L-异亮氨酸键合色谱固定相的制备及其性能
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摘要
利用异氰酸丙基三乙氧基硅烷与L-异亮氨酸反应合成了一种新型的硅烷偶联剂,并进一步将其与硅胶反应制得键合有L-异亮氨酸的亲水色谱固定相。通过核磁共振氢谱表明亮氨酸功能化硅烷偶联剂的成功合成、元素分析表征证明亮氨酸已成功键合到硅胶表面。将其作为亲水模式下的固定相填料填装在150 mm×4. 6 mm不锈钢色谱柱中,以一系列经典的极性小分子作为探针,考察了这些探针分子在固定相上的色谱行为。极性化合物的保留时间随着流动相中有机溶剂含量提高而逐渐增大,表现出典型的亲水保留特征。进一步研究了流动相中乙腈含量、缓冲盐p H值及缓冲盐浓度等因素对分析物在固定相上的保留的影响。在优化了相关参数后,将固定相应用于碱性化合物、水溶性维生素以及核苷类极性物质的分离当中。在等度洗脱下,5种碱性化合物、6种水溶性维生素和8种核苷类物质分别在8、18及25 min内被成功分离。分离结果表明了合成的L-异亮氨酸键合亲水色谱固定相具有较好的色谱性能,在极性化合物的分离上具有良好的应用前景。
L-Isoleucine-bonded silica stationary phase was prepared starting from L-isoleucine and 3-isocyanatopropyltriethoxysilane,and further reaction between the resulting L-isoleucine functionalized silane coupling agents and activated silica. The result of ~1H NMR spectroscopy showed that the expected L-isoleucine functionalized silane was successfully synthesized. The successful grafting was further confirmed by elemental analysis. The synthesized stationary phase was slurry-packed into stainless steel columns( 150 mm ×4. 6 mm). A set of typical polar compounds were employed to evaluate the chromatographic behaviors on the stationary phase in hydrophilic interaction liguid chromatography( HILIC) mode. Increased retention factors were observed for these compounds with the increase of acetonitrile( ACN) content in the mobile phase,demonstrating a typical HILIC retention characteristics. Additionally,the effect of various parameters,such as ACN content,pH values and ionic strength of the mobile phase on the retention of the tested solutes were investigated. Under optimized conditions,5 basic compounds,6 water-soluble vitamins and 8 nucleosides were successfully separated within 8 min,18 min and 25 min,respectively,by isocratic elution. The resulting separation chromatogram exhibits remarkable chromatographic performance, demonstrating the excellent application potential of the L-isoleucine-bonded silica stationary phase in the separation of polar compounds.
L-Isoleucine-bonded silica stationary phase was prepared starting from L-isoleucine and 3-isocyanatopropyltriethoxysilane,and further reaction between the resulting L-isoleucine functionalized silane coupling agents and activated silica. The result of ~1H NMR spectroscopy showed that the expected L-isoleucine functionalized silane was successfully synthesized. The successful grafting was further confirmed by elemental analysis. The synthesized stationary phase was slurry-packed into stainless steel columns( 150 mm ×4. 6 mm). A set of typical polar compounds were employed to evaluate the chromatographic behaviors on the stationary phase in hydrophilic interaction liguid chromatography( HILIC) mode. Increased retention factors were observed for these compounds with the increase of acetonitrile( ACN) content in the mobile phase,demonstrating a typical HILIC retention characteristics. Additionally,the effect of various parameters,such as ACN content,pH values and ionic strength of the mobile phase on the retention of the tested solutes were investigated. Under optimized conditions,5 basic compounds,6 water-soluble vitamins and 8 nucleosides were successfully separated within 8 min,18 min and 25 min,respectively,by isocratic elution. The resulting separation chromatogram exhibits remarkable chromatographic performance, demonstrating the excellent application potential of the L-isoleucine-bonded silica stationary phase in the separation of polar compounds.
引文
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[19]HAN Xiaoqian,LIN Yunyun,LI Zhen,et al. Preparation and Application of Novel Bonded L-Valine and L-Alanine Derived Chiral Stationary Phases[J]. Chinese J Appl Chem,2018,35(1):68-74(in Chinese).韩小茜,蔺云云,李臻,等.新型氨基酸衍生物手性固定相的制备及应用[J].应用化学,2018,35(1):68-74.
[20]Hayriye A,?elik K S,Alt?nda? R,et al. Synthesis,Characterization,and Application of a Novel Multifunctional Stationary Phase for Hydrophilic Interaction/Reversed Phase Mixed-Mode Chromatography[J]. Talanta,2017,174(1):703-714.
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[3]Alpert A J. Hydrophilic-Interaction Chromatography for the Separation of Peptides,Nucleic Acids and Other Polar Compounds[J]. J Chromatogr,1990,499(2):177-196.
[4] Regnier F E,Noel R. Glycerolpropylsilane Bonded Phases in the Steric Exclusion Chromatography of Biological Macromolecules[J]. J Chromatogr Sci,1976,14(7):316-320..
[5]Wu J,Bicker W,Lindner W. Separation Properties of Novel and Commercial Polar Stationary Phases in Hydrophilic Interaction and Reversed-Phase Liquid Chromatography Mode[J]. J Sep Sci,2008,31(9):1492-1503.
[6]Risley D S,Strege M A. Chiral Separations of Polar Compounds by Hydrophilic Interaction Chromatography with Evaporative Light Scattering Detection[J]. Anal Chem,2000,72(8):1736-1739.
[7]Nguyen H P,Schug K A. The Advantages of ESI-MS Detection in Conjunction with HILIC Mode Separations:Fundamentals and Applications[J]. J Sep Sci,2008,31(9):1465-1480.
[8]JIN Gaowa,DING Junjie,CHEN Xue,et al. Separation of Glucoside Compounds by Reversed-Phase and Hydrophilic Interaction Liquid Chromatography[J]. J Instrum Anal,2014,33(2):133-137(in Chinese).金高娃,丁俊杰,陈雪,等.反相/亲水色谱法分析糖苷类化合物[J].分析测试学报,2014,33(2):133-137.
[9]Qiu H,Loukotkov L,Sun P,et al. Cyclofructan 6 Based Stationary Phases for Hydrophilic Interaction Liquid Chromatography[J]. J Chromatogr A,2011,1218(2):270-279.
[10]Guo Y,Gaiki S. Retention Behavior of Small Polar Compounds on Polar Stationary Phases in Hydrophilic Interaction Chromatography[J]. J Chromatogr A,2005,1074(1/2):71-80.
[11]Greco G,Letzel T. Main Interactions and Influences of the Chromatographic Parameters in HILIC Separations[J]. J Sep Sci,2013,51(7):684-693.
[12]SHEN Aijin,GUO Zhimou,LIANG Xinmiao. Development and Application of Hydrophilic Interaction Liquid Chromatographic Stationary Phases[J]. Prog Chem,2014,26(1):10-18(in Chinese).沈爱金,郭志谋,梁鑫淼.亲水作用色谱固定相的发展及应用[J].化学进展,2014,26(1):10-18.
[13]Buszewski B,Noga S. Hydrophilic Interaction Liquid Chromatography(HILIC)—A Powerful Separation Technique[J].Anal Bioanal Chem,2012,402(1):231-247.
[14]Jandera P. Stationary and Mobile Phases in Hydrophilic Interaction Chromatography:A Review[J]. Anal Chim Acta,2011,692(1/2):1-25.
[15]García-Gómez D,Rodríguez-Gonzalo E,Carabias-Martínez R. Stationary Phases for Separation of Nucleosides and Nucleotides by Hydrophilic Interaction Liquid Chromatography[J]. Trend Anal Chem,2013,47:111-128.
[16]CHENG Xiaodong,FENG Yuqi. Preparation and Evaluation of N-Acryloyltris(hydroxymethyl)aminomethane-bonded Chromatographic Stationary Phase[J]. Chinese J Chromatogr,2015,33(9):917-921(in Chinese).成晓东,冯钰锜.三元醇丙烯酰胺键合色谱固定相的制备及评价[J].色谱,2015,33(9):917-921.
[17]GUO Zhimou,ZHANG Xiuli,XU Qing,et al. Stationary Phases for Hydrophilic Interaction Liquid Chromatography and Their Applications in Separation of Traditional Chinese Medicines[J]. Chinese J Chromatogr,2009,27(5):675-681(in Chinese).郭志谋,张秀莉,徐青,等.亲水作用色谱固定相及其在中药分离中的应用[J].色谱,2009,27(5):675-681.
[18]Olsen B A. Hydrophilic Interaction Chromatography Using Amino and Silica Columns for the Determination of Polar Pharmaceuticals and Impurities[J]. J Chromatogr A,2001,913(1/2):113-122.
[19]HAN Xiaoqian,LIN Yunyun,LI Zhen,et al. Preparation and Application of Novel Bonded L-Valine and L-Alanine Derived Chiral Stationary Phases[J]. Chinese J Appl Chem,2018,35(1):68-74(in Chinese).韩小茜,蔺云云,李臻,等.新型氨基酸衍生物手性固定相的制备及应用[J].应用化学,2018,35(1):68-74.
[20]Hayriye A,?elik K S,Alt?nda? R,et al. Synthesis,Characterization,and Application of a Novel Multifunctional Stationary Phase for Hydrophilic Interaction/Reversed Phase Mixed-Mode Chromatography[J]. Talanta,2017,174(1):703-714.
[21]Hemstrom P,Irgum K. Hydrophilic Interaction Chromatography[J]. J Sep Sci,2006,29(12):1784-1821.