Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples

Background: When conducting plant research, the measurement of photosynthetic pigments can provide basic information on the physiological status of a plant. High-pressure liquid chromatography (HPLC) is becoming widely used for this purpose because it provides an accurate determination of a variety of photosynthetic pigments simultaneously. This technique has a drawback compared with conventional spectroscopic techniques, however, in that it is more prone to structural modification of pigments during extraction, thus potentially generating erroneous results. During pigment extraction procedures with acetone or alcohol, the phytol side chain of chlorophyll is sometimes removed, forming chlorophyllide, which affects chlorophyll measurement using HPLC. Results: We evaluated the artifactual chlorophyllide production during chlorophyll extraction by comparing different extraction methods with wild-type and mutant Arabidopsis leaves that lack the major isoform of chlorophyllase. Several extraction methods were compared to provide alternatives to researchers who utilize HPLC for the analysis of chlorophyll levels. As a result, the following three methods are recommended. In the first method, leaves are briefly boiled prior to extraction. In the second method, grinding and homogenization of leaves are performed at sub-zero temperatures. In the third method, N,N'-dimethylformamide (DMF) is used for the extraction of pigments. When compared, the first two methods eliminated almost all chlorophyllide-forming activity in Arabidopsis thaliana, Glebionis coronaria, Pisum sativum L. and Prunus sargentii Rehd. However, DMF effectively suppressed the activity of chlorophyllase only in Arabidopsis leaves. Conclusion: Chlorophyllide production in leaf extracts is predominantly an artifact. All three methods evaluated in this study reduce the artifactual production of chlorophyllide and are thus suitable for pigment extraction for HPLC analysis. The boiling method would be a practical choice when leaves are not too thick. However, it may convert a small fraction of chlorophyll a into pheophytin a. Although extraction at sub-zero temperatures is suitable for all plant species examined in this study, this method might be complicated for a large number of samples and it requires liquid nitrogen and equipment for leaf grinding. Using DMF as an extractant is simple and suitable with Arabidopsis samples. However, this solvent cannot completely block the formation of chlorophyllide in thicker leaves.