Construction And Application Of Liquid Chromatography Systems Based On A New Type Of Stable Flow Micro-nano Pump

Miniaturization and ultrahigh performance are two important trends in the field of modern high performance liquid chromatography,which can improve the separation and analysis efficiency and expand the application.Based on the research of "National Key Scientific Instrument and Equipment Development Projects" by the Ministry of Science and Technology in the "12th Five-Year Plan",various of new micro-nano ultrahigh performance liquid chromatographic pumps were developed and evaluated taking advantage of high precision direct-driven motors.By means of upgrading other components of the chromatographic system to adapt the nanoliter conditions,nano liquid chromatographic system and several kinds of integrated protein analysis platforms were constructed so that samples of proteomics could be analyzed efficiently.Reynolds number of water,methanol and acetonitrile at micro-nano flow rates was studied.On the conditions of 100 n L/min-100 ?L/min flow rates and 0.001"-0.007" tubing inner diameters,the Reynolds number range of the three mobile phase was calculated from0.019 to 91.It was indicated that the mobile phases were steady laminar flow and the mixture of mobile phases was dominated by inefficient molecular diffusion.Liquid compressibility at micro-nano flow rates was analyzed in theory.This illustrated that the compressibility of mobile phase could also affect the flow rate and gradient accuracies of micro-nano pump.The mass method and flowmeter method for measurement of micro-nano flow rates were compared.Results shown that the flowmeter method could measure the instantaneous flow rates which was more suitable for the tests of micro-nano pump.Based on the flowmeter method,the flow pulsations of normal chromatographic pumps at microliter flow rate were studied.The flow pulsations of two types of pumps were reduced from 7% to 1% by a pulsation damper.Based on the high precision direct-driven step motor,a single-head reciprocating micro pump and a double-head reciprocating micro pump were developed.According to the theoretical parameters of the motor,the minimum flow rate and the maximum pressure of the pumps were 6 nL/min and 95 MPa respectively for a 2 mm plunger rod.The evaluation of the single-head reciprocating micro pump without backpressure indicated that the flow rate accuracy was better than 4%,the repeatability RSD and the stability RSD were lower than 0.1% and 1% respectively in the range of 5-50 ?L/min.A inlet check valve with an auxiliary sealing spring and an outlet check valve with double valve balls and seats were designed after optimizing the structure of an original check valve.Testing results shown that the performance of the new inlet check valve was similar with a commercial check valve and the new outlet check valve could achieve better sealing than a commercial check valve.An auxiliary sealing solenoid valve was applied to the pump and the deviation of the pump decreased to 1%.A kind of double-plunger full-stoke variable velocity control software was developed to study the alternation rule of the double-head reciprocating micro pump with different backpressure.By optimizing the intake velocity and alternation time,continuous and stable infusion at the flow rates of 50 ?L/min and 25?L/min under the backpressure of 60 MPa and 28 MPa was realized.Two kinds of direct-driven nano gradient chromatographic pumps in different structure were developed and evaluated for their flow rate accuracies,stabilities and gradients.Results shown that at the flow rate range of 50-1000 nL/min,the accuracy and stability RSD of the nano pump based on check valve structure were better than 3% while the two parameters of the nano pump based on switch valve structure were better than 2%and 3% respectively.By means of optimizing the mix tee inner volume,flow cell volume of detectors and precompression process,etc,the gradient results were improved.The gradient accuracy of the nano pump based on switch valve structure could be better than1%.Photodiode array detectors in two kinds of optical paths were designed.The optical paths and optical components were tested on an optical platform and in a prototype.A high energy and a high resolution of 2.8 nm were obtained in the optical system.Two nano flow cell that could be used in an ultraviolet detector and a photodiode array detector respectively were fabricated and the volumes of them were both lower than 100 nL.Furthermore,two nano liquid chromatographic systems based on manual injection and trapping injection were constructed with developed nano pumps,nano optical detectors and commercial mass spectrometer.For the nano liquid chromatographic system based on manual injection,the RSD(n=7)of retention time was better than 1% for the separation of a standard evaluation solution.Reproducible results could be achieved when BSA digests were separated in gradient in the two nano liquid chromatographic systems.The practical application of the constructed nano liquid chromatographic system was carried out.A extraction method of benzo(a)pyrene in fried chips with supramolecular solvent was developed and a spiked sample was separated by the nano liquid chromatographic system.Less organic solvent consumption and less environmental pollution could be realized in the process of extraction and nano liquid chromatographic analysis which was coincident with the trend of green chemistry.Two kinds of protein analysis platforms based on protein direct injection were established and evaluated.By means of online digestion of protein samples and separation of acquired peptides by nano liquid chromatographic system,sequence coverage of more than 90% for BSA and protein identified numbers of 1032 and 849 for He La cell proteins were obtained.Besides,two kinds of protein analysis platforms based on reversed phase liquid chromatography online fractionation and gel filtration chromatography semi-online fractionation were constructed.The two systems were evaluated by He La cell proteins and yeast cell proteins.Results shown that the identified number for He La cell proteins of the online fractionation system was 60% higher than that of the system without fractionation and 2161 proteins could be identified in yeast cell proteins by the semi-online system.By combining reversed phase chromatography and gel filtration chromatography,a more integrated protein analysis platform based on two-dimensional fractionation was created.The fraction overlap could be further reduced and the protein identified number could be further increased,which were beneficial to the high efficient analysis and deep coverage of protein samples.The construction and successful application of these protein analysis platforms based on nano liquid chromatographic system demonstrated that the nano liquid chromatographic system and the platforms based on it had a great prospect in the proteomics research.