BIOLOGICAL METALS: A BRIEF INTRODUCTION INTO ATOMIC SPECTROSCOPY 7

BSc Forensic Science Program Forensic Toxicology Ildi Fenyvesi 2013 Ildif.BSC@gmail.com

Beginning, Middle and End!

Metals and Us The theoretical basics Samples pretreatment and preservation Instrumentation

ELEMENTS AND THE BODY

The mineral elements constitute a small amount of total body tissues however they are essential to many vital processes. The animal body requires 7 main mineral elements: calcium, magnesium, sodium, potassium, phosphorous, sulphur and chlorine. 60-80% of all inorganic material in the body. What is meant by inorganic?? An inorganic compound which contains a metal are those elements in the periodic table that when ionized, lose electrons and form cations. At least 7 other minerals are important in trace levels: Iron, copper, iodine, manganese, cobalt, zinc and molybdenum. Other elements also play a role, but exact nature is still not fully understood: fluorine, aluminium, boron, selenium, cadmium, chromium.

ELEMENTS & BIOLOGICAL MOLECULES

Important roles in various biological activities

Metalloporphyrins: iron containing biological complex in the transport of oxygen and the mediation in electron transfer chains. Heme group associated with a protein (hemoglobin, myoglobin, cytochromes, catalase, peroxidase).
Cobalt containing biological molecules: Vitamin B12 coenzymes (cobalamins). Metalloenzymes and metal activated enzymes ~ M incorporated into enzyme: carbonic anhydrase and carboxypeptidase (Zn) as well as ascorbic acid oxidase and various tyrosinases (Cu).

Iron (Fe) in hemoglobin.

Zinc in insulin.

METAL IMBALANCES?
1. Metal toxicity occurs more often than one would expect. We are exposed to toxic metals on a day-to-day basis in our environment. Whether heavy metal poisoning comes from pollution, cooking utensils, exposures to waste contaminations, deodorants, pesticides, food sources etc., all have an effect on the human body. 2. Disease states: Rickets which is characterized by a faulty calcification of bones due to low Vit D content in the body and deficiency of calcium and phosphorous in diet (deficiency of Ca = serum levels are low and P=low); Wilsons disease associated with large amounts of copper accumulation in the body (excessive urinary excretion and increased absorption of copper from the intestine and hence accumulation occurs).

TOXICITY AND INORGANIC COMPOUNDS

Increased industrialization of the world has caused the increased use of metals and metallic compounds, hence the exposure of humans and animals to this is greater than before. Occupation, living environment, intentional and unintentional poisonings, consumption of food and water.

ARSENIC
Has properties of both a metal and non metal = semi-metal Used for therapeutic uses and for poisonings 3rd most common element and is not typically mined but is a recoverable by-product of the smelting process of various metals such as copper and lead. Wood preservatives, insecticides (calcium arsenate), herbicides (arsenites), sheep dips, fly paper, rat poisons. Occupational exposure (use of pesticides) as well as through food sprayed with the pesticide, leached from spring water and power plant effluent coming into contact with soil.

ARSENIC: PHARMACODYNAMICS
Elemental Arsenic (As ) is relatively non toxic. Arsenate (As +5) and Arsenite (As+3) are common forms of arsenic with arsenite being more toxic of the three. Arsine gas (most toxic) is AsH3 is a gas formed when hydrogen is generated in the presence of trivalent arsenic industrial organic synthesis and in lead storage battery manufacture Arsenic compounds are primarily absorbed with the respiratory and GI tracts. Breathing (workplace exposure) and percutaneous exposure. >90% of orally consumed arsenic is absorbed (organic arsenic compounds in seafoods are readily absorbed after ingestion)

ARSENIC: PHARMACODYNAMICS
Arsenic is absorbed by diffusion, enters the portal system, circulates to the liver and enters general circulation. The half life of inorganic arsenic = 10hrs and methylated arsenic = 30hrs Arsenic binds to sulfhydryl groups and concentrates in the hair and nails Excreted to a minor degree in sweat and skin. Nails and hair accumulate the metal. Trivalent arsenic is oxidized in vivo to the pentavalent species and the reverse also applies. Urine is the major elimination pathway and accounts for approx 60% absorbed

ARSENIC: TOXICITY
Poisonings are less common but still occur due to the availability of the arsenic containing herbicides and pesticides. Arsine poisonings may occur in occupational settings and poisonings may occur as a byproduct of a chemical reaction. Acute symptoms: GI symptoms including pain, vomiting, discomfort, diarrhea. Skeletal muscle pain and severe thirst is common and in high concentrations results in spasms, stupor and convulsions. Chronic symptoms: muscle weakness, garlic breath and neuropathy.

ARSENIC: APPLICATION
1. Daisy De Melker Case: poisoned her husbands with Strychnine and her son with Arsenic.
http://www.africacrimemystery.co.za/books/fsac/chp6.htm

1. 2. 3. 4.

Arsenic cases are not that common locally International cases: http://www.localhistories.org/ars enic.html Not that popular anymore!

http://www.iol.co.za/sport/arsenic-victim-had-similar-symptoms-1.71748

MERCURY
Drawings on cave walls made with cinnabar (red stone made of mercury sulfide). Mercury is mined and produced as a by-product of gold and bauxite mining. 16th Century: treatment for syphilis, diuretic, antiseptic, laxative. Mercury is the only metal that is liquid at room temp. Metallic or elemental mercury is the main volatile form occurring in air (Hg 0) Inorganic mercury compounds are formed in the Hg+ (mercurous state) and organic compounds are in the Hg 2+ (mercuric state).

MERCURY
Exposure from environment, industrial processes (dry cell batteries and lamps and wiring and production of chlorine etc), thermometers, pigments, lubricating oils and at one stage dental amalgams.

MERCURY: PHARMACODYNAMICS
Mercury vapor inhaled and inorganic salts of mercury is readily absorbed and transported to other organs in the body. 80% of the inhaled dose is absorbed into the circulation with only 2% absorbed percutaneously. The majority of the taken up erythrocytes and is oxidized to the divalent mercuric ion. This then combines with sulfhydryl residues in plasma proteins. 80% of mercury is deposited in the proximal tubules in the kidneys and free mercury crosses the blood brain barrier hence a large proportion of the metal is deposited mainly in the kidney and the brain.

MERCURY: PHARMACODYNAMICS AND TOXICITY

Routes of elimination include faeces (biliary excretion and intestinal secretion) and urine (50%). The half life is 3-5 days as well longer elimination of up to 45 days. (2 compartment model with a rapid and slow phase). Orally (and inhalation) consumed Mercury poses more risks than dermal exposure. Acute toxicity: inhalation of high vapor or dust. Flu-like symptoms and symptoms of interstitial pneumonitis and bronchitis; GI symptoms(burning mouth and throat, nausea, vomiting). Chronic poisoning: neurological symptoms with tremors or the arms and hands (lower concentrations) as well as lower limb involvement (high concentrations). Chronic effects lead to renal tubular injury, loss of memory and tremors.

http://www.guardian.co.uk/commentisfree/2013/jan/10/mercury-poisoning-globalmenace-treaty

MERCURY APPLICATIONS
1. 2. 3. Occupational and environmental exposure concerns. Some forensic cases are still reported. Mercury is not routinely tested for however in a forensic workup it must be suspected and requested specifically.

http://www.ncbi.nlm.nih.gov/pubmed/21646904

LITHIUM
Lightest of metals and was discovered in 1817 by J. Arfuedson (Swedish chemist). Small amounts found in meteorites, soils, tobacco, grains, coffee and milk. Therapeutically: since the 19th century as an anticonvulsant, a sedative (manic patients) and a treatment for gout. 1949 Cade discovered that Lithium carbonate was useful in the treatment of mania. Is used as ongoing treatment for manic depressive disorders. Industry (Lithium Hydride): source of hydrogen, high performance desiccant and a condensing agent in organic synthesis.

LITHIUM: PHARMACODYNAMICS
Rapid and complete absorption of lithium from the GI tract and distribution to the organs. The concentration in CSF is 40-50% of plasma concentration. Approximately 95% is eliminated in the urine (half life 20-24 hrs). 80% of lithium is reabsorbed in the proximal renal tubules. Less than 1% is eliminated in the feces and 4-5% in sweat.

LITHIUM TOXICITY
Lithium hydride is intensively corrosive and can produce skin burns. Inhalation of dust causes strictures of the larynx, bronchi and trachea. Patients taking lithium may develop thyroid enlargement. Acute exposure may produce polyuria, sedation, polydipsia, confusion and coma.

LITHIUM APPLICATIONS
Lithium overdose cases since lithium is prescribed as medication.

http://www.ncbi.nlm.nih.gov/pubmed/20515402

http://www.bipolar-lives.com/lithium-toxicity-symptoms.html

QUESTIONS:
1. What is it? Describe its abundance in nature and where it can be found? 2. How are we exposed to it? Give examples. 3. Describe its absorption, distribution, metabolism and elimination in the human body. 4. What is its toxicity? What are the medical effects of over exposure? 5. Explain a case or case study involving the metal of interest.

SAMPLE PREPARATION SAMPLE PRESERVATION INSTRUMENTS

ANALYTICAL PROCESS

THE BASICS
Atomic Spectroscopy is the technique of analyzing the energy emitted by atoms. All atoms have electrons existing in a most stable state = the ground state = also the lowest energy state. Certain processes can change the energy of the state e.g. adding heat can cause the electrons of an atom to move to a higher energy state or the excited state. The transition from ground to excited state requires the absorption of a unique packet or quanta of energy. When the excited electron returns to the ground state, it emits radiation of a specific wavelength. Each element in the periodic table will absorb and emit light at a specific wavelength.

THE BASICS cont

The energy or wavelength is UNIQUE to an element and hence one wavelength = one element which gives a selective technique. Volatilization of atoms in a flame will cause them to emit or absorb light of specific wavelengths. Basic premise: is that with increasing concentration of metal ions you will increase the amount of energy produced. This energy is electronically transferred into a digital signal which is measured.

SAMPLE PREPARATION
Need some form of pretreatment to bring the sample into solution: slowest and most labour intensive. Method: sample type; element, concentration, analytical technique. Preliminary pretreatment: drying of compound in an oven to get rid of the water content; grinding for homogeneity. Dissolution: no interfering substances must be introduced and usually dissolve into acids or water.

SAMPLE PREPARATION
Digestion: wet or microwave ~ prolonged heating with concentrated mineral acid.

Hydride generation ~ for elements forming volatile hydrides e.g. As, Pb, Sn (acidified sample + sodium borohydride).

SAMPLE PREPARATION
Cold vapour generation ~ Hg where mercury in sample is converted to Hg2+ with HNO3 and H2SO4 and then reduced to elemental Hg with SnCl2 .

Ion Exchange ~ metal ion pre-concentrated on cation exchange resin and then desorbed using acid (removal of metal ion from matrix).

SAMPLE PREPARTION
Solvent Extraction ~ chelate used for the transfer of metal aqueous phase to organic phase eg. Ammonium pyrrolidine dithiocarbamate + methyl isobutyl ketone.

Solids ~ electrothermal vaporization into the graphite furnace oven.

SAMPLE PRESERVATION
Issues: adsorption of trace metals onto glassware. Laboratory ware contamination of samples e.g. airborne Pb and Cu from polythene. Keep acid concentrations of samples high. Use plastic ware. Store in fridge.

HOW DO MEASURE THE AMOUNTS OF ELEMENTS?

ATOMIC ABSORPTIONFLAME ATOMIC ABSORPTION FLAMELESS (FURNACE)
INDUCTIVELY COUPLED PLASMA (ICP)-ATOMIC EMISSION SPECTROSCOPY

ICP-MASS SPECTROMETRY (MS)

Atomic Absorption Spectrometry: FLAME AND FURNACE

A method that measures the concentration of atoms of an element by passing light (emitted by a lamp of that element) through a cloud of atoms from a sample. Basic components: Spectral source emitting atomic resonance line of analyte; atomiser; detector system.

FLAME ATOMIC ABSORPTION SPECTROMETRY (FAAS)

SOURCE: hollow cathode lamp PRE-MIX BURNER SYSTEM ~ NEBULISERLISER ATOMISER: convert all chemical forms of element in the sample solution into free atoms in a gaseous state. All analyte atoms should be converted to free atoms. Absorption depends on density of atoms and absorption path length. 2 types of flame in which atoms are generated: Air/Acetylene (2400C) or a Nitrous Oxide/Acetylene (2600C) generated in a single slot burner head.

AA spectrometers use monochromators and detectors for UV and visible light. The main purpose of the monochromator is to isolate the absorption line from background light due to interferences .

FAAS

FLAME PROPERTIES
Critical aspect because the flame must be sufficient to break down compounds of these elements and provide efficient atomisation of analyte element. Ability to tolerate wide variety of solvents. Low level of background emission and absorption. Low noise and suitable reproducibility. Convenient and safe. Inexpensive operation.

FLAME OBJECTIVE
FACTORS AFFECTING ATOMISATION EFFICIENCY ~ Temperature: greater temp, greater dissociation. Chemical environment in flame (oxidising or reducing). Aspiration rate:spl uptake. Ionisation: high temp causes thermal ionisation (red # neutral atoms=red sensitivity) Physical effects: viscosity, surface tension. INTERCONAL ZONE: hottest, lowest noise = analytical zone.

INTERFERENCES: what can interfere in the analysis

MATRIX CHEMICAL
Element forms thermally stable compound with molecular or ionic species in solution e.g. phosphate Species combine with analyte to give more volatile compounds e.g. EDTA with Ca Occlude analyte into volatile matrix particles

MATRIX PHYSICAL SPECTRAL

Transport of sample solution to flame Due to atomic or molecular fine structure in the spectrum

Viscosity, surface tension of solution

Spectral line overlap e.g. 553.55nm calcium hydroxide + barium

Aspiration, nebulisation or atomisation efficiency

STRENGTHS v WEAKNESSES
STRENGTHS ~ Easy to use Fast Low cost Few interferences overcome by matrix matching Most elements are atomised Automated Good precision reasonable sensitivity WEAKNESSES ~ Require large volume of solutions Require solution form Chemical interferences Low atom concentrations due to dilution effects Safety precautions (explosive gases)

GRAPHITE FURNACE ATOMIC ABSORPTION SPECTROMETRY (GFAAS)

Proposed by Lvov Commercial since 1969 Flameless; replaced by small electrically heated graphite tube/cuvette which is heated to 3000C to generate a cloud of atoms. The higher atom density and longer residence time in the tube improve detection limits.

FLAMELESS

FURNACE PROGRAM
DRY: remove sample solvent, isothermal/ramp heating; do not want spluttering. ASHING: remove sample matrix to leave analyte in a form to atomize efficiently and reproducible; remove organic matter; analyte must be thermally stable to allow for high ash temp (matrix modifier). ATOMISATION: atomize sample as fast as possible to produce sharp reproducible profiles. CLEAN: removal of residual analyte.

FURNACE PROGRAM

INTERFERENCES: MATRIX & SPECTRAL EFFECTS

CHEMICAL
Pyrolsis loss (elemental form in matrix is more volatile than in standards)

PHYSICAL
Micro-droplet delivery

SPECTRAL
Background absorption from molecular species present in the furnace atmosphere e.g. NaCl. Background correction (Zeeman)

Atomisation interferencechemical speciation (Se species differs in nature vs. stds) Vapour phase condensation reactionsrecombination of free atoms in vapour phase to molecular species

Viscosity, surface tension of solution (soaking into the furnace carbon surface) Wetting and spreading of sample in the furnace

INTERFERENCES: MATRIX EFFECTS INTERVENTIONS

Matrix matching of standards. Standard addition in calibration. Isothermal atomization. Matrix Modification (makes analyte less volatile OR makes the matrix more volatile) ~ Palladium Nitrate modifier (volatile analyte less volatile) and Magnesium Nitrate which acts as oxidant to remove organic matter.

STRENGTHS v WEAKNESSES
STRENGTHS ~ Better detection limits Small sample size Few spectral interferences WEAKNESSES ~ Slower analysis time Major chemical interferences Element limitations Limited dynamic range

ICP-AES
Inductively coupled plasma atomic emission spectrometry. Multi-element technique that uses an inductively coupled plasma source (argon). Plasma reaches temperature of 10000C (break molecular bonds high atomization). Plasma configuration can be radial or axial. The sample dissociates into the constituent atoms, exciting them to a level where they emit light of a characteristic wavelength. A detector measures the light emitted specific for atom and intensity indicates concentration.

ICP-AES

SPECTRAL INTERFERENCES
Common Line rich spectra produced by hot plasma source (high resolution spectrometers or use alternate line). Background effects. Physical: nebulizer (viscosity and surface tension effects) and spray chamber.

STRENGTHS v WEAKNESSES
STRENGTHS ~ Easy to use Multi-elements in 1 sample in 1 min and no compromise on precision or detection limit Few chemical interferences Excellent screening abilities, high productivity Solid and organic samples

WEAKNESSES ~ Moderate to low detection Spectral interferences

ICP-MS
Inductively coupled plasma mass spectrometry. Multi-element technique Uses a plasma source to dissociate the sample into its constituent positively charged ions. The ions themselves are detected whereby they are passed through a mass spectrometer and are separated by their mass to charge ratio. Utilize mostly quadrupole mass spectrometers. Isotope detection of elements is possible e.g. Cu (63 and 65).

ICP-MS SETUP

Interferences
SPECTRAL: species have same or similar mass as the analyte (isobaric interferences) e.g. Cr (52) vs. ArC(40+12) or ClOH(35+16). Overcome by using collision cell technology to break molecules. CHEMICAL: presence of argon gas and also solvents used. PHYSICAL: viscosity and surface tension.

STRENGTHS v WEAKNESSES
STRENGTHS ~ Excellent detection limit. Multi-element High productivity Wide dynamic range Isotopic measurements WEAKNESSES ~ High cost Some spectral interferences Method development skill

SUMMARY OF ELEMENTAL ANALYSIS TECHNIQUES

FAAS
DETECTION LIMIT SAMPLE THROUGHPUT

GFAAS SUB-PPB

ICP 1-10PPB 1-60 elements per min 106 0.1-5% Many spectral Medium

ICPMS 1-10PPT All elements per min 108 0.5-4% Few spectral, some matrix small

SUB PPM

10-15s per 3-4min per element element 103 0.1-10% Many chemical Large 102 0.5-10% Many chemical Small

DYNAMIC RANGE PRECISION(SHORT AND LONG TERM) INTERFERENCES SAMPLE VOLUMES REQUIRED

CAPITAL AND RUNNING COSTS

Low

Mediumhigh

High

High

CALIBRATION
Signal caused by the detection of the element in a sample is compared with a set of calibration samples of known content. External calibration is utilized with suitable matrix matching precautions taken and the range of concern taken into account. Quality Assurance: procedures covering the sampling and storage; contamination possibilities and treatment of samples during analytical phase. Sources of Error: unsuitable methodology, contamination, interferences, calibration errors, losses and degradation, incompetence and lack of care.