Preparative size-exclusion chromatography for purification and characterization of colloidal quantum dots bound by chromophore-labeled polymers and low-molecular-weight chromophores
Introduction
Polymers have been extensively used for surface modification of nanocrystals to tune their physical properties, and to impart other functionalities for biological and optical/electrical applications [1], [2]. We are particularly interested in semiconductor nanocrystals, also called quantum dots (QDs) due to their unique size-dependent optoelectronic properties. In previous publications, we reported that poly(2-N,N-dimethylaminoethyl methacrylate) (PDMA) in toluene could replace TOPO from the surface of CdSe/TOPO QDs [3], [4], [5], [6], [7]. This process has been studied in some detail, so that one has an idea of how many polymer chains bind to QDs of certain sizes [5], and how many DMA groups of the polymer participate in replacing each TOPO [7]. Our previous results suggest that PDMA binds as a multidentate ligand to the surface of CdSe QDs. The driving force that contributes to this interaction may come from two effects: one is the entropic gain that derives from the dissociation of TOPO ligands from the QD surfaces, assuming that the entropy loss from the polymer binding to QDs is much smaller. The other is the enthalpic effect from the binding between the tertiary amines of the polymer and the Cd sites on the QD surfaces.
Polymer-coated QDs are normally prepared by treating the QDs with excess polymer in order to saturate the QD surfaces and to avoid undesired bridging, i.e. one polymer chain binding to two or more than two QDs. As a consequence, only a fraction of the added polymers binds to the nanoparticles. For many end-use applications of these materials, one should separate the polymer-bound QDs from the excess free polymer. This is not easy to do.
Several separation methods have been used to characterize the hydrodynamic size of inorganic nanoparticles and to separate polymer-bound nanoparticles from free polymer. These include gel electrophoresis [8], [9], [10], high-performance liquid chromatography (HPLC) [10], [11], [12], and size-exclusion chromatography (SEC) [13], [14]. For example, Parak and co-workers [9] prepared polymer-coated colloidal core–shell CdSe–ZnS QDs initially passivated with a surface layer of trioctylphophine oxide (CdSe–ZnS/TOPO). They synthesized an amphiphilic polymer by linking an amino-functionalized hydrocarbon chain (dodecylamine) and an amino-modified fluorophore (AATO590) to some of the anhydride rings of poly(isobutylene-alt-maleic anhydride) (m ≈ 6000). The procedure of coating core–shell CdSe–ZnS/TOPO QDs with this polymer involved the dispersion of the QD/polymer mixture in chloroform, followed by a drying process and then redispersion in a sodium borate buffer (pH 12). This polymer-coating procedure resulted in some empty polymer micelles in addition to the polymer-coated QDs. These empty micelles could be separated by gel electrophoresis based on the fact that they eluted faster than the polymer-coated particles.
The Wilcoxon group [15] employed HPLC to fractionate TOPO-coated CdSe and core–shell CdSe–ZnS QDs of various sizes using tetrahydrofuran (THF) as the mobile phase. They used a high-resolution column (Polymer Laboratory, model PL50) filled with 5-μm microgel particles of cross-linked polystyrene to separate the nanoparticles. Colvin's group [13] studied the size and size distribution of TOPO-coated CdSe nanocrystals by high-performance SEC using 0.1 M trioctylphosphine in toluene as the mobile phase. Zubarev's group [16], [17] applied centrifugal membrane filtration on regenerated cellulose for the purification of polymer-functionalized gold and silver nanoparticles, and gold nanorods from the residual free polymer chains.
In a previous publication [5], we reported as a proof of concept that SEC could be used to separate CdSe QDs bearing pyrene-labeled poly(2-N,N-dimethylaminoethyl methacrylate) (Py-PDMA) molecules from excess free polymer, and that the SEC traces could be interpreted quantitatively in terms of the average number of polymer molecules that became attached to each QD. In these experiments, the addition of known amounts of QDs to a well-defined polymer solution with a small excess of polymer led to a decrease in the peak area for the polymer. Although high separation efficiency was achieved by using a commercial analytical SEC column, the loading capability of the column was limited by its small dimensions. Larger amounts of purified QD/polymer materials are normally needed for end-use applications. Therefore, it is critical to develop a facile, efficient and cost-effective purification method to fulfill this requirement.
In this article, we describe our attempts to scale up the purification of polymer-bound QDs from excess free polymers using preparative HPLC. Our first experiment employed a column that could be packed manually with commercial porous beads, TOYOPEARL gel. While this separation was effective, we encountered problems of removing the solvent, N-methyl-2-pyrrolidinone (NMP) from the purified sample. Running the column in other solvents, such as absolute ethanol, acetonitrile, tetrahydrofuran, or THF + 2 or 50 vol% triethylamine, as the mobile phase was unsuccessful due to the adherence of the QDs to the TOYOPEARL gel. Nonetheless, the QD particles could be eluted effectively from a preparative column packed with another type of porous beads, Bio-Beads (S-X1) using THF as the mobile phase. We found that this SEC column was efficient for the separation and purification of QDs bearing chromophores from excess free dye. The use of this column for the separation of polymer-coated QDs from free polymer is limited by the unavailability of Bio-Beads with a higher cutoff molecular weight.
Section snippets
Materials and methods
N-Methyl-2-pyrrolidinone (99.9%, HPLC grade) was purchased from Aldrich and used as received. Three sizes of CdSe/TOPO QDs with band-edge absorptions at 519, 580 and 587 nm, respectively, were used in this study. They were synthesized by the procedure reported previously [18]. CdSe–ZnS(625)/hexadecylamine (HDA), with a band-edge absorbance at 625 nm and a diameter of 5 ± 2 nm, was received as a gift from Dr. Margaret Hines at Evident Technology. The QDs as concentrated dispersions (ca. 10 mg/mL in
Results and discussion
The molecular structures of the two chromophore-labeled PDMA homopolymers used in this study are shown in Fig. 2. Both were synthesized by atom transfer radical polymerization (ATRP) initiated from the functionalized chromophores. The first polymer (referred to as Py-PDMA, Mn = 8000 (by GPC), Mw/Mn = 1.2) is labeled at one end by a UV-absorbing pyrene group. The second polymer (Mn = 9800 (by GPC), Mw/Mn = 1.2) contains at one end a naphthalimide group, which absorbs at visible wavelengths. This polymer
Summary
A preparative HPLC column packed with a TOYOPEARL gel, with NMP as the mobile phase, allowed the effective separation of polymer-bound QDs from the excess free polymer in solution. This method is applicable to CdSe QDs of different sizes as well as to core–shell CdSe–ZnS QDs. Re-analysis of the HPLC-purified samples by analytical SEC indicated that both Py-PDMA and Np-PDMA bound strongly to CdSe QDs. No dissociation of the polymers was observed after re-injection of the purified sample into an
Acknowledgments
The authors thank NSERC Canada for their financial support and Dr. M. Hines at Evident Technologies for providing the CdSe/ZnS sample. We also thank Anna Valborg Gudmundsdottir and Dr. Guohua Zhang for the help with the HPLC experiments.
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N, C- 1
Current address: Department of Chemistry, Payame Noor University, Iran.