Separation of Glutathionate-Protected Gold Clusters by Reversed

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Separation of Glutathionate-Protected Gold Clusters by ReversedPhase Ion Pair High-Performance Liquid Chromatography Yoshiki Niihori, Yoshihiro Kikuchi, Daisuke Shima, Chihiro Uchida, Sachil Sharma, Sakiat Hossain, Wataru Kurashige, and Yuichi Negishi Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b03814 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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Industrial & Engineering Chemistry Research

Separation of Glutathionate-Protected Gold Clusters by Reversed-Phase Ion Pair High-Performance Liquid Chromatography Yoshiki Niihori,a Yoshihiro Kikuchi,a Daisuke Shima,a Chihiro Uchida,a Sachil Sharma,a Sakiat Hossain,a Wataru Kurashige,a and Yuichi Negishia,b,* a

Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1−3 Kagurazaka,

Shinjuku-ku, Tokyo 162−8601, Japan. b

Photocatalysis International Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278−8510, Japan.

ACS Paragon Plus Environment

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ABSTRACT. Recent studies have demonstrated that reversed-phase high-performance liquid chromatography (RP-HPLC) is a very effective means of separating thiolate (SR)-protected gold clusters (Aun(SR)m). In the present study, we applied RP-HPLC to the separation of hydrophilic glutathionate (SG)-protected Aun(SG)m clusters. To achieve such separation, an ion pair reagent was dissolved in the solution to form ion pairs with the functional groups on the cluster surfaces, improving the interaction between the cluster surfaces and the hydrophobic stationary phase, representing ion pair chromatography. This technique resulted in the high-resolution separation of Aun(SG)m clusters via RP-HPLC. Experiments at different solution pH values led to slight improvements in the resolution, showing that pH adjustment, which modifies the efficiency of the ion pair formation process, is a useful technique for improving this type of separation.


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1. INTRODUCTION Thiolate (SR)-protected gold clusters (Aun(SR)m)1-11 have received significant attention as functional nanomaterials because of their size-specific physicochemical properties, such as photoluminescence,12−21 catalysis,22−25 and redox behavior,26−28 which are not seen in bulk gold.29−34 Among these materials, hydrophilic Aun(SR)m clusters exhibit a good affinity for biomaterials35−37 and also have other advantages, including high toxic-element recognition in aqueous solutions38 and photoluminescence with high quantum yields.21,39 Therefore, much research has been conducted in this area. In initial studies concerning the isolation of these hydrophilic Aun(SR)m clusters, mixtures were separated with high-resolution by polyacrylamide gel electrophoresis (PAGE) according to the cluster size.13,40−42 This technique allowed the precise isolation of various glutathionate (SG)protected Aun(SG)m clusters, such as Au10(SG)10, Au15(SG)13, Au18(SG)14, Au22(SG)16, Au22(SG)17, Au25(SG)18, Au29(SG)20, Au33(SG)22, and Au39(SG)24.13 Methods of converting unstable clusters to stable clusters by exposure to harsh conditions43 and of controlling the growth rate of such clusters using specific reduction parameters21,44,45 were subsequently established. These methods have allowed us to synthesize several hydrophilic Aun(SR)m clusters in a size-selective manner. Reversed-phase high-performance liquid chromatography (RP-HPLC) is also an effective approach to the high-resolution separation of Aun(SR)m clusters.46−55 In fact, dodecanethiolate (SC12H25)-protected Aun(SC12H25)m clusters have been separated with high-resolution using this method over a wide range of sizes; from Au38(SC12H25)24 to Au~520(SC12H25)~130.48 Furthermore, clusters comprising two types of ligands (SR1, SR2), such as Au24Pd(SR1)18-x(SR2)x, have been separated by RP-HPLC with high resolution according to the ligand combinations49−51 and


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coordination isomers.52 RP-HPLC is also applicable to the separation of hydrophilic Aun(SR)m clusters, as demonstrated by Murray et al. and Choi et al.47 The present study focused on the RP-HPLC separation of Aun(SG)m clusters,13,21,40−42 which represent some of the most widely studied clusters. The surface of the stationary phase of the RP column is hydrophobic, whereas hydrophilic functional groups are present on the cluster surfaces. Therefore, in these separation experiments, an ion pair reagent was dissolved in the cluster solution to form ion pairs with these functional groups.47 This treatment increased the hydrophobicity of the cluster surfaces, such that the clusters were able to properly interact with the hydrophobic stationary phase (representing ion pair chromatography; Figure 1) and could be separated in the RP column with high resolution. An in-depth understanding of the effective experimental conditions for such separations was acquired by performing experimental trials under a variety of conditions.

Figure 1. A diagram showing the basic concept of reversed-phase ion-pair chromatography in the separation of hydrophilic Aun(SR)m clusters.

2. EXPERIMENTAL SECTION 2.1. Chemicals. All chemicals were obtained commercially and used without further purification. Hydrogen tetrachloroaurate tetrahydrate (HAuCl4· 4H2O) was purchased from


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Tanaka Kikinzoku. Gulutathione (GSH), sodium borohydride (NaBH4), 85 w% phosphoric acid (85 w% H3PO4), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), boric acid (H3BO3), and sodium hydroxide (NaOH) were purchased from Wako Pure Chemical Industries, Ltd. Methanol was purchased from the Kanto Chemical Co., Inc. Tetrabutylammonium perchlorate ([(C4H9)4N]ClO4), N-(2mercaptopropionyl)glycinate ((PG)SH), butylamine hydrochloride ([C4H9NH3]Cl), and sodium 1-butanesulfonate (Na[C4H9SO3]) were purchased from the Tokyo Chemical Industry Co., Ltd. Pure Milli-Q water (18.2 MΩ·cm) was generated using a Merck Millipore Direct 3 UV system. 2.2. Preparation of a mixture of Aun(SG)m clusters. A mixture of Aun(SG)m clusters was prepared according to a previously reported method,13 with a slight modification. First, 0.25 mmol of HAuCl4 was dissolved in 50 mL methanol, after which 1.0 mmol of GSH was added. The resulting solution was cooled to 0 °C. An aqueous NaBH4 solution (2.5 mmol/12.5 mL) also at 0 °C was subsequently added and the solution was stirred vigorously for 3 h. The resulting black precipitate was washed with methanol and a mixture of the Aun(SG)m clusters was obtained by re-dispersion in water. 2.3. Preparation of buffer solutions. Three different buffer solutions (pH 1.8, 6.8, or 8.8) were used in this study. A 50 mM pH 1.8 phosphate buffer solution was prepared in two steps. First, 1.70 mL of an aqueous 14.7 M H3PO4 solution were dissolved in 500 mL Milli-Q water. Following this, the solution was combined with an aqueous solution prepared by mixing 1.95 g of NaH2PO4·2H2O and 0.85 mL of an aqueous 14.7 M H3PO4 solution in 500 mL Milli-Q water to adjust the pH to 1.8 (Scheme S1(a)). A 50 mM pH 6.8 phosphate buffer solution was prepared by dissolving 1.95 g of NaH2PO4·2H2O and 4.48 g of Na2HPO4·12H2O in 500 mL of Milli-Q water (Scheme S1(b)).


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Table 1. Substitution of the mobile phase at each pH. pH

mobile phase

substitution time (min)

1.8

50 mM pH 1.8 phosphate buffer solution → 30 mM Na[C4H9SO3] methanol solution

500

6.8

50 mM pH 6.8 phosphate buffer solution → 30 mM [(C4H9)4N]ClO4 methanol solution

500

6.8

50 mM pH 6.8 phosphate buffer solution → 30 mM Na[C4H9SO3] methanol solution

500

8.8

50 mM pH 8.8 borate buffer solution → 30 mM [(C4H9)4N]ClO4 methanol solution

500

A 50 mM pH 8.8 borate buffer solution was prepared in two steps. First, an aqueous 50 mM H3BO3 solution (1.55 g/500 mL) including approximately 500 mg of NaOH was prepared. This solution was mixed with another 50 mM aqueous H3BO3 solution to adjust the pH to 8.8 (Scheme S1(c)). 2.4. HPLC experiments. HPLC trials were conducted with a Shimadzu Prominence HPLC system (DGU-20A3R online degasser, LC-20AD pump, CTO-20AC column oven, and SPDM20A photo diode array detector). The column consisted of stainless steel (250 mm × 4.6 mm inner diameter) packed with 5 μm octadecyl silyl-bonded silica particles with pore size of 175 Å (Hypersil GOLD, Thermo Scientific). The column oven temperature was fixed at 25 °C and the column and detector were allowed to stabilize prior to each trial. To acquire each chromatogram, a 1.5 mg portion of the Aun(SG)m clusters (Figure S1) was dispersed in 10 μL of the appropriate 50 mM buffer solution, after which 10 μL of either a 30 mM ion pair reagent solution in methanol was added to the cluster solutions. The resulting suspension was injected into the chromatograph as the buffer mobile phase was pumped through the instrument at 1 mL/min. After the injection, the mobile phase was gradually replaced using a linear gradient program (Table 1)50−52 to transition from the buffer solution to the methanol solution including the ion pair reagent (Figure S2); this was carried out to reduce the retention


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Scheme 1. The pH-dependent protonation and deprotonation of GSH.56 time of those clusters that would otherwise have had longer elution times. In the case of the experiments at pH 6.8, the mobile phase was transitioned from a 50 mM pH 6.8 phosphate buffer solution to a 30 mM [(C4H9)4N]ClO4 or 30 mM Na[C4H9SO3] methanol solution over 500 min. At pH 8.8, the mobile phase was transitioned from a 50 mM pH 8.8 borate buffer solution to a 30 mM [(C4H9)4N]ClO4 methanol solution over 500 min. At pH 1.8, the mobile phase was transitioned from a 50 mM pH 1.8 phosphate buffer solution to a 30 mM Na[C4H9SO3] methanol solution over 500 min.

3. RESULTS AND DISCUSSION 3.1. Separation at pH 6.8. We initially attempted the separation at pH 6.8. Glutathione contains two carboxyl groups and one amino group, allowing either deprotonation or protonation depending on the pH of the solution (Scheme 1).56 Although the glutathionate contained in the cluster and uncoordinated glutathione would be expected to have slightly different acid dissociation constants (pKa), it was assumed that deprotonation of the two carboxyl groups (to give −COO−) and protonation of the amino group (to give −NH3+) would occur on the cluster surfaces under these conditions (Scheme 1). Thus, both a cationic ion pair reagent and an anionic ion pair reagent could be applicable.


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Figure 2. (a) Chromatogram and (b) optical absorption spectrum of each peak obtained at pH 6.8 using the cationic ion-pair reagent [(C4H9)4N]ClO4. Substitution time = 500 min, concentration of phosphate buffer = 50 mM, concentration of [(C4H9)4N]ClO4 = 30 mM (Figure S3). The potential assignments of the main peak are summarized in Table 2.

Figure 2(a) presents a chromatogram obtained using the cationic ion pair reagent [(C4H9)4N]ClO4 (Figures S3 and S4), in which multiple peaks (I−XIII) appear. The optical absorption spectra of peaks I−XIII are shown in Figure 2(b). On the basis of the comparison with previous reports,13 I, II, III, V, VI, VII, IX, and XI are attributed to Au10(SG)10, Au15(SG)13, Au18(SG)14, Au22(SG)16, Au25(SG)18, Au29(SG)20, Au33(SG)22, and Au39(SG)24, respectively (Table


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Table 2. Potential assignments of the peaks observed in the chromatograms in Figures 2(a) and 4(a). fraction I II III IV V VI VII VIII IX X XI XII XIII I’ II’ III’ IV’ V’ VI’ VII’ VIII’ IX’ X’ XI’ XII’ XIII’ a

retention time (min) 112.2 140.1 150.4 165.9 170.7 173.6 180.1 187.2 190.0 191.3 194.0 199.4 205.7 254.4 266.3 270.0 275.2 277.0 278.2 280.9 283.7 285.4 286.1 287.3 290.1 293.5

potential assignment a Au10(SG)10 Au15(SG)13 Au18(SG)14 − Au22(SG)16 Au25(SG)18 Au29(SG)20 − Au33(SG)22 − Au39(SG)24 − − Au10(SG)10 Au15(SG)13 Au18(SG)14 − Au22(SG)16 Au25(SG)18 Au29(SG)20 − Au33(SG)22 − Au39(SG)24 − −

These assignments are based on a comparison between the optical absorption spectra in this

study (Figures 2(b) and 4(b)) and those in ref. 13. The peaks indicated by a hyphen (−) could not be assigned.

2). Although it is difficult to assign the other peaks at present, these results indicate that Aun(SG)m clusters were separated with high resolution according to the cluster size. Regarding the elution order, smaller clusters were found to elute faster than larger clusters, in agreement with previous studies of hydrophobic clusters.48 In addition, these results agree with the elution order observed during the RP ion pair chromatography of N-acetyl-L-


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Figure 3. Chromatogram obtained from trials at pH 6.8 using the anionic ion-pair reagent Na[C4H9SO3]. Substitution time = 500 min, concentration of phosphate buffer = 50 mM, concentration of Na[C4H9SO3] = 30 mM.

cysteine-protected gold clusters reported by Murray et al.47 These observations indicate that ion pairs were formed between COO− groups and [(C4H9)4N]+ ions on the cluster surfaces, improving the hydrophobicity of the cluster surfaces (Figure 1). The larger the cluster size become, the more hydrophobic functional groups (C4H9) are present on its surfaces. Therefore, the interaction with the stationary phase should become stronger with increasing cluster size, resulting in a longer elution time with increasing size. In contrast, no clear separation was observed in the experiments during which the anionic ion pair reagent Na[C4H9SO3] was used (Figure 3). It appears that ion pairs were not efficiently formed between the NH3+ groups and the [C4H9SO3]− ions. The optical absorption spectra acquired at various retention times suggest that the clusters were eluted in order from largest to smallest under these conditions (Figure S5). In the absence of ion pairs, the polarity of the cluster surfaces is expected to increase with the cluster size, since the number of hydrophilic functional groups on the cluster surfaces increases with size. Therefore, the interaction with the stationary phase would be expected to decrease with increasing cluster size, resulting in shorter retention


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Figure 4. (a) Chromatogram and (b) optical absorption spectrum of each peak obtained during experiments at pH 8.8 using the cationic ion-pair reagent [(C4H9)4N]ClO4. Substitution time = 500 min, concentration of phosphate buffer = 50 mM, concentration of [(C4H9)4N]ClO4 = 30 mM. The potential assignments of the main peaks are summarized in Table 2.

times for larger clusters. We also performed a similar experiment using a buffer solution with a pH of 1.8, which put the carboxyl groups into the COOH state (Scheme 1). The results show that little formation of ion pairs occurred at this pH (Figures S6 and S7), suggesting that the use of the anionic ion pair reagent Na[C4H9SO3] is less effective for the separation of Aun(SG)m clusters. 3.2. Separation at pH 8.8. The above experiments demonstrated that ion pairs are effectively formed between COO− groups and [(C4H9)4N]+ ions, but not between NH3+ and


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Figure 5. Peak separations between Au22 and Au25 in the chromatograms obtained at (a) pH 6.8 (Figure 2(a)) and (b) pH 8.8 (Figure 4(a)) using a cationic ion-pair reagent, [(C4H9)4N]ClO4.

[C4H9SO3]−. Thus, we attempted separation at pH 8.8, at which only deprotonation of the two carboxyl groups should occur (Scheme 1). In these trials, [(C4H9)4N]ClO4 was again used as the cationic ion-pair reagent. Figure 4(a) shows the resulting chromatogram, in which multiple peaks (I’−XIII’) appear. From the optical absorption spectrum of each peak (Figure 4(b)), peaks I’, II’, III’, V’, VI’, VII’, IX’ and XI’ are attributed to Au10(SG)10, Au15(SG)13, Au18(SG)14, Au22(SG)16, Au25(SG)18, Au29(SG)20, Au33(SG)22, and Au39(SG)24, respectively. This result indicates that the clusters have been separated in order of increasing size even under this experimental condition, with the same elution order as that shown in Figure 2(a). In this experiment, the clusters were separated with slightly higher resolution than in the previous trial at pH 6.8. As an example, Au22(SG)16 and Au25(SG)18 were separated by a greater margin at pH 8.8 than at pH 6.8 (Figure 5). At pH 6.8, the protonation of the amino group could


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occur in addition to the deprotonation of the two carboxyl groups (Scheme 1). In contrast, only the deprotonation of the carboxyl groups is anticipated at pH 8.8 (Scheme 1). Therefore, at pH 8.8, the cations are able to more easily approach the cluster surfaces, and thereby more ion pairs ([(C4H9)4N]+COO−) are formed. This effect likely results in an increase in the interactions between the cluster surfaces and the stationary phase (Figure S8), leading to an improvement of the resolution compared to the results at pH 6.8. Although there may be other methods to enhance these interactions, our results demonstrate that changing the efficiency of the ion pair formation by adjusting the pH value of the mobile phase is an effective approach to improve the resolution.

4. CONCLUSIONS This work demonstrated the separation of hydrophilic Aun(SG)m clusters by RP-HPLC. To achieve such separation, ion pairs were formed between the functional groups and the ion pair reagent, [(C4H9)4N]ClO4. In this manner, Aun(SG)m clusters were separated with high resolution. Experimental trials at various pH values optimized the resolution, providing evidence that pH adjustment is effective at improving the separation by changing the ion pairing efficiency. Further preliminary experiments imply that this method is also applicable to the separation of Aun(S(PG))m and Agn(SG)m clusters (Figures S9 and S10). As noted in the introduction, it has become possible to synthesize several hydrophilic Aun(SR)m clusters while controlling their cluster sizes in recent years. However, it remains necessary to employ a post-treatment, such as washing (that is, purification) of the reaction product, to isolate highly pure clusters, unless no impurities are present. In addition, the use of HPLC allows easy evaluation of the distribution and purity of the products in the case that the absorption spectrum of each Aun(SR)m is known.


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Thus, in the future, it is expected that the separation method described in this study could be widely used for the separation and analysis of Aun(SG)m and other hydrophilic Aun(SR)m clusters.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Tel.: +81-3-5228-9145

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This contribution was identified by Prof. Thomas Bürgi (Univ. of Geneva) as the Best Presentation in the session “Nanometal: Synthesis, Structure, Property & Application” of the 2016 ACS Spring National Meeting in San Diego, CA.” This work was supported by JSPS KAKENHI Grants (numbers JP15H00763, JP15H00883, JP16H04099, and 16K17480). Funding from the Nippon Sheet Foundation for Materials Science and Engineering, the Sumitomo Foundation, the Takahashi Industrial and Economic Research Foundation, the Tanaka Kikinzoku Memorial Foundation, the Futaba Electronics Memorial Foundation, and the Sasakawa Foundation is also gratefully acknowledged.


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Supporting Information. Chromatograms acquired under various conditions, optical absorption spectra of the samples at several retention time in Figure 3, the chromatogram obtained at pH 1.8, and other data. This material is available free of charge via the Internet at http://pubs.acs.org.

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TOC graphic Ion Pair Chromatography COO−

(C4H9)4N+

Chromatogram Aun(SG)m

Aun(SG)m C18H37 Stationary phase


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Separation of Glutathionate-Protected Gold Clusters by Reversed

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