Mar. Sci. 653. Paleo-oceanography Lab # _________ Date: ______________ Object: To analyze the Carbon and Oxygen isotopic composition from a given substrate (containing Foraminifera shells) through Mass Spectrometry Requirements: 1. Mass Spectrometer 2. Core sample benthic sediments 4. CO2 Reference gas 5. Carrier gas (Helium) of 3. Orthophosphoric acid Theory: Mass spectrometry: Alfred Nier (1947) developed mass spectrometry techniques allowing the measurement of minute differences in isotopic compositions between natural compounds. It is a powerful analytical technique used to identify unknown compounds with in a sample with the help of known quantity of materials. This technique is also helpful to elucidate the structure and chemical properties of different molecules. The complete process involves the conversion of the sample into gaseous ions, with or without fragmentation, which are then characterized by their mass to charge ratios (m/z) and relative abundances. This technique basically studies the effect of ionizing energy on molecules. It depends upon chemical reactions in the gas phase in which sample molecules are consumed during the formation of ionic and neutral species. In this technique, mass spectrum of isotopic abundances in a given sample is produced through Mass Spectrometer (MS). This spectrum is produced by measuring the relative masses of atoms or molecules present in that sample. This technique is used to determine the abundances of parent and progeny isotopes in naturally radioactive decay systems that have half-lives of geological relevance (years to billions of years). Scientific methods for dating materials of geological interest commonly utilize natural radioactive isotopes that spontaneously transform to progeny isotopes at constant and well-known rates of decay. In order to use this property to estimate absolute ages, it is essential to accurately determine the abundances of both parent and progeny isotopes in a mineral and rock sample that has remained closed to isotopic exchange with its surroundings since its formation. Decay counting can be used to quantify abundances of short-lived radioactive isotopes or elements. Mass Spectrometer (MS): is an instrument used to generate the mass spectrum of isotopes from administered sample. Components of Mass Spectrometer: This instrument is composed of three (3) parts: 1. Ion Source: It produces gaseous ions from the sample Teacher’s Sign: __________________ Page # _______ Mar. Sci. 653. Paleo-oceanography 2. Analyzer: It resolves the ions into their characteristics mass components according to their mass-to-charge ratio 3. Detector System: It detects the ions and recording the relative abundance of each of the resolved ionic species 4. Other necessary components: a. Inlet: It is used to insert sample and pass it to the ion source b. High Vacuum maintenance: Approximately 10-6 to 10-8 mm of mercury is required to maintain high vacuum inside the instrument so that the ions produced in the ionization chamber can move freely without hitting air molecules c. Computer: It controls the instrument, acquire and manipulate data, and compare spectra to reference libraries Working Principle: A mass spectrometer generates multiple ions from the sample under investigation; it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type. The first step in the mass spectrometric analysis of compounds is the production of gas phase ions of the compound, basically by electron ionization. This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on. The ions are separated in the mass spectrometer according to their mass-to-charge ratio, and are detected in proportion to their abundance. A mass spectrum of the molecule is thus produced. It displays the result in the form of a plot of ion abundance versus mass-to-charge ratio. Ions provide information concerning the nature and the structure of their precursor molecule. In the spectrum of a pure compound, the molecular ion, if present, appears at the highest value of m/z (followed by ions containing heavier isotopes) and gives the molecular mass of the compound. Performance: The following stages are involved in mass spectrometry: 1. Ionization: Produce ions from the sample in the ionization source Teacher’s Sign: __________________ Page # _______ Mar. Sci. 653. Paleo-oceanography 2. Acceleration: The ions are accelerated so that they all have the same kinetic energy 3. Deflection: The ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected. The amount of deflection also depends on the number of positive charges on the ion - in other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected. Remember that stream A was most deflected - it has the smallest value of m/z (the lightest ions if the charge is 1+). To bring them on to the detector, you would need to deflect them less - by using a smaller magnetic field (a smaller sideways force). To bring those with a larger m/z value (the heavier ions if the charge is +1) on to the detector you would have to deflect them more by using a larger magnetic field. If you vary the magnetic field, you can bring each ion stream in turn on to the detector to produce a current which is proportional to the number of ions arriving. The mass of each ion being detected is related to the size of the magnetic field used to bring it on to the detector. The machine can be calibrated to record current (which is a measure of the number of ions) against m/z directly. The mass is measured on the 12C scale. The 12C scale is a scale on which the 12C isotope weighs exactly 12 units 4. Separation: Separate these ions according to their mass-to-charge ratio (m/z or m/e) in the mass analyzer. Eventually, fragment the selected ions and analyze the fragments in a second analyzer 5. Detection: Detect the ions emerging from the last analyzer and measure their abundance with the detector that converts the ions into electrical signals. Process the signals from the detector that are transmitted to the computer and control the instrument using feedback 6. Resultant or Output: The output from the chart recorder is usually simplified into a "stick diagram". This shows the relative current produced by ions of varying mass/charge ratio. The stick diagram looks like the following: Note 1: The vertical scale is related to the current received by the chart recorder - and so to the number of ions arriving at the detector: Thus, greater the current, the more abundant the ion. The above diagram is of Molybdenum and these 7 stick lines are showing that Molybdenum has 7 isotopes with relative masses 92, 94, 95, 96, 97, 98 and 100 at 12C scale while all isotopes have +1 charge. The most abundant isotope of Molybdenum is 98. Note 2: If there were also 2+ ions present, you would know because every one of the lines in the stick diagram would have another line at exactly half its m/z value (because, for Teacher’s Sign: __________________ Page # _______ Mar. Sci. 653. Paleo-oceanography example, 98/2 = 49). Those lines would be much less tall than the 1+ ion lines because the chances of forming 2+ ions are much less than forming 1+ ions. Application: It can be used to: 1. 2. 3. 4. Analyze Biomolecules, Glycans, Lipids, Proteins and Polypeptides Detect Marine isotopic composition Determine Ages and Dating the fossils, rocks, sediments, minerals Record paleoclimatic and paleo-oceanographic data etc Oxygen and Carbon Isotopic Studies in Paleo-oceanography: Oxygen and carbon isotopes of foramininfera have been used in paleoceanography studies for decades. Urey and his graduate students developed the use of oxygen isotope composition of calcite as a paleothermometer (Epstein, Buchsbaum, Lowenstam, & Urey, 1953; Urey, Epstein, Lowenstam, & McKinney, 1951). Cesare Emiliani, one of Urey’s students, was the first to use oxygen isotope paleothermometry to reconstruct the glacial–interglacial swings in climate of the late Pleistocene using fossil foramininfera shells from deep-sea sediments (Emiliani, 1955). Although he initially overestimated the glacial–interglacial temperature changes by not adequately taking into account the large changes in the oxygen isotopic composition of seawater, he was the first to use oxygen isotopic records in support of the Milankovitch theory (Emiliani & Geiss, 1959), and was responsible for the initial use of Marine Isotope Stages (MIS) (Emiliani, 1955). Shackleton (1967) revised Emiliani’s interpretation and to conclude that Teacher’s Sign: __________________ Page # _______ Mar. Sci. 653. Paleo-oceanography glacial/interglacial variations in foraminiferal oxygen isotope records were primarily influenced by changes in the oxygen isotopic composition of seawater, rather than temperature. In subsequent years, MIS finally emerged as a basic stratigraphic tool (Shackleton & Opdyke, 1973). So, measuring the oxygen and carbon isotopic composition of fossil foraminiferal calcite has been one of the most effective techniques for reconstructing ocean and climate conditions of past times. Over the last few decades, oxygen and carbon isotopic records derived from measurements of fossil foraminiferal shells have been used to address a large range of questions regarding the evolution and history of the ocean and climate. However, these discoveries were not possible without parallel studies aimed at understanding biological factors, or ‘vital effects’, that cause some species of foraminifera to calcify out of equilibrium with seawater. In addition, the species specific ecology of planktonic and benthic foraminifera has been studied to understand how isotopic records might be interpreted in light of, for example, the seasonality and depth of calcification in the water column (for planktonic species) and in the sediment (for benthic species). Notation and Standards: The stable isotopes of oxygen used in paleoceanographic studies are 16O and 18O, which comprise 99.63 and 0.1995% of the oxygen on Earth, respectively (Faure, 1986). The stable isotopes of carbon are 12C and 13C, which comprise 98.89 and 1.11% of the stable carbon on Earth, respectively (Faure, 1986). Accurate quantification of low abundances of the rare isotopes (18O and 13C) is possible only as ratios to the more common isotopes (18O/16O and 13C/12C) in the sample, as expressed in comparison with the ratios of a known standard. The difference in the ratio of the sample compared with the standard is expressed as a delta (d) value: The d18O and d13C values have concentration units of per thousand, or ‘per mil’ (%) relative to the standard. For example, a d18O value of 1.0o/oo means that the sample has an 18O/16O ratio that is 0.1% greater than the standard, or a d13C value of -25o/oo means that the sample has a 13C/12C ratio that is 2.5% lower than that of the standard. Various standards are used in different laboratories, but all lab standards are calibrated to international reference standards. Originally, they were the ‘‘historical’’ PeeDee Belemnite (Belemnitella americana) shell from the Cretaceous PeeDee formation (PDB) and/or the Standard Mean Ocean Water (SMOW). The need for clarification arose in the 1990s, notably due to the Teacher’s Sign: __________________ Page # _______ Mar. Sci. 653. Paleo-oceanography exhaustion of the original PDB standard and because the definition of the ‘‘Standard Mean Ocean Water’’ was unclear. Coplen (1996) clarified guidelines to report isotopic compositions against standards of the International Atomic Energy Agency of Vienna, the VPDB and VSMOW (i.e., the ‘‘Vienna’’ PDB and SMOW). These guidelines are now largely adopted. Worthy of mention is the fact that most laboratories doing stable isotope studies on foraminifera also use the ‘‘Carrara Marble’’ standard as calibrated by M. Hall (University of Cambridge) against VPDB (d13C= 2.25o/oo; d18O= -1.27o/oo). It should be noted that the reported carbon isotope composition of this ‘‘Carrara Marble’’ is slightly distinct from the value listed in IAEA documents for the Carrara Marble-C1 radiocarbon reference material (d13C= 2.42o/oo). All d18O and d13C values of carbonates, and all d13C values of dissolved inorganic carbon (DIC) of seawater are thus reported relative to VPDB, and all d18O values of water (snow, ice, rain, groundwater, seawater) are reported relative to VSMOW. Typically the analytical errors, or external precision, that are reported in the literature are based on the long term (month to year) reproducibility of a lab standard, and, for calcite are approximately 0.05o/oo for d13C and 0.08o/oo for d18O (both ±1d), or slightly better. The standard values in these expressions are: Standard mean ocean water (SMOW or V-SMOW): 18 16 O/ O = 0.0020052 and for C isotopes, the "Pee Dee belemnite" (PDB): 13C/12C = 0.011238 Procedure: 1. The first step in analyzing the carbon and oxygen isotopic composition of most substrates is to produce carbon dioxide (CO2) gas 2. To determine the d18O and d13C values of foraminifera shells, the shells are dissolved at a given temperature in orthophosphoric acid (see Bowen, 1966; Burman, Gustafsson, Segl, & Schmitz, 2005) to produce CO2 3. To determine the d18O of seawater, CO2 gas is isotopically equilibrated with seawater at a constant temperature following the original procedure of Epstein and Mayeda (1953) 4. To determine the d13C value of dissolved inorganic carbon (DIC) of seawater, CO2 is stripped from the seawater by acidification (e.g., St-Jean, 2003) 5. Once CO2 is isolated, a gas-source stable isotope mass spectrometer with three collectors is typically used to ionize the CO2 gas and to quantify the isotopic ratios (18O/16O and 13 12 C/ C) of the sample CO2 and of an aliquot of CO2 reference gas 6. Recent improvements in mass spectrometers now allow similar measurements in continuous flow mode, using a carrier gas (helium) for CO2 and introducing sequentially standard and sample gases 7. The d18O and d13C values can then be calculated Teacher’s Sign: __________________ Page # _______ Mar. Sci. 653. Paleo-oceanography Exercises and Numerical: 1. An ion is passed through ionization chamber of mass spectrometer with the mass of 56 while the ion is containing 2 positive charges. Calculate Charge to mass ratio (m/z) for that ion. 2. Calculate the concentration of d18O in the sample. When the sample contains the ratio of 18 16 O/ O is 0.002002o/oo while the standard ratio of 18O/16O is 0.0020052o/oo. 3. If the ratio of 13C/12C in the sample is 0.01092 and the standard 13C/12C ratio is 0.011238. Then, calculate the concentration of d13C in the sample. 4. The pyroxene and plagioclase feldspar in a gabbro have d18O values relative to the SMOW standard of 7‰ and 7.7‰ respectively. Calculate the absolute 18O/16O ratios of these two minerals. Use standard ratio of 18O/16O according to SMOW. 5. A sample of Antarctic snow has an 18O/16O ratio of 0.0019250. What is its d18O value? 6. A sample of coral has a d18O value relative to the SMOW standard of 26‰. What is its absolute 18O/16O ratio? 7. The d18O value of PDB calcite relative to the SMOW standard is 30.9‰. Calculate the absolute 18O/16O ratio of PDB calcite. 8. Two (2) ion steams A and B are deflected in ionization chamber of mass spectrometer such that an ion steam A is deflected more than ion steam B. Explain which ion steam is containing less charge to mass ratio as compared to other? 9. Two (2) ion steams A and B with the same charge (i.e. +1) are deflected in ionization chamber of mass spectrometer such that ion steam A has lighter ions while ion steam B has heavier ions. Tell which one will deflect more than other and why? 10. Why is vacuum necessary in mass spectrometer? 11. Look at the stick diagram of Zirconium and explain the following: a. How can we identify the number of isotopes from the diagram? b. How many isotopes of Zirconium and what are their relative masses? c. Which one is the most abundant isotope of Zirconium? d. Which one is the least abundant isotope of Zirconium? e. Which isotopes of Zirconium have similar abundances? f. Why is the current line on 90 m/z huge as compared to the line obtained at 96 m/z? g. The spectrum shows lines for 1+ ions. If there were also peaks for 2+ ions, where would you expect to find them, and what would you predict about their heights relative to the 1+ peaks? Teacher’s Sign: __________________ Page # _______