亲水作用色谱固定相的可控制备及其在枸杞有效成分分离中的应用研究

Although the number of commercially available columns designed specially for HILIC is growing, there is still not a versatile stationary phase like C18 in RPLC. These special separation materials for HILIC demonstrate good selectivity and reproducibility for separation of polar compounds but still cannot meet the requirements of dynamic binding capacity for the application of sample preparation. In our opinion, preparation of new HILIC stationary phase by surface initiated atom transfer radical polymerization method to improve bonding density of the functional groups should be the appropriate way to solve this problem. Four categories based on the charge characteristics of the functional groups, namely, neutral, positively charged, negatively charged and zwitterionic monomers will be synthesized and be polymerized on to the surface of silica. The prepared stationary phase was used for HILIC mode in the separation of nucleobases and nucleosides to evaluation the HILIC performance of the stationary phases. The selectivity of various stationary phases will be evaluated under the same chromatographic conditions using phenolic acids and flavonoids as model compounds, and the relationship between the charge characteristics of the functional groups and the selectivity of various polar stationary phases for HILIC will be evaluated. The separation behaviors and characteristics of overloading for phenolic acids and oligosaccharides in HILIC will be also studied. Based on the chromatograms obtained from HILIC, the bonding capacity and the separation parameters will be optimized. The bonding capacity of the grafted polymer chain will be controlled via controlling the ratio of monomer to initiator or the reaction time. A preparative HPLC was employed to isolate and purify compounds from Lycium barbarum Linnaeus extract. Two separation modes, HILIC and RPLC, were used in preparative HPLC. HILIC mode was adopted to resolve the purification of the compounds with similar hydrophobicity, which were co-eluted under RP mode. Using the two separation modes during the purification process, phenolic acids and oligosaccharides in fruits of Lycium barbarum Linnaeus, will be purified from by using these two separation modes. The results indicated that the multi-dimensions of preparative HPLC performed under HILIC mode and RPLC mode is an efficient method for the isolation and purification of compounds from natural products.

针对现有亲水作用色谱（HILIC）固定相功能基键合量较低的现状，本项目根据HILIC的分离特点，设计四种电性能不同的单体。使用能可控提高功能基键合容量的表面引发原子转移自由基聚合（SI-ATRP）技术，将单体聚合在基材表面，制备HILIC固定相；选用碱基和核苷样品评价固定相的HILIC性能。以酚酸和寡糖标品为表征物,研究固定相功能基电性能和固定相选择性之间的作用机理。研究超载情况下, HILIC固定相对样品的吸附行为及各分离参数的变化规律，结合固定相功能基的键合容量，筛选出可控制备HILIC固定相的最佳工艺条件。使用HILIC与RPLC组成的离线二维色谱模式对枸杞中酚酸和聚糖进行分离制备。本项目提出的可控制备方法也可用于其它类型色谱固定相的制备，为高吸附容量制备色谱柱的研制，提供一种定向可控的制备方法，有重要科学意义和应用前景。

强极性化合物是中药提取物的典型代表，亲水作用色谱法(hydrophilic interaction chromatography, HILIC)，是目前分离强极性化合物的最有效手段，也是目前最有效的强极性和强亲水性样品定性定量分析技术。本项目针对普通化学键合法制备HILIC固定相功能基键合量较低的现状，提出使用能可控提高功能基键合容量的表面引发原子转移自由基聚合（SI-ATRP）技术生产HILIC固定相的方法：将功能单体聚合在硅胶基材表面，制备HILIC固定相。针对溶质与固定相表面功能团之间的选择性作用力是决定溶质保留的事实，设计合成了四种电性能不同的功能单体，完成了四大类电性能不同的功能单体：6-O-甲基丙烯酰基-D-吡喃半乳糖（电中性单体）、2-羟基-3-[4-(羟甲基) -1H-1,2,3-三唑-1-炔基]丙基2-甲基丙烯酸酯(HTMA电离后含正电荷单体)，4-乙烯基四唑（电离后含负电荷单体）和1-羧基-N,N-二甲基-N-丙烯酰胺丙基内盐（含两性离子单体）的制备，并开发出相应的HILIC固定相，研究了其亲水作用色谱分离性能。发现由于表面电性能随溶液pH值调控容易，含两性阴阳离子（zwitterionics）功能基的功能单体在分离方面有优势，进一步设计合成了单体N,N-二甲基-N-丙烯酰氧乙基-N-丙基磺酸铵，甲基丙烯酰氧乙基二甲基乙酸铵，N-乙烯基咪唑-N-丙磺酸盐，3 - (2,3-二羟基丙基) -1-乙烯基咪唑氯盐，2-甲基丙烯酰氨基组胺酸等不同种类含两性离子功能基的功能单体，制备了多种HILIC固定相，研究了所得固定相的分离性能。以酚酸和黄酮化合物标品为表征物,研究固定相功能基电性能和固定相选择性之间的作用机理。结合固定相功能基的键合容量，筛选出可控制备 HILIC 固定相的最佳工艺条件。使用 HILIC 与 RPLC 组成的离线二维色谱，对枸杞水提物采用类组分离的方法进行分离，对其中酚酸和黄酮类化合物进行了分离。本项目提出的分离分析方法，为高吸附容量制备色谱柱的研制提供了一种可靠方法。