ABSTRACT
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 were 1.61 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 mL/min), focus time (3.00, 3.50, 4.00, 4.50 min), transition time (0.5, 1.0, 1.5, 2.0 min), pH 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 self-constructed 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.
Subject(s)
Fractionation, Field Flow , Lipoproteins , Lipoproteins, LDLABSTRACT
OBJECTIVE: To design and construct aset of a symmetric flow field flow fractionation instrument which can be used to separate biomacromolecules, and preliminarily evaluate the instrument performance to provide practical experience for its application to real samples. METHODS: The apparatus was made up of separator and tempered glass. Polycarbonate membrane was used as the accumulated wall of the channel. In order to solve the problem of focusing samples, a novel focus position was developed. The performance of the asymmetric flow field flow fractionation device was preliminarily studied by injecting samples consisting of standard bovine serum albumin and alcohol dehydrogenase. The impacts of three factors at six levels on separation (resolution R) were investigated, including injection/focus time, cross-in flow and cross-out flow. RESULTS: Along with the decrease of focus time and increase of cross-in flow velocity, the resolution increased obviously. CONCLUSION: The self-made asymmetric flow field flow fractionation instrument can successfully separate protein mixtures and it is expected to separate more complex samples.