CENTER FOR ENVIRONMENTAL
BIOINORGANIC CHEMISTRY
UNDERGRADUATE RESEARCH FELLOWSHIP
SUMMER 2005
DEVELOPING A METHODOLOGY FOR THE
MEASUREMENT OF IRON AND COPPER
STABLE ISOTOPE RATIOS IN THE MARINE DIATOM
THALASSIOSIRA OCEANICA
DAVID SEMENIUK
SUPERVISED BY:
Dr. MAITE MALDONADO
Dr. DOMINIQUE WEIS
DEPARTMENT OF EARTH AND OCEAN SCIENCES
UNIVERSITY OF BRITISH COLUMBIA
Stable isotopes of traditional elements (C, N, O, and S) have been used
successfully to trace past and present biogeochemical cycling of these elements,
as well as to model ancient atmospheric and oceanic processes. Transition
metals, such as iron (Fe) and copper (Cu), are thought to produce small massdependent fractionations due to the small differences in mass between their
stable isotopes. However, with the advent of multi-collector inductively coupled
plasma mass spectrometry (MC-ICPMS), it has become possible to measure
transition element isotopic ratios to precisions hitherto impossible.
To date, biologically mediated Fe-fractionation has been investigated exclusively
in bacteria and humans, while Cu isotopic ratios are more scarce and confined
mainly to abiotic systems. Marine phytoplankton require a minimal amount of Fe
to maintain growth, and as such have developed mechanisms for coping with
iron-limitation. Ironlimited phytoplankton have recently been observed to utilize
organically bound Fe by reducing Fe(III) to Fe(II) using a ferric reductase,
followed by oxidation of Fe(II) by a multi-copper containing oxidase, and the
subsequent transport of Fe(III) by an Fe permease (Maldonado et al., in press).
The transport of Fe was Cu-dependent, and so it is likely that phytoplankton have
a considerable effect on the biogeochemical cycling of Fe and Cu isotopes under
both iron replete and limited conditions. The goal of my work this summer was to
develop a method for adequately digesting the sample material, to isolate Fe and
Cu from the sample to reduce possible matrix effects, and finally to perform
preliminary isotopic measurements on at least one sample.
In order to avoid contaminating samples, and thus altering the original isotopic
ratios, rigorous cleaning protocols were developed and followed for all laboratory
equipment used. I learned how different plastic materials (i.e. Teflon, HDPE,
Polypropylene, etc.) have the potential to contaminate a sample, depending on
the elements being analyzed. I also learned how different acids are used to clean
for different elements, and that the most effective method is the sequential
combination of strong acids and a final leaching step in Milli Q water. These
procedures and knowledge will become invaluable during the following year while
completing my undergraduate honours thesis.
Under Fe-replete conditions, Thalassiosira oceanica, an oceanic diatom, was
grown in a fortified recipe of the synthetic ocean water medium Aquil. The cells
were filtered, rinsed with chelexed synthetic ocean water, centrifuged, and
freeze-dried. The lyophilized sample was transferred to a clean PTFE-digestion
vessel, and a mixture 95%/5% (v/v) of concentrated, Ultrapure (Seastar)
HNO3/HF was added to the bomb.
After the microwave digestion, the sample was dried down to remove residual HF
that did not complex as a gas with the siliceous diatom frustules. Once resuspended in a few milliliters of concentrated HNO3, the sample was transferred
to a clean PFA digestion vial, and placed on a hot plate. The combination of heat,
pressure, and concentrated HNO3 completed the digestion of all remaining
organics, as indicated by the evolution of a dark brown gas from the vial. Once
the gas production ceased, the sample was heated to near dryness, and resuspended in 7M HCl to prime it for the anion exchange column chemistry.
All chemistry was performed in the Class 1000 clean labs of the Pacific Center of
Isotope and Geochemical Research (PCIGR) under the guidance of Dr. Jane
Barling and Prof. Dominique Weiss. Firstly, to ensure complete separation of Fe
and Cu from each other and other elements, a multi-element standard was used
to test how effective our
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Elution Volume (mL)
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Pb
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LOAD 6M HCl 1M HCl + 0.001% H2O2
protocol was. The sample was loaded onto the prepared column, and eluted with
6M HCl
first to remove the Cu, then 1M HCl + 0.001% H2O2 to remove the Fe (Figure 1).
Each
cut was collected in a clean polypropylene test tube. A second column was used
to elute
a total Cu and Fe yield into cleaned PFA beakers as would be done during the
actual
sample analysis.
Figure 1: Elution curves for the multi-element standard as the sample load, 6M HCl, and 1M
HCl+0.001%
H2O2 was added to the column.
Element concentrations of each cut were determined using a Thermofinnigan
Element2 High resolution ICPMS. Under the technician’s guidance, I learned the
importance of matrix effects on the analyte, and methods for reducing them as
much as
possible. The majority of the major cations were immediately eluted in the first
few
milliliters, leaving cobalt and zinc (a contaminant) in the Cu cut. The possible
matrix
effects of Co on the Cu analysis will be carried out once the Co concentrations of
the
diatoms are determined. Although approximately 100% of the Fe was collected,
only
83% Cu yield was achieved. Incomplete elution of Fe and Cu has been shown to
induce
mass-dependent fractionation, and therefore the chemistry for the diatom sample
was to
increase the chance of obtaining ≥99% yield. Instead of 6M HCl, 7M HCl +
0.001%
H2O2 was used in hopes of stripping off all the Cu from the column before eluting
the Fe
(Figure 2).
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Elution Volume (mL)
Percent Total Elution
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7M HCl + 0.001% H202 1M HCl + 0.001% H202
Figure 2: Elution of copper and iron from the digested T. oceanica sample.
Again, a significant portion of Cu eluted with the first cut of Fe, and so the Cu
isotope analysis has not been performed yet. The total Co was less than 0.1%
the total
Cu present, and so it likely will not have an effect on the Cu analysis. I plan on
determining a method of isolating 100% of the Cu over the course of my honours
thesis.
Using a Nuplasma MC-ICPMS, Dr. Barling and myself successfully analyzed the
stable
isotope ratio of Fe for the diatom sample as an exponentially corrected ratio
using a Cu
dopant (remaining sample Cu likely has no affect, since <0.1% total Fe). The
ratio was
determined to be approximately 0.3‰ heavier than the in house standard.
Unfortunately,
a sub-sample of the medium was not taken before inoculating it with the culture,
and so
an absolute fractionation value for the experiment cannot be determined.
I have learned that oceanography is one of the most multidisciplinary faculties in
science. It requires a broad understanding of concepts from chemistry, physics,
biology,
and mathematics. My fellowship this summer has allowed me to broaden my
scope to
analytical chemistry and geochemistry, and their potential uses for a student of
biological
oceanography like myself. The work I completed here has peaked my interest in
marine
biogeochemistry, and consequently myself along with two other students have
proposed a
student directed seminar here at UBC on the biological pump. I plan on
continuing my
studies after completing my honours degree by attending graduate school, where
the
knowledge and skills I have obtained through this fellowship will be used.