2. AN INTRODUCTION TO STABLE ISOTOPE DENDROCLIMATOLOGY
3.2 Material and Methods
3.2.1 Sample Selection
We studied the effectiveness of SBrendel method variants using samples of resin-rich trees with potential to make significant contributions to palaeoclimate reconstruction: ponderosa pine, New Zealand kauri and huon pine. Ponderosa pines may live >400 years and have a wide geographic distribution, occurring across much of western North America. Our sample was cored from a ~100 year old specimen growing in northwest Arizona. Kauri and huon pine have more limited natural distributions; they are confined to northernmost New Zealand and southwestern Tasmania, Australia, respectively. However, individual trees can live in excess of 2000 years and the recovery of sub-fossil specimens has allowed construction of discontinuous tree-ring chronologies that extend to ~60,000 and 13,500 years before present, respectively (Hua et al., 2009; Turney et al., 2010). Furthermore, stable isotope dendroclimatology remains an almost untapped area of study in the Southern Hemisphere extra-tropics and by working with huon pine and kauri we aim to facilitate development of new records from this region. Our kauri sample comes from a fallen tree from the Upper Huia Dam region, near Auckland, New Zealand and the huon pine sample from a disk sawn from a fallen tree in western Tasmania.
3.2.2 SBrendel Processing
We obtained approximately 250mg of wood from each of the three tree species which was then shared between our laboratories at The University of Canterbury (UC) and the University of New Mexico (UNM) where a combined total of 6 powdered fractions were created . Each sample was powdered using a either a dremel tool or a saw depending on size and then homogenized through a freeze-milling process or ultrasonic disaggregation, both based on Laumer et al. (2009). For this study we used only the <355μm fraction of the generated powder (following Rinne et al. (2005)).
At each laboratory the fractions were divided into subsamples of ~20 mg plus one with mass ~10 mg.
The 10mg samples remained untreated and were used to characterize the FTIR spectra and stable isotope (δ13C, δ18O) values of whole wood.
The ~20 mg wood samples were each divided into ~20 0.9-1.1 mg subsamples. Each batch of ~1mg subsamples was then subject to either the original small sample SBrendel method (Evans and Schrag, 2004) or one of seven variants (Table 3.1) in dedicated laboratory spaces developed by the authors. The resultant cellulose from each batch was pooled and homogenized.
The next step of the SBrendel method is for samples to be boiled at 120°C in a nitric-acetic acid mixture.
This temperature is approximately the boiling point for 70% nitric acid (120.5°C at 1
atm.;(Sigma-Aldrich, 2012)) and close to the melting point (~130oC) of many brands of polypropylene microcentrifuge tubes (e.g. VWR) which generally show signs of softening at ~125oC (personal experience and Kevin Anchukaitis, personal communication). For these experiments we trialed flip-top and screw-top microcentrifuge tubes. Each has advantages and disadvantages with respect to performance, cost and time taken to open and close. However, given the tendency for flip-top tubes to pop open during boiling Table 3.1: Variants of the SBrendel method to be investigated as part of this project. The SBrendel method of Evans and Schrag (2004), experiment #1, describes boiling wood samples at 120°C in a 120 μL 80% acetic acid and 12 μL 70% nitric acid mixture for ~30 minutes. All experiments were repeated for each wood type at low temperature (115oC) and high temperature (120oC).
Experiment # Acetic Acid (μL) Nitric Acid (μL) Boiling Time (hrs)
1 120 12 0.5
2 120 12 1
3 180 18 0.5
4 180 18 1
5 240 24 0.5
6 240 24 1
7 360 36 0.5
8 360 36 1
Experimental assessment of the purity of α-cellulose
at 120°C, often exploding the contents into the fume hood, this study investigated the influence of boiling temperature on product purity. SBrendel variants performed at UNM using flip-top vials were conducted at 115oC (rather than the 120oC suggested by the SBrendel). The 5oC temperature reduction generally prevents explosion and allows researchers to reduce costs and save time during the four rinse steps in the method. The UC samples were digested at 120oC using screw-top microcentrifuge tubes (Sarsted, 1.5ml). Samples digested at 115oC are coded LT (low temperature) while those digested at 120oC are coded HT (high temperature). Detailed, step-by-step procedures can be found in the supplementary data online (Appendix 8.8). We did not trial at temperatures greater than 120°C due to limitations of reagent boiling temperatures and microcentrifuge tube integrity. In designing the experiment we did not consider whether the effect of altitude on the boiling point of reagents may have played a role in our methods; UC experiments were conducted at near sea-level (~1.0 atm.) while the UNM campus sits at almost 1600m above sea level (0.8-0.85 atm.) where reagent boiling points are likely to be lower.
3.2.3 FTIR
Preliminary work by the authors used visual analysis to determine whether or not the cellulose extraction process resulted in a pure product. However, neither the level of purity, nor the contaminant(s) can be determined quantitatively by this method.
Subsamples of whole wood, the material produced from each experiment, and a commercial α-cellulose (Sigma Aldrich) were analyzed on a Nicolet Nexus 670 at the New MIRA FTIR facility at the University of New Mexico, using both microscope and bench transmission. The former method was preferred as it required <0.1 mg of sample and individual fibers could be selected under the microscope and analyzed separately. Multiple spectra were collected for all samples to minimize potential influence of orientation effects (Hinterstoisser et al., 2001).
Bench transmission was performed on only a sub-set of samples. Approximately ~0.25mg of desiccated sample material is pressed into a potassium bromide disk. In so doing, numerous fibers, in many orientations, are measured simultaneously, providing a ‘bulk’ spectrum of the sample, similar to the Attenuated Total Reflectance (ATR) method used in similar studies (Rinne et al., 2005; Anchukaitis et al., 2008).
3.2.4 Stable Isotope Analysis
The remainder of each sample was used for stable isotope (δ13C, δ18O) analysis. For each of the
experiments and the whole wood, 10 subsamples were analyzed: five for δ13C and five for δ18O. Carbon isotope ratios were determined at the UC Stable Isotope Facility; samples of mass 255 ± 15 μg were loaded into tin capsules and combusted in a hot (1050°C) Costech 4010 EA (Elemental Analyzer) coupled with a DeltaVPlus IRMS (Isotope Ratio Mass Spectrometer) via a ConFlo III open-split continuous-flow interface. The precision of in-house and certified standard materials measured via this method during these experiments was better than 0.15 ‰ (n = 151)..
Oxygen isotopic analysis of experimental products was performed at the UNM Stable Isotope Laboratory.
Samples of mass 255 ± 15 μg were loaded into silver capsules and pyrolized in the hot (1400°C)
alumina:glassy carbon reactor of a TC/EA (Thermal Conversion/Elemental Analyzer). Sample gas (CO) was analyzed in a Finnigan-MAT 252 IRMS via a ConFlo III open-split continuous-flow interface. The precision of in-house and certified standard materials measured via this method during these experiments was better than 0.3‰ (n = 107).