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Xiaojing Liang1 Xusheng Wang1 Haixia Ren1,2 Shengxiang Jiang1 Licheng Wang1 ∗ Shujuan Liu1 1 Key
Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, China 2 Chinese Academy of Sciences, University of the Chinese Academy of Sciences, Beijing, China Received January 3, 2014 Revised March 13, 2014 Accepted March 19, 2014
Research Article
Gold nanoparticle decorated graphene oxide/silica composite stationary phase for high-performance liquid chromatography In the initial phase of this study, graphene oxide (GO)/silica was fabricated by assembling GO onto the silica particles, and then gold nanoparticles (GNPs) were used to modify the GO/silica to prepare a novel stationary phase for high-performance liquid chromatography. The new stationary phase could be used in both reversed-phase chromatography and hydrophilic interaction liquid chromatography modes. Good separations of alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases were achieved in both modes. Compared with the GO/silica phase and GNPs/silica phase, it is found that except for hydrophilicity, large -electron systems, hydrophobicity, and coordination functions, this new stationary phase also exhibited special separation performance due to the combination of 2D GO with zero-dimensional GNPs. Keywords: Gold nanoparticles / Graphene oxide / High-performance liquid chromatography / Stationary phases DOI 10.1002/jssc.201400005
1 Introduction Graphene, a 2D sheet of sp2 -conjugated carbon atoms [1], has recently sparked much research interest because of its high specific surface area (2630 m2 /g) [2], and unique electronic, exceptional mechanical and thermal properties [3]. It is usually considered to be nonpolar and hydrophobic. Graphene oxide (GO), in contrast, contains much more polar moieties, such as C–O–C (epoxide), C–OH, and –COOH groups [4, 5], and is thus more polar and hydrophilic than graphene. These oxygen-containing groups also make GO easy to assemble onto many support surfaces via certain interactions [6–9]. It was reported that GO was grafted onto a silicon substrate covered with 3-aminopropyl triethoxysilane through chemical reactions between the oxygen-containing groups and amine groups [10]. Using the same theory, Liu et al. [11] coated GO onto an amorphous amino silica surface to make a GO@silica composite for SPE. Subsequently, our research group [12] coated GO onto chromatographic 3-aminopropyl silica to make GO/SiO2 microspheres for an HPLC stationary phase. Taking advantage of the reactive oxygen functional groups of GO, further modification can be easily carried out on the surface of GO/SiO2 . We have also investigated the modification of octadecylsilane onto the GO/SiO2 as a new HPLC stationary phase [12], which exhibited both hydropho-
bicity and a large -electron system. Using the above ideas, we can also decorate GO/SiO2 with other materials. Gold nanoparticles (GNPs) possess many special properties, such as long-term stability, high surface-to-volume ratio, easy chemical modification, size-dependent electrical properties, high electrocatalytic activity, compatibility with biomolecules [13, 14], and so on. In addition, organic molecules containing thiol (–SH) or amino (–NH2 ) groups can adsorb spontaneously onto the gold surface to form wellorganized self-assembled monolayers [15]. Because of the simplicity and flexibility of this approach, it has attracted a great deal of attention in various technologies, including catalysis, optical devices, biosensors, drug carriers, high contrast cell imaging [16–20], sample preconcentration [21–26], and separation science [27, 28]. Researchers have applied GNPs in analytical and separation science for a long time. The application of GNPs in CE-based analysis has attracted much attention, mainly because of the enhanced separation resolution [29]. As a pseudostationary phase or stabilized on modified fused-silica capillary, GNPs can separate biomolecules [30–33], neutral steroid drugs [34], and polycyclic aromatic hydrocarbons [35]. GNPs can also be used as novel stationary phases providing high separation efficiencies for a variety of analytes in various separation systems [36], including GC [36–39] and HPLC [40–43]. In recent years, the nanocomposites of 2D GO combined with zero-dimensional metal (e.g., Au, Pd, Pt) nanoparticles (metal NPs-GO nanocomposite) show obvious superiorities,
Correspondence: Dr. Shujuan Liu, Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China E-mail: [email protected] Fax:+86 931 8277088
∗ Additional corresponding author: Licheng Wang, E-mail: [email protected]
Abbreviations: GNP, gold nanoparticle; GO, graphene oxide; HILIC, hydrophilic interaction chromatography
Colour Online: See the article online to view Figs. 1, 6, 7, and 9 in colour.
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which already have been used as electrochemical sensors for the determination of various analytes [44–49]. However, to the best of our knowledge, there is no report on the GNPs-GO nanocomposite as an HPLC stationary phase yet. In the present study, a GNP-decorated GO/silica stationary phase (GNPs/GO/SiO2 ) has been prepared. Characterization indicates that GO and GNPs are both successfully bonded onto the silica microsphere. Significant differences have been found by investigating the chromatographic performances of GO/SiO2 and GNPs/GO/SiO2 columns. The surface of GO/SiO2 is obviously hydrophilic; while GNPs/GO/SiO2 , combining the advantages of GNPs together with GO, exhibits hydrophobicity, hydrophilicity, and structural recognition capability, leading to the possible separation of analytes with electron-donating groups.
2 Materials and methods 2.1 Apparatus and reagents All chromatographic tests were performed on an Agilent 1100 Series modular HPLC system (Agilent Technologies, Inc, USA) with a binary pump, a 20 L sample loop, a UV/Vis detector and an evaporative light-scattering detector. Separations were carried out using columns of 150 × 4.6 mm id. Deionized water and acetonitrile (analytical grade) were filtered through a 0.45 m nylon membrane filter and were degassed ultrasonically prior to use. All the compounds used in experiments were of analytical grade and used without further purification. Silica spheres were synthesized using the polymerization-induced colloid aggregation method in our laboratory. The average particle size was 5 m. The specific surface area, pore volume, and pore diameter were 391 m2 /g, 0.80 m3 /g, and 7.2 nm, respectively. The reaction solvent, toluene, was distilled and dried over sodium before use. Aminopropyltrimethoxysilane and thiopropyltriethoxysilane were of analytical grade and was used without purification. GO (sheet thickness: 0.3–1.2 nm, sheet length: 35 m C: 46.75%) was purchased from Nanoon Nanomaterials Science and Technology (Langfang, China). GNPs were prepared by the chemical reduction of HAuCl4 .
2.2 Synthesis of GNPs Our approach for GNPs preparation was based on the AuIII reduction by citrate according to Ref.[21]. First, 60 mL of 0.01% w/w HAuCl4 aqueous solution was added into a roundbottomed flask and brought to a vigorous boil under constant stirring. Next, 1.2 mL of 1% w/w trisodium citrate aqueous solution was added at once into the flask. Stirring was continued for another 2 min and the solution turned to wine red. Finally, the solution was heated for 15 min continuously. Stirring did not stop until the solution cooled to room temperature. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.3 Synthesis of GNPs/GO/SiO2 The detailed description of GO/SiO2 preparation can be found in our previous paper [11]. Shortly, 15 g of dried aminopropylsilica particles were added to 150 mL GO dispersion (1 mg/mL) under ultrasonic treatment for 5 min and then stirred at 80⬚C for 12 h for the bonding of GO. The GO/SiO2 was washed with deionized water and then dried. A schematic diagram of the synthetic approach for the preparation of GNPs/GO/SiO2 is outlined in Fig. 1. The dried GO/SiO2 (10 g) was suspended in 120 mL of dry toluene and then an excess of thiopropyltriethoxysilane (10 mL) was added. The suspension was stirred and refluxed for 48 h. After refluxing, the sulfhydrylpropyl-modified GO/SiO2 was washed with toluene, ethanol/water (1:1, v/v), and deionized water in turn, and then dried under vacuum for 12 h at 60⬚C. 5 g of dried sulfhydrylpropyl modified GO/SiO2 particles were added to the GNPs sol (50 mL) under ultrasonic treatment for 20 min and subsequently stirred for 3 h for the adsorption of GNPs. After the above process, due to the attraction between GNPs and –SH, the GNPs were adsorbed onto the surface of sulfhydrylpropyl-modified GO/SiO2 . GNPs/GO/SiO2 was dried under vacuum at 60⬚C. GNPs/SiO2 was prepared using the same synthetic route of Fig. 1 but changing GO/SiO2 to SiO2 . 2.4 Characterization of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 The surface morphologies of GO/SiO2 and GNPs/GO/SiO2 were conducted on an S-4800 field emission scanning electron microscope (SEM; Hitachi, Tokyo, Japan). The elemental analysis of aminopropyl silica, GO/SiO2 , sulfhydrylpropylmodified GO/SiO2 were performed on a Vario EL (Elementar, Hanau, Germany). X-ray photoelectron spectroscopy (XPS; VG ESCALAB210, ThermoFisher Scientific, MA, USA) of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 was performed with MgK␣ radiation as the excitation source. 2.5 Column packing GO/SiO2 and GNPs/GO/SiO2 columns (150 × 4.6 mm id) were made from stainless-steel tubing and were downward packed using slurries of the stationary phase in CCl4 . A 40 MPa packing press (6752B-100, Beijing, China) was used and n-hexane was used as the propulsive solvent. When preparing the GNPs/SiO2 column, n-hexane/isopropanol 90:10 v/v was used for slurrying and propelling the GNPs/SiO2 . 2.6 Conditions of chromatographic evaluation All test mixtures were analyzed at 20⬚C at a flow rate of 1.0 mL/min with UV detection (254 nm) or evaporative light-scattering detection. Each analyte was dissolved in deionized water. www.jss-journal.com
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Figure 1. Schematic diagram of preparation of GNP-functionalized GO/SiO2 .
3 Results and discussion 3.1 Characterization of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 The scanning electron micrographs of GO/SiO2 and GNPs/GO/SiO2 are shown in Fig. 2. From Fig. 2A, we can see that there are a lot of flakes appeared on the surface of GO/SiO2 , which means the surface of SiO2 is completely covered by GO sheets. The SEM photograph of GNPs/GO/SiO2 (Fig. 2B and C) shows that besides flakes, a lot of light spots are homogenously distributed on the flakes, and the particle is spherical with diameter of about 20 nm. Those light spots are ascribed to the GNPs assembled on GO/SiO2 . Elemental analysis of aminopropyl silica, GO/SiO2 , and sulfhydrylpropyl-modified GO/SiO2 are listed in Table 1. Carbon elemental analysis data of aminopropyl silica and GO/SiO2 indicate GO is successfully bonded to the surface of aminopropyl silica. Carbon and sulfur elemental analysis data of GO/SiO2 and sulfhydrylpropyl-modified GO/SiO2 illustrate that sulfhydrylpropyl groups are successfully bonded onto the surface of GO/SiO2 . From the elemental analysis data in Table 1, we can also predicate that every bonding step is successful. The XPS pattern of GO/SiO2 in Fig. 3A indicates a shielding of SiO2 spheres with GO nanosheets. Compared with Fig. 3A, there are noticeable peaks of S and Au in the XPS pattern of GNPs/GO/SiO2 and Fig. 3B that indicates bonding of GO/SiO2 with sulfhydrylpropyl groups and GNPs are both successful. 3.2 Chromatographic separation of alkylbenzenes With a mobile phase of acetonitrile/water 27:73 v/v, separations of benzene, toluene, ethylbenzene, cumene, n C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
butylbenzene, and n-amylbenzene on a GO/SiO2 (Fig. 4A), GNPs/GO/SiO2 (Fig. 4B), and GNPs/SiO2 columns (Fig. 4C) have been compared. From the figure, we can see that that these alkylbenzenes can be well separated on GNPs/GO/SiO2 and GNPs/SiO2 columns, and the eluting order is according to their hydrophobic strength, while there is almost no separation effect on the GO/SiO2 column. The reason for this phenomenon can be attributed to the hydrophobic characteristics of the gold surface [41]. Analytes with higher hydrophobicity have a stronger interaction with GNPs/GO/SiO2 and GNPs/SiO2 stationary phases. A lot of polar groups (such as –OH, COOH, C = O) on the GO flakes make the surface of GO/SiO2 very hydrophilic. Compared with GNPs/SiO2 column, due to the presence of hydrophilic GO, alkylbenzenes have shorter retention times on the GNPs/GO/SiO2 column. 3.3 Chromatographic separation of isomerides The separations of isomerides (hydroquinone, resorcinol, and catechol) obtained on the GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 columns with the same mobile phase (acetonitrile/water 5:95 v/v) are shown in Fig. 5. These three isomerides can be separated on the GNPs/GO/SiO2 (Fig. 5B) and GNPs/SiO2 columns (Fig. 5C), and have longer retention times on GNPs/GO/SiO2 column, while they have no resolution on the GO/SiO2 column (Fig. 5A). It had been reported [42] that using modified GNPs for enantioselective recognition of chiral analytes, such as dansyl-D and Lnorvaline, and in the work of He [43], GNPs were used as a stationary phase for separation of dihydroxybenzene positional isomers by CEC. All these above reports illustrated GNPs may possess some structural recognition capability. In theoretical and computational chemistry, gold is “anomalous” due to its very large relativistic effects [50]. The www.jss-journal.com
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Table 1. Elemental analyses data of aminopropyl silica, GO/SiO2 , and sulfhydrylpropyl modified GO/SiO2
Different particles
Aminopropyl silica GO/SiO2 Sulfhydrylpropyl modified GO/SiO2
Elemental analysis data N (%)
C (%)
H (%)
S (%)
1.35 1.32 1.14
3.39 4.76 6.86
1.42 1.41 1.58
0 0 2.01
Figure 3. XPS pattern of GO/SiO2 (A) and GNPs/GO/SiO2 (B).
relativistic effects lead to excellent electronic mobility that makes it easier to use its empty valency shell to form coordinate bonds with atoms that have a lone pair of electrons. In our experiment, the empty valency shell of the GNPs can form a coordinate bond with the O atom of benzenediol. The possible coordination is shown in Fig. 6. The interaction differences lead to separation of the isomerides with an elution order of hydroquinone, resorcinol, and catechol. Resolutions and column efficiencies of isomerides on different columns are listed in Table 2. From Table 2, we can see that resolutions of isomerides on GNPs/GO/SiO2 column are higher than on GNPs/SiO2 column, while isomerides on GNPs/SiO2 column possess higher column efficiencies. The reason is that the coating of GO onto SiO2 will obviously reduce the surface areas of SiO2 , which has been confirmed in our previous article [12], and the decrease of surface areas of stationary phase will reduce the column efficiency.
3.4 Chromatographic separation of nucleosides and nucleobases Figure 2. Scanning electron micrographs of GO/SiO2 particles (A) and GNPs/GO/SiO2 particles (B and C).
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The eluotropic strength of mobile phase manipulates the retention of polar compounds to a large extent. In order to investigate the surface properties of GNPs/GO/SiO2 column, the separation of six polar compounds (thymidine, uridine, cytosine, inosine, xanthine, and 6-chlorouracil) on the GO/SiO2 column and GNPs/GO/SiO2 column are evaluated in Fig. 7. Figure 7A shows that with the increase of the acetonitrile www.jss-journal.com
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Figure 4. Separation of test mixture of benzene (1), toluene (2), ethylbenzene (3), cumene (4), n-butylbenzene (5), and namylbenzene (6). Mobile phase: acetonitrile/water 27:73 v/v. Conditions: GO/SiO2 column (A; 150 × 4.6 mm id), GNPs/GO/SiO2 column (B; 150 × 4.6 mm id), GNPs/SiO2 column (C; 150 × 4.6 mm id), flow rate: 1.0 mL/min; temperature: 20⬚C; injection volume: 20 L, detection: UV at 254 nm.
content (10–50%) in the mobile phase, the k value of these compounds gradually decreased, then the retention of all test compounds gradually increased with increasing the acetonitrile content from 50 to 80%, finally dramatically increased when further increasing acetonitrile above 80%. The retentions depending on the acetonitrile content in mobile phase exhibit U-shaped curves with a minimum of retention for the 50:50 water/acetonitrile mixture on the new column (Fig. 7A). Similar U-shaped curves have been observed with a great many stationary phases and mobile phase [51–54]. U-shaped elution curves indicate mixed retention effects. When the acetonitrile content is 50%, it shows typical characteristics of hydrophilic interaction chromatography (HILIC). Hence, retention at high levels of organic solvents reflected the hydrophilic nature of GO/SiO2 phase. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 5. Separation of test mixture of hydroquinone (1), resorcinol (2), catechol (3). Mobile phase: acetonitrile/water 5:95 v/v. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B), GNPs/SiO2 column (C), other conditions are the same as in Fig. 4.
Figure 6. The mechanism of separation of hydroquinone, resorcinol, and catechol on GNPs/GO/SiO2 column and GNPs/SiO2 column.
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Table 2. Resolutions and column efficiencies of isomerides on different columns
Resolution
GO/SiO2 column GNPs/GO/SiO2 column GNPs/SiO2 column
Column efficiency
Hydroquinone
Resorcinol
Catechol
Hydroquinone
Resorcinol
Catechol
− − −
− 1.58 1.52
− 1.00 0.65
− 4913 24 833
− 3373 22 253
− 1060 8400
Figure 7. Effect of different contents of acetonitrile in mobile phases on retention factors of nucleosides and basic groups. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B); other conditions are the same as in Fig. 4.
As shown in Fig. 7B, except 6-chlorouracil, the retentions of other test compounds only exhibit typical characteristics of HILIC. Exceptionally, the retention of 6-chlorouracil depending on the acetonitrile content in mobile phase exhibits U-shaped curve that indicates mixed retention effects. Except for hydrophilic interaction, we speculate that the unique nanocomposite that combined 2D GO with zerodimensional GNPs can also produce a special interaction with 6-chlorouracil compared with only GO or only GNPs. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 8. Separation of test mixture of thymidine (1), uridine (2), cytosine (3), inosine (4), xanthine (5), and 6-chlorouracil (6). Mobile phase: acetonitrile/0.02 mol/L ammonium acetate solution 75:25 v/v. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B), GNPs/SiO2 column (C), other conditions are the same as in Fig. 4.
Under the HILIC mode, a test mixture of six nucleosides and nucleobases has been investigated on these three columns with a mobile phase of acetonitrile/0.02 mol/L ammonium acetate solution 75:25 v/v, and the separation chromatograms are shown in Fig. 8A, B, and C. It can be seen that all the components are well separated on the GNPs/GO/SiO2 column, but on GO/SiO2 column, xanthine and 6-chlorouracil cannot be separated and the retention time www.jss-journal.com
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of 6-chlorouracil is much shorter. The eluting order is according to their hydrophilic strength, the stronger the hydrophilicity, the longer the retention time. However, on the GNPs/SiO2 column, most of the analytes are not completely separate and the eluting order is totally reversed, which indicates the surfaces of GNPs/SiO2 are hydrophobic and cannot exhibit typical characteristics of HILIC. Figure 8 also shows that the unique nanocomposite has a better separation performance than the single material.
3.5 Chromatographic separation of amino acids To further study the different surface properties of GNPs/GO/SiO2 column, the effect of acetonitrile content in the mobile phase on the retention factors of amino acids (DLleucine, L-tyrosine, proline, glycine, 3-nitro-L-tyrosine, and L-glutamic acid) on GO/SiO2 column and GNPs/GO/SiO2 column are evaluated and the results are shown in Fig. 9A and B. We can see that the retentions of the test compounds (except 3-nitro-L-tyrosine) almost do not change with the increase of the acetonitrile content (10–50%), and when further increasing the acetonitrile content, the retention of the solutes gradually increases. This phenomenon indicates single hydrophilic retention effects. It is because these amino acids are extremely hydrophilic, and they cannot produce any other interactions with these two stationary phases except for hydrophilic interactions. Meanwhile, the increased trend of retention on the GO/SiO2 column is more remarkable than that on the GNPs/GO/SiO2 column, indicating the more hydrophilic surface of GO/SiO2 than that of GNPs/GO/SiO2 , which is consistent with the results in Section 3.2. The retention of 3-nitro-L-tyrosine depends on the acetonitrile content in the mobile phase and exhibits a U-shaped curve, indicating the existence of mixed retention effects. And as 3-nitroL-tyrosine can form an intramolecular hydrogen bond that can cause – interactions between 3-nitro-L-tyrosine and the two columns, the flat structure of the GO/SiO2 surface is better than GNPs/GO/SiO2 , so the – interaction between 3-nitro-L-tyrosine and GO/SiO2 column is much stronger. So, the U-shaped curve obtained with the GO/SiO2 column is more remarkable than that of the GNPs/GO/SiO2 column. Figure 10 shows the separations of these amino acids on the GO/SiO2 (Fig. 10A), GNPs/GO/SiO2 (Fig. 10B), and GNPs/SiO2 columns using acetonitrile/0.2 mol/L ammonium acetate solution (70:30, v/v) as the mobile phase and evaporative light-scattering detection. Compared with the GO/SiO2 column, the analytes have much shorter retention times on the GNPs/GO/SiO2 column and almost no retention on the GNPs/SiO2 column under the HILIC mode. This phenomenon confirms that the surface of GNPs/SiO2 is hydrophobic and that of GO/SiO2 is more hydrophilic than the surface of GNPs/GO/SiO2 . From Fig. 10, we can also see that baseline separation of glycine and 3-nitro-L-tyrosine is not achieved on the GO/SiO2 column, and there is almost no separation effect on the GNPs/SiO2 column, but because of the presence of the nanocomposite of 2D GO combined C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 9. Effect of different contents of acetonitrile in mobile phases on retention factors of amino acids. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B); detection: evaporative light-scattering detection, other conditions are the same as in Fig. 4.
with zero-dimensional GNPs, the six amino acids can be completely separated on the GNPs/GO/SiO2 column.
4 Concluding remarks In this work, a new stationary phase GNPs/GO/SiO2 was prepared by immobilizing GNPs onto the surface of GO-modified silica. The SEM, XPS, and elemental analysis characterizations of GO/SiO2 and GNPs/GO/SiO2 gave eloquent proof of the existence of GO and GNPs. The comparison of chromatographic performances of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 columns clearly indicated that GO/SiO2 exhibited both a large -electron system and hydrophilicity, and GNPs/SiO2 exhibited hydrophobicity and coordination functions. However, GNPs/GO/SiO2 not only combined the properties of GO and GNPs, but also exhibited unique separation performances. Alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases can www.jss-journal.com
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Figure 10. Separation of test mixture of DL-leucine (1), Ltyrosine (2), proline (3), glycine (4), 3-nitro-L-tyrosine (5), and L-glutamic acid (6). Mobile phase: acetonitrile/0.2 mol/L ammonium acetate solution 70:30 v/v. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B), GNPs/SiO2 column (C), detection: evaporative light-scattering detection, other conditions are the same as in Fig. 4.
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be well separated in the presence of the nanocomposite 2D GO combined with zero-dimensional GNPs. Therefore, the utility of the nanocomposite in various practical applications will be further explored in future. Financial support from the National Natural Science Foundation of China (21105107, 21175143, and 20905072) is gratefully acknowledged.
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The authors have declared no conflict of interest.
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Xiaojing Liang1 Xusheng Wang1 Haixia Ren1,2 Shengxiang Jiang1 Licheng Wang1 ∗ Shujuan Liu1 1 Key
Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, China 2 Chinese Academy of Sciences, University of the Chinese Academy of Sciences, Beijing, China Received January 3, 2014 Revised March 13, 2014 Accepted March 19, 2014
Research Article
Gold nanoparticle decorated graphene oxide/silica composite stationary phase for high-performance liquid chromatography In the initial phase of this study, graphene oxide (GO)/silica was fabricated by assembling GO onto the silica particles, and then gold nanoparticles (GNPs) were used to modify the GO/silica to prepare a novel stationary phase for high-performance liquid chromatography. The new stationary phase could be used in both reversed-phase chromatography and hydrophilic interaction liquid chromatography modes. Good separations of alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases were achieved in both modes. Compared with the GO/silica phase and GNPs/silica phase, it is found that except for hydrophilicity, large -electron systems, hydrophobicity, and coordination functions, this new stationary phase also exhibited special separation performance due to the combination of 2D GO with zero-dimensional GNPs. Keywords: Gold nanoparticles / Graphene oxide / High-performance liquid chromatography / Stationary phases DOI 10.1002/jssc.201400005
1 Introduction Graphene, a 2D sheet of sp2 -conjugated carbon atoms [1], has recently sparked much research interest because of its high specific surface area (2630 m2 /g) [2], and unique electronic, exceptional mechanical and thermal properties [3]. It is usually considered to be nonpolar and hydrophobic. Graphene oxide (GO), in contrast, contains much more polar moieties, such as C–O–C (epoxide), C–OH, and –COOH groups [4, 5], and is thus more polar and hydrophilic than graphene. These oxygen-containing groups also make GO easy to assemble onto many support surfaces via certain interactions [6–9]. It was reported that GO was grafted onto a silicon substrate covered with 3-aminopropyl triethoxysilane through chemical reactions between the oxygen-containing groups and amine groups [10]. Using the same theory, Liu et al. [11] coated GO onto an amorphous amino silica surface to make a GO@silica composite for SPE. Subsequently, our research group [12] coated GO onto chromatographic 3-aminopropyl silica to make GO/SiO2 microspheres for an HPLC stationary phase. Taking advantage of the reactive oxygen functional groups of GO, further modification can be easily carried out on the surface of GO/SiO2 . We have also investigated the modification of octadecylsilane onto the GO/SiO2 as a new HPLC stationary phase [12], which exhibited both hydropho-
bicity and a large -electron system. Using the above ideas, we can also decorate GO/SiO2 with other materials. Gold nanoparticles (GNPs) possess many special properties, such as long-term stability, high surface-to-volume ratio, easy chemical modification, size-dependent electrical properties, high electrocatalytic activity, compatibility with biomolecules [13, 14], and so on. In addition, organic molecules containing thiol (–SH) or amino (–NH2 ) groups can adsorb spontaneously onto the gold surface to form wellorganized self-assembled monolayers [15]. Because of the simplicity and flexibility of this approach, it has attracted a great deal of attention in various technologies, including catalysis, optical devices, biosensors, drug carriers, high contrast cell imaging [16–20], sample preconcentration [21–26], and separation science [27, 28]. Researchers have applied GNPs in analytical and separation science for a long time. The application of GNPs in CE-based analysis has attracted much attention, mainly because of the enhanced separation resolution [29]. As a pseudostationary phase or stabilized on modified fused-silica capillary, GNPs can separate biomolecules [30–33], neutral steroid drugs [34], and polycyclic aromatic hydrocarbons [35]. GNPs can also be used as novel stationary phases providing high separation efficiencies for a variety of analytes in various separation systems [36], including GC [36–39] and HPLC [40–43]. In recent years, the nanocomposites of 2D GO combined with zero-dimensional metal (e.g., Au, Pd, Pt) nanoparticles (metal NPs-GO nanocomposite) show obvious superiorities,
Correspondence: Dr. Shujuan Liu, Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China E-mail: [email protected] Fax:+86 931 8277088
∗ Additional corresponding author: Licheng Wang, E-mail: [email protected]
Abbreviations: GNP, gold nanoparticle; GO, graphene oxide; HILIC, hydrophilic interaction chromatography
Colour Online: See the article online to view Figs. 1, 6, 7, and 9 in colour.
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which already have been used as electrochemical sensors for the determination of various analytes [44–49]. However, to the best of our knowledge, there is no report on the GNPs-GO nanocomposite as an HPLC stationary phase yet. In the present study, a GNP-decorated GO/silica stationary phase (GNPs/GO/SiO2 ) has been prepared. Characterization indicates that GO and GNPs are both successfully bonded onto the silica microsphere. Significant differences have been found by investigating the chromatographic performances of GO/SiO2 and GNPs/GO/SiO2 columns. The surface of GO/SiO2 is obviously hydrophilic; while GNPs/GO/SiO2 , combining the advantages of GNPs together with GO, exhibits hydrophobicity, hydrophilicity, and structural recognition capability, leading to the possible separation of analytes with electron-donating groups.
2 Materials and methods 2.1 Apparatus and reagents All chromatographic tests were performed on an Agilent 1100 Series modular HPLC system (Agilent Technologies, Inc, USA) with a binary pump, a 20 L sample loop, a UV/Vis detector and an evaporative light-scattering detector. Separations were carried out using columns of 150 × 4.6 mm id. Deionized water and acetonitrile (analytical grade) were filtered through a 0.45 m nylon membrane filter and were degassed ultrasonically prior to use. All the compounds used in experiments were of analytical grade and used without further purification. Silica spheres were synthesized using the polymerization-induced colloid aggregation method in our laboratory. The average particle size was 5 m. The specific surface area, pore volume, and pore diameter were 391 m2 /g, 0.80 m3 /g, and 7.2 nm, respectively. The reaction solvent, toluene, was distilled and dried over sodium before use. Aminopropyltrimethoxysilane and thiopropyltriethoxysilane were of analytical grade and was used without purification. GO (sheet thickness: 0.3–1.2 nm, sheet length: 35 m C: 46.75%) was purchased from Nanoon Nanomaterials Science and Technology (Langfang, China). GNPs were prepared by the chemical reduction of HAuCl4 .
2.2 Synthesis of GNPs Our approach for GNPs preparation was based on the AuIII reduction by citrate according to Ref.[21]. First, 60 mL of 0.01% w/w HAuCl4 aqueous solution was added into a roundbottomed flask and brought to a vigorous boil under constant stirring. Next, 1.2 mL of 1% w/w trisodium citrate aqueous solution was added at once into the flask. Stirring was continued for another 2 min and the solution turned to wine red. Finally, the solution was heated for 15 min continuously. Stirring did not stop until the solution cooled to room temperature. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.3 Synthesis of GNPs/GO/SiO2 The detailed description of GO/SiO2 preparation can be found in our previous paper [11]. Shortly, 15 g of dried aminopropylsilica particles were added to 150 mL GO dispersion (1 mg/mL) under ultrasonic treatment for 5 min and then stirred at 80⬚C for 12 h for the bonding of GO. The GO/SiO2 was washed with deionized water and then dried. A schematic diagram of the synthetic approach for the preparation of GNPs/GO/SiO2 is outlined in Fig. 1. The dried GO/SiO2 (10 g) was suspended in 120 mL of dry toluene and then an excess of thiopropyltriethoxysilane (10 mL) was added. The suspension was stirred and refluxed for 48 h. After refluxing, the sulfhydrylpropyl-modified GO/SiO2 was washed with toluene, ethanol/water (1:1, v/v), and deionized water in turn, and then dried under vacuum for 12 h at 60⬚C. 5 g of dried sulfhydrylpropyl modified GO/SiO2 particles were added to the GNPs sol (50 mL) under ultrasonic treatment for 20 min and subsequently stirred for 3 h for the adsorption of GNPs. After the above process, due to the attraction between GNPs and –SH, the GNPs were adsorbed onto the surface of sulfhydrylpropyl-modified GO/SiO2 . GNPs/GO/SiO2 was dried under vacuum at 60⬚C. GNPs/SiO2 was prepared using the same synthetic route of Fig. 1 but changing GO/SiO2 to SiO2 . 2.4 Characterization of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 The surface morphologies of GO/SiO2 and GNPs/GO/SiO2 were conducted on an S-4800 field emission scanning electron microscope (SEM; Hitachi, Tokyo, Japan). The elemental analysis of aminopropyl silica, GO/SiO2 , sulfhydrylpropylmodified GO/SiO2 were performed on a Vario EL (Elementar, Hanau, Germany). X-ray photoelectron spectroscopy (XPS; VG ESCALAB210, ThermoFisher Scientific, MA, USA) of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 was performed with MgK␣ radiation as the excitation source. 2.5 Column packing GO/SiO2 and GNPs/GO/SiO2 columns (150 × 4.6 mm id) were made from stainless-steel tubing and were downward packed using slurries of the stationary phase in CCl4 . A 40 MPa packing press (6752B-100, Beijing, China) was used and n-hexane was used as the propulsive solvent. When preparing the GNPs/SiO2 column, n-hexane/isopropanol 90:10 v/v was used for slurrying and propelling the GNPs/SiO2 . 2.6 Conditions of chromatographic evaluation All test mixtures were analyzed at 20⬚C at a flow rate of 1.0 mL/min with UV detection (254 nm) or evaporative light-scattering detection. Each analyte was dissolved in deionized water. www.jss-journal.com
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Figure 1. Schematic diagram of preparation of GNP-functionalized GO/SiO2 .
3 Results and discussion 3.1 Characterization of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 The scanning electron micrographs of GO/SiO2 and GNPs/GO/SiO2 are shown in Fig. 2. From Fig. 2A, we can see that there are a lot of flakes appeared on the surface of GO/SiO2 , which means the surface of SiO2 is completely covered by GO sheets. The SEM photograph of GNPs/GO/SiO2 (Fig. 2B and C) shows that besides flakes, a lot of light spots are homogenously distributed on the flakes, and the particle is spherical with diameter of about 20 nm. Those light spots are ascribed to the GNPs assembled on GO/SiO2 . Elemental analysis of aminopropyl silica, GO/SiO2 , and sulfhydrylpropyl-modified GO/SiO2 are listed in Table 1. Carbon elemental analysis data of aminopropyl silica and GO/SiO2 indicate GO is successfully bonded to the surface of aminopropyl silica. Carbon and sulfur elemental analysis data of GO/SiO2 and sulfhydrylpropyl-modified GO/SiO2 illustrate that sulfhydrylpropyl groups are successfully bonded onto the surface of GO/SiO2 . From the elemental analysis data in Table 1, we can also predicate that every bonding step is successful. The XPS pattern of GO/SiO2 in Fig. 3A indicates a shielding of SiO2 spheres with GO nanosheets. Compared with Fig. 3A, there are noticeable peaks of S and Au in the XPS pattern of GNPs/GO/SiO2 and Fig. 3B that indicates bonding of GO/SiO2 with sulfhydrylpropyl groups and GNPs are both successful. 3.2 Chromatographic separation of alkylbenzenes With a mobile phase of acetonitrile/water 27:73 v/v, separations of benzene, toluene, ethylbenzene, cumene, n C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
butylbenzene, and n-amylbenzene on a GO/SiO2 (Fig. 4A), GNPs/GO/SiO2 (Fig. 4B), and GNPs/SiO2 columns (Fig. 4C) have been compared. From the figure, we can see that that these alkylbenzenes can be well separated on GNPs/GO/SiO2 and GNPs/SiO2 columns, and the eluting order is according to their hydrophobic strength, while there is almost no separation effect on the GO/SiO2 column. The reason for this phenomenon can be attributed to the hydrophobic characteristics of the gold surface [41]. Analytes with higher hydrophobicity have a stronger interaction with GNPs/GO/SiO2 and GNPs/SiO2 stationary phases. A lot of polar groups (such as –OH, COOH, C = O) on the GO flakes make the surface of GO/SiO2 very hydrophilic. Compared with GNPs/SiO2 column, due to the presence of hydrophilic GO, alkylbenzenes have shorter retention times on the GNPs/GO/SiO2 column. 3.3 Chromatographic separation of isomerides The separations of isomerides (hydroquinone, resorcinol, and catechol) obtained on the GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 columns with the same mobile phase (acetonitrile/water 5:95 v/v) are shown in Fig. 5. These three isomerides can be separated on the GNPs/GO/SiO2 (Fig. 5B) and GNPs/SiO2 columns (Fig. 5C), and have longer retention times on GNPs/GO/SiO2 column, while they have no resolution on the GO/SiO2 column (Fig. 5A). It had been reported [42] that using modified GNPs for enantioselective recognition of chiral analytes, such as dansyl-D and Lnorvaline, and in the work of He [43], GNPs were used as a stationary phase for separation of dihydroxybenzene positional isomers by CEC. All these above reports illustrated GNPs may possess some structural recognition capability. In theoretical and computational chemistry, gold is “anomalous” due to its very large relativistic effects [50]. The www.jss-journal.com
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Table 1. Elemental analyses data of aminopropyl silica, GO/SiO2 , and sulfhydrylpropyl modified GO/SiO2
Different particles
Aminopropyl silica GO/SiO2 Sulfhydrylpropyl modified GO/SiO2
Elemental analysis data N (%)
C (%)
H (%)
S (%)
1.35 1.32 1.14
3.39 4.76 6.86
1.42 1.41 1.58
0 0 2.01
Figure 3. XPS pattern of GO/SiO2 (A) and GNPs/GO/SiO2 (B).
relativistic effects lead to excellent electronic mobility that makes it easier to use its empty valency shell to form coordinate bonds with atoms that have a lone pair of electrons. In our experiment, the empty valency shell of the GNPs can form a coordinate bond with the O atom of benzenediol. The possible coordination is shown in Fig. 6. The interaction differences lead to separation of the isomerides with an elution order of hydroquinone, resorcinol, and catechol. Resolutions and column efficiencies of isomerides on different columns are listed in Table 2. From Table 2, we can see that resolutions of isomerides on GNPs/GO/SiO2 column are higher than on GNPs/SiO2 column, while isomerides on GNPs/SiO2 column possess higher column efficiencies. The reason is that the coating of GO onto SiO2 will obviously reduce the surface areas of SiO2 , which has been confirmed in our previous article [12], and the decrease of surface areas of stationary phase will reduce the column efficiency.
3.4 Chromatographic separation of nucleosides and nucleobases Figure 2. Scanning electron micrographs of GO/SiO2 particles (A) and GNPs/GO/SiO2 particles (B and C).
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The eluotropic strength of mobile phase manipulates the retention of polar compounds to a large extent. In order to investigate the surface properties of GNPs/GO/SiO2 column, the separation of six polar compounds (thymidine, uridine, cytosine, inosine, xanthine, and 6-chlorouracil) on the GO/SiO2 column and GNPs/GO/SiO2 column are evaluated in Fig. 7. Figure 7A shows that with the increase of the acetonitrile www.jss-journal.com
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Figure 4. Separation of test mixture of benzene (1), toluene (2), ethylbenzene (3), cumene (4), n-butylbenzene (5), and namylbenzene (6). Mobile phase: acetonitrile/water 27:73 v/v. Conditions: GO/SiO2 column (A; 150 × 4.6 mm id), GNPs/GO/SiO2 column (B; 150 × 4.6 mm id), GNPs/SiO2 column (C; 150 × 4.6 mm id), flow rate: 1.0 mL/min; temperature: 20⬚C; injection volume: 20 L, detection: UV at 254 nm.
content (10–50%) in the mobile phase, the k value of these compounds gradually decreased, then the retention of all test compounds gradually increased with increasing the acetonitrile content from 50 to 80%, finally dramatically increased when further increasing acetonitrile above 80%. The retentions depending on the acetonitrile content in mobile phase exhibit U-shaped curves with a minimum of retention for the 50:50 water/acetonitrile mixture on the new column (Fig. 7A). Similar U-shaped curves have been observed with a great many stationary phases and mobile phase [51–54]. U-shaped elution curves indicate mixed retention effects. When the acetonitrile content is 50%, it shows typical characteristics of hydrophilic interaction chromatography (HILIC). Hence, retention at high levels of organic solvents reflected the hydrophilic nature of GO/SiO2 phase. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 5. Separation of test mixture of hydroquinone (1), resorcinol (2), catechol (3). Mobile phase: acetonitrile/water 5:95 v/v. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B), GNPs/SiO2 column (C), other conditions are the same as in Fig. 4.
Figure 6. The mechanism of separation of hydroquinone, resorcinol, and catechol on GNPs/GO/SiO2 column and GNPs/SiO2 column.
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Table 2. Resolutions and column efficiencies of isomerides on different columns
Resolution
GO/SiO2 column GNPs/GO/SiO2 column GNPs/SiO2 column
Column efficiency
Hydroquinone
Resorcinol
Catechol
Hydroquinone
Resorcinol
Catechol
− − −
− 1.58 1.52
− 1.00 0.65
− 4913 24 833
− 3373 22 253
− 1060 8400
Figure 7. Effect of different contents of acetonitrile in mobile phases on retention factors of nucleosides and basic groups. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B); other conditions are the same as in Fig. 4.
As shown in Fig. 7B, except 6-chlorouracil, the retentions of other test compounds only exhibit typical characteristics of HILIC. Exceptionally, the retention of 6-chlorouracil depending on the acetonitrile content in mobile phase exhibits U-shaped curve that indicates mixed retention effects. Except for hydrophilic interaction, we speculate that the unique nanocomposite that combined 2D GO with zerodimensional GNPs can also produce a special interaction with 6-chlorouracil compared with only GO or only GNPs. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 8. Separation of test mixture of thymidine (1), uridine (2), cytosine (3), inosine (4), xanthine (5), and 6-chlorouracil (6). Mobile phase: acetonitrile/0.02 mol/L ammonium acetate solution 75:25 v/v. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B), GNPs/SiO2 column (C), other conditions are the same as in Fig. 4.
Under the HILIC mode, a test mixture of six nucleosides and nucleobases has been investigated on these three columns with a mobile phase of acetonitrile/0.02 mol/L ammonium acetate solution 75:25 v/v, and the separation chromatograms are shown in Fig. 8A, B, and C. It can be seen that all the components are well separated on the GNPs/GO/SiO2 column, but on GO/SiO2 column, xanthine and 6-chlorouracil cannot be separated and the retention time www.jss-journal.com
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of 6-chlorouracil is much shorter. The eluting order is according to their hydrophilic strength, the stronger the hydrophilicity, the longer the retention time. However, on the GNPs/SiO2 column, most of the analytes are not completely separate and the eluting order is totally reversed, which indicates the surfaces of GNPs/SiO2 are hydrophobic and cannot exhibit typical characteristics of HILIC. Figure 8 also shows that the unique nanocomposite has a better separation performance than the single material.
3.5 Chromatographic separation of amino acids To further study the different surface properties of GNPs/GO/SiO2 column, the effect of acetonitrile content in the mobile phase on the retention factors of amino acids (DLleucine, L-tyrosine, proline, glycine, 3-nitro-L-tyrosine, and L-glutamic acid) on GO/SiO2 column and GNPs/GO/SiO2 column are evaluated and the results are shown in Fig. 9A and B. We can see that the retentions of the test compounds (except 3-nitro-L-tyrosine) almost do not change with the increase of the acetonitrile content (10–50%), and when further increasing the acetonitrile content, the retention of the solutes gradually increases. This phenomenon indicates single hydrophilic retention effects. It is because these amino acids are extremely hydrophilic, and they cannot produce any other interactions with these two stationary phases except for hydrophilic interactions. Meanwhile, the increased trend of retention on the GO/SiO2 column is more remarkable than that on the GNPs/GO/SiO2 column, indicating the more hydrophilic surface of GO/SiO2 than that of GNPs/GO/SiO2 , which is consistent with the results in Section 3.2. The retention of 3-nitro-L-tyrosine depends on the acetonitrile content in the mobile phase and exhibits a U-shaped curve, indicating the existence of mixed retention effects. And as 3-nitroL-tyrosine can form an intramolecular hydrogen bond that can cause – interactions between 3-nitro-L-tyrosine and the two columns, the flat structure of the GO/SiO2 surface is better than GNPs/GO/SiO2 , so the – interaction between 3-nitro-L-tyrosine and GO/SiO2 column is much stronger. So, the U-shaped curve obtained with the GO/SiO2 column is more remarkable than that of the GNPs/GO/SiO2 column. Figure 10 shows the separations of these amino acids on the GO/SiO2 (Fig. 10A), GNPs/GO/SiO2 (Fig. 10B), and GNPs/SiO2 columns using acetonitrile/0.2 mol/L ammonium acetate solution (70:30, v/v) as the mobile phase and evaporative light-scattering detection. Compared with the GO/SiO2 column, the analytes have much shorter retention times on the GNPs/GO/SiO2 column and almost no retention on the GNPs/SiO2 column under the HILIC mode. This phenomenon confirms that the surface of GNPs/SiO2 is hydrophobic and that of GO/SiO2 is more hydrophilic than the surface of GNPs/GO/SiO2 . From Fig. 10, we can also see that baseline separation of glycine and 3-nitro-L-tyrosine is not achieved on the GO/SiO2 column, and there is almost no separation effect on the GNPs/SiO2 column, but because of the presence of the nanocomposite of 2D GO combined C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 9. Effect of different contents of acetonitrile in mobile phases on retention factors of amino acids. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B); detection: evaporative light-scattering detection, other conditions are the same as in Fig. 4.
with zero-dimensional GNPs, the six amino acids can be completely separated on the GNPs/GO/SiO2 column.
4 Concluding remarks In this work, a new stationary phase GNPs/GO/SiO2 was prepared by immobilizing GNPs onto the surface of GO-modified silica. The SEM, XPS, and elemental analysis characterizations of GO/SiO2 and GNPs/GO/SiO2 gave eloquent proof of the existence of GO and GNPs. The comparison of chromatographic performances of GO/SiO2 , GNPs/GO/SiO2 , and GNPs/SiO2 columns clearly indicated that GO/SiO2 exhibited both a large -electron system and hydrophilicity, and GNPs/SiO2 exhibited hydrophobicity and coordination functions. However, GNPs/GO/SiO2 not only combined the properties of GO and GNPs, but also exhibited unique separation performances. Alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases can www.jss-journal.com
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Figure 10. Separation of test mixture of DL-leucine (1), Ltyrosine (2), proline (3), glycine (4), 3-nitro-L-tyrosine (5), and L-glutamic acid (6). Mobile phase: acetonitrile/0.2 mol/L ammonium acetate solution 70:30 v/v. Conditions: GO/SiO2 column (A), GNPs/GO/SiO2 column (B), GNPs/SiO2 column (C), detection: evaporative light-scattering detection, other conditions are the same as in Fig. 4.
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The authors have declared no conflict of interest.
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