UNIVERSITY OF CINCINNATI Date:August 31, 2006________ I, Allison Nichole Krentz_______________________________________, hereby submit this work as part of the requirements for the degree of: Master of Science in: Chemistry It is entitled: Investigation on the Chemical Association of Important Elements in Seaweed Using SEC-ICP-MS This work and its defense approved by: Chair: _Joeseph Caruso _______________ _Patrick Limbach_______________ _Bruce Ault____________________ _______________________________ _______________________________ Investigation on the Chemical Association of Important Elements in Seaweed Using SEC- ICP-MS A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the Requirements for the degree of MASTER OF SCIENCE In the Department of Chemistry of the College of Arts and Sciences By ALLISON NICHOLE KRENTZ B.S., Chemical Technology University of Cincinnati, Cincinnati, Ohio, June 2004 Committee Chair: Dr. Joseph Caruso ii Abstract Seaweeds are found in oceans throughout the world and have been consumed by humans for centuries. There are many commercially available varieties of edible seaweed known for their medicinal properties and food values. However, seaweed can concentrate exceptionally high amounts of heavy elements available from sea, some of which are toxic. Seaweeds have also been used as bioindicators to monitor environmental pollution and for decontamination of some toxic elements from wastewaters [1]. In this study, multielemental speciation analysis of essential and toxic elements in commercially available brown seaweeds such as hiziki and wakame is performed using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry in order to examine the association of trace elements and metals with different molecular weight extracts of seaweed. Total elemental determination using ICP-MS revealed that these seaweeds have high amounts of elements such as As, Sr, Hg, and Zn. Preliminary speciation studies using SEC-ICP- MS reveal that major type of biomolecules responsible for association of these elements in seaweeds are polysaccharides such as alginic acid and fucans. Other biological macromolecules such as proteins and polyphenols also contribute to some extent towards accumulation of trace elements in seaweeds. Other studies have examined trace element concentrations in various extracts of seaweeds but none have analyzed the different trace elements and metals associated with different molecular weight extracts. iii iv Acknowledgements I would like to thank the Department of Chemistry for the opportunity to study chemistry and grow as a scientist and a person. I would like to thank my advisor and mentor Dr. Joseph Caruso for welcoming me into his research group. The amount of support and encouragement I received from Dr. Caruso was more than I could have ever wished for. Dr. Caruso is not just an intelligent and renowned professor, but he is also a kind and caring individual. I would also like to thank my other committee members Dr. Bruce Ault and Dr. Pat Limbach for their input and support. I would also like to thank all of my groupmates, especially Dr. Monika Shah. Monika was a wonderful mentor to me. My groupmates have become great friends who were always there when I needed to vent, a shoulder to cry on, or just help figuring out why something was not working. I would like to thank Dr. Rajiv Soman who was a mentor to me during my undergraduate years at the University of Cincinnati College of Applied Science. Dr. Soman always pushed me to do my best and he encouraged me to attend graduate school. I would also like to thank my family. My husband Mike and my son Evan have been very supportive of me throughout my college education. Without Mike’s support, I never would have graduated with an undergraduate degree, never less a master’s degree. I’m appreciative of Evan for understanding that sometimes Mommy had work to do on the computer, and playing quietly in his room until I finished. I also want to thank Evan for not crying the first time I dropped him off at daycare when I started graduate school (even though I was bawling my eyes out). I want to thank my parents Jim and Debbie Bruning for instilling a good work ethic in me and teaching me the importance of education. I would like to thank my parents and my in laws v Steve and Ann Krentz for babysitting when Mike was out of town so I could study or work late. vi Table of Contents Abstract………………………………………………………………………………………….ii List of Tables……………………………………………………………………………………vi List of Figures…………………………………………………………………………………..vii Introduction……………………………………………………………………………………....1 Experimental……………………………………………………………………………………..9 Instrumentation………………………………………………………………………….9 Reagents and Standards………………………………………………………………..10 Total Element Analysis…………………………………………………………………11 Extraction Procedures………………………………………………………………….12 Chromatographic Conditions………………………………………………………….14 Results and Discussion………………………………………………………………………….15 Total element determination…………………………………………………………...15 Chromatographic Speciation Analysis………………………………………………...16 Conclusions and Future Work…………………………………………………………………28 References……………………………………………………………………………………….31 vii List of Tables Table Page 1. Chromatographic Conditions………………………………………………………………...9 2. Instrumental Parameters and Conditions………………………………………………….10 3. Microwave digestion parameters……………………………………………………………11 4. Total elemental concentrations in µg g -1…………………………………………………...16 5. Approximate Retention times of inorganic elements bound with higher molecular weight species……………………………………………………………………………………19 6. Crude total elemental concentration in µg g-1 of proteinase K …………………………...21 7. Total elemental concentrations of various hiziki extracts in µg g-1……………………….25 8. Total elemental concentrations of various wakame extracts in µg g-1……………………26 viii List of Figures Figure Page 1. Chemical structure of alginate……………………………………………………………….2 2. Chemical structure of disaccharide repeating unit extracted from A. nodosum………….3 3. Polyphloroglucinol containing biphenyl and ether linkages……………………………….4 4. SEC-ICP profiles of various elements in protein extracts………………………………...18 5. Fe chromatogram for protein extracts and proteinase K…………………………………20 6. SEC-ICP profiles of various elements in polyphenol extracts…………………………….22 7. Strontium chromatogram for Hiziki alginate extracts…………………………………….23 8. SEC-ICP profiles of various elements in fucoidan extracts……………………………….24 ix Introduction Seaweeds are found in oceans throughout the world. Seaweeds are extensively used as food by people throughout the world, particularly Japan and Korea, and are also consumed in the form of dietary supplements. Extracts of seaweed are also used throughout the world in food production and can be found in foods such as meat products, baked goods, dairy items, and salad dressings [2]. The elemental content of the seaweed depends on the type of seaweed and the environment in which they are found. The iodine enrichment factor in Laminaria japonica (Kombu) reaches 106 [3]. Strontium, calcium, potassium, magnesium, and sodium have been reported as major elements present in various brown seaweeds[4]. High arsenic content has been reported in various brown seaweeds, particularly Hizikia fusiforme [4-6]. In one study the daily dietary intake values for arsenic, iodine, mercury, and cadmium could be exceeded based on the Japanese algae consumption estimates[6]. There are many studies that have been published on the determination of trace elements in seaweeds[1, 3-14]. The occurrence of high metal content in seaweed is of considerable importance, because there may be a possible toxicological hazard. Certain elements when concentrated in living organisms, are regarded as human toxins. Although few have extracted the different constituents of seaweed for analysis, and none have analyzed the different trace elements and metals associated different molecular weight extracts. Since bioavailability and properties (beneficial and toxic ) of different elements depend upon the physiochemical form in which they are found, it is desirable to determine the chemical form in which they are present. 1 There are three types of seaweeds that are classified by the pigments present, red, brown, and green[15]. Brown seaweeds readily accumulate metals and other trace elements from their environment. There are many important chemical constituents of brown seaweed. Some components of brown seaweed which may play an important role in the accumulation of trace elements are proteins, polyphenols, and anionic polysaccharides such as alginate and fucoidan. Alginate or alginic acid is a major constituent in all brown seaweeds that have been investigated. Alginate is a polysaccharide made up of only uronic acids. Alginate is a heteropolymer that contains two different units, D-mannuronic acid and L-glucuronic acid[15]. Alginates have the unique property of thickening and gelling and in water they thicken and swell, increasing its viscosity. Aliginic acids have the ability to jellify, thicken, emulsify, and stabilize various food and industrial products. For these reasons the main fields were alginates are used are the food, textile, cosmetic, paper, and pharmaceutical industries.[16] The chemical structure for alginate is shown in Figure 1. The M represents the mannuronic acid units and the G represents glucuronic acid units. Figure 1. Chemical structure of alginate taken from http://www.genialab.de/inventory/alginate.htm.