The Research And Applications Of High Performance Liquid Chromatography-Resonance Rayleigh Scattering Detection Technique

High performance liquid chromatography (HPLC) has become an important branch of analytical science with the characteristics of high separation efficiency, rapid analysis, high sensitivity, and widely application, etc. The development of a new high efficient and general detection technique is the major development trend of HPLC. Resonance Rayleigh scattering(RRS)has aroused widespread concerned because of its simplicity and high sensitivity characteristics, but it is not enough for detecting complicated compound. Thus, there is great potential in the incorporation of HPLC and RRS and the incorporation will become a promising analysis technique.At present, there are many proteins and aminoglycoside antibiotic analytic methods. Most of them need to be derivated or detected with short-ultraviolet, which could decrease the accuracy and sensitivity. So the separation and determination of them has great significance in the fields of medical study, quality supervision, biomedicine and other correlative fields. In this thesis, a novel detection technique for high performance liquidchromatography-resonance Rayleigh scattering(HPLC-RRS) has been developed which can be applied to detect real samples. The research results are shown as follow:PartⅠ.A new detection system for high performance liquid chromatography - resonance Rayleigh scattering(HPLC-RRS) has been explored. The feasibility of this technique of HPLC-RRS was studied by using SDS as the probe of Ins,Cyt-c,Lys, HSA,the methodology of HPLC-RRS technique has been researched as well. The comprehensive experimental scheme has been established by building the post-column ion-association model. The technique was applied to detect four proteins successfully, and a better linear relation,high reproducibility and high sensitivity were obtained and will have a perfect perspective.PartⅡ. Proteins were separated and detected by using biebrich scarlet (BS) as the molecular recognition probes with the HPLC-RRS technique which has been established in PartⅠ. The experiment condition was performed on a sinochrom ODS-AP column(150 mm) with gradient elution, the mobile phase was trifluoroacetic acid- acetonitrile. The utility of present method was demonstrated by the separation and determination of four proteins involving Cyt-c, Lys, HSA,γ-Glo in 12 minutes. Combined with BS, the proteins formed larger ion-association complex, the RRS signal was detected atλEx =λEm=376 nm. A linear range was found between concentration and peak area at the range of 0.20～3.0μg·mL-1 for Cyt-c, 0.25～2.5μg·mL-1 for HSA, 1.5～10μg·mL-1for Lys, and 2.0～15μg·mL-1 forγ-Glo, with linear regression coefficients of all above 0.99. When the S/N = 3, the detection limit of the four kinds of proteins were within 2.0～10μg·mL-1. The novel method is first used to determine HSA andγ-Glo in human serum sample synchronously.PartⅢ. A new method for detecting tobramycin (Tob) was established by using the pontamine sky blue as the spectroscopic probe by HPLC-RRS. This method required a separation condition performed on a diamond ODS with equivalent elution. The separation condition and the concentration of the probe have been optimized. The parameter of the experiment condition and post column-probe solution on the sensitivity have been optimized including the pH of the buffer solution, the length of the reaction tube, the probe flow rate. Under the optimal experiment condition, Tob could be separated in 10 minutes. This method had been applied to determine the Tob in tobramycin eye drops and the results were in good agreement with commodity marked. This method provided a reliable method for the production and the quality inspection department with rapidity, simpleness, high sensitivity, good repeatability and detected compound lack of chemical detectable characteristics.PartⅣ.A HPLC-RRS method had been developed for the determination composition of gentamicin by using pontamine sky blue as spectroscopic probes. The separation condition was performed on a Platisil C18, the mobile phase consisted of 0.24 % trifluoroacetic acid-methanol (96.5:3.5, v/v) at a flow rate of 0.5 mL?min-1 and the probe flow rate was 0.08 mL?min-1. The RRS signal was detected atλEx =λEm=356 nm. The utility of presented method was demonstrated by the separation and determination of the composition in the gentamicin within 15 minutes. A detection limit of 6.0～7.2μg·mL-1 was reached and a linear range was found between peak area and concentration at the range of 7.2～115.2μg·mL-1 for C1, 8.2～131.6μg·mL-1 for C1a, 7.5～78.8μg·mL-1 for C2 and 8.5～78.0μg·mL-1 for C2a, with linear regression coefficients of all above 0.99. The correlation coefficients of calibration curves of C1, C1a, C2,C2a, were 0.9928,0.9973,0.9980 and 0.9981,respectively. The method was used to detect commercial sample and the results were in good agreement with specification of both China and European Pharmacopoeia (2005 version). This method can meet the requirement of quality controlling and it provides as a new method for the quality supervision station and medical real-time monitoring the antibiotics components with rapidity, simpleness, high sensitivity and good repeatability.