Communications ADVANCED MATERIALS

[11] For another fluorescence switching system see: J. Daub, M. Beck, A. Knorr, H. Spreitzer, Pure Appl. Chem. 1996,68, 1399.

[12] All conversions were quantitative except the closing of l o to l c and 50 to 5c, which was limited to 85-90 YO of the photostationary state.

Thermally Responsive Rigid Polymer Monoliths" * By Eric C. Peters, Frantisek Svec, and Jean M. J . Frkhet*

The principle of size exclusion lies at the heart of several technologies, including gel-permeation chromatography['] and membrane separations.[21 Typically, both organic and inorganic based solids of defined stable pore structure are used to effect such separations. However, a more powerful approach involves the use of materials that change their permeability in a preprogrammed manner in response to small environmental changes. Such "smart" polymers re- sponsive to changes in pH,131 temperat~re,[~-~] and 1ightl'"l have been reported in the literature, and have been used in applications such as drug delivery,["*'21 encapsulated en- zyme b io rea~ to r s ,~ '~~ and membranes with controlled per- meability.1326-y1 Herein, we describe a method to modify the internal pore surface of rigid poly(glycidy1 methacrylate- co-ethylene dimethacrylate) (GMA-EDMA) monoliths with poly(N-isopropylacrylamide) (PNIPAAm) chains. The grafted chains can either completely block or control the flow through the micrometer-sized pores of the monolith. Further, the changes in the surface polarity accompanying the phase transition of the grafted PNIPAAm can also be used for the selective adsorption of proteins, leading to the novel separation mode of isocratic hydrophobic interaction chromatography.

Poly(N-isopropylacrylamide) is perhaps the best known of a class of temperature-sensitive polymers. At its lower critical solution temperature (LCST) of 32 0C,['41 PNI- PAAm chains undergo a rapid and reversible phase transi- tion from extended hydrated helices below the LCST to collapsed hydrophobic coils above the LCST.['53'61 For ex- ample, hydrogel membranes with temperature- controlled permeability for molecules of different sizes'" and polar- ity'') were prepared directly by the copolymerization of N- isopropylacrylamide (NIPAAm) with comonomer and crosslinking agent. A decrease in the permeability of these membranes was observed at temperatures exceeding the LCST. NIPAAm has also been grafted to pre-made solid supports such as porous glass disks, producing composites whose permeability increased above the LCST.L8.91

[*] Prof. .I. M. J. Frkhct. E. C. Peters, Dr. F. Svec Department of Chemistry. University of California, Berkeley Berkeley, CA 94720-1460 (USA)

[**I Financial support of this research by the Office of Naval Research and the National Institutes of Health (GM-44885) is gratefully acknow- ledged. ECP thanks the Rohm and Haas company for its financial sponsorship.

Similarly, the volume transition exhibited by this polymer in response to changes in temperature was used to control the pore size of beads grafted with NIPAAm in gel permea- tion chr~matography.[~,~] However, since flow in a packed column occurs through the interstitial voids between the packed particles, these materials cannot be used to produce thermal gates. Further, the molecular weights of the grafted polymer chains were reported to be below 10 000 D a l t ~ n s ' ~ ~ or, when not directly measured, could be inferred to be low based on both the polymerization conditions and the slight changes in the observed retention times.l5I

Recently, we have introduced a totally new class of macroporous polymeric materials prepared by a simple molding process.[173181 These porous monoliths are charac- terized by a unique bimodal pore distribution, consisting of large micrometer-sized convective pores and much smaller 10 nm-sized diffusive pores.["] High flow rates through these monoliths can be obtained at low back pressures due to the network of the large canal-like pores that traverse the length of the monolith. Additional flow-through prop- erties can be imparted to these materials by grafting PNI- PAAm chains to their internal pore surface.

The two-step grafting procedure, summarized in Scheme 1, involves the vinylization of the pore surface by reaction of the epoxide moiety 1 with ally1 amine 2, and a subsequent in situ radical polymerization of NIPAAm

IW H2' a7 0 HO NH

(M BAAm) AlBN

2.

Scheme 1.

within these pores. Methylenebisacrylamide (MBAAm) can be added to the polymerization mixture to control the swelling. The composites 3 thus produced change their properties in response to external temperature and, de- pending on the conditions of their preparation, allow the following, which are treated separately below: 0 The occurrence of thermal "on/off" behavior that fully

opens and closes the micrometer-sized pores and con- trols the flow through the device (thermal gate).

0 The control of the flow rate through the device (thermal valve).

0 The switching of surface polarity (thermally controlled hydrophobicitylhydrophilicity). Thermal Gate: The gate effect is demonstrated in

Figure 1. A monolith modified with PNIPAAm was equili- brated with water pumped at a flow rate of 1 mL/min through the column immersed in a bath heated to 40 "C. At this temperature, the chains existed in their collapsed form, and there was little resistance to flow. The column was then

630 0 VCH VerliiRsRe.sellschiif~ mhH, 0-69469 Weinheirn, 1997 0935-9648/97/0806-0630 $ 17.50+.50/0 Adv. Mater. 1997, 9, No. 8

Communications A D V A N C E D MATERIALS

m I B

20

10

0

B

3

i i; B !

0 10 20 30 4 0

Time, min

Fig. 1. Thermal gate behavior of porous poly(glycidy1 methacrylate-co-ethyl- ene dimethacrylate) monolith grafted with poly(N-isopropylacrylamide). Conditions: monolithic disk 10 mm thick, 10 mm i.d., water; flow rate 1 mLi min. PointA: removal of cartridge from 40°C bath; point B: Reimmersion into 40 "C bath.

removed from the bath (pointA), while the flow of water was allowed to continue. Within a few minutes, the back pressure quickly increased as the grafted PNIPAAm chains expanded and filled the pores completely When the back pressure approached the operational limit of the pump, the column was reimmersed in the 40°C bath (point B), and the back pressure returned almost immediately to its origi- nal value. Figure 1 clearly documents that this gate effect is rapid, reversible and reproducible.

In addition to this behavioral evidence, further confirma- tion of the presence of desired grafted PNIPAAm includes: i) the appearance of the amideI (1647 cm-') and amideII (1456 cm-l) bands in the FTIR spectra recorded from KBr disks; ii) an increase of 0.6-0.8 wt.-% in nitrogen content determined by elemental analysis compared to that found for the original allyl amine functionalized monoliths, which corresponds to values of 4.8-6.5 wt.-% of PNIPAAm grafted to the monolith; and iii) that an endotherm at 31.5 "C is observed in the differential scanning calorimetry (DSC) curve of the polymer equilibrated in water over- night. Finally, in order to demonstrate that the PNIPAAm chains were indeed grafted to the monolith's pore surface, a macroporous rod that was not first vinylized with allyl amine was subjected to the same in situ polymerization. The resulting rod column exhibited no temperature-depen- dent gate behavior.

Thermal Valve: According to the Poiseuille-Hagen a decrease in a tube's diameter leads to a decrease

in the flow rate of a liquid through the tube at constant pressure drop, or requires a greater applied pressure to achieve the same flow rate. Therefore, back pressure mea- surements at a constant flow rate can be used to determine changes in the diameter of a tube. Although our monoliths are permeated with pores that have shapes very different from those of a regular tube, increases in the back pressure can be directly measured and used for monitoring changes in the pore size.

Figure 2 shows the effect of temperature on the back pressure profile for a 1 cm thick monolithic block modified with a grafting solution that contained MBAAm (cross- linking monomer) in addition to NIPAAm. Unlike the thermal gate, flow proceeded through the monolith regard-

n

$ u

Q lo - i Y I L

m

L!

0 1 2 3 4 5 6

Flow rate, mL/ min

Fig. 2. Effect of temperature on back pressure in porous poly(glycidy1 methacrylate-co-ethylene dimethacrylate) monolith grafted with poly(N- isopropylacrylamide-co-methylenebisacrylamide). Conditions: monolithic disk 10 mm thick, 10 mm i.d., water, temperature 25°C (empty squares), 40°C (filled squares)

less of whether the grafted chains existed in their extended or collapsed forms. This thermal valve behavior resulted from the fact that the MBAAm crosslinking did not allow the chains to swell to an extent sufficient to fill the pores completely. However, the back pressure at different flow rates was always much higher at a temperature of 25°C than at 40 "C. Thus, grafting in the presence of a crosslinker leads to composite material in which thermal control of flow rate can be effected.

Isocratic Hydrophobic Interaction Chromatography: The change in shape of these grafted polymer chains with tem- perature is accompanied by changes in surface polarity. The extended chains that prevail below the LCST are more hydrophilic, while the collapsed chains that exist above the LCST are more hydrophobic. This change in surface polar- ity has been used to control separation selectivity in reverse phase chromatography run under isothermal condi-

In light of the excellent mass transport properties of our polymeric monoliths and their particularly effective appli- cation in the separation of macromolecules,~23-261 the ther- mally induced change in surface polarity of the grafted composites was also tested to achieve the isocratic chroma- tographic separation of proteins in the hydrophobic inter- action mode. This mild separation technique is based on the interaction of hydrophobic surface patches of a protein with hydrophobic functionalities interspersed within the hydrophilic surface of the stationary phase.[271 Typically, proteins are eluted consecutively using a decreasing gradi- ent of salt concentration, with the more hydrophilic pro-

tions.121,221

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ADVANCED MATERIALS

teins eluting first. The re-equilibration of the column in the initial mobile phase after each analysis is a serious limita- tion for high throughput processes.

We found that the separation of proteins can be achieved at conslant salt concentrations by utilizing the hydropho- bic-hydrophilic transition of the grafted chains of PNI- PAAm that occurs in response to changes in temperature. Figure 3 shows the isocratic separation of carbonic anhy-

3 a a, c m

0 v)

n L

n a

0.009

0.004

0

0 8 16

Retent ion t ime, min

Fig. 3. Temperature-controlled hydrophobic interaction chromatography of carhonic anhydrase (1) and soybean trypsin inhibitor (2) using porous poly- (glycidyl methacrylale-co-ethylene diniethacrylate) monolith grafted with poly(N-isopropylacrvlamide-co-methylenebisacry1amide). Conditions: mo- nolithic disk 10 mm thick, 10 mm i.d., mobile phase 1.4 moliL ammonium SUlfdte in 0.01 mol/L phosphate buffer (pH 7). flow rate 1 mlimin.

drase and soybean trypsin inhibitor. First, thc 1 cm thick monolithic disk modified with the grafting solution con- taining MBAAm was heated to 40 "C, and a mixture of the two proteins was injected into the column in 1.4 mol/L am- monium sulfate solution at a flow rate of 1 mUmin. The more hydrophilic carbonic anhydrase is not retained under these conditions, and elutes immediately from the column. In contrast, the more hydrophobic trypsin inhibitor does not elute even after 10 min. However, once the tempera- ture of the column is lowered to 25 "C, the protein elutes al- most immediately.

In conclusion, we have developed a method for the func- tionalization of the pores of polymeric monoliths with tem- perature-responsive PNIPAAm. Depending on the grafting conditions employed, different functional thermally re- sponsive composites can be produced, such as thermal gates, which completely block flow through the micro- meter-sized pores of the monoliths, and thermal valves, which control flow rate. These materials can also be used for the isocratic hydrophobic interaction chromatography of proteins.

Experimental

All materials were used as received, except for NIPAAm, which was re- crystallized from hexane, and dried at room temperature in vacuo. All monoliths were prepared in 10 mm long, 10 mm i.d. stainless steel car-

Communications

tridges. The reaction mixture consisted of 24 vol.-% GMA, 16 vol.-% EDMA and 60 vol.-% cyclohexanol (porogenic solvent). Azobisisobutyro- nitrile (AIBN, 1 wt.-% with respect to monomers) was dissolved in the mix- ture, and the mixture was purged with nitrogen for 10 min. The columns were filled with the mixture, and the polymerization was allowed to proceed for 20 h at a temperature of 55°C. Upon completion, the columns were fitted with threaded end pieces containing 2 pm frits, and placed inside a corresponding stainless steel holder. The holder was attached to a high-per- ftrrmance liquid chromatography (HPLC) system, and 25 mL tetrahydrofur- an was pumped through the column to remove the porogenic solvent and any soluble compounds that remained in the column at the end of the poly- merization.

The polymer monolith was washed with 25 mL of water and 5 mL of a 50 wt.-% aqueous solution of ally1 amine was then flushed through the col- umn at 1 mLimin using a syringe pump (Sage Instruments, Model 341A). The imbibed column was sealed, and placed in a 60°C bath for 8 h. After the reaction was completed, water was pumped through the column to re- move the excess amine. This was continued until the pH of the effluent re- turned to the value for distilled water.

Two grafting solution were used. The first was used for the preparation of the thermal gate, and consisted of 10 wt:% NIPAAm in benzene. The sec- ond was used for the preparation of the thermal valve, and consisted of 9.9 wt.-% NIPAAm and 0.1 wt.-% MBAAm in benzene. AIBN (1 wt.-% with respect to monomers) was dissolved in each mlxture, and the mixtures were purged with nitrogen for 10 min. The ally1 amine modified column was washed with 5 mL THF (tetrahydrofuran) and 10 mL of benzene. 5 mL of the grafting solution was pumped through the column, the column sealed, and placed in a 60°C bath for 20 h. After the reaction was completed, the column was washed first with THF, and then with water.

A Waters HPLC system consisting of two 510 HPLC pumps and a 486 UV detector was used to carry out the chromatography and hack pressure measurement. The proteins were injected through a Rheodyne 7725 valve loop injector. and monitored at 280 nm. The data were acquired and pro- cessed with Millenium 2010 software (Waters).

Received: November 20.1996 Final version: March 10, 1997

[I] Packing and Stationary Phases in Chromatographic Techniques (Ed: K. K. Unger), Dekker, New York 1990.

[2] Synthetic Membranes: Science, Engineering and Applications (Eds: P. M. Bungay, H. K. Lonsdale, N. M. DePinho), Riedel, Dordrecht 1986.

[3] Y. Okahata, K. Ozaki, T. Seki, J. Chem. Soc., Chem. Cornmiin. 1984, 519.

[4] M. Gewehr, K. Nakamura, N. lse, H. Kitano, Macrornol. Chern. 1992, 193,249.

[S] K. Hosoya, E. Sawada, K. Kimata, T. Araki, N. Tanaka, J. M. J. Frechet, Macromolecules 1994,27,3973.

[6] H. Fiel, Y. H. Bae, J. Feijen, S. W. Kim, J. Mernbr. Sci. 1991, 64,283. [7] T. Ogata, T. Nonaka, S. Kurihara, J. Menrhu. Sci. 1995,103, 159. [8] Y. M. Lee, S. Y. Ihm, J. K. Shim, J. H. Kim. C. S. Cho, Y. K. Sung,

Polymer 1995,36,81. [Y] M. Konno. T. Tsuji, S. Saito, in Polymer Gels (Ed: D. DeRossi).

Plenum Press, New York 1991, p. 173. [lo] K. Ishihara, N. Hamada, S. Kato, I . Shinohara,J. Polym. Scr. A, Polym.

Chem. 1994,222,881. [ 111 L. C. Dong, A. S. Hoffman, ./. Controlled Release 1991, 15. 141. [12] L. C. Dong, Q. Yan, A. S. Hoffman,./. Controlled Release 1992,19,171. [I31 E. Kokufuta, 0. Ogane, H. Ichijo, S. Watanabe, 0. Hirasa, J. Chem.

Soc., Chem Commun. 1992,416, [14] M. Heskins, J.E. Guillet, E. J. James. J. Macromol. Sci., CIzm. 1968,

A2, 1441. [15] Y. H. Bae, T. Okano, S. Kim, J. Polym. Scz., Polym. Phys. 1990,28,923. [16] S. Fujishige, K. Kubota, I. Ando, J. Phys. Chem. 1989. 93, 3311. [17] J. M. J. Frechet, Makromol. Chem., Macrornol. Symp. 1993, 70/71.289. [18] C. Viklund, F. Svec, J. M. J. Frichet, K. Ingrum, Chern. Mateu. 1996,8,

744. [19] F. Svec, J. M. J. Frechet, Chem. Mateu. 1995,7,707. [20] R. B.Bird, W. E. Steward, E. N. Lightfoot, Transport Phenomena,

Wiley, New York 1960. [21] K. Kanazawa, K. Yamamoto, Y. Matsushima, N. Takai, A. Kikuchi, Y.

Sakurai, T. Okano, Anal. Chem. 1996,68,100. [22] K. Hosoya, K. Kimata, T. Araki, T. Tanaka, J. M. J. Frechet, Anal.

Chem. 1995,67,1907. [23] Q. C. Wang, E Svec, J. M. J. Frechet, Anal. Chem. 1993,65,2243. [24] E Svec, J. M. J. FrCchet. Biofechnol. Bioeng. 1995,48,476.

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ADVANCED AATERIALS

Communications

[25] F. Svec, J. M. J. FrCchet, J. Chromutogr. 1992,702,89. [26] M. Petro, F. Svec, J. M. J. FrBchet, Anal. Chem. 1996,68,315. [27] R. E. Shansky, S. I,. Wu, A. Figueroa, B. L. Karger, in HPLC ofBiolo-

gical Mucromolecules, Methods and Applications (Eds: K. M. Good- ing, F. R. Regnicr), Dekker, New York 1990, pp. 95-144.

Structures and Properties of MeDTDM Salts**

By Yohji Misaki," Masateru Taniguchi, Takeshi Miura, Hideki Fujiwara, Tokio Yamabe, Tadashi Kawamoto, and Takehiko Mori"

In the recent development in the field of organic conduc- tor~,[ '-~] unsymmetrical tetrathiafulvalene (TTF) deriva- tives have played an important role because several un- symmetrical TTF, tetraselenafulvalene (TSF), and diselena- dithiafulvalene (STF) derivatives, for example MDT- TTF[4351 and DMET,[4,61 have yielded a large number of supercond~ctors [~-~~ Recently, we have noted 1,3-dithiol-2- ylidene group as a substituent on TTF to develop stable or- ganic metals down to low temperatures, and synthesized many unsymmetrical TTFs fused with 1,3-dithio1-2-ylidenes (DT-TTFS)~~] as well as symmetrical ones.["] As expected, ethylenedithio-substituted DT-TTF derivatives MeD- TET[4*"1 and CPDTET[43'21 (Scheme 1) have afforded many

R = Me, MeDTET MeDTDM 2R =-(CH,)4-, CPDTET

Scheme 1

metallic cation radical salts in which the donors are ar- ranged in the so-called #-type fashion. In this context, mod- ification of DT-TTF is of interest to explore new metallic salts and perhaps superconductors. In particular, substitu- tion of methyl groups is attractive because several methyl- substituted donors have afforded s u p e r c o n d ~ c t o r s . ~ ~ ~ ~ ~ ~ I In this communication we report the structures and physical properties of MeDTDM salts, where MeDTDM is 2-isopro- pylidene-l,3-dithiolo[4,5-d]dimethyl-TTF.~'31

MeDTDM was prepared by a Wittig-Horner reaction of 4,s-( diethoxyphosphinyl)methylenedithio-4',5'-dimethyl- TTF 1 (Scheme 2)'141 with acetone in the presence of

[*I Dr. Y. Misaki, Prof. T. Yamabe, M. Taniguchi, T. Miura, H. Fujiwara Department of Molecular Engineering Graduate School of Engineering Kyoto University Yoshida, Kyoto 606-01 (Japan) Dr. T. Mori. T. Kawamoto Department of Organic and Polymeric Materials Faculty of Engineering, Tokyo Institute of Technology 0-okayama, Tokyo 152 (Japan)

[**I This work was partially supported by a Grant-in-Aid for Scientific Research on a Priority Area (No. 07232219), and Proposal-Based Advanced Industrial Technology R&D Program (No. A-1033).

Me

1 0

Scheme 2

lithium diisopropylamide (LDA) at -70 "C in 40 % yield. The cyclic voltammogram of MeDTDM exhibited three pairs of single-electron redox waves at +0.43, +0.75, and +1.37 V (vs. SCE, in benzonitrile at 25°C). Crystals of cation radical salts were prepared by an electrochemical oxidation of the donor in the presence of the correspond- ing tetrabutylammonium salts in chlorobenzene at a con- stant current of 0.2 PA. Electrical conductivity measure- ments were carried out using the four-probe technique. All the salts measured showed high conductivity, B , ~ = 10'-10* S cm-' (see Table l), most of which exhibited me- tallic temperature dependence (Fig. la). Among the me-

Table 1. Composition and electrical conductivity of MeDTDM salts (MeDTDM . A,).

~r, Is cm-'] [b] Conducting behavior Anion x [a1 c104 0.43 (Cl) 280 Metallic 220 K BF4 - ICI 610 Metallic 220 K ReOl 0.65 (Re) 52 GaCb 0.54 (Ga) 80 E. = 0.045 eV

Metallic 220 K PF6 0.59 (P) 230 A s F ~ 0.61 (As), 0.50 (X) 94 Metallic 220 K

[a] Determined by energy dispersion spectroscopy (EDS) from the ratio of sulfur and the elements designated in parentheses. X designates the value determined from X-ray structure analysis. [b] Room temperature conductivi- ty measured by four-probe technique on a single crystal. [c] The contents of light elements such as B and F were not reliably determined by EDS.

T M = 100 K

0 ' I 0 100 200 300

TIK-

b) ~ 1 0 . ~ I

t R i Qcrn

0 100 200 300 TIK-

Fig. I . Temperature dependence of resistivity of a) MeDTDM salts under ambient pressure and b) (MeDTDM)2AsF, under applied pressures; a pres- sure release of 3 kbar has been subtracted from the room temperature pres- sure values.

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