The Research Of New Method For Extraction And Determination Of Chlorophyll A In Macroalgae

The development of methods for extraction and determination of chlorophyll a in macroalgae were reviewed in this thesis. More than 10 macroalgaes were collected at the coast of Qingdao for research and direct immersion and grinding procedure were used for the comparative study of the chlorophyll a extraction method for macroalgae with different solvents. The effect of molecular structure of organic solvents to the extraction of chlorophyll a in macroalgae was analyzed based on above results and a new method for extraction and determination of chlorophyll a in macroalgae was provided. The differences of pigment compositions and concentrations of Phaeophyceae Undaria pinnatifida with different solvents and extraction methods was analyzed by HPLC and the results of HPLC was compared with those of spectrophotometric method.Some relationships were found between molecular structure and chlorophyll a extraction effect of organic solvents based on the analysis of the results of immersion experiments of representative macroalgae of 3 phyla (Chondrus ocellatus, Undaria pinnatifida and Ulva pertusa) in 12 solvents. The extraction effect of solvent with short carbon chain is better than that with long carbon chain, and the solvents with little branches is better than that with more branches. In same kind of solvent the better one is the one which is symmetrical, water-soluble and has little dipole moment. The solvents with famoyl, carbonyl and hydroxyl function groups are preferable.A new solvent-N,N-dimethylacetamide (DMAC)-was found as it is not only better than 90% acetone but also than DMF. The toxicity of DMAC is far smaller than that of DMF and their price is very close. The formula for calculation of chlorophyll a in macroalgae and pure chlorophyll a solution with DMAC was obtained by the comparative experiments of molar extinction coefficient of pure chlorophyll and wasvalidated with 14 macroalgae:Chlorophyta: chlorophyll a(g/ml)=11.58664-1.88 ?47 Phaeophyta: chlorophyll a(g/ml) =11.14664-0.40 ?30 Rhodophyta: chlorophyll a(g/ml) =11.50664-1.50 ?47-0.08 ?30 8 solvents were selected for HPLC pigment analysis of Undaria pinnatifida. hi these solvents THF, ISP and SBA can make pigment peak distortion and bring some difficulty for qualitative and quantitative analysis and so aren't applicable for HPLC pigment analysis. In DMAC the peak shape is very sharp and the pigments with close elution time can be separated on baseline. It can be sorted by peak shape for solvent performance as below: DMAO90%ACE>MEN>ACE> Ethanol>THF> ISP>SBA.The Chl-a result of the immersion of Ethanol is the highest in all immersion methods and the next is the immersion of DMAC. However, the extraction result of polar pigment (chl-c2 and chl-cl) of Ethanol is less than that of DMAC and other solvents, hi the same way the results of chlc-2 and chlcl especialy chl-c2 in ACE and 90%ACE are lower than that of DMAC either. So DMAC is the best solvent formacroalgae pigments extraction.The macroalgae solution extracted by DMAC were determined by spectrophotometric method and HPLC method separately, and it is showed that the resultsof these two methods are same.