| Enteromorpha prolifera is a kind of economic seaweed with strong fertility,and it is widely distributed in temperate areas of the intertidal zone and inland waters. In recent years,"green tide"have occurred frequently in the coast of China, which have made a great negative impact on marine ecosystem and aquaculture. We should actively explore the way of rational utilization of the resource for Enteromorpha prolifera "green tide", in order to"transform original wastes into treasure", which is benefit to mankind. Enteromorpha prolifera is a good resource of natural pigments. Unfortunately, few studies have been done for analysis of natural pigments in Enteromorpha prolifera, which has negatively influence on the utilities of pigments in Enteromorpha prolifera. In this thesis, new method for the determination of chlorophylls, lutein in Enteromorpha prolifera and preparation of sodium copper chlorophyllin are studied, which lays the theoretical and practical foundation on reasonable utilization of natural pigment. The main studies and results are outlined as follows:1. The research state and trend about Enteromorpha prolifera, chlorophyll and lutein are summarized, which maily included the utilization of Enteromorpha prolifera, the physicochemical properties and physiological functions of lutein and chlorophyll.2. A new high performance liquid chromatography-diode-array detector–electrospray ionization-time of flight mass spectrometry (HPLC-DAD-ESI- TOF/MS) method was established for the identification and quantification of chlorophyll a and chlorophyll b in Enteromorpha prolifera. The proposed method was applied to quantify chlorophyll a and chlorophyll b in Enteromorpha prolifera from Qingdao seashore, which could provide the scientific basis for research and development of natural chlorophyll. Methanol was used to extract the target compounds in Enteromorpha prolifera by ultrasonic-assisted extraction, and the HPLC-DAD-ESI-TOF/MS conditions were as following: Diamonsil-C18 (4.6 mm×150 mm) column, MeOH (with 0.2% acetic acid) and acetone as the mobile phase with linear gradient elution, flow-rate is 0.8 mL/min, DAD (430 nm) and ESI-TOF/MS (in positive mode) detector, injection volume is 10μL. The results indicated that the developed method can eliminate the interference from chlorophyll derivatives and be used for the determination of chlorophyll a and b in Enteromorpha prolifera with a good resolution, precision, reproducibility, recovery and linearity between 0.50～500 mg/L (R2=0.9999). The total contents of chlorophyll a and b are about 1% of dry weight of Enteromorpha prolifera from Qingdao seashore, which showed that Enteromorpha prolifera is a good material for development of natural chlorophyll.3. High performance liquid chromatography- atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MSn) was used for rapid identification of chlorophylls and their derivatives in Enteromorpha prolifera. The fragmentation pathways of chlorophylls and their derivatives were also studied, which could provide a scientific basis for study degradation behavior of chlorophylls in Enteromorpha prolifera. The crude extract of Enteromorpha prolifera was separated by Eclipse XDB-C18 column(4.6 mm×150 mm, 5μm)and detected with DAD and APCI-MS detectors in series. A gradient elution was carried out using water, MeOH(with 0.5% acetic acid) and acetone. Eight compounds (chlorophyll b, chlorophyll a, hydroxypheophytin b, hydroxypheophytin b', hydroxypheophytin a, hydroxypheophytin a', pheophytin a and pheophytin a') in Enteromorpha prolifera could be rapidly identified by the molecular weight data obtained with APCI-MS combined with the literatures. On the other hand, the fragment patterns of eight compounds were preliminary explored based on APCI-MS2 fragment information. We get three rules: 1) The parent ions of chlorophyll a, b and their derivatives are easy to fragment into smaller daughter ions and neutral fragments (m/z 278), and it is possible to form acidic ester compounds by the McLafferty rearrangement. 2) Daughter ions could be easily generated by lose off m/z 296, 351, 369, 384 neutral fragments for hydroxypheophytin. 3) Structural isomer have the same fragmentation pathways.4. A new method based on high performance liquid chromatography-diode-array detector-electrospray ionization-ion trap mass spectrometry (HPLC- DAD-ESI-IT-MS) was developed for rapid identification and quantification of lutein in Enteromorpha prolifera, and it was was used to determine the content of lutein in Enteromorpha prolifera from Qingdao seashore. Enteromorpha prolifera sample was infiltrated firstly by 1.0 g/L of VC solution for minutes, then lutein was extracted through ultrasonic-assisted extraction. In this way, lutein could maintain stability in extracting solvent. The crude extract of Enteromorpha prolifera was separated by Eclipse XDB-C18 column(4.6 mm×150 mm, 5μm). MeOH (with 0.2% acetic acid) and water as the mobile phase, linear gradient elution, detection length of Vis is 450 nm. The results indicated that the developed method can be used for the determination of lutein in Enteromorpha prolifera with a good resolution and linearity between 0.10 ~20.00 mg/L (R2=0.9994). The content of lutein in Enteromorpha prolifera from Qingdao seashore is in the range of 2.15～2.28 mg/g and about 0.22% of dry weight, which show that Enteromorpha prolifera is rich in lutein.5. To study the technology of preparing sodium-copper chlorophyllin from Enteromorpha prolifera and provide basis for developing and utilizing natural chlorophyll. Firstly, the main instrumental parameters for ultrasonic-assisted extraction of chlorophylls from Enteromorpha prolifera were optimized, moreover, sodium copper chlorophyllin was obtained through the processing technology of saponification and copper replacing. Then, sodium copper chlorophyllin prepared from Enteromorpha prolifera was preliminarily identified through ultraviolet-visible absorption spectra and infrared spectrum, and effects of acidity, heat, light, and alkali on the stability of sodium copper chlorophyllin was studied. The results showed that the sodium copper chlorophyllin is metallic, blackish green powder, and it is soluble in water and slightly soluble in ethanol, methanol, chloroform. Also it is stable in 90℃below and certain acid or alkali condition. However, its photostability is weaker, it should be made to avoid solar irradiation during using process, and storage in the shade. |
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