Environmental Responsive Chromatography - J-Stage

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The temperature of observation cell was controlled with a deviation of ± 0.02. °C with a LAUDA RC20 waterbath. Chromatographic Apparatus was followed. The.
ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT 2001 © The Japan Society for Analytical Chemistry

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Environmental Responsive Chromatography Eri AYANO1, Hideko KANAZAWA, 1† Akihiko KIKUCHI2, and Teruo OKANO2 †1

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Department of Physical Chemistry, Kyoritsu College of Pharmacy, Shibakoen, Minato, Tokyo 105-8512, Japan (E-mail:[email protected]) Institute of Biomedical Engineering, Tokyo Women's Medical College, Kawadacho, Shinjuku, Tokyo 16 2-8666, Japan We designed a novel polymeric chromatography system that utilizes linear, pH/temperature-sensitive polymers of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm). The copolymer showed hydrophillic-hydrophobic phase transitions in response to changes of temperature as PNIPAAm but the transition temperatures were affected by changes in pH. In the chromatographic system using the copolymer modified packing materials, we investigated to separate acid drugs and nucleotides by temperature with ionizable groups. The surface properties and functions of stationary phases were controlled by the external stimuli. Therefore it easily changes an interaction of solute with the surface with constant aqueous mobile phase. The retention on the copolymer modified stationary phase remarkably changed by changing temperature and pH. Environmental responsive chromatography would be highly useful for biopolymer separation as well as nucleotides. The method is useful for various separations in life science. (Received on August 7, 2001; Accepted on September 13, 2001 )

Recently, polymers that display a physicochemical response to stimuli are widely utilized for drug delivery system, cell culture substrate, and bioconjugate. Stimuli studied to date include chemical substances and changes in temperature, pH and electric field. However, there have been few reports for the use of these stimulus-responsive polymers in chromatographic separation. We reported a new method of HPLC using packing materials modified with a temperature responsive polymer, poly (N-isopropylacrylamide) (PNIPAAm). 1-5 PNIPAAm has the sharpest phase transition of the class of thermosensitive N-alkyl acrylamide polymers. This feature makes it the most suitable for studies and practical applications. We designed and synthesized novel temperature-responsive polymers with additional sites responsive to other environmental stimuli such as changes in pH. They showed hydrophilic-hydrophobic phase transitions in response to changes of temperature as PNIPAAm but the transition temperatures were affected by changes in pH. The surface properties and functions of stationary phases were controlled by the external stimuli. Stimuli-responsive (sensitive) polymers, which change their structure and physical properties in response to external signals, comprise a new set of materials with interesting applications in biomaterials science and technology. Novel chromatography system may be achieved using stimuli responsive polymers, which alter their structure and physical properties in response to external environmental change. These methods are expected to be applicable for the separation of pharmaceuticals, nucleoic acid, peptides and proteins. In the work presented here, we have designed and synthesized a pH-sensitive polymer, which is a copolymer of NIPAAm and DMAPAAm. Experimental The

chain

transfer

polymerization

technique

was

employed in order to prepare carboxyl-terminated copolymer. 2,2'-Azobisisobutyronitrile and 3-mercaptopropionic acid were used as an initiator and a chain transfer agent, respectively. PNIPAAm and its copolymer were synthesized and grafted to aminopropyl silica by activated ester-amine coupling and they were used as packing materials1, 2. An average of molecular weights of the polymers used was 5000. The lower critical solution temperature (LCST) was determined by change of optical transmittance. The transmittances at 500 nm of NIPAAm copolymer solutions (5 mg/mL) were measured at various temperatures and pH using a spectrophotometer (Hitachi U-3000). The temperature of observation cell was controlled with a deviation of ± 0.02 °C with a LAUDA RC20 waterbath. Chromatographic Apparatus was followed. The polymer-grafted silica support was packed into a stainless-steel column (length: 150 mm x 4.6 mmI.D.). The column was connected to an HPLC system (HITACHI Model L-6200 intelligent pump; L-4000 UV-monitor, D-2500 data processor). The elution behaviors of samples were recorded at a flow-rate of 1 mL/min at various temperatures. Results and Discussion NIPAAm polymers exhibit a lower critical solution temperature (LCST). Aqueous solutions of these polymers are soluble below their LCST and precipitated above their LCST. The LCST is dependent on pH for polymers with ionizable groups because of a change in hydrophilicity with ionization and electrostatic repulsion that cause a shift in the LCST. The desired LCST profile can be obtained as a function of pH by the copolymerization of NIPAAm with hydrophobic (butyl methacrylate, BMA) and hydrophilic ionizable comonomers (DMAPAAm). The LCST of poly (NIPAAm-co-DMAPAAm) was around 57 °C at pH 3 and around 43 °C at pH 7. This allows the manipulation of both pH and temperature for

ANALYTICAL SCIENCES 2001, VOL.17 SUPPLEMENT

(a)

Mobile phases Temperatureresponsive column

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Hydrophobic Temperature gradient Detector

Fig.1 Environmental-responsive chromatography using polymer modified surfaces

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changing properties of polymer chains. As the DMAPAAm content in the copolymer increases, the colymer chains become more hydrophilic because of the ionizable groups. Increase in the hydrophilicity of copolymer chains leads to the rise in LCST. The both influence of pH and temperature on the property of ionizable and thermosensitive polymers have been investigated. Poly (NIPAAm-co-BMA-co-DMAPAAm) terpolymers of varying mol ratio have been synthesized. The LCST of this comonomer was around 28 °C at pH 3 and around 40 °C at pH 7. This allows the manipulation of both pH and temperature for changing properties of polymer chains. We designed a novel chromatography system that utilizes a random copolymer of NIPAAm, BMA and DMAPAAm, which retains its pH-controlled temperature sensitivity. This is one of the advantages of utilizing a pH-sensitive polymer that is also temperature sensitive. The temperature- and pH-dependent elution profiles of oligonucleotides were investigated. The elution behavior of oligonucleotides on NIPAAm-co-BMA and NIPAAm-co-BMA-co-DMAPAAm copolymer-modified silica columns were obtained varying pH, as shown in Fig. 2. On poly(NIPAAm-co-BMA) modified column which surface is a non-charged, oligonucleotides are not retained because of the deprotonation of solutes regardless of temperature. In contrast oligonucleotides were retained on Poly (NIPAAm-co-BMA-co-DMAPAAm) modified column at pH 4.5 mainly due to the electrostatic interaction. At 10 °C polymer chain is hydrated to expand, thus ionizable groups interact with olgonucleotides. A new concept of chromatography, environmentally responsive chromatography, is proposed. There is numerous ways to optimize selectivity and improve resolution in HPLC. The most common approach for altering the selectivity is varying the organic composition in the mobile phase. The newly developed pH/temperature-responsive stationary phases are useful in method development as an extra tool to optimize selectivity by adjusting both of the temperature and pH rather than changing the mobile phase composition. These results show that the column modified with poly(NIPAAm-co-BMA-co-DMAPAAm) could respond to both temperature and pH to modulate retention of charged compounds using aqueous mobile phase. Thus, the unique properties of the pH/temperature-responsive polymer make it a novel chromatography system for separation of biomolecule such as peptide, protein and nucleotide. The ability of the proposed environmentally-responsive polymer modified stationary phase to separate the solutes without the use of

1. dCGTC, 2. dAGTC, 3. dTGTC , 4. dGGTC

Absorbance 265nm

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Fig.2 Chromatograms of oligonucleotides. Flow-rate:1 ml/min, Detection:265 nm. Eluent: (a), (c) Na2HPO4/citric acid (I= 0.1, pH3.0); (b), (d) Na2HPO4 (I= 0.1, pH4.5) Column: (a), (b) P(NIPAAm95%-co-BMA5%), (c),(d) P(NIPAAm95%-co-BMA5%-co-DMAPAAm5%) organic solvent is advantageous from the point of view of keeping biological activity, environmental reasons, and economical cost of mobile phases. References 1. H. Kanazawa, K. Yamamoto, Y. Matsushima, N. Takai, A. Kikuchi, Y. Sakurai, T. Okano, Anal. Chem. 1996, 68, 100. 2. H. Kanazawa, Y. Kashiwase, K. Yamamoto, Y. Matsushima, A. Kikuchi, Y. Sakurai, T. Okano, Anal. Chem. 1997, 69, 823. 3. H. Kanazawa, Y. Kashiwase, K. Yamamoto, Y. Matsushima, N. Takai, A. Kikuchi, Y. Sakurai, T. Okano, J.Pharm. Biomed. Anal. 1997, 15, 1545. 4. H. Kanazawa, K. Yamamoto, Y. Matsushima, K.Oikawa, A. Kikuchi, T. Okano, T.Environmental Sciences. 2000, 7, 047.

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H. Kanazawa, T. Sunamoto, Y. Matsushima, A. Kikuchi, T. Okano, T. Anal. Chem. 2000, 72, 5961.