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Journal of Chromatography A, 1216 (2009) 5011–5019

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

reparative size-exclusion chromatography for purification and characterizationf colloidal quantum dots bound by chromophore-labeled polymers andow-molecular-weight chromophores

ingfeng Wang, Ghasem Rezanejade Bardajee1, Sandeep Kumar, Mark Nitz,regory D. Scholes ∗, Mitchell A. Winnik ∗∗

epartment of Chemistry, University of Toronto, 80 St. George Street, Toronto, M5S 3H6 Ontario, Canada

r t i c l e i n f o

rticle history:eceived 13 November 2008eceived in revised form 18 April 2009ccepted 20 April 2009vailable online 3 May 2009

a b s t r a c t

We explore the use of preparative size-exclusion chromatography (SEC) and high-performance liquidchromatography (HPLC) to purify quantum dots (QDs) after surface modification. In one example, in whichBio-Beads (S-X1) were used as the packing material for the preparative SEC column, CdSe QDs treatedwith a functional coumarin dye could be separated from the excess free dye by using tetrahydrofuran

eywords:igh-performance liquid chromatographyize-exclusion chromatographyuantum dotsolymers

(THF) as the mobile phase. This column was unable to separate polymer-coated QDs from free polymer(M ∼ 8000) because of the relatively low cutoff mass of the column. Here a preparative HPLC columnpacked with TOYOPEARL gel allowed the effective separation of polymer-bound QDs from the excess freepolymer by using N-methyl-2-pyrrolidinone (NMP) as the mobile phase. When other solvents such asabsolute ethanol, acetonitrile, THF, and THF–triethylamine mixtures were used as the eluent, QDs stuckto the column. While NMP was an effective medium to remove excess free polymer from the QDs, it was

rified

hromophores difficult to transfer the pu

. Introduction

Polymers have been extensively used for surface modifica-ion of nanocrystals to tune their physical properties, and tompart other functionalities for biological and optical/electricalpplications [1,2]. We are particularly interested in semiconductoranocrystals, also called quantum dots (QDs) due to their uniqueize-dependent optoelectronic properties. In previous publications,e reported that poly(2-N,N-dimethylaminoethyl methacrylate)

PDMA) in toluene could replace TOPO from the surface ofdSe/TOPO QDs [3–7]. This process has been studied in some detail,o that one has an idea of how many polymer chains bind to QDsf certain sizes [5], and how many DMA groups of the polymerarticipate in replacing each TOPO [7]. Our previous results sug-est that PDMA binds as a multidentate ligand to the surface of

dSe QDs. The driving force that contributes to this interaction mayome from two effects: one is the entropic gain that derives fromhe dissociation of TOPO ligands from the QD surfaces, assuminghat the entropy loss from the polymer binding to QDs is much

∗ Corresponding author.∗∗ Corresponding author. Tel.: +1 416 978 6495; fax: +1 416 978 0541.

E-mail addresses: gscholes@chem.utoronto.ca (G.D. Scholes),winnik@chem.utoronto.ca (M.A. Winnik).1 Current address: Department of Chemistry, Payame Noor University, Iran.

021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.04.060

QDs to more volatile solvents and maintain colloidal stability.© 2009 Elsevier B.V. All rights reserved.

smaller. The other is the enthalpic effect from the binding betweenthe tertiary amines of the polymer and the Cd sites on the QDsurfaces.

Polymer-coated QDs are normally prepared by treating the QDswith excess polymer in order to saturate the QD surfaces and toavoid undesired bridging, i.e. one polymer chain binding to two ormore than two QDs. As a consequence, only a fraction of the addedpolymers binds to the nanoparticles. For many end-use applicationsof these materials, one should separate the polymer-bound QDsfrom the excess free polymer. This is not easy to do.

Several separation methods have been used to charac-terize the hydrodynamic size of inorganic nanoparticles andto separate polymer-bound nanoparticles from free polymer.These include gel electrophoresis [8–10], high-performance liquidchromatography (HPLC) [10–12], and size-exclusion chromatog-raphy (SEC) [13,14]. For example, Parak and co-workers [9]prepared polymer-coated colloidal core–shell CdSe–ZnS QDs ini-tially passivated with a surface layer of trioctylphophine oxide(CdSe–ZnS/TOPO). They synthesized an amphiphilic polymer bylinking an amino-functionalized hydrocarbon chain (dodecy-

lamine) and an amino-modified fluorophore (AATO590) to someof the anhydride rings of poly(isobutylene-alt-maleic anhydride)(m ≈ 6000). The procedure of coating core–shell CdSe–ZnS/TOPOQDs with this polymer involved the dispersion of the QD/polymermixture in chloroform, followed by a drying process and then redis-

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ersion in a sodium borate buffer (pH 12). This polymer-coatingrocedure resulted in some empty polymer micelles in addition tohe polymer-coated QDs. These empty micelles could be separatedy gel electrophoresis based on the fact that they eluted faster thanhe polymer-coated particles.

The Wilcoxon group [15] employed HPLC to fractionate TOPO-oated CdSe and core–shell CdSe–ZnS QDs of various sizes usingetrahydrofuran (THF) as the mobile phase. They used a high-esolution column (Polymer Laboratory, model PL50) filled with-�m microgel particles of cross-linked polystyrene to separatehe nanoparticles. Colvin’s group [13] studied the size and size dis-ribution of TOPO-coated CdSe nanocrystals by high-performanceEC using 0.1 M trioctylphosphine in toluene as the mobile phase.ubarev’s group [16,17] applied centrifugal membrane filtration onegenerated cellulose for the purification of polymer-functionalizedold and silver nanoparticles, and gold nanorods from the residualree polymer chains.

In a previous publication [5], we reported as a proof of concepthat SEC could be used to separate CdSe QDs bearing pyrene-abeled poly(2-N,N-dimethylaminoethyl methacrylate) (Py-PDMA)

olecules from excess free polymer, and that the SEC traces coulde interpreted quantitatively in terms of the average number ofolymer molecules that became attached to each QD. In thesexperiments, the addition of known amounts of QDs to a well-efined polymer solution with a small excess of polymer led to aecrease in the peak area for the polymer. Although high separationfficiency was achieved by using a commercial analytical SEC col-mn, the loading capability of the column was limited by its smallimensions. Larger amounts of purified QD/polymer materials areormally needed for end-use applications. Therefore, it is critical toevelop a facile, efficient and cost-effective purification method toulfill this requirement.

In this article, we describe our attempts to scale up theurification of polymer-bound QDs from excess free polymerssing preparative HPLC. Our first experiment employed a columnhat could be packed manually with commercial porous beads,OYOPEARL gel. While this separation was effective, we encoun-ered problems of removing the solvent, N-methyl-2-pyrrolidinoneNMP) from the purified sample. Running the column in other sol-ents, such as absolute ethanol, acetonitrile, tetrahydrofuran, orHF + 2 or 50 vol% triethylamine, as the mobile phase was unsuc-essful due to the adherence of the QDs to the TOYOPEARL gel.onetheless, the QD particles could be eluted effectively from areparative column packed with another type of porous beads, Bio-eads (S-X1) using THF as the mobile phase. We found that thisEC column was efficient for the separation and purification of QDsearing chromophores from excess free dye. The use of this col-mn for the separation of polymer-coated QDs from free polymer

s limited by the unavailability of Bio-Beads with a higher cutoffolecular weight.

. Experimental

.1. Materials and methods

N-Methyl-2-pyrrolidinone (99.9%, HPLC grade) was purchasedrom Aldrich and used as received. Three sizes of CdSe/TOPO QDsith band-edge absorptions at 519, 580 and 587 nm, respectively,ere used in this study. They were synthesized by the procedure

eported previously [18]. CdSe–ZnS(625)/hexadecylamine (HDA),ith a band-edge absorbance at 625 nm and a diameter of 5 ± 2 nm,

as received as a gift from Dr. Margaret Hines at Evident Technol-gy. The QDs as concentrated dispersions (ca. 10 mg/mL in toluene)ere purified by precipitation in methanol and followed by redis-ersion in THF before use. The synthesis of pyrene-labeled PDMAPy-PDMA) was described previously [5].

1216 (2009) 5011–5019

TOYOPEARL gel (HW-65F, 45 �m) was purchased from TOSOHBioscience LLC, Montgomeryville, PA. This gel consists of methacry-late copolymer beads in 20% aqueous ethanol. The particle size ofthese TOYOPEARL SEC resins is in the range of 30–60 �m. Their M.W.limit is 1 × 106. This type of gel swells properly in polar solvents.

Bio-Beads (S-X1, 200–400 mesh) were purchased from Bio-Rad Laboratories, Hercules, CA. These beads are neutral, porousstyrene/divinylbenzene copolymer beads. They have a M.W. exclu-sion limit of 14,000 and the M.W. operating range is 600–14,000.They swell properly in non-polar solvents such as toluene, methy-lene chloride and THF.

An Omnifit Replacement Glass column (volume(mL) = 4.9087 × bed length (cm)) was purchased from Bio-chemValve Inc., Boonton, NJ, USA.

The analytical SEC measurements were performed by using anAM Gel Linear/5 exclusion column (American Polymer StandardsCorporation) and a Viscotek VE-1121 GPC solvent pump. The flowrate was 0.6 mL/min with NMP (HPLC grade) as the mobile phase.Two detectors were used: a Viscotek VE-3210 UV–vis detector anda Waters 410 differential refractometer (RI).

Uultracentrifugation experiments were performed using a Beck-man Optima XL-80K Ultracentrifuge (GMI Inc., Ramsey, MN, USA).

2.2. Synthesis of 2-((2-isobutyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)(methyl)amino)ethyl2-bromo-2-methylpropanoate (BQMEBM)

The details of the synthesis and characterization of com-pound (1) (Fig. 1) were reported previously [19]. Anhydrousdichloromethane (50 mL), compound (1) (0.326 g, 1 mmol) andtriethylamine (0.41 mL, 3 mmol) were added to a three-neck150 mL round-bottom flask equipped with a condenser, magnetand a gas inlet. 2-Bromoisobutyryl bromide (0.4 mL, 3 mmol) indichloromethane (5 mL) was added drop wise into the mixture at0 ◦C and stirred for 30 min. It was then warmed to room tempera-ture and stirred for 7 more hours. The completion of the reactionwas monitored by TLC with n-hexane–acetone (3:1) as eluent. Thereaction was quenched with water (5 mL), washed three times withsaturated NaHCO3 (10 mL each time) and water (10 mL). The result-ing organic layer was dried over anhydrous MgSO4, filtered andevaporated in vacuum. Purification by column chromatography onsilica gel (first fraction) gave the desired product as a green oil(yield: 72%, 0.34 g).

IR (film) �max cm−1: 1737; 1695; 1656; 1589; 1380. 1H NMR(400 MHz, acetone-d6): ı 0.94 (d, J = 6.8 Hz, 6H); 1.79 (s, 6H); 2.21(m, 1H); 3.18 (s, 3H); 3.80 (t, J = 5.4 Hz, 2H); 3.96 (d, J = 7.5 Hz, 2H);4.51 (t, J = 5.5 Hz, 2H); 7.42 (d, J = 8.4 Hz, 1H); 7.78 (dd, J = 7.2 Hz,8.6 Hz, 1H); 8.43 (d, J = 8.4 Hz, 1H); 8.51 (dd, J = 1.2 Hz, 7.2 Hz, 1H);8.64 (dd, J = 1.2 Hz, 8.6 Hz, 1H). 13C NMR (100 MHz, acetone-d6):ı 19.7; 27.2; 40.8; 46.4; 55.1; 56.1; 56.5; 63.0; 115.6; 115.9; 123.2125.4; 126.0; 130.0; 130.6; 130.9; 132.0; 156.2; 163.5; 164.1; 170.9.HRMS (EI, m/z) calcd. for C23H27N2O4Br 474.1154 (M+), found474.1158.

2.3. Synthesis of naphthalimide-labeledpoly(2-N,N-dimethylaminoethyl methacrylate) (Np-PDMA)

Cu(I)Br (39 mg, 0.27 mmol) and 1,1,4,7,10,10-hexamethyltriethylenetetramine (62 mg, 0.27 mmol) were placedinto a 50 mL Schlenk flask fitted with rubber septa and degassedby three freeze–pump–thaw cycles. In another flask, DMA (1.06 g,

6.75 mmol) and BQMEBM (0.13 g, 0.27 mmol) were mixed withstirring and purged with N2 for 30 min to remove oxygen. Themixture was then transferred to the Schlenk flask via a canulato initiate the polymerization under N2. The polymerization wasmaintained at room temperature for 15 min. The mixture became

M. Wang et al. / J. Chromatogr. A 1216 (2009) 5011–5019 5013

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ery viscous and the polymerization was terminated by adding ca.0 mL THF. Then the mixture was passed through a column of silicao remove the copper catalyst. After most solvent was evaporated,he rest of the solution was precipitated in n-hexane. The solidroduct was dissolved in THF again and precipitated in n-hexane,nd the process was repeated for four cycles. Finally, the resultingolymer was dried at 40 ◦C under vacuum (ca. 10 Torr) for 3 days.ield: 60%. The monomer conversion was measured by gravimetry.n = 5500 (by 1H NMR); Mn = 9800 (by GPC), PDI = 1.2.

.4. HPLC and column packing

The HPLC system comprised a Waters 1525 binary HPLC pumpnd a Waters 2487 dual absorbance detector. NMP was used as theolvent. The flow rate was 0.5 mL/min.

To pack the preparative column, we placed a small amount10–20 mL) of absolute ethanol in an Omnifit replacement glassolumn to prevent bubble formation at the base of the poured col-mn packing. The TOYOPEARL gel was diluted half with absolutethanol and then poured into the column slowly along the innerolumn wall. Then we closed the column outlet, and adjusted theevel of liquid in the column such that 1–2 cm of liquid was abovehe level of the bed. Then the column adjuster and column connec-or were positioned near the frit end of the endpiece. The endpieceas pushed slowly into the column until the column connector

ould be screwed onto the column. Then the column adjuster wasurned clockwise slowly to move the plunger to the desired positionn the tube. Finally the column was connected to the HPLC and NMPas run through the column to rinse out the ethanol and water that

etained in the column. Bed length = 7.6 cm, bed vol = 13.4 mL.

.5. Purification of CdSe(519) QDs/Np-PDMA by preparative SEC

Np-PDMA (38 mg, Mn = 9800 (by GPC), Mw/Mn = 1.2) was dis-olved in THF (5 mL). Then purified CdSe(519)/TOPO (5 mL,.8 mg/mL, in THF) was added to the polymer solution. After stirringvernight at room temperature, an aliquot of the mixture (6 mL)as withdrawn and subjected to sonication for 30 min in an ultra-

onic cleaning bath (Branson 1510, 60 W). The mixture was thenoncentrated (to ca. 1 mL) by a rotary evaporator. Then hexane (ca.mL) was added to precipitate the QD/polymer under stirring. After

ecanting the supernatant, the slurry was redispersed in 1 mL ofHF.

An aliquot of CdSe(519)/Np-PDMA (1.0 mL, CCdSe = 23 mg/mL,polymer = 23 mg/mL, in THF) was injected onto the HPLC column.he two channels of UV–vis detector were set at 400 and 519 nm,espectively. Fractions were collected over time for analysis by ana-ytical SEC and spectroscopy.

olin-6-yl)(methyl)amino)ethyl 2-bromo-2-methylpropanoate (compound 2).

2.6. Purification of CdSe(580) QDs/Py-PDMA by preparative SEC

Py-PDMA (83 mg, Mn = 8000 (by GPC), Mw/Mn = 1.2) was dis-solved in THF (5 mL). Then purified CdSe(580)/TOPO (5 mL,12.6 mg/mL, in THF) was added to the polymer solution. Afterstirring for 1 h at room temperature, 4 mL of the mixture was with-drawn and concentrated (to ca. 1 mL) by a rotary evaporator. Thenhexane (ca. 7 mL) was added to precipitate the QD/polymer understirring. After decanting the supernatant, the slurry was redispersedin 1 mL of THF.

An aliquot of CdSe(580)/Py-PDMA(0.5 mL, CCdSe = 25.2 mg/mL, Cpolymer = 33.2 mg/mL, in THF) wasinjected onto the HPLC column. The two channels of the UV–visdetector were set at 346 and 580 nm, respectively. Fractions werecollected over time for analysis by analytical SEC and spectroscopy.

2.7. Synthesis of 7-(diethylamino)-2-oxo-N-(2-(piperazinyl)ethyl)-2-chromene-3-carboxamide(CMPP)

In a round-bottom flask equipped with a magnet stirrer, a solu-tion of 7-diethylaminocoumarin-3-carboxylic acid succinimidylester (3) (100 mg, 0.27 mmol) in 1,4-dioxane (10 mL) was added,followed by addition of a ten-fold excess of the 1-(2-aminoethyl)-piperazine (0.35 mL, 2.7 mmol). The reaction mixture was stirredfor 3 h at room temperature, and the course of the reaction wasmonitored using TLC on silica gel with MeOH:MeCN:Et3N (3:1:0.2)as eluent. The product was recovered after dilution of the dioxanesolution with water, by extraction into CH2Cl2. The organic phasewas washed with 5% NaOH and dried over anhydrous MgSO4. Theorganic solvent was evaporated under vacuum and the product wasseparated by column chromatography on silca gel and dried undervacuum at room temperature for 10 h (yield: 66%, 66 mg).

m.p. = 64–65 ◦C. IR (film) �max cm−1: 3333; 2969; 1701; 1618;1584; 1514; 1352; 1135. 1H NMR (400 MHz, CD3Cl3): ı 1.21 (t,J = 7.2 Hz, 6H); 2.47 (bs, 5H); 2.57 (t, J = 6.8 Hz, 2H); 2.90 (t, J = 5.2 Hz,4H); 3.42 (q, J = 7.2 Hz, 4H); 3.53 (m, 2H); 6.46 (d, J = 2.4 Hz, 1H); 6.61(dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H); 8.67 (s, 1H); 9.02(bs, 1H). 13C NMR (100 MHz, CDCl3): ı 12.61; 36.78; 45.23; 45.66;53.22; 57.26; 96.76; 108.55; 110.05; 110.66; 131.27; 148.1; 152.65;157.82; 162.79; 163.3. HRMS (EI, m/z) calc. for C20H29N4O3 373.2234(M++1), found 373.2238.

2.8. Purification of CdSe QDs bound by low-molecular-weight

chromophores

To pack the preparative column, the Bio-Beads were first swollenin THF for ca. 8 h. Then ca. 20 mL of THF was placed in a clean glasscolumn (inner diameter = 2 cm) with a filter and a Teflon valve at the

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ottom (A photograph of the column is shown in Fig. 9A). Then thewollen bead/THF dispersion was transferred slowly to the column.fter a certain length of densely packed column was achieved, theixture of QDs and dye was added. Finally a thin layer of sand (ca.cm) was added to the top of the column.

The sample of CdSe(587)/CMPP was prepared by mixing CMPPye (1.0 mg/mL, in 1 mL of THF) with CdSe(587)/TOPO (1.3 mg/mL,

n 2 mL of THF). The mixture was stirred in the dark at room tem-erature for 12 h. Finally this mixture was loaded onto the packedolumn for purification.

. Results and discussion

The molecular structures of the two chromophore-labeledDMA homopolymers used in this study are shown in Fig. 2. Bothere synthesized by atom transfer radical polymerization (ATRP)

nitiated from the functionalized chromophores. The first poly-er (referred to as Py-PDMA, Mn = 8000 (by GPC), Mw/Mn = 1.2) is

abeled at one end by a UV-absorbing pyrene group. The secondolymer (Mn = 9800 (by GPC), Mw/Mn = 1.2) contains at one end aaphthalimide group, which absorbs at visible wavelengths. Thisolymer is referred to as Np-PDMA.

Replacement of the TOPO ligands of CdSe/TOPO QDs by PDMAas several important consequences. It introduces polar functionalroups at the QD surface and provides loops and tails of the poly-er that protrude into the solvent. The first factor allows the QDs

o form stable colloidal solutions in a variety of polar solvents suchs alcohols, acetone and acetonitrile, and renders the QDs insolu-le in non-polar solvents such as hexane. The second factor leads tolarge increase in the hydrodynamic radius of the QDs in solutionithout affecting the QD diameter as seen by transmission electronicroscopy (TEM) [4,5]. The small size of QDs makes them difficult

o purify by sedimentation via centrifugation, followed by redis-ersion. With the increased hydrodynamic cross section of QDsith polymeric ligands, sedimentation by centrifugation is moreifficult. For example, we found that even 40,000 rpm in an ultra-entrifuge at 25 ◦C was insufficient to sediment PDMA-treated CdSe

Fig. 2. Chemical structures of Py-PDMA and Np-PDMA.

1216 (2009) 5011–5019

QDs dispersed in ethanol. In one experiment, a fraction of the solu-tion was sedimented, but the sediment adhered strongly to the wallof the centrifuge tube and could not be redispersed in ethanol, evenwith the help of sonication. Thus we turned to chromatographicmethods for separation of the QDs from excess polymer.

3.1. Characterization of CdSe(519)/Np-PDMA by analytical SEC

Before attempting the purification of polymer-coated QDs bypreparative HPLC, we first tested the feasibility of this approachby analytical SEC, since analytical columns resolve the QDs and freepolymer better than preparative columns. The sample was preparedby adding purified CdSe(519)/TOPO QDs to a solution of excessNp-PDMA in THF. This mixture was stirred in the dark overnightbefore injection into an analytical SEC column packed in NMP. Forcomparison, an aliquot of the same mixture was treated in an ultra-sonic cleaning bath (Branson 1510, 60 W) for 0.5 or 1.0 h to examinewhether this treatment could promote ligand exchange.

Fig. 3A shows the analytical chromatograms of CdSe(519)/Np-PDMA mixtures before sonication compared to those of the sampleafter 0.5 and 1.0 h sonication. The wavelength of the UV–vis detectorwas set at 400 nm so that the elution of both the QDs and Np-PDMAcan be detected. A curious feature in all these chromatograms is thebimodal peak appearing in the range of 8–10 min, in addition to asingle peak at 13.9 min. We note that the area ratio of the peak at8.8 min to the one at 9.4 min increased with the increase of thesonication time. There was no detectable change for the peak at13.9 min after the sonication treatment of the mixture.

To try to understand the nature of the bimodal peak shownin Fig. 3A, we examined the behavior of CdSe(519)/TOPO itself bythe same analytical SEC as mentioned above. Three samples weremeasured here. The first was the unpurified CdSe(519)/TOPO (withexcess free TOPO) dispersed in THF. The second was the same QDsbut purified by one precipitation in methanol followed by redisper-sion in THF. Most of the free TOPO ligands were removed by thispurification process. The third sample was the purified QDs thatwere treated in an ultrasonic bath (Branson 1510, 60 W) for 0.5 h.All of these samples were filtered through a 0.2 �m filter before theSEC analysis. Fig. 1B shows the analytical chromatograms of thesethree samples. A single narrow peak was observed at 9.5 min forthe unpurified CdSe(519)/TOPO QDs (trace a in Fig. 3B). In contrast,for the sample purified by one precipitation in methanol and thenredispersed in THF, a new shoulder at 8.9 min appeared in addi-tion to the main peak at 9.5 min (trace b in Fig. 3B). This changewas accompanied by a slight red shift (2 nm) of both the band-edgeabsorbance and photoluminescence (PL) of the QDs (Fig. S3, sup-porting information). The shoulder in trace b of Fig. 3B increasedwhen the purified sample was subjected to sonication for 0.5 h(trace c, Fig. 3B). One can also see that the peak at 8.9 min in thesample of purified and sonicated CdSe(519)/TOPO (trace c, Fig. 3B)appeared broader than that of CdSe(519)/Np-PDMA sonicated forthe same time (trace b, Fig. 3A).

The single sharp peak at 9.5 min in the analytical chromatogramof the unpurified CdSe(519)/TOPO QDs (trace a in Fig. 3B) indicatesthe narrow size distribution of these nanoparticles, consistent withthe results of TEM measurements (Fig. S1, supporting information).The removal of free TOPO and the subsequent sonication resulted inthe appearance of a new peak at 8.9 min in the chromatogram. Wewere unable to detect any changes by TEM (Fig. S1, supporting infor-mation) or by absorption or photoluminescence spectroscopy(Fig. S3, supporting information) that accompanied this change in

SEC elution profile.

We interpret the results described above to indicate that thepeak at 9.5 min in the analytical chromatograms corresponds tothe individual CdSe(519)/TOPO QDs. The second peak at 8.9 minis presumably due to larger species, which are finite-size aggre-

M. Wang et al. / J. Chromatogr. A 1216 (2009) 5011–5019 5015

Fig. 3. (A) Analytical SEC traces of unpurified CdSe(519)/Np-PDMA mixture before and after sonication for 0.5 and 1.0 h, respectively. The area ratio of the QD peak at 8.8 minto the one at 9.4 min increased with the increase of sonication time. The peak at 13.9 min corresponding to excess Np-PDMA remained nearly unchanged after the sonication.T etecto( THF.e sampi

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he chromatograms shown in figure (B) and (C) were both monitored by a UV–vis dtrace b) purification by one precipitation in methanol followed by redispersion inxcess TOPO by one precipitation in methanol. Trace c corresponds to the purifiedncreased upon sonication.

ates of the QD particles. The increase of the particle aggregationpon sonication could be attributed to the partial dissociation ofOPO molecules from the QD surfaces, enhancing the tendency forhe nanoparticles to stick together. Moreover, the polymer (Np-DMA), with an elution peak at 13.9 min, is well separated fromhe CdSe(519) QDs in the analytical SEC column. The interparticleggregation of CdSe(519) caused by sonication could not be pre-ented by the presence of excess free polymer. In addition, theseesults imply that SEC is a sensitive method to monitor the state

f aggregation of QDs. To support this assertion, we note that theolvin group [13] demonstrated that high-performance SEC is sen-itive to changes as small as 1 Å for the hydrodynamic diameter ofhiol-capped CdSe QDs.

ig. 4. (A) Chromatograms of CdSe(519)/Np-PDMA eluted in a preparative HPLC column.he dark trace was monitored by the UV–vis channel of 519 nm in which only the QD peaf 400 nm in which both the QD peak and polymer peak (at 53.2 min) can be discerned. (BC) A series of fractionated CdSe(519)/Np-PDMA that were collected from the preparativeV–vis detector at 400 nm. Only the QD peak was observed for the fractions from f1 to f5.

races in the range of 12–16 min are shown in figure (D).

r at 400 nm. (B) Analytical SEC traces of CdSe(519)/TOPO before (trace a) and afterA new shoulder appeared at 8.9 min (trace b) in the sample after removal of mostle after 0.5 h treatment in an ultrasonic bath. The intensity of the peak at 8.9 min

3.2. Purification of CdSe(519)/Np-PDMA by preparative HPLC andthe subsequent analysis of fractionated samples by analytical SEC

To test the idea of larger-scale purification of QDs after ligandexchange, we turned to examine the use of preparative HPLC col-umn (bed length = 7.6 cm; bed volume = 13.4 mL) packed with theTOYOPEARL gel (HW-65F, 45 �m) to scale up the separation of theQDs from excess free polymer. Fig. 4A shows the preparative HPLCchromatograms of CdSe(519)/Np-PDMA eluting in NMP. The peak of

the QD elution was detected over elution times of 17.0–34.4 min bymonitoring the band-edge absorbance of the QD at 519 nm, wherethe naphthalimide of Np-PDMA has no absorption. This peak wasbimodal as seen by analytical SEC (Fig. 3A), although the resolu-

A two-channel dual wavelength UV–vis detector was used to monitor the elution.k appeared at 17.0–34.4 min. The gray trace was monitored by the UV–vis channel) A HPLC chromatogram of CdSe/ZnS core/shell QDs mixed with excess Np-PDMA.HPLC and then re-injected into an analytical SEC. The elution was monitored by aThe presence of excess Np-PDMA started to appear in f6 and f7. The magnified SEC

5016 M. Wang et al. / J. Chromatogr. A 1216 (2009) 5011–5019

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shows the preparative HPLC traces of the CdSe(580)/Py-PDMA mix-ture eluted in NMP. Only one peak at 27.6 min was observed bythe two-channel dual-wavelength UV–vis detector set at 580 nmthat corresponds to the band-edge absorption of the QDs. The

Fig. 6. (A) Chromatograms of CdSe(580)/Py-PDMA eluted in a preparative HPLCcolumn. A two-channel dual wavelength UV–vis detector was used to monitor theelution. The gray trace was monitored by the UV–vis channel of 580 nm in which

ig. 5. UV–vis absorption (A) and PL (B) spectra of a fractionated CdSe(519)/Np-dSe(519)/TOPO and Np-PDMA alone. All the PL spectra were collected by excitatio

ion for the HPLC column is lower. For the 400 nm channel of theV–vis detector, at which both the QD and naphthalimide of Np-DMA absorption can be detected, two peaks were observed. Ones the QD peak at 17.0–34.4 min, the other, centered at 53.2 min,orresponds to the elution of excess Np-PDMA. Similar effectiveeparation was also achieved by preparative HPLC for a mixture ofdSe/ZnS core/shell QDs and Np-PDMA (Fig. 4B).

In order to verify the separation efficiency of the preparativePLC approach, a series of fractions were collected and character-

zed by analytical SEC as well as by absorption and PL spectroscopy.ig. 4C shows a series of chromatograms of the fractionated sam-les of CdSe(519)/Np-PDMA, which were collected over time fromhe preparative HPLC (Fig. 4A) and then re-injected into an analyt-cal SEC. One can see that only the elution of CdSe(519) QDs wasbserved for fractions f1–f5 that correspond to the elution from0 to 32 min in Fig. 4A. Two peaks associated with the QD elutionere observed in all of these fractionated samples. The main peak

s located at 8.4 min, accompanied by a small shoulder at 9.3 min.here was no detectable free polymer in fractions of f1 to f5 (Fig. 4D).evertheless, the elution of excess free Np-PDMA started to appear

n fractions f6 and f7, as shown in Fig. 4C and D. In addition, thentensity of the QD peak at 9.3 min relative to the one at 8.4 minncreased from f1 to f7.

Fig. 5 presents the UV–vis absorption and PL spectra of one rep-esentative fractionated sample of CdSe(519)/Np-PDMA, i.e. f5. Thepectra of CdSe(519)/TOPO in THF and Np-PDMA in NMP are alsohown for comparison. Above 480 nm, the sample of f5 shows auperimposable absorption spectrum to that of CdSe(519)/TOPO.elow 480 nm, a slight enhanced absorption was observed in theample of f5. The maximum PL (at 534 nm) of f5 shows a slight redhift (4 nm) in comparison with the maximum PL (at 530 nm) ofdSe/TOPO.

The enhanced absorption of f5 relative to that of CdSe(519)/TOPOight be evidence for the presence of Np-PDMA on the QDs. Com-

ared to the UV–vis spectrum of SEC-purified CdSe/Py-PDMA asescribed in the following section, the absorption feature of theaphthalimide in f5 is less obvious, possibly due to the muchroader adsorption band of naphthalimide in comparison with theharp S0 → S2 absorption of pyrene and smaller molar extinctionoefficient of the naphthalimide group in comparison. Fig. 5B showshe emission spectra of f5 as well as Np-PDMA and CdSe(519)/TOPO.he identification of the naphthalimide in f5 from these emissionpectra is more complicated due to the overlap of the emissionands of the dye and the QDs.

.3. Purification and analysis of CdSe(580)/Py-PDMA

To test the scope of the preparative HPLC for purification ofther QD/polymer mixtures, we examined the purification pro-

, i.e. f5, collected from the preparative HPLC in comparison with the spectra of0 nm.

cess with another sample, CdSe(580)/TOPO and Py-PDMA. Fig. 6A

only the QD peak appeared at 27.6 min. The dark trace was monitored by the UV–vischannel of 346 nm in which both the QD peak and polymer peak (at 48.5 min) can bediscerned. (B) A series of chromatograms of fractionated CdSe(580)/Py-PDMA thatwere collected from the preparative HPLC and then re-injected into an analyticalSEC. The elution was monitored by a UV–vis detector at 346 nm. Only the QD peakwas observed for all the fractions from f1 to f7.

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hannel of the UV–vis detector at 346 nm, which correspondso S0 → S2 absorption of pyrene, detected two peaks at 27.6 and8.5 min, respectively. These results indicate that the peak at7.6 min corresponds to the elution of the QDs, while the othereak at 48.5 min corresponds to excess Py-PDMA. The QD peaketected by the 346 nm channel is much higher (exceeding the

imit of the detector) than the one detected by the 580 nm channelecause: (1) QDs show higher absorbance at shorter wavelengths;2) both the QDs and Py-PDMA contribute to the absorption at46 nm.

Again, a series of CdSe(580)/Py-PDMA samples fractionated byhe preparative HPLC column were collected and characterizedy analytical SEC as well as by absorption spectroscopy. Fig. 6Bresents a series of analytical SEC traces of the fractionated sam-les collected from the preparative HPLC. The UV–vis detectorf the analytical SEC was set at 346 nm so that the absorptionf both the QDs and the polymer could be detected. One canee that only the QD peak, at 9.5 min, appeared for all the frac-ions (f1–f7) corresponding to the elution time 22–37 min in thePLC chromatogram (Fig. 6A). Two conclusions could be obtained

rom this result. First, there was no detectable excess Py-PDMAn these purified samples, indicating a high efficiency of the sep-ration process. Second, the polymer (Py-PDMA) binds stronglyo the QDs. There was no detectable dissociation of the poly-

er from the QD surfaces during elution in the SEC column,hich is consistent with the results that we reported previously

5].In addition, a second peak (or shoulder) appeared at 8.8 min in

he analytical SEC traces of fractions f1–f3. Then they disappearedn the fractions f4–f7. This result suggests that the preparative col-mn is able to separate the non-aggregated CdSe/Py-PDMA fromhe aggregates that co-elute in early fractions.

Fig. 7 shows the UV–vis absorption spectra of two represen-ative fractionated samples, f1 and f6, of CdSe(580)/Py-PDMA inMP. Characteristic pyrene absorption bands at 346 and 329 nmere observed for both purified samples. Similar pyrene absorp-

ion was also observed in other fractionated samples including2–f5 and f7. These peaks, although small, provide clear evidenceor the polymer binding to the QD surfaces. The QD absorptionn f1 and f6 is superimposable above 400 nm, except for a slighted shift (4 nm) of the band-edge absorption in f1 compared tohat in f6 (inset, Fig. 7). Together with the small shoulder appear-

ng in the analytical SEC trace of f1 (Fig. 6B), this shift implies theresence of a small amount of QD aggregation in this fractionatedample.

ig. 7. UV–vis absorption spectra of fractionated samples, f1 and f6, show the char-cteristic pyrene absorption bands at 346 and 329 nm, respectively. The inset showshe magnified spectra in the range of 480–630 nm in which a slight blue shift (4 nm)as observed in f6 in comparison with f1.

Fig. 8. (A) The molecular structure of CMPP. (B) UV–vis absorption and fluorescence(FL) spectra of CMPP in THF.

3.4. Removal of NMP solvent from the fractionated QD/polymeradduct

The results described above indicate that when NMP (99.9%,HPLC grade) was used as the eluent, a preparative HPLC columnpacked with the TOYOPEARL gel (HW-65F, 45 �m) allows effectiveseparation of polymer-stabilized CdSe QDs of various sizes as well ascore–shell CdSe–ZnS QDs from the excess free polymer (Py-PDMAor Np-PDMA). Up to 23 mg of CdSe(519) QD material mixed withthe same weight of Np-PDMA polymer could be purified by oneinjection into this column with a bed length of 7.6 cm and a bedvolume of 13.4 mL.

The next question is how to isolate the QD/polymer adductand then disperse it into another solvent for further processing.Removal of NMP under vacuum was difficult because of its highboiling point. In addition, dialysis against absolute ethanol by usinga cellulose membrane (Mw cutoff = 1000) led to precipitation ofsome of the QDs. Thus we tried another method, precipitatingthe HPLC-fractionated CdSe(580)/Py-PDMA adduct by addition of anon-solvent, for example, n-hexane. To increase the miscibility ofNMP with n-hexane, THF was added to the solution of NMP beforethe addition of n-hexane. We took the fraction 4 of CdSe(580)/Py-PDMA (Fig. 4B) as an example. All of the QDs precipitated in amixture of NMP/THF/n-hexane at a volume ratio of 1:0.3:8. The pre-cipitate was separated by centrifugation (13,000 rpm, 5 min). Thesupernatant was decanted and then THF or toluene (ca. 3 mL) wasadded to the sediment. However, the sediment could not be redis-persed even after sonication in an ultrasonic cleaning bath (Branson1510, 60 W) for ca. 30 min. In contrast, precipitation of the samesample in the presence of excess Py-PDMA, using the same proce-dure described above, resulted in easy redispersion of the QDs intoluene. These results imply that the presence of excess polymeris important to maintain the colloidal stability of the QDs duringprocessing via precipitation. Further work is needed to learn howto transfer purified QDs from NMP to other solvents.

3.5. Purification and analysis of CdSe QDs modified by7-(diethylamino)-2-oxo-N-(2-(piperazin-1-yl)ethyl)-2H-

chromene-3-carboxamide(CMPP)

Although both the CdSe and core–shell CdSe–ZnS QDs elutedproperly in the preparative column packed with TOYOPEARL gel

5018 M. Wang et al. / J. Chromatogr. A 1216 (2009) 5011–5019

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ig. 9. (A) A photograph of CdSe(587)/CMPP in a preparative SEC column packed wdSe(587)/CMPP in an analytical SEC column run in NMP before and after purificati

nd NMP as the mobile phase, they stuck to the column when otherolvents such as absolute ethanol, acetonitrile, THF, and THF + 2 or0 vol% triethylamine were used as the eluent. As a consequence,e examined a different type of porous beads, Bio-Beads (S-X1)ith a molecular-weight cutoff of 14,000 (the highest one currently

vailable), as the packing material for the preparative HPLC column.his column allows CdSe QDs to elute smoothly in THF as the mobilehase. Unfortunately, due to the lower exclusion limit of this pack-

ng material, it could not separate the QDs from either the Py-PDMAr the Np-PDMA.

We tested the Bio-Beads (S-X1) column for its ability to sepa-ate QDs modified with a fluorescent dye by ligand exchange fromxcess free dye, 7-(diethylamino)-2-oxo-N-(2-(piperazinyl)ethyl)--chromene-3-carboxamide (CMPP). This dye is a derivative of aoumarin containing a piperazine moiety that may bind to CdSe QDsy ligand exchange. The structure of this chromophore is shown inig. 8A. The free dye in THF shows a maximum absorption at 410 nmnd an emission peak at 444 nm (Fig. 8B).

Bio-Beads (S-X1) swell properly in non-polar (e.g. toluene) andid-polar solvents (e.g. THF). THF was chosen as the eluent. Fig. 9 A

hows a photograph of the elution of CdSe(587)/CMPP in a prepar-tive SEC column with an inner diameter of ca. 2 cm and a length ofa. 50 cm. The separation of the QD from the excess chromophoreould be monitored visually based on their different colors. ThusPLC associated with a pump and a UV–vis detector is not needed in

his case. Fig. 9B shows the SEC chromatograms of CdSe(587)/CMPPun in NMP before and after purification by the preparative SEColumn. For the sample before purification, both the QD peak (at.1 min) and a bimodal peak of excess CMPP (at 14.8–18.1 min)ere observed. The broad and bimodal peak of CMPP is presum-

bly induced by some intermolecular aggregation of this moleculen NMP. In contrast, only a weak peak of excess CMPP appeared inhe purified sample that was collected from the preparative SEColumn. This result indicates that most of the excess CMPP waseparated by the preparative SEC column. The presence of a smallmount of excess CMPP may originate from some dissociation ofhe molecules from the QD surface after elution from the prepara-ive SEC column. This dissociation was not observed for the purifiedamples of either CdSe(580)/Py-PDMA or CdSe(519)/Np-PDMA. Inhese examples, the polymer binds strongly as a multidentate lig-

nd to the QDs. However, dissociation may occur for CdSe/CMPP,here only monodentate or bidentate anchoring of the molecule isossible.

The UV–vis absorption spectrum of the SEC-purifieddSe(587)/CMPP in THF shows an intense additional absorption

-Beads (S-X1) in THF. The elution band of the QDs is circled. (B) Chromatograms of

peak due to the chromophore of CMPP, as shown in Fig. S4 (support-ing information). This absorbance includes the contributions fromboth the dye molecules adsorbed on the QDs and the small amountof free dye molecules that may have dissociated from the QDsurface. In addition, the absorption spectrum of CdSe(587)/CMPPabove 450 nm, including the region of the band edge, is superim-posable on that of CdSe(587)/TOPO, indicating no size change ofthe QDs after ligand exchange and the SEC purification.

4. Summary

A preparative HPLC column packed with a TOYOPEARL gel,with NMP as the mobile phase, allowed the effective separationof polymer-bound QDs from the excess free polymer in solution.This method is applicable to CdSe QDs of different sizes as well asto core–shell CdSe–ZnS QDs. Re-analysis of the HPLC-purified sam-ples by analytical SEC indicated that both Py-PDMA and Np-PDMAbound strongly to CdSe QDs. No dissociation of the polymers wasobserved after re-injection of the purified sample into an analyti-cal SEC column. The elution of QDs in the TOYOPEARL-gel packedcolumn is sensitive to the solvent. When analogous experimentswere run using solvents such as absolute ethanol, acetonitrile,THF, and THF–triethylamine mixtures, the QDs did not elute fromthe column. These observations suggest that the polymer-coatedQDs adhered to the packing material. Thus, at the present time,only NMP has been found to be an effective solvent for this chro-matographic separation. A disadvantage of NMP as a solvent forpreparative scale separations is the difficulty of transferring the QDsto other solvents for further experiments. NMP is high boiling anddifficult to remove by evaporation. Attempts to use precipitationfollowed by redispersion have so far been unsuccessful. For exam-ple, an HPLC-purified CdSe(580)/Py-PDMA sample in NMP couldbe precipitated in a solvent mixture of NMP/THF/n-hexane. Theprecipitate, however, could not be redispersed in toluene or THF,even after sonication. Further experiments are needed to find suit-able chromatography solvents to replace NMP for processing of thepurified QD/polymer material.

The QDs do not stick to the packing material when Bio-Beads(S-X1) were used as the packing material for the preparative SECcolumn with THF as the mobile phase. The molecular weight cutoff

for these beads was not high enough to resolve the polymer-coatedQDs from excess free polymer. Nevertheless, this column is veryeffective at separating CdSe QDs modified with a coumarin dyefrom the excess free chromophore. The differences we observed inQD elution are certainly related to differences in the chemical com-

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ositions of these packing materials. The TOYOPEARL gel consistsf methacrylate copolymer beads, while the Bio-Beads are neutral,orous styrene/divinylbenzene copolymer beads. We imagine thathese two types of materials have different adhesion tendencies forhe PDMA polymer and the PDMA-coated QDs, and that these inter-ctions are sensitive to the choice of solvent chosen as the mobilehase.

cknowledgments

The authors thank NSERC Canada for their financial support andr. M. Hines at Evident Technologies for providing the CdSe/ZnS

ample. We also thank Anna Valborg Gudmundsdottir and Dr. Guo-ua Zhang for the help with the HPLC experiments.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.chroma.2009.04.060.

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