丙酮浸提法测定油松和杨树叶片叶绿素含量比丙酮研磨提取法和二甲基亚砜-丙酮两步快速提取法偏低10%～30%；叶绿素 a/b 比也存在一定差异。而对于小麦和油菜三种方法测定结果基本一致。树木叶片叶绿素含量结果偏低的主要原因是丙酮浸提后的叶片材料中仍有少量褐色(杨树)或浅黄色物质(油松)。上述残余材料中的色素在65℃下用二甲基亚砜再次浸提，或者常温下用二甲基亚砜进行研磨提取，测定吸光度发现上述残余材料中的色素对叶绿素 a 和 b 的吸光度具有显著干扰，导致吸光度增大，测定结果偏高。丙酮浸提法不会将干扰物质溶出，或者溶出较少，叶绿素含量测定最为准确，适合含有次生有色物质的树木叶片的叶绿素含量测定。%Our results in this paper showed that chlorophyll content of Pinus trees and Populus trees determined by direct extraction method with acetone was lower 10%~30% than that of the grinding method with acetone or two-step extraction with dimethyl sulfoxide (DMSO) and acetone. The ratio of chlorophyll a to chlorophyll b also had obvious variability among the three methods. However, there was no obvious difference among three method in chlorophyll content of wheat and rape. Small amount of brown substance in Populus trees leaves and yellow substance in Pinus trees leaves were the main causes resulting in the low chlorophyll content determined by direct extraction method. The residual substance in above plant material were extracted with DMSO at 65℃ or extracted with DMSO by grinding method, and their absorbance at 663.6 nm, 646.6 nm were measured. The absorbance results showed that the residual substance in leaves had obvious interference on the absorbance of chlorophyll a and chlorophyll b, which results in an increase of chlorophyll absorbance and measured results. The colored secondary substances could not be extracted by direct extraction method with acetone, or little be extracted. So the measured results of chlorophyll content by direct extraction method with acetone is the most accurate method among the three methods. Chlorophyll content of tree leaves with colored secondary substances should be determined using birect extraction method.
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