基于非对称流场流分离系统优化脂蛋白的分离条件

Optimization of the experimental condition on separating lipoproteins by self-constructed asymmetrical flow field-flow fractionation

目的:使用自组装非对称流场流分离(asymmetrical flow field-flow fractionation,AF4)仪器对脂蛋白的分离条件进行优化,分析仪器的分离性能并验证载液黏度对分离效果的影响。方法:使用2种标准蛋白(碳酸酐酶和甲状腺球蛋白)初步验证自组装非对称流场流分离仪器的性能,标定分离通道相关参数(压制后的分离通道实际高度),进而通过四水平五因素的正交设计试验进行脂蛋白的分离条件优化,通过直观分析和方差分析评价差异具有统计学意义的影响因素,并对这些影响因素进行深入验证分析。结果:通过调节流速条件及载液条件得到碳酸酐酶和甲状腺球蛋白混合样品的分离图,根据流体动力学原理及Stoke’s公式计算得到分离通道高度为164μm。使用正交设计试验得到优化后的分离条件可实现高密度脂蛋白与低密度脂蛋白大于1.50的分离度(分离度为1.61和1.58),所选取的5种主要影响因素及其水平分别为分离总流速(3.00,3.50,4.00,4.50 m L/min)、聚焦时间(3.00,3.50,4.00,4.50 min)、过渡时间(0.5,1.0,1.5,2.0 min)、载液p H(6.8,7.00,7.20,7.40)、载液黏度(使用不同浓度的羟丙基甲基纤维素调节载液黏度,其浓度分别为0.00%,0.03%,0.06%,1.00%),正交设计试验结果显示,5种主要因素中载液黏度对分离度影响最大,结果差异具有统计学意义。进一步分析载液黏度的影响,发现增大黏度可有效提高分离度,但会带来增大负峰的作用。结论:自组装非对称流场流分离仪器可根据颗粒的粒径差异对脂蛋白进行有效分离,在优化的条件下可实现高、低密度脂蛋白的基线分离,载液黏度对分离度的影响差异具有统计学意义。

Objective： To analyze and characterize the separation effectiveness of self-constructed asymmetrical flow field-flow fractionation system on proteins and lipoproteins,to achieve the optimization of the experimental conditions when separating lipoproteins by orthogonal design test and to investigate the carrier viscosity＇s influence on separation effectiveness. Methods： The evaluation of asymmetrical flow field-flow fractionation separation capacity was conducted by using two standard proteins（ carbonic anhydrase and thyroglobulin）. Under the optimized separation conditions of carbonic anhydrase and thyroglobulin,the channel actual thickness（ after assembling,the actual thickness of separation channel was less than initial thickness） was calculated by the analytes＇ elution time based on the hydrokinetic theory.With orthogonal design test the optimized experimental conditions were studied and statistical analysis was carried on to find out the factors with statistical significance which needed further exploration. Results：According to the hydrodynamics principle and Stoke＇s function,the channel actual thickness was measured to be 164 μm by separating the two standard proteins,carbonic anhydrase and thyroglobulin,under proper experimental conditions. By the optimization based on orthogonal design test,base-line separation（ the resolution had to be higher than 1. 50） was achieved. The resolutions of the two experiments were1. 61 and 1. 58. According to previous study/pre-study and supporting theory,in the orthogonal design test,the total 5 factors were integrated for comprehensive investigation： the total flow rate（ 3. 00,3. 50,4. 00,4. 50 m L/min）,focus time（ 3. 00,3. 50,4. 00,4. 50 min）,transition time（ 0. 5,1. 0,1. 5,2. 0 min）,p H of the carrier fluid（ 6. 8,7. 00,7. 20,7. 40） and viscosity of the carrier fluid hydroxypropylmethylcellulose concentration： 0. 00%,0. 03%,0. 06%,1. 00%）. Among the 5 factors,viscosity was found to have the statistical significance on separation effectiveness which was further investigated.The resolution of high density lipoprotein and low density lipoprotein was increased by the increasing viscosity which also caused more obvious negative spikes. Conclusion： The separating capacities of selfconstructed asymmetrical flow field-flow fractionation system on lipoproteins were verified to be effective and an optimized experimental condition was found to achieve the base-line separation of high density lipoprotein and low density lipoprotein. Viscosity of the carrier fluid was proved to have the statistical significance on lipoprotein separation.