| Capillary separation technologies in liquid phase mainly includes capillary liquid chromatography(CLC), capillary electrophoresis(CE) and capillary electrochromatography(CEC). Pressurized CEC(pCEC) is a typical capillary separation technique, featuring a dual separation mechanism of both CHPLC’s and CE. It has the characteristics of high column efficiency, high selectivity, high resolution and fast speed, also the advantages of precise quantification and gradient elution. It has been widely used in the fields of life science, pharmaceutical, food safety and environmental monitoring. Nowadays, detectors suitable for p CEC include UV, laser-induced fluorescence, electrochemical detectors and mass spectrometry. Unlike high performance liquid chromatography(HPLC), capillary liquid separation system is lacking a universal detector.Since 1960’s or 70’s, separation technique of HPLC has been rapidly developing. It stimulated the study of ELSD as a universal detector. The universality was shown in aspects of its independence of either the material structure or optical properties, therefore ELSD is able to detect almost any substances with lower volatility than the mobile phase. ELSD is very suitable for capillary separation system detection, as it contributes little to chromatographic peak widening. It is therefore of great significance to develop the ELSD as a universal detector for pCEC. ELSD mainly adapts to the conventional HPLC, and it does not accommodate the eluent flow rate of capillary liquid separation system(in micro- or nano liter/min). Our objective is to develop and optimize a micro-fluidic ELSD(μELSD) suitable for p CEC. The development of μELSD has been achieved, and the platform of μELSD coupled to pCEC was established. In the thesis, micro-fluidic nebulization, evaporation, sheathing and light scattering system are further investigated and optimized. The performance of μELSD has been improved significantly. The μELSD coupled to pCEC are applied in the separation and detection of six active components, e.g. platycodin D, peimisine, oleanolic acid, imperialine, peimine and peiminine in Chuanbei Pipa Syrups, thus demonstrating the practicability of pCEC-μELSD instrument. The development and optimization of μELSD has broken new ground of wider applications for capillary liquid separation technologies, such as p CEC, CLC, CEC, CE.There are five chapters in this thesis, in which the main contents are as following:The first chapter described the essential structure, operating principle, and advantage of ELSD. Then the study of capillary separation system and its combination with relevant detectors was summarized. In addition, Chuanbei Pipa Syrups and its pharmacological effects and analytical methods of effective components were briefly introduced.In chapter two, micro-fluidic nebulization and evaporation system were investigated and optimized respectively. Firstly, the nebulization system was further investigated by reducing the inner diameter(i.d.) of the nozzle from 470 μm to 370 μm and the wall thickness of the capillary spray needle from 155 μm to 65 μm. The trailing factor of the peak was reduced from 3.6 to 1.2, the flow rate of the corresponding carrier gas decreased from 2.0 L/min to 0.25 L/min, the signal response was improved five times. Secondly, the fluid field and trajectories of the droplets subsequent to nebulization were investigated via computational fluid dynamics(CFD) to validate the nebulization designing and implied that the inner diameter of the drift tube should be optimized. The optimal i.d. of the evaporative tube was c.a. 12 mm, and verified that the inspiration gained from the CFD. The evaporative tube was evenly heated by heating resistance wire wrapped around and temperature was steadily controlled by the PID temperature control. Evaporation for μELSD could be accomplished at relatively low temperatures that facilitated detection for volatile or thermally sensitive analytes and enlarged the application range.Chapter three investigated a newly designed sheath gas module that was suitable for capillary separation and a light scattering chamber. The optimal sheath gas flow rate was 0.5 L/min, a higher signal to noise ratio, a better peak symmetry and stability were achieved. The investigation of laser power showed that the baseline noise was proportional to signal response as the laser power increasing. A slant light trap decreased the background absorption, expanded the linear range.A platform of μELSD coupled to p CEC was built and applied to the separation and detection of six active components, e.g. platycodin D, peimisine, oleanolic acid, imperialine, peimine and peiminine in Chuanbei Pipa Syrups in the fourth chapter. The evaluation of pCEC-μELSD system demonstrated that μELSD accommodated an effluent flow rate of 0.1 to 2.0 μL/min, the RSDs of the peak area were 1%(n=6), the fine linearity of glucose ranged from 0.1 to 20 ng and the LOD was as low as 100 pg. The confirmatory experiments investigating the effects of μELSD on peak broadening validated that it contributes little to chromatographic peak broadening. The simultaneous analysis of six active components by pCEC-μELSD was performed on a C18 capillary column with mobile phase A: acetonitrile and mobile phase B: 10 mmol/L formic acid-triethylamine aqueous solution(pH 11.0) in gradient elution. The optimal conditions were as follows: column flow rate: 1.02 μL/min, applied voltage: +10 k V, nebulizing gas flow rate: 0.4 L/min and evaporative temperature: 40℃. Six active components in Chuanbei Pipa Syrups were successfully separated in 10 min with the excellent linearity of four orders of magnitude. The limit of detection(LOD) reached to the pg level, and the recoveries were between 97.9% and 103.0%. The precision, repeatability and stability indicated a good reproducibility for the determination. The results sufficiently verified the feasibility and practicality of pCEC-μELSD platform and showed the advantage of separation and determination of the active ingredients in complicated traditional Chinese medicine.In chapter 5, we summarized the development and advances, as well as provided our view on the outlook and prospect of the pCEC-μELSD technology. |
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