Rapid Sample Preparation

14 Jan 2004
Dear Colleagues,
I am looking for a rapid and simple recipe for alpha-cellulose extraction applicable for small tree-ring samples. I found an article written by O. Brendel et al., 2000 entitled "A Rapid and Simple Method to Isolate Pure Alpha-Cellulose" this morning. This paper was published on Phytochemical Analyisis 11, 7-10 (2000). Has anyone here tried that method yet? Would
you please share your comments?
I appreciate your inputs,
Dr Zheng-Hua Li
306 G&G Building
Dept of Geological Sciences
University of Tennessee
Knoxville, TN 37996
Tel: 1-865-974-9622
Fax: 1-865-974-2368


14 Jan 2004
Dear Zheng-Hua:
We are putting together a methods comparision paper for isotope analyses of different types (14C, 13C and 18O) and one of the methods we have used is the Brendel et al. (2000) method and two modifications of this method. The results are currently being wirtten-up for publication (working title: Cellulose extraction techniques: a comparative analysis of their uses for 13C, 14C and 18O isotopic analysis, by Gaudinski, Dawson, Quideau, Trumbore, Roden, Schuur , Hu, Sandquist, Sidall, Zheng, Xiaomei Zu . . . .perhaps others).

The conclusions from our analyses raise concerns about using the Brendel method for use with 18O and 14C isotopes in particular and as a method in general. Using wood, root and leaf samples from a diversty of plants we've found that the Brendel method appears to produce the least pure form of cellulose according to our stable isotope and 13C-NMR analysis and produces 18O values that are enriched relative to values one gets if they use the holo- and alpha-cellulose soxhlet methods. While it produces 13C results that are often similar to the alpha- and holo-cellulose soxhlet methods (especially for wood) the results are inconsistent. Modifiing the Brendel method helps with many of these problems, but our analyses of wood, leaf and root cellulose shows that the soxhlet method still produces the most reliable results. Several labs now have a "rapid" batch soxhlet method that makes it easier to process many samples in a relatively short time.

We know that others have used the Brendel method and seem generally pleased with it, but our results are showing us that it is not the best method if you want the highest possible precision for stable isotope analyses and it produces erroneous results for 14C.

Todd Dawson
Professor of Biology
Director, Center for Stable Isotope Biogeochemistry,
Department of Integrative Biology,
Valley Life Sciences Building,
University of California - Berkeley
Berkeley, CA 94720 USA
my office: 510/642-6090
my labs phone/FAX: 510/642-1054
my dept. FAX: 510/643-6264
website: http://ib.berkeley.edu/labs/dawson/

Center for Stable Isotope Biogeochemistry
Main lab phone: 510/643-1748
Managers office phone: 510/643-1749
CSIB website: http://ib.berkeley.edu/groups/biogeochemistry/


4 Jan 2004
Hi Zheng,
How small? We routinely us the modified Green (1963) method for smaller samples (anything from 10 mg to about 45 mg of raw wood). The method I use at my lab is from Uni Bern, but it is most similar to Loader et al. (1997). This approach works fine, and you just need some glassware, I believe I have part number somewhere for Kontes or Kimble to make the vials. Let me know if you need it.

Green, J. W. (1963). Wood cellulose. Methods in Carbohydrate Chemistry. R. L. Whistler. New York, Academic Press. III: 9-20.

Loader, N. J., I. Robertson, et al. (1997). "An improved technigue for the batch processing of small whole wood samples to a-cellulose." Chemical Geology 136: 313-317.

William Anderson
Assistant Professor
Earth Sciences Department and Southeast Environmental Research Center
Florida International University

Mailing address:
Earth Sciences Dept., PC 344
11200 S.W. 8th Street
Miami, FL 33199
office: 305 348 2693, sec. 348 3572
SERC stable isotope lab: 305 348 3044
fax: 305 348 3877
email: andersow@fiu.edu


16 Jan 2004
Dear Dr. Li, Todd, Dr. Anderson, Dr. Feng,
We (Pascale Poussart, Dan Schrag and I) have been using a protocol slightly modified from the Brendel procedure since mid-2000. The protocol and isotopic data are described in a preprint (GCA, in press, 2004; preprint available from: http://www.ltrr.arizona.edu/~mevans/PrePub.html).
This protocol is scaled for samples of order 1mg. The results we obtain for c13 and o18 on dense tropical woods are not statistically different from those we get using the protocol described by Brendel et al., (2000).

Additional results for both oxygen and carbon (stable and radio-) analyses have just been published in EPSL (please contact Pascale Poussart at Harvard, poussart@eps.harvard.edu, for a reprint).

We have not made any purity tests of the product, such as are described in Brendel's paper for c13, and described by Todd in his email. Mike Lott made some intercomparisons between this modified Brendel method and the Jayme-Wise extraction in summer of 2002. He found no significant difference in results for o18 measurements using continuous flow analysis. We not not found any problems with reproducibility of measurements for either o18 or c13, within typical measurement precision of 0.2-0.3 permil SMOW on a continuous flow instrument.

Todd, I would be interested to discuss a draft of your manuscript on method intercomparison. I am especially interested in why and how radiocarbon results (but not c13?) are affected by the Brendel protocol, but not by other techniques. I would also like to see how your purity results differ from Oliver's. Is this mainly due to the diverse and variable mixture of C compounds in soils and other heterogeneous samples compared to something relatively simple, like wood, or is it in the extraction chemistry?

We have found both reasonable and strange radiocarbon results after processing wood via modified-Brendel. On wood from Costa Rica from the post-bomb period, we found prebomb (500-1000BP) ages which were replicated within c14 error by WHOI and CAMS radiocarbon labs. I chalked these results up to an unknown but large fossil radiocarbon contamination; maybe due to alpha-cellulose extraction? But in wood from Indonesia, Pascale Poussart has published radiocarbon results which are consistent within error with an age model developed from oxygen isotope data. (See our EPSL and GCA papers for the hypothesis behind this approach, based on work of Roden et al.) Then again, Pascale has also found radiocarbon ages which are too young, based on samples from crossdated (annual-ringed) teaks from Indonesia. Pascale can provide more details on those results and her thinking on their interpretation.

-Mike Evans


18 Jan 2004
I agree with John's comments (by the way, thank you for the lengthy treatment, John. That level of commentary is much more useful than a simple statement of preference.) The only benefit I see to the Brendel technique is completion of the extraction process in a single day. I can
picture a circumstance where a researcher is not available for the short time required in the morning and evening, for 5-6 days, as required by the Soxhlet/bleaching technique (and has no student help).

I regularly use two soxhlet setups at one time (a larger soxhlet would serve the same purpose). The number of samples processed in each soxhlet is determined by the mean sample size, but I usually extract 80-100 samples in less than a week. If necessary, two extractions can be
overlapped, because the soxlet step is completed at the end of the 2nd day. In this case, you would roughly double the number of samples processed per week.

Ed Wright
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