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Development of a sensitive [delta]¹5 N analysis for photopigments

UNCW Author/Contributor (non-UNCW co-authors, if there are any, appear on document)
Nicolas D. Ballew (Creator)
The University of North Carolina Wilmington (UNCW )
Web Site:
Robert Hancock

Abstract: A method for the oxidation and nitrogen isotopic analysis of photopigments (chlorophyll and phaeophytin) has been developed. Homogenous single source materials (spinach and diatom culture) as well as mixed matrix (estuarine water) natural samples were tested with this approach. This method first uses extraction and purification of photopigments by column chromatography, followed by UV irradiation to convert photopigment N to nitrate and nitrite in the presence of hydrogen peroxide. d15N of bulk samples and chlorophyll extracts were determined with an Isotope Ratio Mass Spectrometer (IRMS) with an Elemental Analysis (EA) Interface. Quantification of post-oxidation products (nitrate and nitrite) and determination of oxidation efficiency were performed using ion chromatography. For isotopic analysis, the resulting nitrate/nitrite was converted to N2O at the USGS Reston Stable Isotope Laboratory (RSIL) using a strain of bacteria Pseudomonas aureofaciens, which lacks N2O-reductase activity. The N2O is analyzed on an IRMS to provide the d15N ratio of the product. Comparisons were made between bulk source material (spinach and diatom), the respective extracted photopigments, and the post-oxidation products. Isotopic offsets of homogenous samples were isotopically depleted by a range of 3 to 6 ‰ from bulk to extract and again from extract to post-oxidiation. This stepwise lightening of the isotopic signature was consistent with isotopic fractionation during each of these steps, although no clear Rayleigh fractionation relationship was discernable. Natural mixed matrix samples exhibited irreproducible isotopic results following both extractions and oxidation. We suggest that these results were due at least, in part, to very high amounts of co-extracted compounds potentially extracting other nitrogen sources and/or severely depressing oxidation efficiencies of photopigment nitrogen. Although isotopic offsets for each step of the mixed matrix samples varied considerably, they all yielded end products which were substantially isotopically depleted (up to -12‰) relative to bulk isotopic values of the starting material.

Additional Information

A Thesis Submitted to the University of North Carolina at Wilmington in Partial Fulfillment of the Requirement for the Degree of Masters of Science
Language: English
Date: 2009
Chlorophyll--Analysis, Oxidation
Chlorophyll -- Analysis