| HPLC is an important branch of chromatography, the mobile phase is liquid, using high-pressure infusion systems, the mixed solvents of different polarity or different proportions, buffer solvents are all pumped into the fixed phase column. In the column the components are separated and flow into the detector in order to achieve the specimen analysis. In addition to the HPLC stationary phase and mobile phase, an important way to promote the development of HPLC techniques is designing a new type of detector. RRS is a new spectrum analysis technique developed in recent years. It has high sensitivity (relative to the fluorescence spectrometry), and has1-2orders of magnitude lower detection limit than absorption spectroscopy. Therefore it is suitable for the analysis and detection of trace and ultra-trace substances, and it applies to the the reaction system with weak binding force.As long as the reaction can lead to the changes of molecular size, morphology, hydrophobicity, molecular conformational or the occurrence of absorption-scattering, fluorescence-scattering resonance energy transfer reaction system is usually lead to the significant changes of RRS, so it is suitable for the biochemical and pharmacal analysis. However, RRS has low selectivity, does not identify substances with similar functional groups. So we can take advantage of the high selectivity of HPLC to fill the vacancies of the RRS technology. At present, only a few analytical chemists take note of this new method.This article established the the idea of HPLC-RRS hyphenated technology and designed the post-column homogeneous device. This strategy combined the high separation capacity of HPLC and the sensitivity of RRS, which designed RRS method as an on-line technology. The method was validated in the detection of different drugs, and its kinetics was studied in this article, the quantum chemistry and dynamics of this method was supplemented, respectively. HPLC-RRS analysis and HPLC detector development are of great significance. It is undoubtedly that HPLC-RRS is of great significance for the analysis of RRS and the development of HPLC detector. This dissertation consists of four chapters:introduction, research reports (mainly includes the following chapter2～4three chapters).Chapter1:IntroductionThe introduction section briefly describes development of HPLC, a brief overview of the RRS, the design of the post-column homogeneous devices, the "interface" of the HPLC and homogeneous devices, the research and application of HPLC-RRS technology, as well as the research of HPLC-RRS technology and vision for the future.Chapter2:The dynamics of a novel method coupling high performance liquid chromatography with resonance Rayleigh scatteringThe dynamics of a novel method coupling high performance liquid chromatography (HPLC) with resonance Rayleigh scattering (RRS) is represented in the paper. The method was employed in the detection of fluoroquinolones of urine samples from healthy human beings, by forming ion-association complexes between the components separated from HPLC and [Ce(OH)3]+as the molecular recognition probe. The RRS signal was measured at atλex=λem=365nm. It was applied to detect three FQs and obtained satisfactory results. The RRS spectral characteristics of the analytes and the kinetics of flow rate, proportion of organic phase, reaction time and the aggregation level of ion-association complexes were investigated, which provided a new basis for the development of the hyphenated techniques. It was established that the presence of HPLC-RRS would open up a new field in the determination of analytes with the absence of UV or fluorescence.Chapter3:Description and validation of coupling high performance liquid chromatography with resonance Rayleigh scattering in aminoglycosides determinationIn view of the fact that many substances generally exhibit very little ultraviolet absorbance and the absence of native fluorescence, a new strategy with simple instrumentation and excellent analytical performance combining high performance liquid chromatography (HPLC) with resonance Rayleigh scattering (RRS) was developed. It was validated for the quantification of aminoglycosides (AGs). This fact was also carefully calculated by quantum chemistry. However, the sensitivity was probably limited by the volume of flow-through cell. Therefore, the result calls for a suitable one to ensure optimal RRS signal. Interestingly, when serum or urine samples of analytes were analyzed by this method, they were all well resolved without any interference, which would hold a new perspective to be applied in the determination of substances in biological matrix.Chapter4:Study on an on-line chromatographic strategy with resonance light scattering and its experimental validation Light scatter at the wavelength close to the absorbance maximum as a detection method in a flowing stream was presented. In weak acidic medium, CR combined with TOB to form an ion-association complex by electrostatic interaction, hydrophobicity and charge transferring effect. Throwing off the immovable RRS technique, the proposed strategy allows insights into the HPLC-RRS and has overcome the challenge of detecting analytes lack of useful spectroscopic and electrochemical properties. We had performed a detailed computational chemistry theoretical study on the mechanism of the ion-association complex and ESI-MS/MS to demonstrate the reliability of HPLC-RRS method. Such coupling seems to be a dramatic improvement compared with other detection techniques without any laborious steps. |
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