Study On The Novel On-line Sample Preconcentration Techniques And New FI-CE System

Since the early 1980s, the theory and application research of high performance capillary electrophoresis (HPCE) has proved to be one of the most active fields in analytical chemistry because of its high resolution, high efficiency, short analysis time, low cost and sample consumption. In addition, in order to solve the problems of non-continuous injection mode and low precision in CE, flow injection-capillary electrophoresis (FI-CE) has been developed and applied to various fields. However, because of the large sample consumption, FI-CE was confined in some practical application. Furthermore, the short inner diameter (dozens of micro-meter) of the capillary in CE or FI-CE leads to a low sensitivity when using an UV detector, which is still one of the main issues in CE and FI-CE. Hitherto, one of the leading questions that analytical workers focus on is the improvement of CE in sensitivity. In order to decrease the sample consumption in FI-CE and further improve the concentration sensitivity of CE and FI-CE, the following major innovative work was performed in this dissertation on the basis of the previous literatures: 1. Modified Micelle to Solvent Stacking-Capillary Zone Electrophoresis (MSS-CZE) was developed based on methanol assisted micelle collapse for the first time, and it was applied to focus and separate ephedrine and berberine in urine sample.2. Based on the micelle collapse technique, modified Micelle to Solvent Stacking was applied to Nonaqueous Capillary Electrophoresis (MSS-NACE), and the method was used to focus and separate berberine and jatrorrhizine in urine sample.3. A novel cross-H-channel interface was constructed to improve the flow injection-capillary electrophoresis (FI-CE). The performances and practical application of the new FI-CE were investigated.4. An on-line method for the determination of aesculin and aesculetin alkaline hydrolysis rate constants was established using sweeping-flow injection-micellar electrokinetic chromatography (Sweeping-FI-MEKC).This dissertation consists of five chapters.In chapter 1, the combination of FI and CE was detailedly discussed from the necessity, the development, the basic principle, the interface and its practical application. And, the on-line preconcentration methods based on chromatographic effects in CE were reviewed.In chapter 2, the co-solvent of methanol-water was used to facilitate the sodium dodecyl sulfate (SDS) micelles collapse, thereby inducing the on-line sample focusing technique of modified micelle to solvent stacking (MSS). To demonstrate this stacking method, the mechanism of micelles collapse in co-solvent was discussed. The details of the required conditions were investigated and the optimum conditions were:running buffer,20 mM H3BO3-20 mM NaH2PO4 (pH 4.0); micellar sample matrix,20 mM SDS-20 mM H3BO3-20 mM NaH2PO4 (pH 4.0); co-solvent buffer,20 mM H3BO3-20 mM NaH2PO4 in methanoi/water (90:10, v/v). The validity of the developed method was tested by determination of alkaloid compounds (ephedrine and berberine) in urine sample. Under above conditions, this proposed method afforded limits of detection (LODs) of 0.5 and 1.1 ng/mL with 300 and 1036-fold improvements in sensitivity for ephedrine and berberine, respectively, within 15 min.In chapter 3, the new on-line sample preconcentration method of modified micelle to solvent stacking (MSS) in nonaqueous capillary electrophoresis (NACE) was established for the first time. In this proposed MSS-NACE, sodium dodecyl sulfate (SDS) micelles transport, release, and focus analytes from the sample matrix to the running buffer using methanol as its solvent. After the focusing step, the focused analytes were separated via NACE. The focusing mechanism and influencing factors were discussed using berberine (BBR) and jatrorrhizine (JTZ) as model compounds. Under the optimum conditions, this method was applied to derermination of BBR and JTZ in urine, and afforded limits of detection (S/N= 3) of 0.002μg/mL and 0.003μg/mL for BBR and JTZ, respectively. In contrast to conventional NACE, the sensitivity was improved 128-153-fold.In chapter 4, a new cross-H-channel interface was constructed to improve flow injection-capillary electrophoresis (FI-CE) technique. The sample requirement of this novel FI-CE system was reduced distinctly and usual sample dilution in the sample transport process was obviously decreased compared to the typical FI-CE, thereby spontaneously enhancing the sensitivity. Moreover, because of the unique structure of the cross-H-channel interface, field amplified sample stacking (FASS) and high-speed CE were skillfully combined to further improve the sensitivity and to shorten separation time. Under the optimum conditions, this new FI-CE was applied to determination ephedrine (E) and pseudoephedrine (PE) in human urine quantitatively. Up to 45 repeated injections per hour and clearly baseline separation of E and PE in less than 1 min were achieved, giving limits of detection (LODs) of 0.23 and 0.21μg/mL for E and PE, respectively, and yielding relative standard deviation (RSD) values of the migration time and the peak height (n = 5) of 2.6% and 3.1% for E,2.3% and 3.3% for PE, respectively. In contrast to typical FI-CE, approximately 8-250-fold decreases in sample volume requirement,7-fold shortening in separation time and 50-fold improvements in sensitivity were obtained.In chapter 5, an on-line method for the determination of aesculin and aesculetin hydrolysis rate constants was established using sweeping-flow injection-micellar electrokinetic chromatography (Sweeping-FI-MEKC). With this method, all the analytes in the sample were well separated within 5 min, and a sampling frequency of 12 per hour was achieved. At this sampling frequency, a rate constant could be obtained within 30 min. In contrast to off-line methods, it was quite time-saving. And the aesculin hydrolysis mixture solution was directly injected and analyzed without quenching the reaction, thus the information of the hydrolysis process could be obtained from the electrophorogram of a sequence injection. Under the optimum conditions, the hydrolysis rate constants for aesculin at 25,30,35,40 and 45℃using 0.1 M KOH as hydrolyste were obtained as 3.65x10"2/min,5.24×10-2/min,7.12×10-2/min,10.53×10-2/min and 16.27×10-2/min, respectively. The activation energy for aesculin hydrolysis was 58.57 kJ/mol. The hydrolysis rate constants for aesculetin at 15,20,25,30, and 35℃using 10 mM KOH as hydrolyste were obtained as 2.26×10-2/min,2.85×10-2/min,3.55×10-2/min,4.38×10-2/min and 5.29×10-2/min, respectively. The activation energy for aesculin hydrolysis was 31.55 kJ/mol.