An offline sample preparation system and water exchange reaction method for the measurement of &#948;2H of non-exchangeable hydrogen in organic matter&#160;
Authors: Gudasz Cristian , Lundholm Jonas , Geibrink Erik , Öquist Mats , Karlsson Jan 
Published: March 2022

&lt;p&gt;The measurement of &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H of non-exchangeable-H (&lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&lt;/sub&gt;) in organic matter (OM) by isotope-ratio mass spectrometry is often hampered by the difficulties in controlling H isotope exchange of the exchangeable H fraction (f&lt;sub&gt;ex&lt;/sub&gt;) and removal of residual moisture. The determination of &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#160;&lt;/sub&gt;in organic matter requires control of the isotopic composition of f&lt;sub&gt;ex&lt;/sub&gt;. This can be achieved through dual water H isotope exchange experiments. However, these experiments are laborious, sensitive to the method used (e.g. prior sample treatment, temperature, time) and are costly. This has resulted in a wide range of reported f&lt;sub&gt;ex &lt;/sub&gt;for known isotopic references. Moreover, it is not always clear that samples are completely dry following the H exchange experiments, leading to even larger variations. The &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#160;&lt;/sub&gt;data is typically used in ecological studies for source attribution due to the large observed separation between contributing end members. However, it is not clear to what degree the analytical errors in &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H determined by incomplete H isotope exchange of f&lt;sub&gt;ex&amp;#160;&lt;/sub&gt;and the residual moisture impact the source attribution. Here we developed a simple offline sample preparation system, the Isobox, and a protocol for the measurement of &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#160;&lt;/sub&gt;in natural OM as well as pure organic compounds. We performed dual water H isotopic exchange experiments with both liquid and vapor water at near 0 and 105&amp;#176;C respectively. We analyzed three keratin reference materials (KHS, CBS and USGS42), two amino acids (Isoleucine and Threonine), along with caffeine (USGS62) and polyethylene (IAEA-CH-7) as drying references. We have also used natural samples of demineralized soil and green algae to create known mixtures to test these methods and their analytical uncertainty impact on the source attribution. We show that the liquid water exchange experiments led to f&lt;sub&gt;ex &lt;/sub&gt;close to the theoretical expectations for both keratin and pure compounds. Depending on the research question careful determination with controlled dual water procedure for the determination of &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;n&amp;#160;&lt;/sub&gt;may be required. However, simple sample treatment with exposure to a single isotopically known water can be used to derive &lt;em&gt;&amp;#948;&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;H for source attribution. The offline sample preparation system and equilibration method we developed is simple, accurate and cost effective and can be implemented in virtually any laboratory for the analysis of a wider range of OM types.&lt;/p&gt;