorporated in the polymers. Figure 3. An example of a NMR spectrum of a MIM initial solution (System 3 E) using 1,4-dioxane as the reference standard in deuterated DMSO. Refer to Figure 2 for assignment of peaks.
14.00
1.00 0.98
12.00 10.00
[[TOAU]b/[TOAU]0
I(std)/I(EGDMA)
0.96
8.00 6.00 4.00 y = 0.1182x ± 0.0099 + 0.002 R² = 0.9967
2.00
20.0
40.0
60.0 80.0 [std]/[EGDMA]
100.0
MIPs
0.92
NIPs
0.90
The sensitivity of the NMR method is sufficient to discriminate the template binding by the NIMs and the MIMs showing imprinting effect.
0.88 0.86 0.84 0.82
0.00 0.0
0.94
120.0
Figure 5. Calibration curve for EGDMA using the proton signal of O-CH2 , peak 1. Calibration curves for each components were obtained by the internal standard method using 1,4-dioxane.
Time binding analyses for System 2 showed that the maximum binding of the MIMs is achieved in 20 minutes.
0.80 5
15
20
25
30
60
90
120
mins Figure 6. Time binding analysis curve for System 3.
Conclusion Quantitative NMR is an efficient method for the characterisation of MIMs from copolymer composition to rebinding studies. Analyses of the rebinding capacities of the MIMs using NMR is a time and cost-effective alternative to HPLC, i.e. no mobile phase needed and no separation of the microspheres necessary. References: 1. Ye, L.; Springer Berlin Heidelberg: 2015, p 1. 2. Komiyama, M., Takeuchi T., Mukawa, T. and Asanuma, H. Molecular Imprinting, from Fundamentals to Applications; Wiley-VCH: Germany, 2003.
Acknowledgements: Dr. Monica Rossignoli, Discipline of Chemistry, University of Newcastle
