Capillary Coated with Graphene and Graphene Oxide Sheets as

Article pubs.acs.org/ac

Capillary Coated with Graphene and Graphene Oxide Sheets as Stationary Phase for Capillary Electrochromatography and Capillary Liquid Chromatography Qishu Qu,* Chenhao Gu, and Xiaoya Hu Jiangsu Key Laboratory of Environmental Materials and Environmental Engineering, College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China S Supporting Information *

ABSTRACT: Graphene oxide (GO) nanosheets were immobilized onto the capillary wall using 3-aminopropyldiethoxymethyl silane as coupling agent. Graphene coated column (G@column) was fabricated by hydrazine reduction of GO modified column. Scanning electron microscopy (SEM) images provided visible evidence of the GO grafted on the capillary wall. Energy dispersive X-ray spectrometry (EDS) indicated the high coverage of the GO on the capillary wall. The G@column exhibited a pH-dependent electroosmotic flow (EOF) from anode to cathode in the pH range of 3−9 while the graphene oxide coated column (GO@column) showed a constant EOF. Both GO@column and G@ column were evaluated for open-tubular capillary electrochromatography (OT-CEC). The GO@column was also evaluated for open-tubular capillary liquid chromatography (OTCLC). Good separation of the tested neutral analytes on the GO@column was achieved on the basis of a typical reversed-phase behavior. On the contrary, G@column showed poor separation performance because of the strong π−π stacking and hydrophobic interactions between graphene and polyaromatic hydrocarbons. The high coverage of GO improved the column phase ratio which makes the GO@column promising for OT-CLC separation. Five of the major known proteins including three glycoisoforms of ovalbumin in chicken egg white were identified in a single run on the GO@column with phosphate buffer (5 mM, pH 7.0) and an applied voltage of 20 kV. The run-to-run, day-to-day, and column-to-column reproducibilities are evaluated by calculating the relative standard deviations (RSDs) of the EOF in OT-CEC and retention time of naphthalene in OT-CLC, respectively. These RSD values were found to be less than 3%.

G

chromatography (HPLC). It was found that the retention mechanism of PGC is different to that of C18 phase. The unique properties of PGC make it a promising stationary phase for the resolution of various types of compounds. Because of these reasons, more carbon based materials, such as carbon nanotubes (CNTs)29 and C60 fullerene,30 have been used as stationary phases shortly after they were found. As two of carbon based materials possessing the superior qualities, G and GO were expected to be used as novel stationary phases for chromatographic separation. However, both G and GO cannot be used directly as packing materials for chromatography because the aggregated graphene sheets are not mechanically strong enough to withstand the high pressure generated in the packing process (ca. 52 MPa). Besides, the irregular morphology of graphene sheets would make the separation performance greatly decreased. Therefore, Wang and Yan developed a method to incorporate GO nanosheets into monolithic capillary column for capillary electrochromatography (CEC).31 It was found that the CEC separation performance was improved greatly due to the increased interaction between the stationary phase and the test

raphene (G), which is considered as the basic building block of all graphitic forms (including carbon nanotubes, graphite, and fullerene C60), is a single-atom-thick and twodimensional sp2 carbon networking material.1,2 Graphene oxide (GO) is a chemically modified graphene sheet with a giant aromatic macromolecule containing reactive oxygen functional groups on their basal planes and edges such as epoxide, hydroxy, and carboxylic acid. Compared with other graphitic forms, G and GO exhibit excellent mechanical, electrical, thermal, and optical properties.3−11 Therefore, G and GO have found potential applications like field-effect transistors,12,13 sensors,14,15 electrochemical devices,16 polymer nanocomposites,17 batteries,18 ultracapacitors,5 and so forth. Since the large delocalized π-electron system of G can form a strong π-stacking interaction with the benzene ring,8,19 G and GO have been explored for solid-phase microextraction of pyrethroid pesticides in natural water samples20 and polycyclic aromatic hydrocarbons in water and soil samples.21 In addition, owing to the very high specific surface area (theoretical value 2630 m2/ g)5 of G and GO, they were also a good candidate as an adsorbent.22−27 Carbon based materials have long been used as stationary phase because of the pioneer work of Knox et al.28 They reported that porous graphitized carbon (PGC) could be used for both gas chromatography and high performance liquid © 2012 American Chemical Society

Received: August 15, 2012 Accepted: September 19, 2012 Published: September 19, 2012 8880

dx.doi.org/10.1021/ac3023636 | Anal. Chem. 2012, 84, 8880−8890

Analytical Chemistry

Article

Figure 1. Schematic representation of the fabrication processes of GO and G-coated capillary columns.

■

EXPERIMENTAL SECTION Chemicals and Materials. Analytical grade hydrazine hydrate (85%), phosphorus pentoxide (P2O5), hydrogen peroxide (H2O2), potassium permanganate (KMnO4), potassium persulfate (K2S2O8), sulphuric acid (H2SO4), thiourea, toluene, naphthalene, biphenyl, 2-methylnaphthalene, acenaphthene, aniline, pyridine, N,N-dimethylaniline, catechol, phenol, 1-naphthol, and HPLC-grade methanol (MeOH) were all purchased from Shanghai Chemical Reagent, Inc. of Chinese Medicine Group (Shanghai, China). Avidin, ovotransferrin, ovalbumin, ovomucoid, ovoflavoprotein, and lysozyme were obtained from Sigma (St. Louis, MO). All chemicals were used without any further purification. Water used in all of the experiments was Robust pure water purchased from a supermarket. Fused silica capillary (75 μm i.d. × 365 μm o.d.) was purchased from Yongnian Rui-feng Fiber Plant (Handan, China). Graphite powder (99.95%, particle size ≤30 μm) and 3-aminopropyldiethoxymethyl silane (3-AMDS) were purchased from Aladdin Chemistry (Shanghai, China). Synthesis of Graphene Oxide. Graphene oxide was synthesized from natural graphite according to the modified Hummers method.51 Briefly, graphite powder was preoxidized in a mixture of concentrated H2SO4, K2S2O8 and P2O5 at 80 °C for 4.5 h. The product was diluted with pure water, and then washed with water and dried under room temperature. The obtained preoxidized graphite was added into cold concentrated H2SO4, and then, KMnO4 was added slowly under stirring with an ice-bath to control the temperature below 20 °C. The mixture was stirred for 4 h. Then, H2O2 and pure water were added to terminate the reaction. The resulting solution was filtrated, washed with 1.2 M HCl solution, and dried finally to give a brown solid. The as-prepared graphene oxide was dispersed in water at 1 mg mL−1 and ultrasonicated

compounds, such as polycyclic aromatic hydrocarbons and anilines. Capillary electrochromatography is an emerging micro column electroseparation technique which combines the high selectivity of HPLC and the high efficiency of capillary electrophoresis (CE) and is often chosen for separation because of its potential for high separation efficiency and the minimal consumption of chemicals. Packed column, opentubular column, and monolithic column are the columns commonly used in CEC. Among all the CEC columns, open tubular column is the most used column because of the ease of preparation, the absence of frits formation and particles packing, and simple instrumental handling. However, the lowphase ratio of open-tubular capillary columns is the primary drawback and restricts their wide application in chromatographic separations. To resolve this problem, some new approaches including sol−gel-derived phases,32 etching,33 porous layers,34−38 and nanoparticle phases39−49 have been developed to increase the surface area of the columns in opentubular capillary electrochromatography (OT-CEC) separations. Since G and GO possess very high surface area, coating the capillary wall with graphene would greatly increase the phase ratio of open-tubular column and consequently improve the resolving power. In this article, we report the first example of directly using G and GO as coating material for OT-CEC and open-tubular capillary liquid chromatography (OT-CLC) separation. Graphene oxide sheets were immobilized onto the capillary wall through chemical reaction of carboxy group with the amine group to increase the stability of the coating.8,24,50 Graphene coated column was then obtained by hydrazine reduction of GO modified column. The chromatographic performance of graphene oxide and graphene coated column were evaluated, and the application of GO coated column to chicken egg white was also investigated. 8881



Article

Figure 2. FTIR spectrum of GO (A) and TEM image of GO (B).

acid solutions. The mobile phase was obtained by mixing the phosphate solution with the appropriate amount of water and MeOH. Chicken eggs were obtained from the local supermarket. The egg white and egg yolk were separated. The egg white was diluted with an 8-time volume of phosphate buffer solution (20 mM, pH 7.6). Prior to carrying out the chromatographic separation, all solutions were filtered through a 0.22 μm membrane filter (Schleicher & Schuell, Dassel, Germany) and degassed in an ultrasonic bath for 15 min before use. Characterization. The general morphology of the graphene oxide on the surface wall was characterized by field emission scanning electron microscopy (FESEM, Hitachi S-4800 II, 15 kV). The surface content of the graphene oxide on the capillary wall was characterized by energy dispersive X-ray spectrometry (EDS, Hitachi S-4800 II, 20 kV). The microstructure of graphene oxide was characterized by transmission electron microscopy (TEM, Philips Tecnai-12, 120 kV). Fourier transform infrared (FTIR) spectra of GO were recorded with a Varian 610-IR (Varian) using the KBr pellet method. The UV−vis analysis was performed by a UV spectrometer (Shimadzu UV-2550).

at 100 W for 10 min to obtain a clear dispersion of GO. This GO dispersion could keep stable over four months. Preparation of Capillary Columns Coated with GO and G. Figure 1 presents the strategy employed to prepare capillary columns coated with GO and G. There are three main steps in these fabrication procedures. For the fabrication of GO-coated capillary column (GO@column): (a) A bare fused silica capillary was flushed with 1 M NaOH for 3 h, water for 30 min, 1 M HCl for 30 min, water for 1 h, and acetone for 30 min, respectively. (b) After drying with nitrogen, the capillary was flushed with 200 μL of 3-AMDS toluene solution (1 vol %) to modify the inner surface of the capillary column through a covalent interaction between hydroxy groups and 3-AMDS. The capillary was kept at room temperature (25 °C) for 30 min and then flushed with nitrogen for 10 min. (c) To produce a thin layer of GO, the capillary was subsequently flowed with 100 μL of 0.2 wt % aqueous GO, after which the capillary was sealed at both ends and heated at 70 °C for 2 h. The heated capillary was purged with nitrogen and then washed with water for 30 min. Thus prepared GO coated capillary column was denoted as “one layer of GO”. Operations of (b) and (c) were allowed to repeat for one time to increase the surface coverage. Thus prepared GO coated capillary column was denoted as “two layers of GO”. For the fabrication of G-coated capillary column (G@ column): The GO-coated column prepared as described above was conditioned in the GC injector under nitrogen at 60 °C for 2 h to strengthen the GO coating. After treatment with heat, a mixture of 5.0 mL of water, 20.0 μL of hydrazine (35 wt % in water) and 35.0 μL of ammonia solution (28 wt % in water) was injected into the GO-coated column.52 The capillary was then sealed at both ends and heated at 70 °C for 18 h to obtain the G@column. Chromatographic Separation. A Beckman MDQ P/ACE system (Beckman, Fullerton, CA) with an on-column DAD detector was used for all OT-CEC and OT-CLC experiments. Stock solutions of 1.0 mg mL−1 of each sample were prepared in methanol. Standard working solution was prepared by diluting the standard stock solution with MeOH−H2O (50/50, v/v). A stock background electrolyte was prepared by dissolving an exact amount of phosphate in water. The pH value of the phosphate solution was adjusted to the range of 3.0−9.0 by the addition of 0.1 M NaOH or 1.0 M phosphoric

■

RESULTS AND DISCUSSION Capillary Column Coated with GO. As shown in Figure 2A, the bands centered at 3370 and 1366 cm−1 were attributed to the O−H stretching and O−H vibration of the C−OH groups, respectively. The band centered at 1618 cm−1 corresponded to the O−H bending of C−OH. The CO stretching of the −COOH was observed as band at 1721 cm−1. The band centered at 1065 cm−1 was associated with the stretching of the C−O bond.53 The presence of these vibrations confirmed that GO nanosheets have been successfully synthesized. The TEM image of GO suspension in water shows that the as-produced GO nanosheets are entangled with each other, showing a disordered and wrinkled paper-like structure (Figure 2B).24 The typical thin wrinkled structure represents a curled and corrugated morphology of GO which is ascribed to the disruption of the planar sp2 carbon sheets by the introduction of sp3-hybridized carbon upon oxidation.54 Figure 3A shows the SEM image of the inner surface of bare fused silica capillary column. It can be seen that the inner wall surface of the bare fused silica capillary treated with 1 M NaOH 8882



Article

Figure 3. Typical SEM images of bare fused silica capillary wall treated with 1 M NaOH for 3 h (A), capillary wall coated with one layer of GO (B), and capillary wall coated with two layers of GO (C). (a) The cross section of the cut capillary columns. (b) The enlarged coated inner surface of the capillary columns.

quartz are shown in Figure 4. It can be seen that no characteristic absorptions for the quartz and only some weak adsorptions for the quartz modified with 3-AMDS are observed within the range from 200 to 400 nm (Figure 4A,B). However, the GO coated quartz exhibit a characteristic strong absorption with a maximum wavelength at 242 nm (Figure 4C,D). UV−vis spectra of the GO@column indicate UV absorption increased as the number of layers of GO coating increased (Figure 4C,D). The increase of spectra intensity further confirms a successful growth of GO coating. The surface composition of elements in GO@column, except for hydrogen, was measured using EDS, and the approximate atom and element percentage analysis results are given in Table 1. Compared with the capillary only modified with 3-AMDS, the content of carbon was obviously enhanced for GO@ column, indicating the successful immobilization of GO

for 3 h is smooth and only a little small holes can be found existed on the capillary wall. After coating, the inner wall surface became visibly roughened (Figure 3B,C). This is a clear indication of the immobilization of GO onto the capillary wall. It also can be observed that the roughness of capillary surface in Figure 3C is significantly higher than that in Figure 3B, indicating increased surface coverage of GO. The assembly of GO coatings onto the capillary wall was further monitored by UV−vis absorption spectroscopy. Since it is not convenient to obtain UV−vis spectra of GO on the capillary wall, in this experiment, fused silica capillary columns were replaced with quartz slides whose character is the same as fused silica capillary column. Except for that, the modification processes on the quartz were the same as those used for coating capillary wall. The absorption spectra of quartz, 3-AMDS modified quartz, one layer of GO coated quartz, and two layers of GO coated 8883



Article

above, at most pH values, the EOF mobilities for the columns coated with GO are slower than those of bare column and G@ column possesses the slowest EOF mobility because of the reduced negative charges on the capillary wall (Figure 5B). From the Figure 5B, it can be found that the EOF mobilities of all the columns are stable above pH 7. When the pH value is below 7, the EOF mobilities for both bare column and G@ column decreased when the pH value decreased from 7 to 3 while it decreased only slightly for GO@column. Since the pKa of phenolic hydroxyl groups was determined to be about 8,55 the magnitude of the EOF mobility on G@column and GO@ column would mainly depend on the number of ionized carboxy groups on the capillary wall when the pH value is smaller than 7. However, it is well-known that the dispersion of G is poor because of lacking of hydrophilic groups such as hydroxy groups and carboxy groups.25,52,56,57 Therefore, for G@column, the number of ionized carboxy groups would decrease rapidly with decreasing pH value from 7 to 3. As a result, the EOF mobility of G@column decreased quickly with decreasing pH value. For GO@column, the number of carboxy groups on GO@column is larger than that on G@column. Thus, the EOF mobility on GO@column decreased slightly when the pH value decreased from 7 to 3. The similar experimental results were obtained by Li et al.52 They found that the negative charge density of G decreased rapidly when the pH value is less than 7 while the negative charge density of GO decreased quickly only when the pH value is less than 4.5. It should be noted that in our experiments, G and GO nanosheets were covalently attached onto the capillary wall. Therefore, the agglomerate of G and GO nanosheets caused by the reduced electrostatic repulsion would not occur when the pH value of the buffer was gradually reduced.58 Thus, more ionized groups were exposed to the solution at low pH value. As a result, the effect of the pH value on the EOF mobility of G and GO@column, especially the latter, was not as significant as that reported by Li et al.52 The nearly constant EOF mobility on GO@column confirmed that the coverage of the GO on the capillary wall was high. Otherwise, the EOF mobility on GO@ column would decrease a lot as that of G@column. OT-CEC Separation on GO@column and G@column. Figure 6 shows the electrochromatograms for the separation of three neutral analytes using GO and G coated columns. The resolutions, number of theoretical plates, retention factors, and asymmetry factors of sample components were compared between the GO@column and G@column by using different content of MeOH in the mobile phase (Table 2). It can be seen that the separation performance of GO@column was better than that of G@column. Three neutral analytes can be baseline separated (Rs = 1.58) at 30% MeOH on GO@column. The theoretical plate number is 51104 for naphthalene and 17754 for biphenyl at 30% MeOH, respectively. In contrast, when G@ column was used for separation, the peaks of naphthalene and biphenyl were always overlapped although different percentages of MeOH were used. In addition, G@column gave the more asymmetric broad peaks for polyaromatic hydrocarbons

Figure 4. UV−vis spectra of bare quartz (A), quartz modified with 3AMDS (B), quartz coated with one layer of GO (C), and quartz coated with two layers of GO (D).

nanosheets on the surface of capillary wall modified with amino groups. Furthermore, consistent with the results obtained from the SEM and UV−vis, the C content in GO@column increased from 26.31% (wt %) for the column coated with one layer of GO (Figure 3B) to 33.48% (wt %) for the column coated with two layers of GO (Figure 3C). These results demonstrate again that more GO nanosheets could be bonded onto the capillary wall through the layer-by-layer strategy. Effect of Organic Modifier and pH on the EOF. In OTCEC, the transport of mobile phase through the capillary is achieved by the electroosmotic flow (EOF). The EOF, μeo, is defined as LL μeo = d t Vto Where Ld is the distance from injector to detector, Lt is the total capillary length, t0 is the migration time of the EOF marker, and V is the applied voltage. In this study, thiourea was used as EOF marker, which was expected to be unretained by the stationary phase coated onto the capillary wall. Knowing the characteristics of the μeo will be helpful to understand the separation behavior and to select optimal separation conditions. Figure 5 shows the effect of MeOH percentage and buffer pH on the EOF mobility for various capillary columns. Since carboxy groups are only exist on the border of the GO, the negative charge density would decrease obviously when the capillary wall was covered with GO sheets. As can be seen from Figure 5A, and as was expected, the GO@column shows lower EOF mobility than bare capillary. After the GO was reduced into G, the negative charge density was decreased further. So the lowest EOF mobility was obtained on the G@column. The effect of the pH value on the EOF mobility for various columns was carried out by using the buffer with different pH value according to the descending order. Same as that already stated

Table 1. Element Analyses of Columns Modified with 3-AMDS and GO@column by EDS capillary wall coated with 3-AMDS

one layer of GO

two layers of GO

element

C

Si

O

C

Si

O

C

Si

O

atom (%) element (wt %)

30.98 18.96

40.40 57.68

28.62 23.36

39.00 26.31

27.81 43.87

33.19 29.82

46.12 33.48

19.73 33.50

34.15 33.02

8884



Article

Figure 5. Effect of MeOH percentage and buffer pH on the EOF mobility for various columns. (A) MeOH−5 mM Na2HPO4 (pH 7.0). (B) MeOH−5 mM Na2HPO4 (50:50). Other experimental conditions: capillary column, 60 cm (50 cm effective length) × 75 μm i.d.; temperature, 25 °C; detection, 254 nm; separation voltage, 20 kV; injection, 0.5 psi ×3 s. Thiourea was used as EOF marker.

Figure 6. Effect of MeOH percentage on the resolution of solutes in OT-CEC on the capillary column coated with one layer of GO and G. Experimental conditions: electrolyte, MeOH-5 mM phosphate buffer, pH 7.0. All other separation conditions were identical to Figure 5. Peak identities: 1, Thiourea; 2, naphthalene; 3, biphenyl.

Table 2. Comparison of OT-CEC Performance on the GO@column and G@columnc N (plates/m) a

column

MeOH% (% v/v)

Rs

GO@ column

30 40 50 50 60 70

1.58 0.84 -

G@ Column

As b

k

naphthalene

biphenyl

naphthalene

biphenyl

naphthalene

biphenyl

51104 110301 86982 173 3260 14081

17754 61481 -

0.06 0.03 0.02 0.36 0.10 0.04

0.12 0.05 0.02 -

5.00 32.0 30.6 23.8

5.82 -

a

Resolution between naphthalene and biphenyl. bAs, asymmetry factor; As = W0.05/ (2d). W0.05, width of the peak at 1/20 of the peak height. d, distance between the perpendicular from the peak maximum and the leading edge of the peak at 1/20 of the peak height. cConditions are the same as those in Figure 5.

(PAHs), as shown in Figure 6. The As for naphthalene is as high as 23.8 even though 70% MeOH was used while the As for naphthalene on GO@column at 30% MeOH is only 5.00. These experimental results can be attributed to the fact that G which contains much less polar moieties, such as hydroxy, expoxy, and carboxy groups, has a more nonpolar and hydrophobic character than GO.57 The strong π−π stacking

interaction and the hydrophobic effect between PAHs and the highly delocalized conjugate system of the π-electron on the graphene surface makes G having a greater affinity for PAHs than GO.8,19−21 As a result, severe peak tailing was appeared when G@column was used. However, although GO provided more ionized groups than G, the peak tailing on GO@column is still very serious (Table 2). It indicates that the hydro8885



Article

phobicity and π−π electrostatic stacking property of GO also gives a strong affinity to carbon-based ring structure molecules.24 Therefore, when capillary column modified with two layers of GO was used for separation, severe peak tailing same as that occurred on G@column were obtained (Supporting Information, Figure S1). In order to achieve high separation performance, capillary column modified with only one layer of GO was used for the further study. Plots of the logarithm of the retention factor (log k) of naphthalene and biphenyl versus concentration of methanol under OT-CEC and OT-CLC mode were shown in Figure 7. It

The GO@column was also applied for the separation of basic and acidic analytes. As shown in Figure 8A, three basic analytes could be separated on GO@column, but they were not baseline separated. It is well-known that the electrostatic interactions between positively charged basic compounds and negatively charged silica based stationary phase would lead to the decreased separation performance. However, at pH 7, aniline, pyridine, and N,N-dimethylaniline are all neutral since the pKa’s of their conjugate acids are 4.63, 5.19, and 5.15, respectively. Therefore, the poor separation performance of GO@column would not caused by the electrostatic interactions between these basic compounds and GO, although GO contains a lot of negatively charged functional groups such as hydroxy and carboxy groups. However, the large number of carboxy groups on the border of GO could form strong hydrogen bonding interactions between basic analytes and negatively charged GO and this is the most possible reason for the poor separation performance of GO@column for the basic compounds. To further demonstrate the applicability of GO@column, three polar compounds were separated using OT-CEC mode. From the Figure 8B, it can be found that the three acidic compounds, catechol (pKa = 9.5), phenol (pKa = 10), and 1-naphthol (pKa = 9.3) were separated with no obvious peak tailing. Loading Capacity. The effect of sample loading on separation was studied by injecting different sample concentrations of naphthalene, ranging from 0.05 to 1.0 mg mL−1 while the injected sample volume was fixed (∼ 15 nL). The maximum loading capacity of GO@column is defined as the amount of sample injected when the corresponding peak width at half-height (w1/2) is increased by 10% over the peak width at low sample amounts. It can be seen from the Figure 9 that the w1/2 for naphthalene at 0.6 mg mL−1 increased by 10% over the peak width at 0.1 mg mL−1. So the loading capacity of GO@ column for naphthalene was 0.5 mg mL−1 (∼ 58.5 pmol). It is reported that the loading capacity of a capillary column (50 cm × 10-μm i.d.) coated with a film of silica was 1.5 pmol for naphthalene.32 Considering that the inner surface of the capillary column we used was 7.5 times larger than that of Guo et al., the effective loading capacity of GO@column was 5.2 times (58.5/(7.5 × 1.5) = 5.2) larger than that of the column modified with a film of silica. The higher loading capacity of

Figure 7. Relatationship between MeOH percentage and retention factors of aromatic compounds for GO@column operated under OTCEC and OT-CLC mode, respectively. Conditions of the separation under OT-CEC mode are the same as in Figure 5. Conditions of the separation under OT-CLC mode are the same in Figure 8 except different amounts of MeOH were used.

can be seen that the linear correlation coefficients calculated from all the four plots were very high (R2 > 0.99), suggesting that the chromatographic retention mechanism of the GO@ column is basically a reversed-phase behavior in both OT-CEC and OT-CLC separation mode.

Figure 8. Electrochromatogram of the separation of three basic compounds (A) and acidic compounds (B). Experimental conditions: (a) electrolyte, MeOH−5 mM phosphate buffer (20:80), pH 7.0; separation voltage, 20 kV. (b) electrolye, MeOH−5 mM phosphate buffer (40:60), pH 7.0; separation voltage, 15 kV. Other conditions are the same as in Figure 5. 8886



Article

separation of four PAHs in OT-CLC and OT-CEC mode, respectively. It can be seen that four PAHs can be well separated in both OT-CLC and OT-CEC mode. Since the phase ratio in open-tubular column is very low, open-tubular column is rarely used in LC mode. The successful separation of four PAHs in OT-CLC mode confirmed again that the coverage of GO on the capillary wall was high which was consistent with the results obtained from the EDS. Meanwhile, the strong interaction between GO and analytes might also contribute a lot to the successful separation of PAHs in OTCLC mode. Table 3 shows the comparison of the retention time, number of theoretical plates, and resolution of four PAHs in OT-CLC and OT-CEC mode. It can be seen from Table 3 that the plates and resolutions of the compounds obtained in OT-CEC were higher than those obtained in OT-CLC, except the acenaphthene whose plates obtained in OT-CEC were slightly less than those of in OT-CLC. The possible reason for the enhanced separation performance in OT-CEC can be attributed to the “plug-like” flow profile of EOF that the band broadening was significantly reduced during the chromatographic process. Separation of Proteins. A real biological sample of chicken egg white was selected to demonstrate the applicability of the GO@column to complex samples using OT-CEC separation mode. The component of chicken egg white is dominated by ovalbumin (pI 4.5), ovotransferrin (pI 6.0), ovomucoid (pI 4.1), ovoflavoprotein (pI 4.0), lysozyme (pI 10.7), and avidin (pI 10.0), which all have very different molecular weights (14 300−77 700 Da).59 At pH values greater than pI, analytes carry the negative charge, while at pH values less than pI, analytes carry the positive charge. In our experiments, pH values were changed from 7 to 9. In this pH range, ovalbumin, ovotransferrin, ovomucoid, and ovoflavoprotein are negatively charged while lysozyme and avidin are positively charged. As shown in Figure 11, both basic proteins and acidic proteins in egg white can be separated in a single run by using different pH values. The peaks were identified by the standard addition method. The successful elution of basic proteins indicates that the electrostatic interaction is greatly suppressed by the high-surface coverage of the GO on the capillary wall. Otherwise, they would be adsorbed onto the capillary wall and could not be eluted out. At least seven peaks were detected at pH 7 and three glycoisoforms of ovalbumin were observed. Hydrophobic interactions, such as π−π interactions, derived from the π-electron moiety of the GO nanosheets with the hydrophobic residues of the protein species might help a lot for the separation. It suggests that this column is a highly promising device for the separation of glycoprotein isoforms.60 When the pH was increased above 7, only two isoform peaks were obtained. In addition, the resolution was found decreased with the increased pH value.

Figure 9. Loading capacity test of a 50 cm (injection to detection) × 75-μm i.d. GO@column using naphthalene. Experimental conditions: electrolyte, MeOH−5 mM phosphate buffer, pH 7.0; separation voltage, 30 kV. All other separation conditions were identical to Figure 5.

GO@column can be attributed to the high surface area of GO and high surface coverage of GO on the capillary wall. OT-CLC Separation on GO@column. The separation performance of GO@column in OT-CLC mode was also evaluated. Figure 10 shows the chromatograms of the

Figure 10. Separation of the PAHs compounds in OT-CLC with 0.5 psi inlet pressure and OT-CEC with 25 kV. Experimental conditions: mobile phase, MeOH 30% for OT-CLC; MeOH−5 mM phosphate buffer (30:70), pH 7.0 for OT-CEC. Other conditions are the same as in Figure 5. Peak identities: 1, toluene; 2, naphthalene; 3, 2methylnaphthalene; 4, acenaphthene.

Table 3. Comparison of Retention Time, Column Efficiency, and Resolution of Test Compounds in OT-CLC and OT-CEC Modes toluene tra N Rs b

naphthalene

2-methylnaphthalene

acenaphthene

OT-CLC

OT-CEC

OT-CLC

OT-CEC

OT-CLC

OT-CEC

OT-CLC

OT-CEC

15.62 32226 -

11.84 79649 -

16.56 16642 1.52

12.54 68603 2.59

17.79 11264 1.50

13.64 12164 1.98

19.02 8129 1.00

15.04 6943 1.28

Retention time (min). bResolution, Rs = [tr2 − tr1]/[0.5 × (W1 + W2)], where tr1 and tr2 are the retention time of each analytes, W1 and W2 are the peak widths of each analytes at the baseline.

a

8887



Article

Figure 11. Separation of chicken egg white in a single run on GO@column. Experimental conditions: electrolytes, 5 mM phosphate buffer; applied voltage, 20 kV; detection 214 nm; pressure injection, 0.5 psi for 3 s; temperature, 25 °C. Analytes peaks: 1, avidin; 2, lysozyme; 3, ovotransferrin; 4, ovalbumin (a, b, and c: glycoisoforms of OVA); 5, ovomucoid; 6, ovoflavoprotein.

Table 4. Reproducibilities of GO@column (n = 5)a

These results may be attributed to the increased migration rate of the samples which makes the interaction between the GO and analytes decreased. However, main peaks were still obtained in a shorter migration time but with lower resolution values. Stability of GO@column. The reproducibility of a graphene coating plays an important role in the determination of column performance. The reproducibilities were evaluated on the basis of the relative standard deviation (RSD) of EOF under OT-CEC mode and retention time for naphthalene under OT-CLC mode obtained from five replicate analyses. The run-to-run, day-to-day, and column-to-column RSDs shown in Table 4 were all below 3%, which indicated the columns had a good stability. The GO@column endured more than 300 runs under OT-CEC mode and at least 80 runs under OT-CLC mode with 1.0 psi inlet pressure. The long lifetime can be attributed to the chemically bonding of GO onto the inner surface of capillary wall. Furthermore, the long lifetime of GO@column under OT-CLC mode demonstrated again that the GO was immobilized onto the capillary wall via covalent bonding. Coatings formed through physical adsorption could not last for such a long time in pressure driven flow.

OT-CEC run-to-run day-to-day column-to-column

OT-CLC

EOF (min)

RSD (%)

tr (min)b

RSD (%)

13.20 13.07 13.98

0.63 1.25 2.37

16.55 16.42 16.86

0.21 0.37 1.72

a Experimental conditions of OT-CEC: electrolyte, MeOH−5 mM phosphate buffer (50/50, v/v), pH 7.0; separation voltage, 20 kV. Experimental conditions of OT-CLC: inlet pressure, 0.5 psi; mobile phase, MeOH/H2O = 3:7 (v/v). Other conditions are the same as in Figure 3. bRetention time of naphthalene.

■

CONCLUSIONS Capillary columns with a stable graphene and graphene oxide coating were fabricated by a simple procedure with good reproducibility. Effective separation of neutral, basic, and proteins were obtained on the GO@column. In addition, the high coverage of GO on the capillary surface makes the GO@ column also suitable for open-tubular capillary chromatography separation. The G@column, however, exhibited poor separation performance because of the high affinity of PAHs to the hydrophobic groups on the graphene surfaces. This specific retention in G@column is helpful more to achieve unique adsorption behavior while the relative weak interaction 8888



Article

(18) Zhou, X. S.; Yin, Y. X.; Wan, L. J.; Guo, Y. G. Chem. Commun. 2012, 48, 2198−2200. (19) Allen, M. J.; Tung, V. C.; Kaner, R. B. Chem. Rev. 2010, 110, 132−145. (20) Chen, J. M.; Zou, J.; Zeng, J. B.; Song, X. H.; Ji, J. J.; Wang, Y. R.; Ha, J.; Chen, X. Anal. Chim. Acta 2010, 678, 44−49. (21) Zhang, S. L.; Du, Z.; Li, G. K. Anal. Chem. 2011, 83, 7531− 7541. (22) Tang, L.; Wang, J. Z.; Loh, K. P. J. Am. Chem. Soc. 2010, 132, 10976−10977. (23) Gulbakan, B.; Yasun, E.; Shukoor, M. I.; Zhu, Z.; You, M. X.; Tan, X. H.; Sanchez, H. N.; Powell, D. H.; Dai, H. J.; Tan, W. H. J. Am. Chem. Soc. 2010, 132, 17408−17410. (24) Liu, Q.; Shi, J. B.; Sun, J. T.; Wang, T.; Zeng, L. X.; Jiang, G. B. Angew. Chem., Int. Ed. 2011, 50, 5913−5917. (25) Zhao, G. X.; Jiang, L.; He, Y. D.; Li, J. X.; Dong, H. L.; Wang, X. K.; Hu, W. P. Adv. Mater. 2011, 23, 3959−3963. (26) Liu, J. W.; Zhang, Q.; Chen, X. W.; Wang, J. H. Chem.Eur. J. 2011, 17, 4864−4870. (27) Liu, Q.; Shi, J. B.; Cheng, M. T.; Li, G. L.; Cao, D.; Jiang, G. B. Chem. Commun. 2012, 48, 1874−1876. (28) Knox, J. H.; Kaur, B. J. Chromatogr. 1986, 352, 3−25. (29) Valcárcel, M.; Cárdenas, S.; Simonet, B. M. Anal. Chem. 2007, 79, 4788−4797. (30) Chambers, S. D.; Holcombe, T. W.; Svec, F.; Fréchet, J. M.J. Anal. Chem. 2011, 83, 9478−9484. (31) Wang, M. M.; Yan, X. P. Anal. Chem. 2012, 84, 39−44. (32) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511−2516. (33) Hayes, J. D.; Malik, A. Anal. Chem. 2001, 73, 987−996. (34) Matyska, M. T.; Pesek, J. J.; Katrekar, A. Anal. Chem. 1999, 71, 5508−5514. (35) Huang, X.; Zhang, J.; Horváth, C. J. Chromatogr., A 1999, 858, 91−101. (36) Saridara, C.; Mitra, S. Anal. Chem. 2005, 77, 7094−7097. (37) Kubáň, P.; Dasgupta, P. K.; Pohl, C. A. Anal. Chem. 2007, 79, 5462−5467. (38) Münch, A. S.; Seidel, J.; Obst, A.; Weber, E.; Mertens, F. O. R. L. Chem.Eur. J. 2011, 17, 10958−10964. (39) Boyce, M.; Breadmore, M. C.; Macka, M.; Doble, P.; Haddad, P. R. Electrophoresis 2000, 21, 3073−3080. (40) O’Mahony, T.; Owens, V. P.; Murrihy, J. P.; Guihen, E.; Holmes, J. D.; Glennon, J. D. J. Chromatogr., A 2003, 1004, 181−193. (41) Yang, L.; Guihen, E.; Holmes, J. D.; Loughran, M.; O’Sullivan, G. P.; Glennon, J. D. Anal. Chem. 2005, 77, 1840−1846. (42) Liu, F. K.; Hsu, Y. T.; Wu, C. H. J. Chromatogr., A 2005, 1083, 205−214. (43) Hsieh, Y. L.; Chen, T. H.; Liu, C. P.; Liu, C. Y. Electrophoresis 2005, 26, 4089−4097. (44) Dong, X. L.; Wu, R. A.; Dong, J.; Wu, M. H.; Zhu, Y.; Zou, H. F. Electrophoresis 2008, 29, 3933−3940. (45) Qu, Q. S.; Zhang, X. X.; Shen, M.; Liu, Y.; Hu, X. Y.; Yang, G. J.; Wang, C. Y.; Zhang, Y. K.; Yan, C. Electrophoresis 2008, 29, 901−909. (46) Qu, Q. S.; Liu, D. P.; Mangelings, D.; Yang, C.; Hu, X. Y. J. Chromatogr., A 2010, 1217, 6588−6594. (47) Chen, J. L.; Lin, Y. C. Electrophoresis 2010, 31, 3949−958. (48) Hamer, M.; Yone, A.; Rezzano, I. Electrophoresis 2012, 33, 334− 339. (49) Ma, T. Y.; Li, H.; Tang, A. N.; Yuan, Z. Y. Small 2011, 7, 1827− 1837. (50) Song, S. Y.; Chu, R. Q.; Zhou, J. F.; Yang, S. R.; Zhang, J. Y. J. Phys. Chem. C 2008, 112, 3805−3810. (51) Hummers, W. S.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339−1339. (52) Li, D.; Müller, M. B.; Gilje, S.; Kaner, R. B.; Wallace, G. G. Nat. Nanotechnol. 2008, 3, 101−105. (53) Stankovich, S.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Carbon 2006, 44, 3342−3347. (54) Meyer, J. C.; Geim, A. K.; Katsnelson, M. I.; Novoselov, K. S.; Booth, T. J.; Roth, S. Nature 2007, 446, 60−63.

occurring between GO and analytes is benefit for the normal compounds as well as proteins.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Address: College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China. E-mail: [email protected]. Fax: +86-514-87975244. Tel: +86-51487975590. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by NSFC (20975090), Jiangsu Government-sponsored Scholarship Project, the foundation of the Excellence Science and Technology Invention Team in Yangzhou University, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

■

REFERENCES

(1) Novoselov, K. S.; Geim, A. K.; Morozov, S. V.; Jiang, D.; Zhang, Y.; Dubonos, S. V.; Grigorieva, I. V.; Firsov, A. A. Science 2004, 306, 666−669. (2) Geim, A. K. Science 2009, 324, 1530−1534. (3) Lee, C.; Wei, X.; Kysar, J. W.; Hone, J. Science 2008, 321, 385− 388. (4) Bunch, J. S.; van der Zande, A. M.; Verbridge, S. S.; Frank, I. W.; Tanenbaum, D. M.; Parpia, J. M.; Graighead, H. G.; McEuen, P. L. Science 2007, 315, 490−493. (5) Stoller, M. D.; Park, S.; Zhu, Y.; An, J.; Ruoff, R. S. Nano Lett. 2008, 8, 3498−3502. (6) Chen, H. Q.; Muller, M. B.; Gilmore, K. J.; Wallace, G. G.; Li, D. Adv. Mater. 2008, 20, 3557−3561. (7) Zhu, Y. W.; Murali, S.; Cai, W. W.; Li, X. S.; Suk, J. W.; Potts, J. R.; Ruoff, R. S. Adv. Mater. 2010, 22, 3906−3924. (8) Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. Chem. Soc. Rev. 2010, 39, 228−240. (9) Mkhoyan, K. A.; Contryman, A. W.; Silcox, J.; Stewart, D. A.; Eda, G.; Mattevi, C.; Miller, S.; Chhowalla, M. Nano Lett. 2009, 9, 1058−1063. (10) Paredes, J. I.; Villar-Rodil, S.; Martínez-Alonso, A.; Tascón, J. M. D. Langmuir 2008, 24, 10560−10564. (11) Hontoria-Lucas, C.; Lopez-Peinado, A. J.; Lopez-Gonzalez, J. D. D.; Rojas-Cervantes, M. L.; Martin-Aranda, R. M. Carbon 1995, 33, 1585−1592. (12) Gilje, S.; Han, S.; Wang, M.; Wang, K. L.; Kaner, R. B. Nano Lett. 2007, 7, 3394−3398. (13) Li, X.; Wang, X.; Zhang, L.; Lee, S.; Dai, H. Science 2008, 319, 1229−1232. (14) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (15) Yang, W.; Ratinac, K. R.; Ringer, S. P.; Thordarson, P.; Gooding, J. J.; Braet, F. Angew. Chem., Int. Ed. 2010, 49, 2114−2138. (16) Schedin, F.; Geim, A. K.; Morozov, S. V.; Hill, E. W.; Blake, P.; Katsnelson, M. I.; Novoselov, K. S. Nat. Mater. 2007, 6, 652−655. (17) Ramanathan, T.; Abdala, A. A.; Stankovich, S.; Dikin, D. A.; Herrera-Alonso, M.; Piner, R. D.; Adamson, D. H.; Schniepp, H. C.; Chen, X.; Ruoff, R. S.; Nguyen, S. T.; Aksay, I. A.; Prud’Homme, R. K.; Brinson, L. C. Nat. Nanotechnol. 2008, 3, 327−331. 8889



Article

(55) Konkena, B.; Vasudevan, S. J. Phys. Chem. Lett. 2012, 3, 867− 872. (56) Zhang, S.; Shao, Y. Y.; Liao, H. G.; Engelhard, M. H.; Yin, G. P.; Lin, Y. H. ACS Nano 2011, 5, 1785−1791. (57) Compton, O. C.; Nguyen, S. T. Small 2010, 6, 711−723. (58) Wang, X. L.; Bai, H.; Shi, G. Q. J. Am. Chem. Soc. 2011, 133, 6338−6342. (59) Awadé, A. C.; Efstathiou, T. J. Chromatogr., B 1999, 723, 69−74. (60) Legaz, M. E.; Pedrosa, M. M. J. Chromatogr., A 1996, 719, 159− 170.

8890


Capillary Coated with Graphene and Graphene Oxide Sheets as

Recommend Documents