Comparative Analysis of Cellulose Preparation Techniques for Use with <sup>13</sup>C, <sup>14</sup>C, and <sup>18</sup>O Isotopic Measurements

A number of operationally defined methods exist for pretreating plant tissues in order to measure C, N, and O isotopes. Because these isotope measurements are used to infer information about environmental conditions that existed at the time of tissue growth, it is important that these pretreatments remove compounds that may have exchanged isotopes or have been synthesized after the original formation of these tissues. In stable isotope studies, many pretreatment methods focus on isolating “cellulose” from the bulk tissue sample because cellulose does not exchange C and O isotopes after original synthesis. We investigated the efficacy of three commonly applied pretreatment methods, the Brendel method and two variants of the Brendel method, the Jayme−Wise method and successive acid/base/acid washes, for use on three tissue types (wood, leaves, roots). We then compared the effect of each method on C and O isotope composition (<sup>13</sup>C, <sup>14</sup>C, <sup>18</sup>O), C and N content, and chemical composition of the residue produced (using <sup>13</sup>C nuclear magnetic resonance (NMR)). Our results raised concerns over use of the Brendel method as published, as it both added C and N to the sample and left a residue that contains remnant lipids and waxes. Furthermore, this method resulted in <sup>18</sup>O values that are enriched relative to the other methods. Modifying the Brendel method by adding a NaOH step (wash) solved many of these problems. We also found that processed residues vary by tissue type. For wood and root tissues, the <sup>13</sup>C NMR spectra and the <sup>18</sup>O and <sup>13</sup>C data showed only small differences between residues for the Jayme−Wise and modified Brendel methods. However, for leaf tissue, <sup>13</sup>C NMR data showed that Jayme−Wise pretreatments produced residues that are more chemically similar to cellulose than the other methods. The acid/base/acid washing method generated <sup>13</sup>C NMR spectra with incomplete removal of lignin for all tissues tested and both isotopic, and <sup>13</sup>C NMR results confirmed that this method should not be used if purified cellulose is desired.