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Polymer 53 (2012) 466e470

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Polymer

journal homepage: www.elsevier .com/locate/polymer

Synthesis, separation and characterization of knotted ring polymers

Yutaka Ohta a, Masahide Nakamura b, Yushu Matsushita a, Atsushi Takano a,*

aDepartment of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japanb Scientific Instruments Division, Shoko Scientific Co., Ltd., 1-3-3 Azaminominami, Aoba-ku, Yokohama, Kanagawa 225-0012, Japan

a r t i c l e i n f o

Article history:Received 5 September 2011Received in revised form7 December 2011Accepted 18 December 2011Available online 21 December 2011

Keywords:Ring polymerKnotInteraction chromatography

* Corresponding author. Tel.: þ81 52 789 3211; faxE-mail address: atakano@apchem.nagoya-u.ac.jp (

0032-3861/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.polymer.2011.12.034

a b s t r a c t

Knotted ring polystyrene (PS) with molecular weight of 380 k was successfully synthesized by intra-molecular cyclization reaction in cyclohexane under extremely diluted condition. Crude product wasconfirmed to include linear precursor molecule, single ring molecules, and various intermolecular-reacted byproducts by SEC and interaction chromatography characterizations. The crude product wasfractionated repeatedly several times by high performance interaction chromatography and finallyhighly-purified knotted ring molecules were obtained. It has been found by SEC-MALS that the chaindimension of the knotted ring polymers is evidently smaller than those of linear and the trivial ringpolymer, while knotted polymer molecules have the same absolute molecular weight as the corre-sponding counterparts.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Ring polymers show specific structures with no chain ends andthey have been attracting many researchers. In addition to theo-retical [1e8] and simulational researches [9e13], experimentalinvestigations on ring polymers were conducted in accordancewith the progress of synthetic methods for several decades[8,14e43]. A ring polymer has topological isomers, e.g., a concate-nated ring and knotted ring polymers and they themselves are alsointeresting targets in polymer science. However, experimentaltrials to prepare these polymers have hardly been proceededbecause of the difficulty in synthesizing these complicated mole-cules by regular synthetic approaches.

The methods of preparing ring polymers can be divided intoseveral types. For example, ring expansion reaction of small cyclicmolecule [14e17], intermolecular coupling reaction betweena polymer with two carbanion end groups and a bifunctionalcoupling reagent [8,18e24] and simpler intramolecular couplingreaction [25e31] were developed in several decades. Thanks tosuch progress in synthetic methods, fundamental properties suchas solution and viscoelastic properties of ring polymers can beelucidated in recent years. Additionally, these synthetic methodsfor ring polymers lead to the simultaneous syntheses of theirtopological isomers. In short, topological isomers of a ring polymercan be also produced by bimolecular coupling or intramolecular

: þ81 52 789 3210.A. Takano).

All rights reserved.

end-to-end coupling reactions taken place statistically. Synthesis ofa catenated polymer with relatively high molecular weight isconducted by an intramolecular ring closure reaction naturallyhooked on a ring polymer under diluted condition [44,45], whileknotted ring molecules can be possibly prepared by an end-to-endintramolecular reaction occurred under the condition that themolecule possesses self-entanglement. Little has been reported forknotted ring polymers except for the ring DNAs and oligomers.Knotted ring DNAs have been synthesized and observed by TEM byLiu et al. [46,47] and Krasnow et al. [48]. However DNA is regardedas a rigid chain due to inter- and/or intramolecular interaction bycomplementary base pairs. To elucidate the topological effects onpolymer chain behavior free from the inter- and/or intramolecularinteractions, knotted ring polymers consisting of flexible chains arerequired. Formation of knotted oligomers using metal templatesyntheses have been reported by many researchers [49e58], butthey cannot be used for the investigation of topological effects onpolymer chain as well. Recently Ohta et al. reported the formationof knotted ring polystyrenes in cyclization reaction product of tel-echelic polystyrene with high molecular weight (Mw ¼ 380k)synthesized in cyclohexane, which is a theta solvent of polystyrene[59]. Direct observation of knotted ring polymers by an atomic forcemicroscopy was reported by Schappacher et al. [60], however, theseparation (isolation) and characterization of these polymers arenot achieved successfully even today, so that several properties arenot known yet so far.

Recently a novel liquid chromatography technique called “liquidchromatography at critical condition” (LCCC) was developed andthis method enables to analyze and separate the polymers with

Fig. 1. SEC chromatograms of linear precursor (black curve), cyclized products in THF(red curves) and that in cyclohexane (blue curves) of (a) Crude products and (b) SEC-fractionated products. L1 denotes non-reacted linear precursor, R1 does intra-molecular coupling ring polymer, while LX and RX mean intermolecular-coupled linearor ring X-mers.

Y. Ohta et al. / Polymer 53 (2012) 466e470 467

differentmolecular architectures [24,32e34]. Moreover, interactionchromatography (IC) is useful for analysis and separation of ringpolymers with high molecular weight [30]. By applying thesemethods to the preparation of ring chains, we can prepare ringpolymers and can even determine their purity quantitatively. Herewe report on the synthesis and separation of knotted ring polymersby using these high-performance liquid chromatography methodscoupled with light scattering techniques.

2. Experimental

2.1. Materials and sample preparation

Industrial grade of tetrahydrofuran (THF) for SEC analysis andSEC fractionation was purchased from Daishin Chemical Co. Ltd.and used as received. Acetonitrile and dichloromethane were alsopurchased from Kishida Chemical Co. Ltd. and used as received forLCCC and IC analyses. For polymer syntheses, GR grade of THF werepurchased from Hayashi Pure Chemical Inc., Ltd. and purified usinganthracene soduim in glass apparatus in vacuo, being used for bothpolymerization and cyclization reactions. EP grade of cyclohexanewas purchased from Kishida Chemical Co. Ltd. and distilled withn-butyl lithium and then sealed in a glass apparatus. The purifica-tion of styrene and 1,1-diphenylethylene (DPE) were conducted asthe same manner reported previously [61].

The telechelic polystyrene (PS) with the molecular weight of3.8 � 105 having a DPE type double bond on both ends wassynthesized by an anionic polymerization and followed by endcapping reaction [29]. The telechelic polymers were diluted withTHF as a good solvent and also with cyclohexane as a poor solventat concentration of ca. 0.05%. Potassium naphthalenide as a cycli-zation reagent in THF was added into the solutions of the telechelicPSs and stirred for 12 h, and the reaction temperatures were 25 �Cin THF and 34 �C in cyclohexane, respectively. Latter condition isnear the q-temperature of the solvent for PS. The products thusobtained were precipitated into an excess amount of methanol toremove naphthalene and the other chemical residues, followed byfreeze-drying from dioxane solutions. The details of experimentalprocedures were reported previously [59].

2.2. Characterization

The SEC analyses and SEC fractionation experiments were con-ducted by using an HPLC pump, DP-8020 of Tosoh Ltd., a UVdetector, UV-8020 of Tosoh Ltd., and a Rheodyne 7125 injectorequipped with a 100 mL sample loop. A set of three polystyrene gelcolumns (TSK-gel G5000HHR, 7.8 mm(I.D.) � 300 mm, particle sizeis 5 mm and pore size is 65 nm) of Tosoh Co. Ltd. was used for thehigher resolution analyses. The eluent used was THF and the flowrate was 1.0 mL/min. The column temperature was kept at 40 �C bya column oven, CO-8020 of Tosoh Co. Ltd.

The IC analyses and IC fractionation experiments were carriedout by using the same apparatuses, i.e. a pump, a UV detector andan injector as for SEC. A set of two ODS gel columns (Inertsil WP300C18, 4.6 mm(I.D.) � 25 mm, particle size is 5 mm and pore size is30 nm) of GL Science Inc. was used for the separation of eachcomponent. The eluent used was the mixture of acetonitrile anddichloromethane (42/58, v/v) and the flow rate was 0.5 mL/min.The column temperature was precisely controlled at 27.5 �C bya hand-made column jacket and a cooling circulator, HAAKEPhoenix II C25P. In this condition LCCC for linear polystyrenes wasobserved at 30.5 �C.

The SEC-MALS systemwas also consisted of an HPLC pump (DP-8020), a column oven (CO-8020) and three polystyrene gel columns(TSK-gel G5000HHR) of Tosoh Co. Ltd. The eluent used was THF and

the flow rate was 1.0 mL/min. The column temperature was kept at40 �C. AMALS detector, DAWNHELEOS-II of Wyatt Technology, wasused to measure scattering intensities, where wavelength of thelaser light adopted is 658 nm, and temperature of light scatteringcell was kept at 40 �C. Optilab rEX differential refractometer wasconnected to the SEC system described above for SEC-MALSmeasurements.

3. Results and discussion

The samples used and compared were two cyclization productsfrom a telechelic PS with molecular weight of 380k in a goodsolvent (THF) and in a poor solvent (cyclohexane). Our maininterest is in separation of unknotted ring polymer chains, which isdefined as “trivial” ring here, and “knotted “ones included in thecyclization products.

Fig.1(a) compares SEC chromatograms of linear precursor (blackcurve), cyclized product in THF (red curve) and that in cyclohexane(blue curve). From these curves, we clearly notice that the dimer-ized linear and ring components (L2, R2), trimerized components(L3, R3), and also multicoupling components were produced bycyclization reactions in two solvents in addition to an intra-molecular cyclic component (R1). These intermolecular byproductswere fractionated out from the crude product by preparative SECfractionation. Fig. 1(b) shows the chromatograms of a fractionatedproduct cyclized in THF (red) and that in cyclohexane (blue) afterSEC fractionation. Referring two chromatograms in Fig. 1(a), it isclear that intermolecular byproducts were thoroughly removedfrom both cyclization products by successful fractionation. In

Y. Ohta et al. / Polymer 53 (2012) 466e470468

addition, we can notice the small difference in two chromatogramsat the elution volume of around 21.0e21.8 min in Fig. 1(b), that is,two peaks are not overlapping completely, and the ring formed incyclohexane (blue curve) is spread to lower molecular weight sideslightly, and hence has apparently wider molecular weight distri-bution than the ring formed in THF (red curve). In these SEC-fractionations, the quantitative recovery yields of the fractionatedproduct from the cyclization reaction product in THF (0.25 g) was

Fig. 2. IC chromatograms of (a) Linear precursor (black curve), SEC-fractionatedproduct from cyclized sample in THF (red curve), that in cyclohexane (blue curve)and that for toluene (dashed black curve), (b) SEC-fractionated product from cyclizedsample in THF (red curve), that in cyclohexane (blue curve). Figure (c) Compares threefractions for areas #1, #2 and #3 pointed out in figure (b). (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version ofthis article.)

0.10 g (40%), while the yield of another fractionated product fromthe cyclization reaction product in cyclohexane (0.25 g) was 0.13 g(52%).

Furthermore, IC analyses of two fractionated samples wereconducted to isolate ring polymers. Fig. 2(a) compares IC chro-matograms of the fractionated cyclization products reacted in THF(red) and that in cyclohexane (blue) together with that of linearprecursor (black), and also that of toluene as a solvent peak (dashedblack line). From the comparison, it is apparent that both products(red and blue curves) contain solvent eluted at around 8 mL, andlinear polystyrene with molecular weight of 380 k appears ataround 12 mL. Two chromatograms also contain peaks eluted at16 mL, which are associated with single ring polymers because ofthe stronger interaction of a ring molecule than a linear one to thestationary phase. Moreover if we compare two curves carefully, anadditional shoulder is conceived at around 17.5e22 mL in thechromatogram of the fractionated product reacted in cyclohexane.This shoulder might be due to the knotted molecules, which shouldhave higher segment density than a linear molecule and evenhigher than a regular (trivial) ringmolecule, in otherwords, knottedmolecules have stronger adsorption interaction than a linear andalso a trivial ring molecules to the stationary phase.

To separate trivial and knotted ring molecules, IC fractionationwas conducted from two SEC-fractionated products. The fraction-ation areas were shown as #1, #2 and #3 designated in Fig. 2(b),where the double vertical bars indicate the boundaries of eachfraction. The quantitative recovery yield of fraction #1 from theSEC-fractionated cyclization product reacted in THF (100 mg) was

Fig. 3. IC chromatograms of (a) Fraction #3, and (b) Fraction #4 obtained by thesuccessive IC fractionation.

Table 1Molecular characteristics and composition of the topological isomers in eachsample.

Sample 10�3 Mwa Rg

a/nm Mole fractionb

Linear Trivial Knotted

Linear 380 24.7 100 e e

#1 383 18.4 5 95 e

#2 377 18.0 6 94 e

#3 378 16.8 4 25 71#4 380 16.0 e 6 94

a Determined by SEC-MALS in THF.b Determined by IC.

Y. Ohta et al. / Polymer 53 (2012) 466e470 469

35 mg, while the recovery yields of fractions #2 and #3 from theSEC-fractionated cyclization product reacted in cyclohexane(100mg) were 3.6 mg and 24mg, respectively. Fig. 2(c) compares ICchromatograms of the three fractions obtained, i.e. fraction #1(pink), #2 (sky blue) and #3 (violet), together with that of the linearprecursor (black). It is evident that the curves for the fraction #1(pink) and #2 (sky blue) are almost overlapping, possibly indicatingthat they could be ‘trivial’ ring molecules. On the other hand frac-tion #3 (violet) consists of the same component appeared at16.0 mL in fraction #1 and #2 and also an additional componenteluted at higher elution volume,18.9 mL, indicating the molecule infraction #3 show stronger adsorption interaction than a trivial ringto the stationary phase. This new peak could be the strong evidenceof “knotted rings”. From the peak areas of the IC chromatogramswecan estimate the content of two ring molecules in each sample. Thefractions #1 and #2 contain mostly trivial ring molecule, however,the fraction #3 contains 71% of knotted molecules.

In order to obtain more purified knotted ring sample, additionalIC fractionation was conducted for fraction #3. Fig. 3(a) shows theIC chromatogram of the fraction #3 (violet) with the IC fraction-ation area pointed out by double vertical bars, and the new fractionis designated as fraction #4. The final quantitative recovery yield offraction #4 from fraction #3 (2.5 mg) was approximately 1.2 mg.Fig. 3(b) compares IC chromatograms of the trivial ring (the fraction#1, pink) and a highly-purified ring (the fraction #4, green). Fromthis green curve, wewere able to estimate high fraction of the mainpeak, i.e., 94%, which must be composed of knotted ring molecules.

SEC-MALS analysis of IC-fractionated products were carriedout to compare the absolute Mws and Rgs. Fig. 4(a) shows SEC

Fig. 4. SEC chromatograms of linear precursor (black curve), the fraction #1(red curve) and the fraction #4 (purple curve). Figure (a) Exhibits absolute molecularweight of linear molecule, fraction #1 and #4, while figure (b) Compares Rgs of thosesamples. (For interpretation of the references to colour in this figure legend, the readeris referred to the web version of this article.)

chromatograms of linear (black), trivial ring (fraction #1, pink) andhighly-purified knotted ring (fraction #4, green) together with thecorresponding Mw plots. This figure expresses that absolute Mwsare almost the same one another. Rg value of the fraction #4 (green)is compared with those of the trivial ring (fraction #1, pink) andlinear precursor (black) in Fig. 4(b). It is evident that the highly-purified knotted ring (fraction #4, green) has definitely smaller Rg

than the trivial ring (fraction #1, pink), hence we are confident thatknotted ring molecules with high purity have been prepared by thepresent experimental procedure.

Table 1 summarizes absolute molecular weights and radii ofgyration for linear precursor and four kinds of ring polymers(fraction #1-#4), and also the composition of the topologicalisomers in each sample estimated from the area analyses of the ICchromatograms. It is obvious that molecular weight is the sameamong linear, trivial ring and knotted rings within experimentalerrors, while the radii of gyration are clearly different. Stochasti-cally trefoil-type knot might be mostly formed. However we do notknowwhich types of knots are formed in the isolated sample at themoment. Presently we are synthesizing the knotted ring poly-isoprenes by the same synthetic strategy to transform it intobrushed ring polymer chains for future observation by an atomicforce microscopy as performed by Schappacher et al. [60].

In conclusion, knotted ring polymers, which have smaller chaindimensions than a trivial ring polymer, were synthesized success-fully by intramolecular cyclization reaction of long linear chains ina poor solvent, and they can be isolated from linear molecule andtrivial ring molecule by multi-step IC fractionation procedure.

Acknowledgments

Y.O. acknowledges for the financial support from the Global COEProgram entitled “Establishment of COE for Elucidation and Designof Materials and Molecular Functions” (G-COE in chemistry,Nagoya), which has been selected as one of the programs sponsoredby the Ministry of Education, Culture, Sports, Science and Tech-nology of Japan.

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