DOI: 10.1002/chem.201302746
A Sulfonic-Azobenzene-Grafted Silica Amphiphilic Material: A Versatile Stationary Phase for Mixed-Mode Chromatography Hongdeng Qiu,*[a] Mingliang Zhang,[a] Tongnian Gu,[a] Makoto Takafuji,[b] and Hirotaka Ihara*[b] Abstract: A novel sulfonic-azobenzene-functionalized amphiphilic silica material was synthesized through the preparation of a new sulfonic azobenzene monomer and its grafting on mercaptopropyl-modified silica by a surface-initiated radical chain-transfer reaction. The synthesis was confirmed by infrared spectra, elemental analysis, and thermogravimetric analysis. This new material was successfully applied as a new kind of mixed-mode stationary phase in liquid chromatography. This allows an exceptionally flexible adjustment of retention and selectivity by tuning the experimental conditions.
The distinct separation mechanisms were outlined by selected examples of chromatographic separations in the different modes. In reversed-phase liquid chromatography, this new stationary phase presented specific chromatographic performance when evaluated using a Tanaka test mixture. Seven dinitro aromatic isomers, four steroids, and seven flavonoids were separated successfully in simple reversed-phase Keywords: amphiphiles · analytical methods · azo compounds · liquid chromatography · silica
Introduction With the development of chromatographic science, mixedmode chromatography (MMC) has emerged as a new type of chromatography that can behave as more than one interaction mode with solutes.[1] The main advantage of MMC over conventional single-mode chromatography is that two or more completely different kinds of analyte can be determined using the same column with different chromatographic modes. The advancement of liquid chromatography has mostly relied on the development of new stationary phases.[2] Therefore, more and more new mixed-mode or multimode stationary phases have been found recently.[3] For example, Wijekoon et al. prepared a zwitterionic sta[a] Prof. H. Qiu, M. Zhang, T. Gu Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province Lanzhou Institute of Chemical Physics, CAS Lanzhou, 730000 (P.R. China) Fax: + 86-931-8277088 E-mail: [email protected] [b] Assoc. Prof. M. Takafuji, Prof. H. Ihara Department of Applied Chemistry and Biochemistry Kumamoto University 2-39-1 Kurokami, Kumamoto 860-8555 (Japan) Fax: + 81-96-3423662 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201302746.
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mode. This stationary phase can also be used in hydrophilic interaction chromatography because of the existing polar functional groups; for this, nucleosides and their bases were used as a test mixture. Interestingly, the same nucleosides and bases can also be separated in per aqueous liquid chromatography using the same stationary phase. Three ginsenosides including Rg1, Re, and Rb1 were successfully separated in hydrophilic mode. There is the potential for more applications to benefit from this useful column.
tionary phase based on 4-propylaminomethyl benzoic acid for multimode liquid chromatography.[3i] Liu et al. prepared a mixed-mode stationary phase based on click amino-modified silica for ion chromatography (IC) and hydrophilic interaction liquid chromatography (HILIC).[3j] In other examples, ionic liquids were immobilized on silica through covalent bonding or a polymerized grafting method and used as a new kind of stationary phase for different chromatographic modes.[4] Methyl orange (MO) was employed as a counter anion of the poly(ionic liquid)-modified silica stationary phase, which plays a key role in enhancing the shape selectivity to polycyclic aromatic hydrocarbons (PAHs) because of the existence of rigid functionalized azobenzene. In that case, long-chain alkylimidazoliumfunctionalized silica particles were prepared first and then the counteranions were changed from bromide to MO by means of ionic self-assembly before column packing[5] or in situ anion exchange after column packing.[6] The cooperation effect of the azobenzene group of MO on the enhancement of the chromatographic selectivity is very clear. We were strongly stimulated to prepare a new stationary phase with the azobenzene group and mainly investigate the performance of the azobenzene structure. Therefore, a new sulfonic azobenzene monomer was synthesized and immobilized on the silica by using a facile method described in this work. This newly prepared stationary phase not only presents an enhanced selectivity to PAHs and steroids in reversed-phase liquid chromatography
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FULL PAPER (RPLC), but also can be used to separate nucleosides and bases in HILIC mode and by per aqueous liquid chromatography (PALC). Seven flavonoids and three ginsenosides were also effectively separated in RPLC and HILIC mode, respectively.
Results and Discussion Synthesis and characterization: The silica surface-modification procedures are simple and facile as outlined in Scheme 1. In brief, the synthesis includes the preparation of an azobenzene monomer (MA-AzO) and the grafting of the monomer on 3-mercaptopropyl-modified silica.
Scheme 1. Synthesis of the sulfonic-azobenzene-modified silica stationary phase.
Figure 1. Thermogravimetric curves of a) Sil-MPS and b) Sil-PAzO.
16.3 % over the same temperature range, which indicates that the organic content clearly increased. These mass losses are consistent with the immobilized amounts estimated by elemental analyses. Infrared spectroscopy is another useful tool that can be used to identify the chemical modifications of compounds. Some clear differences in wave numbers and in the intensities of the absorption bands were observed in the spectra of Sil-MPS and Sil-PAzO as shown in Figure 2. In the spectrum for the Sil-PAzO surface, clear characteristic signals at 1601 and 1504 cm1 are attributed to the stretching vibration of the phenyl bond of sulfonic azobenzene, which confirms the anchoring of the organic molecule onto the silica surface.[9]
The elemental content (%) of Sil-MPS and Sil-PAzO was C 4.42, H 1.27 and C 9.40, H 1.76, N 1.28, respectively. The degree of surface coverage for Sil-MPS and Sil-PAzO was calculated from the following equations [Eqs. (1) and (2)][7]: Sil-MPS ½mmol m2 ¼
%C ¼ 4:34 36 ð1 %C %HÞ S
Sil-PAzO ½mmol m2 ¼
ð1Þ
%N ¼ 0:87 56 ð1 %C %H %NÞ S ð2Þ Figure 2. Diffuse reflectance infrared Fourier transform (DRIFT) spectra of a) Sil-MPS and b) Sil-PAzO.
in which %C, %H, and %N represent the percentages of carbon, hydrogen, and nitrogen, respectively, and S is the surface area. The amount of mercaptopropyl and imidazolium moieties attached to the silica surface can thus be calculated as 4.34 mmol m2 for Sil-MPS and 0.87 mmol m2 for Sil-PAzO. Thermogravimetric curves are usually used to determine thermal stability and to confirm the amount of immobilized compounds on the silica surface. The weight loss observed between 200 and 600 8C can be associated with the loss of the organic groups attached to the silica surface.[8] As shown in Figure 1, Sil-MPS presented a mass loss of about 5.9 % from 200 to 600 8C. After covalently bonding with the azobenzene monomer, Sil-PAzO showed a mass loss of about
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RPLC mode: It is known that conventional octadecylsilane (ODS) or other alkylated organic stationary phases can recognize the hydrophobicity of solutes in HPLC, which is measured by the selectivity of the stationary phase for the methylene group. This reflects the possibility that the phase may be able to separate two molecules that differ only by a methylene group, for example, amylbenzene and butylbenzene. The retention mode as well as the extent of the hydrophobic interactions among the solutes and the packing materials in HPLC can be determined by retention studies using alkylbenzenes as solutes.[10] The relationship between the retention factor (log k) and the water/1-octanol partition coef-
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ficient (log Po/w) for Sil-PAzO, ODS, and polyACHTUNGRE(styrene)-grafted silica (Sil-Stn) columns are compared in Figure S1 in the Supporting Information. It was observed that Sil-PAzO showed a higher retention for PAHs than for alkylbenzenes. For instance, the log Po/w of naphthacene (5.71) is much smaller than that of decylbenzene (7.36), but the log k value of naphthacene (0.65) is clearly higher than that of decylbenzene (0.46). A similar selectivity to PAHs and alkylbenzenes was also found in Sil-Stn, but for ODS, log k versus log Po/w plots for alkylbenzenes and PAHs were almost on the same line. The Tanaka test mixture is usually used to effectively characterize the chromatographic performance of a new stationary phase.[11] As shown in Figure 3, a comparable separa-
dinitronaphthalene, 1,5-dinitronaphthalene, and 1,3-dinitronaphthalene were separated. The retention orders of these analytes on Sil-PAzO and ODS columns are a little different as shown in Figure S3 in the Supporting Information. Under the same chromatographic conditions, a mixture of four steroids including estriol, 17a-estradiol, 17b-estradiol, and estrone were successfully separated in Sil-PAzO within six minACHTUNGREutes as shown in Figure 4. The positions of the isomer pair 17a-estradiol and 17b-estradiol were baseline-separated within 5 min on Sil-PAzO, which can be a challenge for the ODS column.[12] As shown in Figure 4, the same mixture cannot be separated on the ODS column under the same chromatographic conditions and the retention order of the last three analytes was reversed.
Figure 3. Separation of the Tanaka test mixture: 1) uracil, 2) phenol, 3) caffeine, 4) butylbenzene, 5) o-terphenyl, 6) amylbenzene, and 7) triphenylene with a) ODS and b) Sil-PAzO columns. Mobile phase: a) methanol/water (85:15, v/v) and b) methanol/water (70:30, v/v). T = 30 8C; flow rate = 1 mL min1; detection: l = 254 nm.
tion of the Tanaka test mixture, which included uracil, phenol, caffeine, butylbenzene, o-terphenyl, amylbenzene, and triphenylene, was performed by the ODS and Sil-PAzO columns. Two different points can be observed clearly. The first one is that the retention of caffeine is even stronger than that of butylbenzene and amylbenzene in Sil-PAzO, which can be due to the existence of a sulfonic group in this new phase. The second one is that the retention order of oterphenyl and amylbenzene is different among the ODS and Sil-PAzO columns. That is because the aromatic azobenzene group can induce strong p–p interactions in the PAHs. It can also be proven that the planar selectivity was enhanced when comparing the selectivity of o-terphenyl and triphenylene on Sil-PAzO (separation factor (atriphenylene/o-terphenyl) = 2.44) and ODS (atriphenylene/o-terphenyl = 1.36) columns. The effect of the methanol content on the retention factors of the Tanaka test mixture is shown in Figure S2 in the Supporting Information. The retention of uracil, phenol, and caffeine was not influenced by the content of methanol, however, the retention of hydrophobic analytes was affected very much. Using methanol/water 65:35 (v/v) as the mobile phase, seven dinitro aromatic isomers including o-dinitrobenzene, p-dinitrobenzene, m-dinitrobenzene, 2,4-dinitrotoluene, 1,8-
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Figure 4. Separation of four steroids: 1) estriol, 2) 17a-estradiol, 3) 17bestradiol, and 4) estrone with a) Sil-PAzO and b) ODS columns with methanol/water (65:35, v/v). Other chromatographic conditions are the same as those in Figure 3.
This column can also be used to separate natural products such as flavonoids. Usually, separation of flavonoids in a RPLC system has relied on an ODS column.[13] The structures of the flavonoids used in this study are listed in Figure S4 in the Supporting Information. The effect of the pH of the buffer on the separation of the flavonoids was investigated as shown in Figure 5. When the pH value was decreased from 6.8 to 5, the retention of luteolin and naringe-
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Figure 5. The effect of pH on the separation of myricetin (~), naringenin (&), and luteolin (*). The mobile phases were mixed to 30 % acetonitrile and 70 % ammonium acetate (20 mmol L1) with different pH values adjusted by using acetic acid. Other chromatographic conditions are the same as those in Figure 3.
nin increased. If the pH decreased again, from 5 to 4, the retention of luteolin and naringenin decreased. However, the retention of myricetin was a little different, with the strongest retention time observed when the pH was 6. It is worth noting that the tailing phenomena are heavy when the pH values are above 5. The chromatographic peaks improve when the pH of the buffer is decreased. This should be attributed to the restrained ionization of sulfonic acid. The best separation of the seven flavonoids, including myricetin, naringenin, quercetin, luteolin, kaempferol, isorhamnetin, and baicalein, was obtained with 20 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (78:22, v/v) as shown in Figure 6b. The effects of acetonitrile and buffer concentration were investigated. On increasing the buffer concentration or the acetonitrile content, the retention of the flavonoids decreased as shown in Figure 6a and c, respectively. HILIC and PALC modes: This developed stationary phase was not only used in the RPLC mode for the separation of alkylbenzenes, PAHs, steroids, dinitrobenzenes, and dinitronaphthalenes, but was also used in the HILIC mode to separate bases, nucleosides, and ginsenosides. As a powerful separation technique, HILIC provides an alternative approach to effectively separate small polar compounds on a polar stationary phase.[14] The sulfonic and other polar groups of Sil-PAzO make it appropriate to be used as a polar stationary phase for application in HILIC mode. Separation of ethylbenzene, uracil, and cytosine on Sil-PAzO was obtained with 20 mmol L1 ammonium acetate/acetonitrile (10:90, v/v) as the mobile phase (Figure S5 in the Supporting Information). The relationships of the retention factors of ethylbenzene, uracil, and cytosine with different proportions of 20 mmol L1 ammonium acetate and acetonitrile as eluent were investigated as shown in Figure 7. The retention of ethylbenzene was not affected on increasing the amount of acetonitrile from 80 to 95 %, and that of uracil gets stronger little by little, however, the retention of cytosine drastically increased, especially when the content of acetonitrile was increased from 90 to 95 % in the mobile phase. It is a classical
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Figure 6. Separation of seven flavonoids: 1) myricetin, 2) naringenin, 3) quercetin, 4) luteolin, 5) kaempferol, 6) isorhamnetin, 7) baicalein with a) 100 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (78:22, v/v), b) 20 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (78:22, v/v) and c) 20 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (75:25, v/v) as mobile phases. RT; flow rate: 1 mL min1; detection: l = 360 nm.
Figure 7. Relationship of the retention factor of ethylbenzene (&), uracil (*), and cytosine (~) with different proportions of 20 mmol L1 ammonium acetate and acetonitrile as eluent. Other chromatographic conditions are the same as those in Figure 3.
example that demonstrates that a polar stationary phase can be used in HILIC mode. The HILIC applications of Sil-PAzO can be identified from the following separation of seven bases and nucleosides including uracil, theophylline, uridine, adenosine, adenine, cytosine, and guanosine. As shown in Figure 8, they were successfully separated within 10 min using 20 mmol L1 ammonium acetate/acetonitrile (10:90, v/v) as the mobile phase. On increasing the proportion of acetonitrile, the re-
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different on the two modes. This may provide an opportunity for wide application of this new phase for PALC. This column also can be used in other applications in HILIC mode. For example, the separation of three ginsenosides including Rg1, Re, and Rb1 as shown in Figure 10.
Figure 8. Separation of seven bases and nucleosides: 1) uracil, 2) theophylline, 3) uridine, 4) adenosine, 5) adenine, 6) cytosine, and 7) guanosine with different mobile phases. Mobile phases: a) 20 mmol L1 ammonium acetate/acetonitrile (10:90, v/v); b) 20 mmol L1 ammonium acetate/ acetonitrile (7.5:92.5, v/v); c) 20 mmol L1 ammonium acetate/acetonitrile (5:95, v/v). Other chromatographic conditions are the same as those in Figure 3.
tention factors and separation factors were increased correspondingly. By using 20 mmol L1 ammonium acetate/acetonitrile (7.5:92.5, v/v) as the mobile phase, 10 bases and nucleosides including 5-bromouracil, uracil, theophylline, uridine, purine, adenosine, adenine, cytosine, cytidine, and guanosine were separated very well as shown in Figure 9a.
Figure 10. Separation of three ginsenosides: 1) Rg1, 2) Re, and 3) Rb1 with a) acetonitrile/water (85:15, v/v) and b) acetonitrile/water (90:10, v/v) as mobile phases. RT; flow rate: 1 mL min1; detection: l = 203 nm.
These ginsenosides are difficult to separate on the ODS column, and a long time and gradient elution is usually necessary. However, these ginsenosides can be separated completely within ten minutes on the Sil-PAzO column with acetonitrile/water (85:15, v/v) as the mobile phase. With the increase of acetonitrile content, the retention of the ginsenosides increased correspondingly, which can be used to prove that these chromatographic separations arise as a result of the HILIC mode.
Conclusion
Figure 9. Separation of 10 bases and nucleosides: 1) 5-bromouracil, 2) uracil, 3) theophylline, 4) uridine, 5) purine, 6) adenosine, 7) adenine, 8) cytosine, 9) cytidine, and 10) guanosine with a) 20 mmol L1 ammonium acetate/acetonitrile (7.5:92.5, v/v) and b) 20 mmol L1 ammonium acetate as the mobile phase. Other chromatographic conditions are the same as those in Figure 3.
After HILIC separation, Sil-PAzO was also creatively used in PALC mode to separate the same 10 bases and nucleosides as described above. Whereas a large amount of acetonitrile is used in HILIC mode, only a small amount of organic solvent or no organic solvent can be used in PALC mode.[15] PALC mode should be encouraged to be used for economical reasons and for preventing pollution through the use of large amounts of organic solvent. As shown in Figure 9b, the 10 bases and nucleosides were surprisingly separated on Sil-PAzO with 20 mmol L1 ammonium acetate as the mobile phase. The retention orders of the samples are
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A new mixed-mode stationary phase based on sulfonic azobenzene grafted on silica was prepared and characterized. This stationary phase was evaluated by the separations of the Tanaka test mixture, dinitro aromatic isomers, PAHs, and steroids in RPLC. The same phase was also used to separate bases and nucleosides in HILIC and PALC modes. It is clear that Sil-PAzO presents different or a specific selectivity to the analytes compared with the commonly used ODS column. This new stationary phase is a mixed-mode stationary phase owing to the existence of multifunctionalized groups. It is foreseeable that this phase can also be used as a strong cation-exchange mode owing to the electrostatic interaction of the sulfonic groups. This new amphiphilic material holds promise for more applications, not only as a new versatile stationary phase, but also as a solid extraction adsorbent and in other chemical fields.
Experimental Section Materials and reagents: p-[(p-Aminophenyl)azo]benzenesulfonic acid was obtained from City Chemical LLC (West Haven, USA). 2-Isocyana-
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toethyl methacrylate was purchased from TCI America. Azobisisobutyronitrile (AIBN) was obtained from Nacalai tesque, Inc. (Kyoto, Japan) and purified by recrystallization from methanol before use. 3-Mercaptopropyltrimethoxysilane (MPS) was purchased from Azmax (Chiba, Japan). Porous silica particles as a support (diameter 5 mm, pore size 120 , specific surface area 300 m2 g1) were obtained from YMC (Kyoto, Japan). Standard reference material (SRM) 869b, column selectivity test mixture for liquid chromatography, and SRM 1647e, priority pollutant polycyclic aromatic hydrocarbons were obtained from the Standard Reference Materials Program (NIST, Gaithersburg, MD). Estriol, estrone, and 17b-estradiol were obtained from Sigma (St Louis, MO, USA). 17aEstradiol was purchased from Dr. Ehrenstorfer (Augsburg, Germany). 5Bromouracil, uracil, theophylline, uridine, purine, adenosine, adenine, cytosine, cytidine, and guanosine were purchased from Sigma–Aldrich, Inc. All dinitro aromatic isomers, alkylbenzenes, and PAHs were commercially available and used without any purification. Synthesis of Sil-PAzO: First, the polymerizable sulfonic azobenzene monomer was prepared as follows: p-[(p-Aminophenyl)azo]benzenesulfonic acid (9.0 g, 30.2 mmol) was added to the flask. Then water (180 mL) and methanol (180 mL) were both added to the flask to dissolve p-[(p-aminophenyl)azo]benzenesulfonic acid with ultrasonication. After the compound had completely dissolved, 6-fold amounts of 2-isocyanatoethyl methacrylate were added dropwise to the solution at 0 8C. The reaction mixture was stirred at room temperature overnight. The solvent was removed by rotary evaporation under reduced pressure. And then the obtained solid was washed with ethanol and dried under vacuum to give a solid yellow powder (8.5 g). C19H20N4O6S: 1H NMR (D2O, 400 MHz): d = 7.75–7.77 (d, 2 H), 7.60–7.62 (d, 2 H), 7.49–7.51 (d, 2 H), 7.14–7.16 (d, 2 H), 5.95 (s, 1 H), 5.51 (s, 1 H), 4.07–4.08 (d, 2 H), 3.33 (s, 2 H), 1.72 ppm (s, 3 H). 3-Mercaptopropyl-modified silica was prepared by the reaction of silica gel and MPS mixed in toluene as described in our previous report.[5–7] Dried silica gel (12.8 g) was placed in a dried flask and dispersed in toluene. Then MPS (6.4 g) was added and the reaction mixture was heated at reflux for 48 h. The MPS-modified silica (Sil-MPS) was filtered, carefully washed with toluene, chloroform, ethanol/water (1:1, v/v), water, methanol, and diethyl ether, and dried under vacuum at room temperature. Finally, Sil-MPS (6.0 g) was added to a 100 mL three-neck round-bottomed flask. The same amount of MA-AzO monomer was dissolved in dimethylformamide (DMF; 30 mL) and added to the flask, followed by the addition of 1 % AIBN. The mixture was stirred at 65 8C overnight. The obtained azobenzene-modified silica (Sil-PAzO) particles were filtered and washed consecutively with DMF, ethanol/water mixture, water, methanol, and diethyl ether. After drying under vacuum, the resultant Sil-PAzO was used for characterization or packed into the stainless-steel column (150 mm 4.6 mm inside diameter (i.d.)). Characterization: The elemental compositions of MPS and azobenzene grafted on silica materials were determined by elemental analysis using a Yanaco CHN Corder MT-6 apparatus (YANACO Co., Ltd., Kyoto, Japan). Thermogravimetric analysis was conducted on a Seiko Exstar 6000 TG/DTA 6200 thermal analyzer (Seiko Instruments Inc., Chiba, Japan) in static air from 35 to 800 8C with a heating rate of 10 8C min1. DRIFT spectra were performed on a FT/IR-4100 with an accessory DR PRO410M (JASCO Co., Ltd, Tokyo, Japan) in the range 4000–400 cm1. Chromatographic conditions: Sil-PAzO was packed into a stainless-steel column (150 4.6 mm i.d.) using methanol/water (1:1, v/v) as the propulsive solvent. A commercial ODS column (Inertsil ODS-3, 150 4.6 mm i.d.) used as the reference was obtained from GL Science (Tokyo, Japan). Another column based on polyACHTUNGRE(styrene)-grafted silica (Sil-Stn) prepared in our lab was used as the reference in the reverse phase. HPLC-grade methanol and acetonitrile were used as components of the mobile phase. Millipore water was used during the experiments. The analytes were directly dissolved in methanol or water. The chromatographic system (JASCO, Tokyo, Japan) consisted of a LC-NetII/ADC communication device, a DG-2080-53 3 Line degasser, a PU-2080 Plus Intelligent HPLC pump, a UV-2075 Plus Intelligent UV/Vis detector, a CO-2065 Plus column oven, and a Rheodyne injector with a 20 mL sample loop. Chromatographic data were obtained by using a JASCO ChromNAV Chroma-
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tography Data System. The flow rate was 1.0 mL min1 and the injection volume was 5 mL. The detection wavelength was UV light: l = 203 nm for ginsenosides, 360 nm for flavonoids, and 254 nm for the others. The retention time of D2O was used as the void volume (t0) marker (the absorption for D2O was measured at 400 nm, which is actually considered as the injection shock). The retention factor (k) of an analyte was calculated according to the equation: k = (tRt0)/t0, in which tR is the retention time of the analyte. The separation factor (a) is the ratio of the retention factors for the two solutes being analyzed. The water/1-octanol partition coefficient (log Po/w), usually used to represent molecular hydrophobicity, was determined from the retention factor with the ODS column stated above as log Po/w = 3.759 + 4.207 log k (r = 0.99997), according to the procedure described in our previous work.[16]
Acknowledgements H. Qiu acknowledges the support of the “Hundred Talents Program” of Chinese Academy of Science. This work was also partly supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grant-in-Aid for Program for Fostering Regional Innovation.
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[7] a) H. Qiu, A. K. Mallik, T. Sawada, M. Takafuji, H. Ihara, Chem. Commun. 2012, 48, 1299 – 1301; b) H. Qiu, S. Jiang, M. Takafuji, H. Ihara, Chem. Commun. 2013, 49, 2454 – 2456. [8] a) J. A. A. Sales, F. P. Faria, A. G. S. Prado, C. Airoldi, Polyhedron 2004, 23, 719 – 725; b) S. Radi, A. Attayibat, A. Ramdani, M. Bacquet, J. Appl. Polym. Sci. 2010, 117, 3345 – 3349. [9] a) F. Jiang, J. Qu, H. Chen, Chin. J. Chem. 2009, 27, 2079 – 2084; b) Z. Dong, N. M. Seemann, N. Lu, Y. Song, J. Phys. Chem. B 2011, 115, 14912 – 14918; c) R. Gimnez, M. Millaruelo, M. PiÇol, J. L. Serrano, A. ViÇuales, R. Rosenhauer, T. Fischer, J. Stumpe, Polymer 2005, 46, 9230 – 9242. [10] a) M. M. Rahman, M. Takafuji, H. R. Ansarian, H. Ihara, Anal. Chem. 2005, 77, 6671 – 6681; b) M. M. Rahman, M. Czaun, M. Takafuji, H. Ihara, Chem. Eur. J. 2008, 14, 1312 – 1321. [11] a) H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N. Tanaka, Anal. Chem. 1996, 68, 3498 – 3501; b) K. Nakanishi, N. Tanaka, Acc. Chem. Res. 2007, 40, 863 – 873. [12] A. K. Mallik, H. Qiu, M. Takafuji, H. Ihara, Anal. Bioanal. Chem. 2012, 404, 229 – 238. [13] a) K. V. Casteele, H. Geiger, C. F. Van Sumere, J. Chromatogr. 1982, 240, 81 – 94; b) W. Bi, C. H. Yoon, K. H. Row, J. Liq. Chromatogr. Relat. Technol. 2013, 36, 2029 – 2043; c) C. Van Sumere, P. Fach, K. V. Casteele, L. De Cooman, E. Everaert, R. De Loose, W. Hutse-
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A Sulfonic-Azobenzene-Grafted Silica Amphiphilic Material: A Versatile Stationary Phase for Mixed-Mode Chromatography Hongdeng Qiu,*[a] Mingliang Zhang,[a] Tongnian Gu,[a] Makoto Takafuji,[b] and Hirotaka Ihara*[b] Abstract: A novel sulfonic-azobenzene-functionalized amphiphilic silica material was synthesized through the preparation of a new sulfonic azobenzene monomer and its grafting on mercaptopropyl-modified silica by a surface-initiated radical chain-transfer reaction. The synthesis was confirmed by infrared spectra, elemental analysis, and thermogravimetric analysis. This new material was successfully applied as a new kind of mixed-mode stationary phase in liquid chromatography. This allows an exceptionally flexible adjustment of retention and selectivity by tuning the experimental conditions.
The distinct separation mechanisms were outlined by selected examples of chromatographic separations in the different modes. In reversed-phase liquid chromatography, this new stationary phase presented specific chromatographic performance when evaluated using a Tanaka test mixture. Seven dinitro aromatic isomers, four steroids, and seven flavonoids were separated successfully in simple reversed-phase Keywords: amphiphiles · analytical methods · azo compounds · liquid chromatography · silica
Introduction With the development of chromatographic science, mixedmode chromatography (MMC) has emerged as a new type of chromatography that can behave as more than one interaction mode with solutes.[1] The main advantage of MMC over conventional single-mode chromatography is that two or more completely different kinds of analyte can be determined using the same column with different chromatographic modes. The advancement of liquid chromatography has mostly relied on the development of new stationary phases.[2] Therefore, more and more new mixed-mode or multimode stationary phases have been found recently.[3] For example, Wijekoon et al. prepared a zwitterionic sta[a] Prof. H. Qiu, M. Zhang, T. Gu Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province Lanzhou Institute of Chemical Physics, CAS Lanzhou, 730000 (P.R. China) Fax: + 86-931-8277088 E-mail: [email protected] [b] Assoc. Prof. M. Takafuji, Prof. H. Ihara Department of Applied Chemistry and Biochemistry Kumamoto University 2-39-1 Kurokami, Kumamoto 860-8555 (Japan) Fax: + 81-96-3423662 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201302746.
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mode. This stationary phase can also be used in hydrophilic interaction chromatography because of the existing polar functional groups; for this, nucleosides and their bases were used as a test mixture. Interestingly, the same nucleosides and bases can also be separated in per aqueous liquid chromatography using the same stationary phase. Three ginsenosides including Rg1, Re, and Rb1 were successfully separated in hydrophilic mode. There is the potential for more applications to benefit from this useful column.
tionary phase based on 4-propylaminomethyl benzoic acid for multimode liquid chromatography.[3i] Liu et al. prepared a mixed-mode stationary phase based on click amino-modified silica for ion chromatography (IC) and hydrophilic interaction liquid chromatography (HILIC).[3j] In other examples, ionic liquids were immobilized on silica through covalent bonding or a polymerized grafting method and used as a new kind of stationary phase for different chromatographic modes.[4] Methyl orange (MO) was employed as a counter anion of the poly(ionic liquid)-modified silica stationary phase, which plays a key role in enhancing the shape selectivity to polycyclic aromatic hydrocarbons (PAHs) because of the existence of rigid functionalized azobenzene. In that case, long-chain alkylimidazoliumfunctionalized silica particles were prepared first and then the counteranions were changed from bromide to MO by means of ionic self-assembly before column packing[5] or in situ anion exchange after column packing.[6] The cooperation effect of the azobenzene group of MO on the enhancement of the chromatographic selectivity is very clear. We were strongly stimulated to prepare a new stationary phase with the azobenzene group and mainly investigate the performance of the azobenzene structure. Therefore, a new sulfonic azobenzene monomer was synthesized and immobilized on the silica by using a facile method described in this work. This newly prepared stationary phase not only presents an enhanced selectivity to PAHs and steroids in reversed-phase liquid chromatography
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FULL PAPER (RPLC), but also can be used to separate nucleosides and bases in HILIC mode and by per aqueous liquid chromatography (PALC). Seven flavonoids and three ginsenosides were also effectively separated in RPLC and HILIC mode, respectively.
Results and Discussion Synthesis and characterization: The silica surface-modification procedures are simple and facile as outlined in Scheme 1. In brief, the synthesis includes the preparation of an azobenzene monomer (MA-AzO) and the grafting of the monomer on 3-mercaptopropyl-modified silica.
Scheme 1. Synthesis of the sulfonic-azobenzene-modified silica stationary phase.
Figure 1. Thermogravimetric curves of a) Sil-MPS and b) Sil-PAzO.
16.3 % over the same temperature range, which indicates that the organic content clearly increased. These mass losses are consistent with the immobilized amounts estimated by elemental analyses. Infrared spectroscopy is another useful tool that can be used to identify the chemical modifications of compounds. Some clear differences in wave numbers and in the intensities of the absorption bands were observed in the spectra of Sil-MPS and Sil-PAzO as shown in Figure 2. In the spectrum for the Sil-PAzO surface, clear characteristic signals at 1601 and 1504 cm1 are attributed to the stretching vibration of the phenyl bond of sulfonic azobenzene, which confirms the anchoring of the organic molecule onto the silica surface.[9]
The elemental content (%) of Sil-MPS and Sil-PAzO was C 4.42, H 1.27 and C 9.40, H 1.76, N 1.28, respectively. The degree of surface coverage for Sil-MPS and Sil-PAzO was calculated from the following equations [Eqs. (1) and (2)][7]: Sil-MPS ½mmol m2 ¼
%C ¼ 4:34 36 ð1 %C %HÞ S
Sil-PAzO ½mmol m2 ¼
ð1Þ
%N ¼ 0:87 56 ð1 %C %H %NÞ S ð2Þ Figure 2. Diffuse reflectance infrared Fourier transform (DRIFT) spectra of a) Sil-MPS and b) Sil-PAzO.
in which %C, %H, and %N represent the percentages of carbon, hydrogen, and nitrogen, respectively, and S is the surface area. The amount of mercaptopropyl and imidazolium moieties attached to the silica surface can thus be calculated as 4.34 mmol m2 for Sil-MPS and 0.87 mmol m2 for Sil-PAzO. Thermogravimetric curves are usually used to determine thermal stability and to confirm the amount of immobilized compounds on the silica surface. The weight loss observed between 200 and 600 8C can be associated with the loss of the organic groups attached to the silica surface.[8] As shown in Figure 1, Sil-MPS presented a mass loss of about 5.9 % from 200 to 600 8C. After covalently bonding with the azobenzene monomer, Sil-PAzO showed a mass loss of about
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RPLC mode: It is known that conventional octadecylsilane (ODS) or other alkylated organic stationary phases can recognize the hydrophobicity of solutes in HPLC, which is measured by the selectivity of the stationary phase for the methylene group. This reflects the possibility that the phase may be able to separate two molecules that differ only by a methylene group, for example, amylbenzene and butylbenzene. The retention mode as well as the extent of the hydrophobic interactions among the solutes and the packing materials in HPLC can be determined by retention studies using alkylbenzenes as solutes.[10] The relationship between the retention factor (log k) and the water/1-octanol partition coef-
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ficient (log Po/w) for Sil-PAzO, ODS, and polyACHTUNGRE(styrene)-grafted silica (Sil-Stn) columns are compared in Figure S1 in the Supporting Information. It was observed that Sil-PAzO showed a higher retention for PAHs than for alkylbenzenes. For instance, the log Po/w of naphthacene (5.71) is much smaller than that of decylbenzene (7.36), but the log k value of naphthacene (0.65) is clearly higher than that of decylbenzene (0.46). A similar selectivity to PAHs and alkylbenzenes was also found in Sil-Stn, but for ODS, log k versus log Po/w plots for alkylbenzenes and PAHs were almost on the same line. The Tanaka test mixture is usually used to effectively characterize the chromatographic performance of a new stationary phase.[11] As shown in Figure 3, a comparable separa-
dinitronaphthalene, 1,5-dinitronaphthalene, and 1,3-dinitronaphthalene were separated. The retention orders of these analytes on Sil-PAzO and ODS columns are a little different as shown in Figure S3 in the Supporting Information. Under the same chromatographic conditions, a mixture of four steroids including estriol, 17a-estradiol, 17b-estradiol, and estrone were successfully separated in Sil-PAzO within six minACHTUNGREutes as shown in Figure 4. The positions of the isomer pair 17a-estradiol and 17b-estradiol were baseline-separated within 5 min on Sil-PAzO, which can be a challenge for the ODS column.[12] As shown in Figure 4, the same mixture cannot be separated on the ODS column under the same chromatographic conditions and the retention order of the last three analytes was reversed.
Figure 3. Separation of the Tanaka test mixture: 1) uracil, 2) phenol, 3) caffeine, 4) butylbenzene, 5) o-terphenyl, 6) amylbenzene, and 7) triphenylene with a) ODS and b) Sil-PAzO columns. Mobile phase: a) methanol/water (85:15, v/v) and b) methanol/water (70:30, v/v). T = 30 8C; flow rate = 1 mL min1; detection: l = 254 nm.
tion of the Tanaka test mixture, which included uracil, phenol, caffeine, butylbenzene, o-terphenyl, amylbenzene, and triphenylene, was performed by the ODS and Sil-PAzO columns. Two different points can be observed clearly. The first one is that the retention of caffeine is even stronger than that of butylbenzene and amylbenzene in Sil-PAzO, which can be due to the existence of a sulfonic group in this new phase. The second one is that the retention order of oterphenyl and amylbenzene is different among the ODS and Sil-PAzO columns. That is because the aromatic azobenzene group can induce strong p–p interactions in the PAHs. It can also be proven that the planar selectivity was enhanced when comparing the selectivity of o-terphenyl and triphenylene on Sil-PAzO (separation factor (atriphenylene/o-terphenyl) = 2.44) and ODS (atriphenylene/o-terphenyl = 1.36) columns. The effect of the methanol content on the retention factors of the Tanaka test mixture is shown in Figure S2 in the Supporting Information. The retention of uracil, phenol, and caffeine was not influenced by the content of methanol, however, the retention of hydrophobic analytes was affected very much. Using methanol/water 65:35 (v/v) as the mobile phase, seven dinitro aromatic isomers including o-dinitrobenzene, p-dinitrobenzene, m-dinitrobenzene, 2,4-dinitrotoluene, 1,8-
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Figure 4. Separation of four steroids: 1) estriol, 2) 17a-estradiol, 3) 17bestradiol, and 4) estrone with a) Sil-PAzO and b) ODS columns with methanol/water (65:35, v/v). Other chromatographic conditions are the same as those in Figure 3.
This column can also be used to separate natural products such as flavonoids. Usually, separation of flavonoids in a RPLC system has relied on an ODS column.[13] The structures of the flavonoids used in this study are listed in Figure S4 in the Supporting Information. The effect of the pH of the buffer on the separation of the flavonoids was investigated as shown in Figure 5. When the pH value was decreased from 6.8 to 5, the retention of luteolin and naringe-
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Figure 5. The effect of pH on the separation of myricetin (~), naringenin (&), and luteolin (*). The mobile phases were mixed to 30 % acetonitrile and 70 % ammonium acetate (20 mmol L1) with different pH values adjusted by using acetic acid. Other chromatographic conditions are the same as those in Figure 3.
nin increased. If the pH decreased again, from 5 to 4, the retention of luteolin and naringenin decreased. However, the retention of myricetin was a little different, with the strongest retention time observed when the pH was 6. It is worth noting that the tailing phenomena are heavy when the pH values are above 5. The chromatographic peaks improve when the pH of the buffer is decreased. This should be attributed to the restrained ionization of sulfonic acid. The best separation of the seven flavonoids, including myricetin, naringenin, quercetin, luteolin, kaempferol, isorhamnetin, and baicalein, was obtained with 20 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (78:22, v/v) as shown in Figure 6b. The effects of acetonitrile and buffer concentration were investigated. On increasing the buffer concentration or the acetonitrile content, the retention of the flavonoids decreased as shown in Figure 6a and c, respectively. HILIC and PALC modes: This developed stationary phase was not only used in the RPLC mode for the separation of alkylbenzenes, PAHs, steroids, dinitrobenzenes, and dinitronaphthalenes, but was also used in the HILIC mode to separate bases, nucleosides, and ginsenosides. As a powerful separation technique, HILIC provides an alternative approach to effectively separate small polar compounds on a polar stationary phase.[14] The sulfonic and other polar groups of Sil-PAzO make it appropriate to be used as a polar stationary phase for application in HILIC mode. Separation of ethylbenzene, uracil, and cytosine on Sil-PAzO was obtained with 20 mmol L1 ammonium acetate/acetonitrile (10:90, v/v) as the mobile phase (Figure S5 in the Supporting Information). The relationships of the retention factors of ethylbenzene, uracil, and cytosine with different proportions of 20 mmol L1 ammonium acetate and acetonitrile as eluent were investigated as shown in Figure 7. The retention of ethylbenzene was not affected on increasing the amount of acetonitrile from 80 to 95 %, and that of uracil gets stronger little by little, however, the retention of cytosine drastically increased, especially when the content of acetonitrile was increased from 90 to 95 % in the mobile phase. It is a classical
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Figure 6. Separation of seven flavonoids: 1) myricetin, 2) naringenin, 3) quercetin, 4) luteolin, 5) kaempferol, 6) isorhamnetin, 7) baicalein with a) 100 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (78:22, v/v), b) 20 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (78:22, v/v) and c) 20 mmol L1 ammonium acetate (pH 4.0)/acetonitrile (75:25, v/v) as mobile phases. RT; flow rate: 1 mL min1; detection: l = 360 nm.
Figure 7. Relationship of the retention factor of ethylbenzene (&), uracil (*), and cytosine (~) with different proportions of 20 mmol L1 ammonium acetate and acetonitrile as eluent. Other chromatographic conditions are the same as those in Figure 3.
example that demonstrates that a polar stationary phase can be used in HILIC mode. The HILIC applications of Sil-PAzO can be identified from the following separation of seven bases and nucleosides including uracil, theophylline, uridine, adenosine, adenine, cytosine, and guanosine. As shown in Figure 8, they were successfully separated within 10 min using 20 mmol L1 ammonium acetate/acetonitrile (10:90, v/v) as the mobile phase. On increasing the proportion of acetonitrile, the re-
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different on the two modes. This may provide an opportunity for wide application of this new phase for PALC. This column also can be used in other applications in HILIC mode. For example, the separation of three ginsenosides including Rg1, Re, and Rb1 as shown in Figure 10.
Figure 8. Separation of seven bases and nucleosides: 1) uracil, 2) theophylline, 3) uridine, 4) adenosine, 5) adenine, 6) cytosine, and 7) guanosine with different mobile phases. Mobile phases: a) 20 mmol L1 ammonium acetate/acetonitrile (10:90, v/v); b) 20 mmol L1 ammonium acetate/ acetonitrile (7.5:92.5, v/v); c) 20 mmol L1 ammonium acetate/acetonitrile (5:95, v/v). Other chromatographic conditions are the same as those in Figure 3.
tention factors and separation factors were increased correspondingly. By using 20 mmol L1 ammonium acetate/acetonitrile (7.5:92.5, v/v) as the mobile phase, 10 bases and nucleosides including 5-bromouracil, uracil, theophylline, uridine, purine, adenosine, adenine, cytosine, cytidine, and guanosine were separated very well as shown in Figure 9a.
Figure 10. Separation of three ginsenosides: 1) Rg1, 2) Re, and 3) Rb1 with a) acetonitrile/water (85:15, v/v) and b) acetonitrile/water (90:10, v/v) as mobile phases. RT; flow rate: 1 mL min1; detection: l = 203 nm.
These ginsenosides are difficult to separate on the ODS column, and a long time and gradient elution is usually necessary. However, these ginsenosides can be separated completely within ten minutes on the Sil-PAzO column with acetonitrile/water (85:15, v/v) as the mobile phase. With the increase of acetonitrile content, the retention of the ginsenosides increased correspondingly, which can be used to prove that these chromatographic separations arise as a result of the HILIC mode.
Conclusion
Figure 9. Separation of 10 bases and nucleosides: 1) 5-bromouracil, 2) uracil, 3) theophylline, 4) uridine, 5) purine, 6) adenosine, 7) adenine, 8) cytosine, 9) cytidine, and 10) guanosine with a) 20 mmol L1 ammonium acetate/acetonitrile (7.5:92.5, v/v) and b) 20 mmol L1 ammonium acetate as the mobile phase. Other chromatographic conditions are the same as those in Figure 3.
After HILIC separation, Sil-PAzO was also creatively used in PALC mode to separate the same 10 bases and nucleosides as described above. Whereas a large amount of acetonitrile is used in HILIC mode, only a small amount of organic solvent or no organic solvent can be used in PALC mode.[15] PALC mode should be encouraged to be used for economical reasons and for preventing pollution through the use of large amounts of organic solvent. As shown in Figure 9b, the 10 bases and nucleosides were surprisingly separated on Sil-PAzO with 20 mmol L1 ammonium acetate as the mobile phase. The retention orders of the samples are
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A new mixed-mode stationary phase based on sulfonic azobenzene grafted on silica was prepared and characterized. This stationary phase was evaluated by the separations of the Tanaka test mixture, dinitro aromatic isomers, PAHs, and steroids in RPLC. The same phase was also used to separate bases and nucleosides in HILIC and PALC modes. It is clear that Sil-PAzO presents different or a specific selectivity to the analytes compared with the commonly used ODS column. This new stationary phase is a mixed-mode stationary phase owing to the existence of multifunctionalized groups. It is foreseeable that this phase can also be used as a strong cation-exchange mode owing to the electrostatic interaction of the sulfonic groups. This new amphiphilic material holds promise for more applications, not only as a new versatile stationary phase, but also as a solid extraction adsorbent and in other chemical fields.
Experimental Section Materials and reagents: p-[(p-Aminophenyl)azo]benzenesulfonic acid was obtained from City Chemical LLC (West Haven, USA). 2-Isocyana-
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toethyl methacrylate was purchased from TCI America. Azobisisobutyronitrile (AIBN) was obtained from Nacalai tesque, Inc. (Kyoto, Japan) and purified by recrystallization from methanol before use. 3-Mercaptopropyltrimethoxysilane (MPS) was purchased from Azmax (Chiba, Japan). Porous silica particles as a support (diameter 5 mm, pore size 120 , specific surface area 300 m2 g1) were obtained from YMC (Kyoto, Japan). Standard reference material (SRM) 869b, column selectivity test mixture for liquid chromatography, and SRM 1647e, priority pollutant polycyclic aromatic hydrocarbons were obtained from the Standard Reference Materials Program (NIST, Gaithersburg, MD). Estriol, estrone, and 17b-estradiol were obtained from Sigma (St Louis, MO, USA). 17aEstradiol was purchased from Dr. Ehrenstorfer (Augsburg, Germany). 5Bromouracil, uracil, theophylline, uridine, purine, adenosine, adenine, cytosine, cytidine, and guanosine were purchased from Sigma–Aldrich, Inc. All dinitro aromatic isomers, alkylbenzenes, and PAHs were commercially available and used without any purification. Synthesis of Sil-PAzO: First, the polymerizable sulfonic azobenzene monomer was prepared as follows: p-[(p-Aminophenyl)azo]benzenesulfonic acid (9.0 g, 30.2 mmol) was added to the flask. Then water (180 mL) and methanol (180 mL) were both added to the flask to dissolve p-[(p-aminophenyl)azo]benzenesulfonic acid with ultrasonication. After the compound had completely dissolved, 6-fold amounts of 2-isocyanatoethyl methacrylate were added dropwise to the solution at 0 8C. The reaction mixture was stirred at room temperature overnight. The solvent was removed by rotary evaporation under reduced pressure. And then the obtained solid was washed with ethanol and dried under vacuum to give a solid yellow powder (8.5 g). C19H20N4O6S: 1H NMR (D2O, 400 MHz): d = 7.75–7.77 (d, 2 H), 7.60–7.62 (d, 2 H), 7.49–7.51 (d, 2 H), 7.14–7.16 (d, 2 H), 5.95 (s, 1 H), 5.51 (s, 1 H), 4.07–4.08 (d, 2 H), 3.33 (s, 2 H), 1.72 ppm (s, 3 H). 3-Mercaptopropyl-modified silica was prepared by the reaction of silica gel and MPS mixed in toluene as described in our previous report.[5–7] Dried silica gel (12.8 g) was placed in a dried flask and dispersed in toluene. Then MPS (6.4 g) was added and the reaction mixture was heated at reflux for 48 h. The MPS-modified silica (Sil-MPS) was filtered, carefully washed with toluene, chloroform, ethanol/water (1:1, v/v), water, methanol, and diethyl ether, and dried under vacuum at room temperature. Finally, Sil-MPS (6.0 g) was added to a 100 mL three-neck round-bottomed flask. The same amount of MA-AzO monomer was dissolved in dimethylformamide (DMF; 30 mL) and added to the flask, followed by the addition of 1 % AIBN. The mixture was stirred at 65 8C overnight. The obtained azobenzene-modified silica (Sil-PAzO) particles were filtered and washed consecutively with DMF, ethanol/water mixture, water, methanol, and diethyl ether. After drying under vacuum, the resultant Sil-PAzO was used for characterization or packed into the stainless-steel column (150 mm 4.6 mm inside diameter (i.d.)). Characterization: The elemental compositions of MPS and azobenzene grafted on silica materials were determined by elemental analysis using a Yanaco CHN Corder MT-6 apparatus (YANACO Co., Ltd., Kyoto, Japan). Thermogravimetric analysis was conducted on a Seiko Exstar 6000 TG/DTA 6200 thermal analyzer (Seiko Instruments Inc., Chiba, Japan) in static air from 35 to 800 8C with a heating rate of 10 8C min1. DRIFT spectra were performed on a FT/IR-4100 with an accessory DR PRO410M (JASCO Co., Ltd, Tokyo, Japan) in the range 4000–400 cm1. Chromatographic conditions: Sil-PAzO was packed into a stainless-steel column (150 4.6 mm i.d.) using methanol/water (1:1, v/v) as the propulsive solvent. A commercial ODS column (Inertsil ODS-3, 150 4.6 mm i.d.) used as the reference was obtained from GL Science (Tokyo, Japan). Another column based on polyACHTUNGRE(styrene)-grafted silica (Sil-Stn) prepared in our lab was used as the reference in the reverse phase. HPLC-grade methanol and acetonitrile were used as components of the mobile phase. Millipore water was used during the experiments. The analytes were directly dissolved in methanol or water. The chromatographic system (JASCO, Tokyo, Japan) consisted of a LC-NetII/ADC communication device, a DG-2080-53 3 Line degasser, a PU-2080 Plus Intelligent HPLC pump, a UV-2075 Plus Intelligent UV/Vis detector, a CO-2065 Plus column oven, and a Rheodyne injector with a 20 mL sample loop. Chromatographic data were obtained by using a JASCO ChromNAV Chroma-
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tography Data System. The flow rate was 1.0 mL min1 and the injection volume was 5 mL. The detection wavelength was UV light: l = 203 nm for ginsenosides, 360 nm for flavonoids, and 254 nm for the others. The retention time of D2O was used as the void volume (t0) marker (the absorption for D2O was measured at 400 nm, which is actually considered as the injection shock). The retention factor (k) of an analyte was calculated according to the equation: k = (tRt0)/t0, in which tR is the retention time of the analyte. The separation factor (a) is the ratio of the retention factors for the two solutes being analyzed. The water/1-octanol partition coefficient (log Po/w), usually used to represent molecular hydrophobicity, was determined from the retention factor with the ODS column stated above as log Po/w = 3.759 + 4.207 log k (r = 0.99997), according to the procedure described in our previous work.[16]
Acknowledgements H. Qiu acknowledges the support of the “Hundred Talents Program” of Chinese Academy of Science. This work was also partly supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grant-in-Aid for Program for Fostering Regional Innovation.
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