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Novel additives for the separation of organic acids by ion-pair chromatography
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Chinese Chemical Letters 21 (2010) 453–456 www.elsevier.com/locate/cclet
Novel additives for the separation of organic acids by ion-pair chromatography Ying Ying Zhong a, Wen Fang Zhou a, Zhen Zhen Hu a, Mei Lan Chen b, Yan Zhu a,* a
b
Department of Chemistry, Zhejiang University, Hangzhou 310028, China Biology and Environment Engineering College of Zhejiang Shuren University, Hangzhou 310015, China Received 17 July 2009
Abstract This paper proposes the use of novel surfactant additives for the separation of organic acids by ion-pair chromatography and studies the influences of surfactants on the chromatographic separation behaviors. Researches have been carried out on both silica gel matrix and polymer supporters in order to compare the two ordinary kinds of stationary phases, and the phenomenon is similar. Separation is based on differences in the stabilities of analyte–additive complexes in solution. Retention times of analytes can be varied over a large range by varying the additive concentration. The results indicate that the additives of proper concentrations can reduce the retention times of different organic acids while the resolution remains the same. The larger the molecular weight is, the greater the shift of the retention time is. This greatly expands the scope of macromolecular polar compounds that can be separated by ion-pair chromatography with the advantages of retention times being greatly reduced. # 2009 Yan Zhu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Organic acids; Ion-pair chromatography; Surfactant; Mobile phase additives
After more than one hundred years’ development, chromatographic separation has been one of the most widely used methods in analytical field ever since it first came up in 1903. As known to us all, the properties of the two basic components, stationary phase and mobile phase, determine the principle and method of chromatographic separation, but the modification of stationary phase is not so easy for it is usually expensive, while the change of mobile phase is quite convenient. More and more new chromatographic separation techniques have been established by changing the mobile phase, such as micellar chromatography [1,2], inclusion chromatography [3,4], and ion-pair chromatography [5–7]. In all types of chromatography, the change of mobile phase influences the separation a lot, including the resolution, plate number, retention time and so on. Different kinds of additives have been utilized in the mobile phase of almost every kinds of chromatography, especially organic additives [8,9], chiral additives [10] and surfactants [11,12]. Meanwhile, the addition of ion-pair reagent makes it possible to analyze polar organic molecules including organic and inorganic ions by liquid chromatography, and the detection of polar macromolecular compounds is realized as well, which helps to separate biological molecules using ordinary liquid chromatography [13,14].
* Corresponding author. E-mail address: [email protected] (Y. Zhu). 1001-8417/$ – see front matter # 2009 Yan Zhu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.12.032
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Y.Y. Zhong et al. / Chinese Chemical Letters 21 (2010) 453–456
In the present paper, the effect of surfactants in mobile phase is studied using ion-pair chromatography under different concentrations. A dramatic decrease in retention time was observed with several of the additives being studied and sharp peaks were obtained with resolutions being unaffected. Both organic and inorganic stationary phases have been tried, and the results are similar, which means that the new additives can be widely used in ion-pair liquid chromatography whatever the stationary phase is. 1. Experimental Standard solutions were prepared by dissolve 0.1000 0.3% g standard samples in 5 mL methanol and scaled to 100 mL by 18.2 MV deionized water, then sonicated for 20 min, and stored at 4 8C. Other sample solutions were prepared by diluting appropriate aliquots of stock solutions with deionized water to give final solute concentration in the range of 5–40 mg/mL. Samples were then degassed by ultrasonic and filtrated with a 0.22 mm membrane before injection. The mobile phase was prepared by adding 5 mmol/L tetrabutyl ammonium hydroxide (TBAOH) and 20 mmol/L NaH2PO4 into mixed solvents of water and acetonitrile, containing proper concentration of surfactant as an additive. The pH value was then adjusted to 6.95 by adding phosphoric acid, then filtrated by 0.45 mm membrane and degassed for 30 min using ultrasonic before use. And the Tween series such as Tween 20, Tween 40, Tween 60 and Tween 80 were used as surfactants. Ultimate 3000 HPLC with autosampler (Dionex, USA) were used at a wavelength of 220 nm and columns used are Dionex IonPac NS1 Column (4 mm i.d. 150 mm), Dalian Johnsson CenturySIL C18 Column (4.6 mm i.d. 150 mm) and Dalian Sipore Tigerkin C18 Column (4.6 mm i.d. 150 mm), with a flow rate 1.0 mL/min. Column temperature is 25 8C, and the injection volume is 10 mL. The ion-pair reagent is TBAOH with concentrations ranging from 5 mmol/L to 20 mmol/L. 2. Results and discussion Ion-pair chromatography is based on a liquid–liquid partition technique usually called ion-pair extraction or ionpair partition. Ionized organic compounds can be extracted into organic phases as ion-pairs with counter ions of suitable hydrophobicity. The effect of surfactants on chromatographic separation has been studied after the optimization of mobile phase. Fig. 1(a) represents a chromatogram obtained without surfactant on a silica gel matrix column, while Fig. 1(b) represents the use of 1 mmol/L Tween 20 on the same column. Comparing these two chromatograms, it is obvious to found that the retention times of strong retention compounds in Fig. 1(b) are much shorter than in Fig. 1(a). Elution of all compounds changes from 33.5 min to less than 23.0 min without affect of the separation efficiency. Hence, the addition of surfactant accelerated the separation, and saved analytical times and reagents, which accorded with the demands of green chemistry. On the other hand, similar results are gained on polymer matrix columns when the same surfactant of different concentrations is used as mobile phase additive (Fig. 2(a)). The addition of surfactant under proper concentration may shorten retention times of analytes as well as improve the peak shapes without decrease of the resolution, especially for strong retention compounds. However, if too much surfactant is added, the resolution will decrease and some compounds cannot be separated. Besides, different surfactants in Tween series have been tested as mobile phase additives (Fig. 2(b)), among which Tween 20 and Tween 40 have similar effects on separation. Tween 60 and Tween 80 have shortened the retention times too greatly to separate all 10 organic acids. Thus, only Tween 20 and Tween 40 are good choices to be used as mobile phase additives. In ion-pair chromatography, there is an electric double layer between the mobile phase and stationary phase. Counter ions and organic additives can be adsorbed onto hydrophobic stationary phase. It is impossible for the counter ions with same charge to cover the surface of stationary phase completely according to repulsion principle. Hence, a balance will be generated between these two phases. Sample molecules with opposite charges can be attracted by counter ions and establish an electrostatic balance between mobile phase and stationary phase. Thus, the separation of polar compounds can be realized. Surfactant ions can move into the inner layer of the electric double layer and also be adsorbed onto stationary phase. They compete with organic additives for the adsorption sites on stationary phase and establish a dynamic balance with the solutes in order to accelerate the separation. Moreover, the surfactant can inhibit
Y.Y. Zhong et al. / Chinese Chemical Letters 21 (2010) 453–456
455
Fig. 1. Chromatograms of 13 organic acids separated by (a) traditional mobile phase without additives and (b) novel mobile phase with 1 mmol/L Tween 20 on silica gel supporters. Peaks identified as: (1) ascorbic acid, (2) 3,5-dihydroxybenzoic acid, (3) 4-hydroxybenzoic acid, (4) syringic acid, (5) 3-hydroxybenzoic acid, (6) tropic acid, (7) 2,4-dihydroxybenzoic acid, (8) 2,5-dihydroxybenzoic acid, (9) benzoic acid, (10) 4-methylbenzenesulfonic acid, (11) 2-iodobenzoic acid, (12) salicylic acid, and (13) 2-(2,4,5-trifluorophenyl)acetic acid.
Fig. 2. Retention times of 10 organic acids under (a) different concentrations of Tween 20 and (b) different surfactants of Tween series of same concentration. Samples identified as: (1) ascorbic acid, (2) 4-hydroxybenzoic acid, (3) caffeic acid, (4) 2-methylbenzoic acid, (5) 2,5dihydroxybenzoic acid, (6) 2-iodobenzoic acid, (7) 2-(2-chlorophenyl)acetic acid, (8) sulfosalicyclic acid, (9) 2-(2,4,5-trifluorophenyl)acetic acid, (10) salicylic acid, (11) D-( )mandelic acid, (12) 2-(3,4-dimethoxyphenyl)acetic acid, (13) 3-(3,4-dimethoxyphenyl)acrylic acid, (14) 4methylbenzene-1-sulfonyl chloride, (15) 3-(3-nitrophenyl)acrylic acid, (16) 4-iodobenzoic acid, and (17) 3-(4-chlorophenyl)acrylic acid.
the analytes from going into the inner side of electric double layer because of steric effect. When the size of analyte increases, the effect will enhance. Thus, the strong retention compounds are more changeable after the addition of surfactants. 3. Conclusion As a novel additive of mobile phase, the use of low concentration surfactant can greatly accelerate the separation in ion-pair chromatography. Accordingly, the defects in traditional micellar chromatography, such as the decline of column efficiency and resolution can be overcome. In a word, it is an easy and convenient method to fasten the separation of polar organic macromolecules by ion-pair chromatography. Acknowledgments This research was financially supported by National Natural Science Foundation of China (No. 20775070), Zhejiang Qianjiang Project of Science and Technology for Competent People (No. 2008R10028), Zhejiang Provincial Natural Science Foundation of China (Nos. R4080124, Y4080064) and Zhejiang Provincial Analysis and Testing Foundation of China (No. 2007F70061).
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Y.Y. Zhong et al. / Chinese Chemical Letters 21 (2010) 453–456
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
M.J. Ruiz-a´ngel, J.R. Torres-Lapasio´, M.C. Garcı´a-a´lvarez-Coque, et al. J. Chromatogr. A 1216 (2009) 3199. M.J. Ruiz-a´ngel, S. Carda-Broch, J.R. Torres-Lapasio´, et al. J. Chromatogr. A 1216 (2009) 1798. A.A. Sakalgaonkar, S.R. Mirgane, R.P. Pawar, Chromatographia 68 (2008) 143. D.Y. Chen, S.M. Jiang, Y.Y. Chen, et al. J. Pharm. Biomed. Anal. 34 (2004) 239. C.E. Doneanu, W.B. Chen, J.C. Gebler, Anal. Chem. 81 (2009) 3485. T. Cecchi, P. Passamonti, J. Chromatogr. A 1216 (2009) 1789. R.P. Patel, C. Narkowicz, G.A. Jacobson, Anal. Biochem. 387 (2009) 113. S. Lopez-Grio, M.C. Garcia-Alvarez-Coque, W.L. Hinze, et al. Anal. Chem. 72 (2000) 4826. D.P. Thomas, J.P. Foley, J. Chromatogr. A 1205 (2008) 36. S.L. Ma, S. Shen, N. Haddad, et al. J. Chromatogr. A 1216 (2009) 1232. X. Li, J.S. Fritz, Anal. Chem. 68 (1996) 4481. X. Li, J.S. Fritz, J. Chromatogr. A 728 (1996) 235. M.J. Dickman, J. Chromatogr. A 1076 (2005) 83. S. Aburuz, J. Millership, J. McElnay, J. Chromatogr. B 798 (2003) 203.
Chinese Chemical Letters 21 (2010) 453–456 www.elsevier.com/locate/cclet
Novel additives for the separation of organic acids by ion-pair chromatography Ying Ying Zhong a, Wen Fang Zhou a, Zhen Zhen Hu a, Mei Lan Chen b, Yan Zhu a,* a
b
Department of Chemistry, Zhejiang University, Hangzhou 310028, China Biology and Environment Engineering College of Zhejiang Shuren University, Hangzhou 310015, China Received 17 July 2009
Abstract This paper proposes the use of novel surfactant additives for the separation of organic acids by ion-pair chromatography and studies the influences of surfactants on the chromatographic separation behaviors. Researches have been carried out on both silica gel matrix and polymer supporters in order to compare the two ordinary kinds of stationary phases, and the phenomenon is similar. Separation is based on differences in the stabilities of analyte–additive complexes in solution. Retention times of analytes can be varied over a large range by varying the additive concentration. The results indicate that the additives of proper concentrations can reduce the retention times of different organic acids while the resolution remains the same. The larger the molecular weight is, the greater the shift of the retention time is. This greatly expands the scope of macromolecular polar compounds that can be separated by ion-pair chromatography with the advantages of retention times being greatly reduced. # 2009 Yan Zhu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Organic acids; Ion-pair chromatography; Surfactant; Mobile phase additives
After more than one hundred years’ development, chromatographic separation has been one of the most widely used methods in analytical field ever since it first came up in 1903. As known to us all, the properties of the two basic components, stationary phase and mobile phase, determine the principle and method of chromatographic separation, but the modification of stationary phase is not so easy for it is usually expensive, while the change of mobile phase is quite convenient. More and more new chromatographic separation techniques have been established by changing the mobile phase, such as micellar chromatography [1,2], inclusion chromatography [3,4], and ion-pair chromatography [5–7]. In all types of chromatography, the change of mobile phase influences the separation a lot, including the resolution, plate number, retention time and so on. Different kinds of additives have been utilized in the mobile phase of almost every kinds of chromatography, especially organic additives [8,9], chiral additives [10] and surfactants [11,12]. Meanwhile, the addition of ion-pair reagent makes it possible to analyze polar organic molecules including organic and inorganic ions by liquid chromatography, and the detection of polar macromolecular compounds is realized as well, which helps to separate biological molecules using ordinary liquid chromatography [13,14].
* Corresponding author. E-mail address: [email protected] (Y. Zhu). 1001-8417/$ – see front matter # 2009 Yan Zhu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.12.032
454
Y.Y. Zhong et al. / Chinese Chemical Letters 21 (2010) 453–456
In the present paper, the effect of surfactants in mobile phase is studied using ion-pair chromatography under different concentrations. A dramatic decrease in retention time was observed with several of the additives being studied and sharp peaks were obtained with resolutions being unaffected. Both organic and inorganic stationary phases have been tried, and the results are similar, which means that the new additives can be widely used in ion-pair liquid chromatography whatever the stationary phase is. 1. Experimental Standard solutions were prepared by dissolve 0.1000 0.3% g standard samples in 5 mL methanol and scaled to 100 mL by 18.2 MV deionized water, then sonicated for 20 min, and stored at 4 8C. Other sample solutions were prepared by diluting appropriate aliquots of stock solutions with deionized water to give final solute concentration in the range of 5–40 mg/mL. Samples were then degassed by ultrasonic and filtrated with a 0.22 mm membrane before injection. The mobile phase was prepared by adding 5 mmol/L tetrabutyl ammonium hydroxide (TBAOH) and 20 mmol/L NaH2PO4 into mixed solvents of water and acetonitrile, containing proper concentration of surfactant as an additive. The pH value was then adjusted to 6.95 by adding phosphoric acid, then filtrated by 0.45 mm membrane and degassed for 30 min using ultrasonic before use. And the Tween series such as Tween 20, Tween 40, Tween 60 and Tween 80 were used as surfactants. Ultimate 3000 HPLC with autosampler (Dionex, USA) were used at a wavelength of 220 nm and columns used are Dionex IonPac NS1 Column (4 mm i.d. 150 mm), Dalian Johnsson CenturySIL C18 Column (4.6 mm i.d. 150 mm) and Dalian Sipore Tigerkin C18 Column (4.6 mm i.d. 150 mm), with a flow rate 1.0 mL/min. Column temperature is 25 8C, and the injection volume is 10 mL. The ion-pair reagent is TBAOH with concentrations ranging from 5 mmol/L to 20 mmol/L. 2. Results and discussion Ion-pair chromatography is based on a liquid–liquid partition technique usually called ion-pair extraction or ionpair partition. Ionized organic compounds can be extracted into organic phases as ion-pairs with counter ions of suitable hydrophobicity. The effect of surfactants on chromatographic separation has been studied after the optimization of mobile phase. Fig. 1(a) represents a chromatogram obtained without surfactant on a silica gel matrix column, while Fig. 1(b) represents the use of 1 mmol/L Tween 20 on the same column. Comparing these two chromatograms, it is obvious to found that the retention times of strong retention compounds in Fig. 1(b) are much shorter than in Fig. 1(a). Elution of all compounds changes from 33.5 min to less than 23.0 min without affect of the separation efficiency. Hence, the addition of surfactant accelerated the separation, and saved analytical times and reagents, which accorded with the demands of green chemistry. On the other hand, similar results are gained on polymer matrix columns when the same surfactant of different concentrations is used as mobile phase additive (Fig. 2(a)). The addition of surfactant under proper concentration may shorten retention times of analytes as well as improve the peak shapes without decrease of the resolution, especially for strong retention compounds. However, if too much surfactant is added, the resolution will decrease and some compounds cannot be separated. Besides, different surfactants in Tween series have been tested as mobile phase additives (Fig. 2(b)), among which Tween 20 and Tween 40 have similar effects on separation. Tween 60 and Tween 80 have shortened the retention times too greatly to separate all 10 organic acids. Thus, only Tween 20 and Tween 40 are good choices to be used as mobile phase additives. In ion-pair chromatography, there is an electric double layer between the mobile phase and stationary phase. Counter ions and organic additives can be adsorbed onto hydrophobic stationary phase. It is impossible for the counter ions with same charge to cover the surface of stationary phase completely according to repulsion principle. Hence, a balance will be generated between these two phases. Sample molecules with opposite charges can be attracted by counter ions and establish an electrostatic balance between mobile phase and stationary phase. Thus, the separation of polar compounds can be realized. Surfactant ions can move into the inner layer of the electric double layer and also be adsorbed onto stationary phase. They compete with organic additives for the adsorption sites on stationary phase and establish a dynamic balance with the solutes in order to accelerate the separation. Moreover, the surfactant can inhibit
Y.Y. Zhong et al. / Chinese Chemical Letters 21 (2010) 453–456
455
Fig. 1. Chromatograms of 13 organic acids separated by (a) traditional mobile phase without additives and (b) novel mobile phase with 1 mmol/L Tween 20 on silica gel supporters. Peaks identified as: (1) ascorbic acid, (2) 3,5-dihydroxybenzoic acid, (3) 4-hydroxybenzoic acid, (4) syringic acid, (5) 3-hydroxybenzoic acid, (6) tropic acid, (7) 2,4-dihydroxybenzoic acid, (8) 2,5-dihydroxybenzoic acid, (9) benzoic acid, (10) 4-methylbenzenesulfonic acid, (11) 2-iodobenzoic acid, (12) salicylic acid, and (13) 2-(2,4,5-trifluorophenyl)acetic acid.
Fig. 2. Retention times of 10 organic acids under (a) different concentrations of Tween 20 and (b) different surfactants of Tween series of same concentration. Samples identified as: (1) ascorbic acid, (2) 4-hydroxybenzoic acid, (3) caffeic acid, (4) 2-methylbenzoic acid, (5) 2,5dihydroxybenzoic acid, (6) 2-iodobenzoic acid, (7) 2-(2-chlorophenyl)acetic acid, (8) sulfosalicyclic acid, (9) 2-(2,4,5-trifluorophenyl)acetic acid, (10) salicylic acid, (11) D-( )mandelic acid, (12) 2-(3,4-dimethoxyphenyl)acetic acid, (13) 3-(3,4-dimethoxyphenyl)acrylic acid, (14) 4methylbenzene-1-sulfonyl chloride, (15) 3-(3-nitrophenyl)acrylic acid, (16) 4-iodobenzoic acid, and (17) 3-(4-chlorophenyl)acrylic acid.
the analytes from going into the inner side of electric double layer because of steric effect. When the size of analyte increases, the effect will enhance. Thus, the strong retention compounds are more changeable after the addition of surfactants. 3. Conclusion As a novel additive of mobile phase, the use of low concentration surfactant can greatly accelerate the separation in ion-pair chromatography. Accordingly, the defects in traditional micellar chromatography, such as the decline of column efficiency and resolution can be overcome. In a word, it is an easy and convenient method to fasten the separation of polar organic macromolecules by ion-pair chromatography. Acknowledgments This research was financially supported by National Natural Science Foundation of China (No. 20775070), Zhejiang Qianjiang Project of Science and Technology for Competent People (No. 2008R10028), Zhejiang Provincial Natural Science Foundation of China (Nos. R4080124, Y4080064) and Zhejiang Provincial Analysis and Testing Foundation of China (No. 2007F70061).
456
Y.Y. Zhong et al. / Chinese Chemical Letters 21 (2010) 453–456
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
M.J. Ruiz-a´ngel, J.R. Torres-Lapasio´, M.C. Garcı´a-a´lvarez-Coque, et al. J. Chromatogr. A 1216 (2009) 3199. M.J. Ruiz-a´ngel, S. Carda-Broch, J.R. Torres-Lapasio´, et al. J. Chromatogr. A 1216 (2009) 1798. A.A. Sakalgaonkar, S.R. Mirgane, R.P. Pawar, Chromatographia 68 (2008) 143. D.Y. Chen, S.M. Jiang, Y.Y. Chen, et al. J. Pharm. Biomed. Anal. 34 (2004) 239. C.E. Doneanu, W.B. Chen, J.C. Gebler, Anal. Chem. 81 (2009) 3485. T. Cecchi, P. Passamonti, J. Chromatogr. A 1216 (2009) 1789. R.P. Patel, C. Narkowicz, G.A. Jacobson, Anal. Biochem. 387 (2009) 113. S. Lopez-Grio, M.C. Garcia-Alvarez-Coque, W.L. Hinze, et al. Anal. Chem. 72 (2000) 4826. D.P. Thomas, J.P. Foley, J. Chromatogr. A 1205 (2008) 36. S.L. Ma, S. Shen, N. Haddad, et al. J. Chromatogr. A 1216 (2009) 1232. X. Li, J.S. Fritz, Anal. Chem. 68 (1996) 4481. X. Li, J.S. Fritz, J. Chromatogr. A 728 (1996) 235. M.J. Dickman, J. Chromatogr. A 1076 (2005) 83. S. Aburuz, J. Millership, J. McElnay, J. Chromatogr. B 798 (2003) 203.