SEM-EDS Technique Employed in Evaluating the AggregationBehaviors of Amphiphilic ABC-Type Triblock Copolymers inMixed Solvents With Tuned PolaritiesJING XU,1 ZHAOSHENG HOU,2 XINDE TANG,3 JINYONG CHENG,1 AND TIANDUO LI1*1Key Laboratory of Fine Chemicals of Shandong Province, Shandong Institute of Light Industry, Jinan 250353,People’s Republic China2College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China3Institute of New Materials, Shandong Jiaotong University, Jinan 250023, China

KEY WORDS SEM-EDS technique; amphiphilic triblock copolymers; aggregation behavior

ABSTRACT In this paper, a scanning electron microscopy-energy dispersive spectroscopy(SEM-EDS) technique has been developed for evaluating the aggregation structure of amphiphilicfluorinated ABC-type triblock copolymers MeOPEO16-PSt220-PFHEA22 in mixed solvents with dif-ferent polarities. The polarities of mixed solvents can be tuned by changing volume ratios of tolu-ene, anhydrous ethanol, and distilled water, which leads to the changes in morphology and size ofself-assembled colloidal particles of the copolymers in the system. The aggregation behaviors of thecopolymers are revealed by SEM, transmission electron microscopy (TEM), and correspondingSEM-EDS techniques. The variations in concentrations of O and F elements over the thickness ofcopolymers particles give direct evidence for a better understanding of the arrangement of eachblock segment of copolymers in solution. And the technique can also help to explain the aggrega-tion structure of micro- or nanomaterial with shell-core structure. Microsc. Res. Tech. 74:1076–1082, 2011. VVC 2011 Wiley Periodicals, Inc.

INTRODUCTION

Recently, much attention has been paid on theamphiphilic block copolymers with fluorocarbon–hydrocarbon hybrid architectures because of theunique characteristics of fluoropolymer such as highthermal and mechanical stability, low surface energy,oil- and water-repellency, and very interesting surfaceproperties (Genzer and Efimenko, 2000; Luning et al.,2001). So, the kinds of copolymers have great manypotential applications in many fields including con-trolled-release system, antifouling coatings, drug deliv-ery system, and gene therapy (He et al., 2008; Townsin,2003). The ability to form a range of self-assembledaggregates is also one of the most important propertiesof amphiphilic block copolymers (Blanazs et al., 2009;Exerowa et al., 1997; Li et al., 2004, 2005; Tang et al.,2009). Most of the previous solution self-assembly stud-ies of block copolymers are performed for AB diblockcopolymers or ABC amphiphilic triblock copolymerswithout the segment of fluorocarbon–hydrocarbonhybrid architecture (Dupont et al., 2009; Holoubeket al., 2009; Lin et al., 2009; Smith et al., 2010). In thispaper, the triblock copolymers are amphiphilic fluori-nated ABC-type triblock copolymers MeOPEO16-PSt220-PFHEA22, consisting of 16 MeOPEO units, 220PSt units, and 22 PFHEA units, and the aggregationbehaviors of the copolymers in mixed solvent systemswith tuned polarities are researched.

The techniques of transmission electron microscope(TEM) and atomic force microscopy (AFM) havebeen used to observe the morphology and size ofself-assembled colloidal particles of copolymers (Dupont

et al., 2009; Fustin et al., 2005; Geng et al., 2005;Smith et al., 2010; Tang et al., 2009). On the basis ofour previous report that the morphology and size of col-loidal particles can be investigated by conventionalscanning electron microscopy (SEM), a morphologicaltransition process of self-assembly aggregates of thecopolymers in mixed solvent systems is revealed bySEM combined with TEM in this study.

Energy dispersive spectroscopy (EDS) is a good mea-surement technique to analyze the concentrations ofelements (Baalousha et al., 2006; Saleh et al., 2003).And the typical feature of EDS analysis is that thedepth of penetration into the colloidal particle of elec-tron beam will increase with the increasing accelerat-ing voltage. Based on the principle, a SEM-EDS tech-nique has been developed for evaluating the aggrega-tion structure of copolymers in solution. The variationsin concentrations of O and F elements over the thick-ness (the distance between a top point/pole and somecross section on a particle) of copolymers particles areanalyzed by SEM-EDS, which give direct evidence for abetter understanding of the arrangement of each blocksegments of copolymers in solution. And the techniquecan also be used to explain the aggregation structure ofmicro- or nanomaterial with shell-core structure.

*Correspondence to: Tianduo Li, Key Laboratory of Fine Chemicals of Shan-dong Province, Jinan 250353, China. E-mail: litianduo@163.com

Received 19 November 2010; accepted in revised form 25 January 2011

Contract grant sponsor: Science and Technology Development Plan of Shan-dong Province of China; Contract grant number: 2007GG10003048.

DOI 10.1002/jemt.20997

Published online 29 April 2011 inWiley Online Library (wileyonlinelibrary.com).

VVC 2011 WILEY PERIODICALS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 74:1076–1082 (2011)

MATERIALS AND METHODSTriblock Copolymers Samples

According to our and others’ papers, (Kamigaitoet al., 2001; Tang et al., 2009; Xia et al., 1999; Zhu andFord, 2008), an amphiphilic fluorinated ABC-type tri-block copolymers (MeOPEO16-PSt220-PFHEA22) com-posed of poly (ethylene oxide) monomethyl ether(MeOPEO, Mn 5 750), polystyrene (PSt), and poly (per-fluorohexylethyl acrylate) (PFHEA) was synthesizedby atom transfer radical polymerization (ATRP). Thechemical structure and molecular weight were con-firmed and measured by 1H NMR and gel permeationchromatography (GPC), as shown in Figure 1.

Triblock Copolymers Solution Preparation

The triblock copolymers investigated in this workcould not be directly dissolved in water. Therefore, co-polymer samples were first dissolved in anhydrous tol-uene to prepare a homogeneous solution at a concen-tration of 2.9 mg/mL. Anhydrous ethanol was gradu-ally added to the toluene solution and the polymersolution was shaken slightly. After an interval of 0.05mL of ethanol addition, the polymer solution wasallowed to sit at room temperature for at least 20 minto reach equilibrium (Tang et al., 2009). Then, waterwas slowly added into the mixed system with an inter-val of 0.05 mL and the polymer solution was alsoallowed to sit at room temperature for at least 20 minto reach equilibrium.

SEM/TEM Characterization

The TEM measurement was performed on a JEM-100CXII microscope (Hitachi, Japan) with an acceler-ating voltage of 100 KV. To prepare the TEM samples,a droplet of the aggregate suspension was set on a cop-

per grid for a few minutes. Excess solution was blottedaway with a strip of filter paper, and the sample gridwas dried under air atmosphere. To obtain the SEMsamples, the slide segments (0.5 cm 3 0.5 cm), whichwere small glass slides cut from a 1.0-mm-thick glassslide, were immersed in the aggregate suspension nextto each other and deposited for 2 min. And then, smallmolecules and most of the water were removed byinfrared lamps (250 W) for 30 s (the distance betweenthe sample and infrared lamp was about 10 cm). Last,the specimens should be continuously dried at roomtemperature to remove the residual water. All the driedsamples were coated with gold (5–10 nm thickness)using a Sputter Coater SCD-005 (TEC, England)and then observed under a Quanta-200 ESEM (FEI,Holland).

SEM-EDS Characterization

The concentration distributions of elements O and Fover the thickness of the copolymers particles wereanalyzed by SEM-EDS technique. EDS analysis wasperformed by EDAX instrument (USA). After themorphologies of colloidal particles for the copolymers(MeOPEO16-PSt220-PFHEA22) were exhibited by SEM(gold layer: 5–10 nm thickness), a proper region wasselected in which the colloidal particles were arrangeduniformly. The initial value of accelerating voltage (2.0KV) was determined based on the results of the calcu-lation for the overvoltage ratio (2.5) of elements O andF with analysis of Monte Carlo Simulation. Then, theconcentration of elements (O, F) were recorded with anaccelerating voltage increasing from 2.0 to 4.0 KV withan interval of 0.1 KV and the concentration-voltagecurves were obtained.

RESULTS AND DISCUSSION

The molecular architecture of block copolymers hada decisive impact on the shape of the aggregate mor-phology whatever the selective solvent, organic sol-vents or water. And the strength of hydrogen-bondinginteractions was highly sensitive to the polarity of sol-vent (Hadjichristidis et al., 2005). In this section, wefocused on the effect of the ratio of toluene/ethanol/water on the aggregate morphologies.

Reverser Micelles

The self-assembly aggregates were formed by gradu-ally adding ethanol to a homogeneous solution of thecopolymers (MeOPEO16-PSt220-PFHEA22) in toluene.The method had been used in many literatures to pre-pare crew-cut micelles from amphiphilic block copoly-mers (Zhang and Eisenberg, 1995, 1996). At the vol-ume ratio of toluene/ethanol being 1:2, the reversemicelles with spherical and spindle aggregates wereformed with a size range from 125 nm to 746 nm, asshown in Figures 2A (SEM image) and 2B (shell-corestructure, TEM image). Our analysis speculated thatintramolecular hydrogen bonds among MeOPEOchains caused them to form the core, and the PSt andPFHEA chains were not segregated but well mixed inthe shell of the spherical and spindle aggregates. Dueto the polarization function of ethanol, PFHEA chainsshould be in the corona of the small spherical aggre-gates which then seemed to gather into large spindleaggregates (Tang et al., 2005).

Fig. 1. Chemical structure of the ABC-type fluorinated triblockcopolymers.

Abbreviations:

AFM atomic force microscopy

ATRP atom transfer radical polymerization

GPC gel permeation chromatography

PFHEA poly (perfluorohexylethyl acrylate)

PSt polystyrene

SEM-EDS scanning electron microscopy-energy dispersive spec-

troscopy

TEM transmission electron microscopy.

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Based on the speculation, MeOPEO chains formedthe core, and PSt and PFHEA chains were in the shell/coronal layer of the spherical aggregates, and the con-centration of element O should have a obvious peak inthe distribution tendency curve. For the concentrationof element F, the distribution tendency should havetwo obvious peaks over the thickness of the copolymersparticles. The 3D model described for representing ouridea was shown in Figure 3, and the volume occupiedby different block chains was described based on themolecular weight of them.

A rough calculation indicated (overvoltage ratio: 2.5)that characteristic X-ray of elements O and F could beexcited when the accelerating voltage was lager than1.3 and 1.6 KV, respectively. Based on a simple analysisof Monte Carlo Simulation, the relationship betweenthe depth of penetration into the colloidal particle ofelectron beam and the value of accelerating voltagewas obtained. Thus, the operation conditions for SEMwere confirmed: the value of accelerating voltageincreased from 2.0 to 4.0 KV with an interval of 0.1 KV,

and spot size (electron beam) was 6.9 nm. As deter-mined by EDS (working mode: Spectrum), the distribu-tion tendency curve of the concentration of element Oover accelerating voltage was shown in Figure 4 (realline). When accelerating voltage was lower than 2.5KV, the concentration of element O was fluctuant witha small range (3.65–3.3 wt%), which was caused byinterface between gold layer and samples surface.When accelerating voltage was 2.5 KV, the concentra-tion of element O began to increase and reached thelargest value (5.4 wt%) at 3.0 KV, and then the valuedecreased with accelerating voltage increasing. At 3.6KV, the concentration of element O reached the lowestvalue (3.56 wt%) and then kept fluctuation in the rangeof 3.56–3.95 wt%. So, our analysis speculated that elec-tron beam had already passed through the whole parti-cle when the accelerating voltage was 4.0 KV.

The distribution tendency curve of the concentrationof element F over accelerating voltage was also shownin Figure 4 (dashed line). When accelerating voltage

Fig. 2. SEM (A) and TEM (B) images of aggregates formed from triblock copolymer at volume ratio of toluene/ethanol being 1:2.

Fig. 3. The 3D model of reverser micelles with spindle aggregatesformed by MeOPEO16(red)-PSt220(yellow)-PFHEA22(blue), and thelength of lines represented the weight percent of elements O and F,respectively. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.] Fig. 4. The distribution curves of the concentrations of elements O

and F over accelerating voltage for reverser micelles.

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was lower than 2.6 KV, the concentration of element Fwas fluctuant with a small range (3.26–3.59 wt%).When accelerating voltage was higher than 2.6 KV, theconcentration of element F began to increase andreached the largest value (6.65 wt%) at 3.1 KV. Afterthat the value gradually decreased to 5.38 wt% (3.4KV) and then increased to another largest value (6.12wt%) at 3.6 KV. When the accelerating voltageincreased from 3.7 to 4.0 KV, it kept fluctuating with asmall range (5.36–5.62 wt%), which meant that elec-tron beam had already passed through the particle.

The results of quantitative microanalysis of SEM-EDS were consistent with our speculation: the reversemicelles were believed to be composed of MeOPEOchains as the core and PFHEA chains as the corona,and PSt chains were a linkage between MeOPEOchains and PFHEA chains.

Micelles

At the volume ratio of toluene/ethanol being 1:3,spindle aggregates had disappeared and some higherlevel aggregates formed from these small micelles, as

Fig. 5. Aggregates morphologies observed by SEM images (A–E) and TEM image (F) formed from the copolymers at volume ratio of toluene/ethanol being 1:3, 1:5, 1:8, 1:10, 1:15 and 1:15, respectively.

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shown in Figure 5A. With continuous addition of etha-nol, up to the volume ratio of toluene/ethanol being 1:5,the polymer chains formed heterogeneous colloidalspheres with a size of around 500 nm, as shown in Fig-ure 5B. At the volume ratio of toluene/ethanol decreas-ing to 1:8, our TEM (Fig. 5F) and SEM (Fig. 5C) analy-sis indicated that the triblock copolymers formedmicelles (shell-core structure) with bead-chain-likeaggregates and the size of beads was in a range from150 to 500 nm. It could be speculated that ethanol wasgood solvent for MeOPEO chains, but a poor solvent forthe PSt and PFHEA block, and with the increasing ra-tio of ethanol, PSt, and PFHEA chains segregated outfrom the solvent phases. Thus, the micelles werebelieved to be composed of PSt and PFHEA chains asthe core and MeOPEO chains as the corona. And also,we could deduce that PSt chains were a linkagebetween PFHEA chains and MeOPEO chains. With theincreasing volume ratio of ethanol (toluene/ethanol:1:10), the bead-chain-like aggregates were alsoobserved by SEM (Fig. 5D), but the size of the beadsbecame uniform (about 200 nm), which attributed tothe increasing strength of hydrogen bond betweenethanol and MeOPEO chains. With further increasingratio of ethanol, the number of colloidal particles wasdecreasing but the morphology had no great change(Fig. 5E). Above all, the size and shape of the micellaraggregates strongly depended on the balance of freeenergy of the core-solvent interface and on the freeenergies of the shell and the core (Hamley, 1998).

With increasing volume ratio of ethanol, we assumedthat MeOPEO chains formed shell and coronal layer,and PSt and PFHEA chains formed core of the spheri-cal aggregates. Thus, the concentrations of element F,O over the thickness of the copolymers particles shouldhave an obvious peak and two obvious peaks, respec-tively. The 3D model described for representing theidea was shown in Figure 6.

Based on the similar simple analysis of Monte CarloSimulation, the operation conditions for SEM-EDXwere confirmed, which was the same with that of re-verser micelles. The distribution curve of the concen-tration of element O over accelerating voltage wasshown in Figure 7 (real line). When accelerating volt-

age was lower than 2.2 KV, the concentration of ele-ment O was fluctuant with a thin range (2.86–3.05wt%), which might be caused by surface adsorption ofsamples. The concentration of element O began toincrease when accelerating voltage was higher than2.2 KV, and reached the largest value (4.76 wt%) withaccelerating voltage increasing to 2.6 KV. After that,the concentration of element O decreased graduallyup to the accelerating voltage increasing to 3.0 KV.Then, the concentration increased again and reachedanother largest value (3.62 wt%) with acceleratingvoltage being 3.3 KV. When the accelerating voltagewas higher than 3.3 KV, the concentration decreasedand kept fluctuation with a small range, which meantthat electron beam had already passed through theparticle.

The dashed line in Figure 7 was the distributioncurve of concentration of element F over acceleratingvoltage. When accelerating voltage was lower than 2.7KV, the concentration of element F was fluctuant witha small range (2.91–2.09 wt%). It reached the largestvalue (5.09 wt%) with accelerating voltage increasingto 2.9 KV, and after that, the concentration of elementF gradually decreased to 4.57 wt% and kept fluctuationin a small range.

The results of quantitative microanalysis of SEM-EDS were also consistent with our speculation: MeO-PEO chains formed shell layer, and PSt and PFHEAchains formed core.

It should be noticed that the weight percent of ele-ment O/F examined by EDS had difference from theactual concentration of them in the copolymers. Onereason was that characteristic X-ray of light elements,such as C/O/N/F, easily led to surface adsorption whichcaused the data be different from actual values.Another was that the density of the sample observed inthe study was not uniform. So, we could not providethe actual concentration of element O/F, but we coulddescribe the distribution trendy of the concentration ofelement O/F over the thickness of the copolymers par-ticles and directly reveal the arrangement of blockchains in different aggregates. Thus, SEM-EDS analyt-ical method was a good measurement to understand

Fig. 6. The 3D model of micelles with spherical aggregates formedby MeOPEO16(red)-PSt220(yellow)-PFHEA22(blue) and the length oflines represented the weight percent of elements O and F, respec-tively. [Color figure can be viewed in the online issue, which is avail-able at wileyonlinelibrary.com.]

Fig. 7. The distribution curves of the concentration of elements Oand F over accelerating voltage for micelles.

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the arrangement of different block chains during self-assembly process.

Vesicular

The various reported morphologies were primarily aresult of the inherent molecular curvature and howthis influenced the packing of the copolymers chains:self-assembled nanostructures could be targetedaccording to a dimensionless ‘‘packing parameter’’ p,which was defined in the equation: p 5 V/aolc, where Vwas the volume of the hydrophobic chains, ao was theoptimal area of the head group, and lc was the length ofthe hydrophobic tail (Blanazs et al., 2009; Dupontet al., 2009). Therefore, the packing parameter of agiven molecule usually dictated its most likely self-assembled morphology. As a general rule, sphericalmicelles were favored when p � 1/3, cylindricalmicelles when 1/3 � p � 1/2, enclosed membrane struc-tures (vesicles, also known as ‘‘proteopolymersomes’’)when 1/2 � p � 1, and reverse micelles when p � 1. Sol-

vation effect on MeOPEO chains in aqueous solutionwas effective to expand the ‘‘heads’’ of a micelle andadjust p in terms of the classical geometric-packing pa-rameter theory. And hydrophobic effect for PSt chainsand PFHEA chains in aqueous solution contributed toform vesicular aggregates for amphiphilic copolymers.Thus, we anticipated a morphological transition for thecopolymers under the condition of addition water tosystem. Polymer chains formed membrane with net-work-like structure when the volume ratio of toluene/ethanol/water was 1:3:8 (Fig. 8A). Vesicular aggregateswith a size of around 200 nm were formed when thevolume ratio of toluene/water/ethanol was 1:8:8 (Fig.8B). In the case, the system polarity was improved con-tinuously through addition water to solution, and withthe ratio of water increasing (the volume ratio of tolu-ene/ethanol/water was larger than 1:8:10) the struc-ture of vesicles had no obvious change but the size ofvesicles increased obviously (Fig. 8C), and this struc-ture of vesicles was also observed by TEM (Fig. 8D).

Fig. 8. Aggregates morphologies observed by SEM images (A–C) and TEM image (D) formed from the copolymers at volume ratio of tolu-ene/ethanol/water being 1:3:8, 1:8:8, 1:8:10 and 1:8:8, respectively.

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After a series of experiments, we found that membranewith network-like structure could always be obtainedwhen the volume ratio of toluene/ethanol was lagerthan 1:5 whatever the amount of water, and vesicularaggregates could be obtained when the volume ratio oftoluene/ethanol was lower than 1:5 whatever theamount of water.

CONCLUSIONS

In this work, a SEM-EDS technique has been devel-oped for evaluating the aggregation structure ofamphiphilic fluorinated ABC-type triblock copolymersMeOPEO16-PSt220-PFHEA22 in mixed solvents withdifferent polarities. The system polarity can be tunedcontinuously by changing volume ratio of the solventsincluding toluene, anhydrous ethanol, and distilledwater, and then a morphological transition process ofself-assembly aggregates for the amphiphilic blockcopolymers in the system are revealed by SEM, TEMimages, and corresponding SEM-EDS patterns. Thevariations in concentration distributions of O and Felements over the thickness of copolymers particlesgive a direct evidence for well understanding thearrangement of each block segments in self-assemblyaggregation of copolymers in solution.

ACKNOWLEDGMENTS

The authors thank EDAX Company for providing theanalysis software of Monte Carlo Simulation.

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