Biological Metals

7/28/2019 Biological Metals

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BIOLOGICAL METALS: A BRIEF

INTRODUCTION INTO ATOMIC

SPECTROSCOPY 7

BSc Forensic Science ProgramForensic Toxicology

Ildi Fenyvesi

2013

[email protected]


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Beginning, Middle and End!

Metals and Us

The theoretical basics

Samples pretreatment and preservation Instrumentation


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ELEMENTS AND THE BODY

The mineral elements constitute a small amount of total bodytissues however they are essential to many vital processes.

The animal body requires 7 main mineral elements: calcium,magnesium, sodium, potassium, phosphorous, sulphur andchlorine.

60-80% of all inorganic material in the body. What is meant byinorganic??

An inorganic compound which contains a metal are thoseelements in the periodic table that when ionized, loseelectrons 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.


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ELEMENTS & BIOLOGICAL MOLECULES

Important roles in various biological activities

Metalloporphyrins: iron containing biological complex in the transport ofoxygen and the mediation in electron transfer chains. Heme groupassociated with a protein (hemoglobin, myoglobin, cytochromes, catalase,peroxidase).

Cobalt containing biological molecules: Vitamin B12 coenzymes(cobalamins).

Metalloenzymes and metal activated enzymes ~ M incorporated intoenzyme: carbonic anhydrase and carboxypeptidase (Zn) as well as ascorbicacid oxidase and various tyrosinases (Cu).

Iron (Fe) in hemoglobin.

Zinc in insulin.


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METAL IMBALANCES?

1. Metal toxicity occurs more often than one would expect. We are exposedto toxic metals on a day-to-day basis in our environment. Whether heavymetal poisoning comes from pollution, cooking utensils, exposures towaste contaminations, deodorants, pesticides, food sources etc., all havean effect on the human body.

2. Disease states: Rickets which is characterized by a faulty calcification ofbones due to low Vit D content in the body and deficiency of calcium andphosphorous in diet (deficiency of Ca = serum levels are low and P=low);Wilsons disease associated with large amounts of copper accumulationin the body (excessive urinary excretion and increased absorption ofcopper from the intestine and hence accumulation occurs).


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ARSENIC: PHARMACODYNAMICS

Elemental Arsenic (As ) isrelatively non toxic.

Arsenate (As +5) and Arsenite(As+3) are common forms ofarsenic with arsenite being

more toxic of the three. Arsine gas (most toxic) is AsH3

is a gas formed whenhydrogen is generated in thepresence of trivalent arsenic industrial organic synthesis

and in lead storage batterymanufacture

Arsenic compounds areprimarily absorbed with therespiratory and GI tracts.

Breathing (workplaceexposure) and percutaneous

exposure. >90% of orally consumed

arsenic is absorbed (organicarsenic compounds inseafoods are readily absorbedafter ingestion)


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ARSENIC: PHARMACODYNAMICS

Arsenic is absorbed bydiffusion, enters theportal system, circulatesto the liver and entersgeneral circulation.

The half life of inorganicarsenic = 10hrs andmethylated arsenic =30hrs

Arsenic binds tosulfhydryl groups andconcentrates in the hairand nails

Excreted to a minordegree in sweat and skin.

Nails and hair accumulatethe metal.

Trivalent arsenic isoxidized in vivo to thepentavalent species andthe reverse also applies.

Urine is the major

elimination pathway andaccounts for approx 60%absorbed


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ARSENIC: TOXICITY

Poisonings are lesscommon but still occurdue to the availability ofthe arsenic containing

herbicides and pesticides. Arsine poisonings may

occur in occupationalsettings and poisonings

may occur as a by-product of a chemicalreaction.

Acute symptoms: GIsymptoms including pain,vomiting, discomfort,diarrhea. Skeletal muscle

pain and severe thirst iscommon and in highconcentrations results inspasms, stupor andconvulsions.

Chronic symptoms:muscle weakness, garlicbreath and neuropathy.


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ARSENIC: APPLICATION

1. Daisy De Melker Case: poisonedher husbands with Strychnine and

her son with Arsenic.

http://www.africacrime-

mystery.co.za/books/fsac/chp6.htm

1. Arsenic cases are not that

common locally2. International cases:

3. http://www.localhistories.org/ars

enic.html

4. Not that popular anymore!

http://www.iol.co.za/sport/arsenic-victim-had-similar-symptoms-1.71748
http://www.africacrime-mystery.co.za/books/fsac/chp6.htmhttp://www.africacrime-mystery.co.za/books/fsac/chp6.htmhttp://www.localhistories.org/arsenic.htmlhttp://www.localhistories.org/arsenic.htmlhttp://www.localhistories.org/arsenic.htmlhttp://www.localhistories.org/arsenic.htmlhttp://www.africacrime-mystery.co.za/books/fsac/chp6.htmhttp://www.africacrime-mystery.co.za/books/fsac/chp6.htmhttp://www.africacrime-mystery.co.za/books/fsac/chp6.htmhttp://www.africacrime-mystery.co.za/books/fsac/chp6.htm


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MERCURY

Exposure from environment, industrial

processes (dry cell batteries and lamps and

wiring and production of chlorine etc),

thermometers, pigments, lubricating oils andat one stage dental amalgams.


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MERCURY: PHARMACODYNAMICS

Mercury vapor inhaled and inorganic salts of mercuryis readily absorbed and transported to other organs inthe 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 thencombines with sulfhydryl residues in plasma proteins.

80% of mercury is deposited in the proximal tubules inthe kidneys and free mercury crosses the blood brainbarrier hence a large proportion of the metal isdeposited mainly in the kidney and the brain.


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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; GIsymptoms(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.


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MERCURY APPLICATIONS

1. Occupational and environmentalexposure concerns.

2. Some forensic cases are still

reported.

3. Mercury is not routinely tested

for however in a forensic workup

it must be suspected and

requested specifically.

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

menace-treaty

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


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LITHIUM

Lightest of metals andwas discovered in 1817 byJ. Arfuedson (Swedishchemist).

Small amounts found inmeteorites, soils, tobacco,grains, coffee and milk.

Therapeutically: since the19th century as an

anticonvulsant, a sedative(manic patients) and atreatment for gout.

1949 Cade discoveredthat Lithium carbonatewas useful in thetreatment of mania. Isused as ongoingtreatment for manicdepressive disorders.

Industry (LithiumHydride): source of

hydrogen, highperformance desiccantand a condensing agent inorganic synthesis.


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LITHIUM: PHARMACODYNAMICS

Rapid and completeabsorption of lithiumfrom the GI tract anddistribution to the

organs.

The concentration inCSF is 40-50% of plasmaconcentration.

Approximately 95% iseliminated in the urine(half life 20-24 hrs).

80% of lithium isreabsorbed in theproximal renal tubules.

Less than 1% iseliminated in the fecesand 4-5% in sweat.


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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.


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LITHIUM APPLICATIONS

Lithium overdose cases since lithium isprescribed as medication.

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

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


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QUESTIONS:

1. What is it? Describe its abundance in nature andwhere 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.


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ANALYTICAL PROCESS

SAMPLE PREPARATION

SAMPLE PRESERVATION

INSTRUMENTS


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THE BASICS

Atomic Spectroscopy is the technique of analyzing the energy emitted byatoms.

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 cancause the electrons of an atom to move to a higher energy state or theexcited state.

The transition from ground to excited state requires the absorption of aunique packet or quanta of energy.

When the excited electron returns to the ground state, it emits radiationof a specific wavelength.

Each element in the periodic table will absorb and emit light at a specificwavelength.


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THE BASICS cont

The energy or wavelength is UNIQUE to anelement and hence one wavelength = oneelement which gives a selective technique.

Volatilization of atoms in a flame will cause themto emit or absorb light of specific wavelengths.

Basic premise: is that with increasingconcentration of metal ions you will increase the

amount of energy produced. This energy iselectronically transferred into a digital signalwhich is measured.


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SAMPLE PREPARATION

Need some form of pretreatment to bring the sampleinto solution: slowest and most labour intensive.

Method: sample type; element, concentration,analytical technique.

Preliminary pretreatment: drying of compound in anoven to get rid of the water content; grinding forhomogeneity.

Dissolution: no interfering substances must beintroduced and usually dissolve into acids or water.


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SAMPLE PREPARATION

Cold vapour generation ~ Hg where mercury in sample isconverted to Hg2+ with HNO3 and H2SO4 and then reduced toelemental Hg with SnCl2 .

Ion Exchange ~ metal ion pre-concentrated on cationexchange resin and then desorbed using acid (removal of

metal ion from matrix).


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SAMPLE PREPARTION

Solvent Extraction ~ chelate used for the transfer ofmetal aqueous phase to organic phase eg.Ammonium pyrrolidine dithiocarbamate + methylisobutyl ketone.

Solids ~ electrothermal vaporization into the graphitefurnace oven.


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SAMPLE PRESERVATION

Issues: adsorption of trace metals ontoglassware.

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.


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HOW DO MEASURE THE AMOUNTS OF

ELEMENTS?

ATOMICABSORPTION-FLAME

ATOMICABSORPTION FLAMELESS(FURNACE)

INDUCTIVELY COUPLEDPLASMA (ICP)-ATOMICEMISSIONSPECTROSCOPY

ICP-MASSSPECTROMETRY

(MS)


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FLAME ATOMIC ABSORPTION

SPECTROMETRY (FAAS)

SOURCE: hollow cathode lamp

PRE-MIX BURNER SYSTEM ~NEBULISERLISER

ATOMISER: convert all chemical

forms of element in the samplesolution into free atoms in agaseous state. All analyte atomsshould be converted to freeatoms.

Absorption depends on density of

atoms and absorption pathlength.

2 types of flame in which atomsare generated: Air/Acetylene(2400C) or a NitrousOxide/Acetylene (2600C)generated in a single slot burnerhead.

AA spectrometers usemonochromators anddetectors for UV and visiblelight. The main purpose of themonochromator is to isolate

the absorption line frombackground light due tointerferences .


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FAAS


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FLAME PROPERTIES

Critical aspect because theflame must be sufficient tobreak down compounds ofthese elements and provideefficient atomisation ofanalyte element.

Ability to tolerate wide varietyof solvents.

Low level of backgroundemission and absorption.

Low noise and suitable

reproducibility. Convenient and safe.

Inexpensive operation.


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FLAME OBJECTIVE

FACTORS AFFECTING ATOMISATIONEFFICIENCY ~

Temperature: greater temp,greater dissociation.

Chemical environment in flame(oxidising or reducing).

Aspiration rate:spl uptake.

Ionisation: high temp causesthermal ionisation (red # neutralatoms=red sensitivity)

Physical effects: viscosity, surfacetension.

INTERCONAL ZONE: hottest,

lowest noise = analytical zone.


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INTERFERENCES: what can interfere in the

analysis

MATRIX

CHEMICAL

MATRIX PHYSICAL SPECTRAL

Element forms thermally

stable compound with

molecular or ionic

species in solution e.g.phosphate

Transport of sample

solution to flame

Due to atomic or

molecular fine structure in

the spectrum

Species combine with

analyte to give more

volatile compounds e.g.

EDTA with Ca

Viscosity, surface tension

of solution

Spectral line overlap e.g.

553.55nm calcium

hydroxide + barium

Occlude analyte into

volatile matrix particles

Aspiration, nebulisation

or atomisation efficiency


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STRENGTHS v WEAKNESSES

STRENGTHS ~ Easy to use

Fast

Low cost

Few interferences overcomeby matrix matching

Most elements areatomised

Automated

Good precision reasonablesensitivity

WEAKNESSES ~ Require large volume of

solutions

Require solution form

Chemical interferences Low atom concentrations

due to dilution effects

Safety precautions(explosive gases)


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GRAPHITE FURNACE ATOMIC ABSORPTION

SPECTROMETRY (GFAAS)

Proposed by Lvov

Commercial since 1969

Flameless; replaced by small electrically

heated graphite tube/cuvette which is heatedto 3000C to generate a cloud of atoms.

The higher atom density and longer residence

time in the tube improve detection limits.


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FLAMELESS


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FURNACE PROGRAM

DRY: remove sample solvent, isothermal/rampheating; do not want spluttering.

ASHING: remove sample matrix to leave analyte in aform to atomize efficiently and reproducible; remove

organic matter; analyte must be thermally stable toallow for high ash temp (matrix modifier).

ATOMISATION: atomize sample as fast as possible toproduce sharp reproducible profiles.

CLEAN: removal of residual analyte.


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FURNACE PROGRAM


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INTERFERENCES: MATRIX & SPECTRAL

EFFECTS

CHEMICAL PHYSICAL SPECTRALPyrolsis loss (elemental

form in matrix is more

volatile than in standards)

Micro-droplet delivery Background absorption

from molecular species

present in the furnace

atmosphere e.g. NaCl.

Atomisation interference-

chemical speciation (Se

species differs in nature

vs. stds)

Viscosity, surface tension

of solution (soaking into

the furnace carbon

surface)

Background correction

(Zeeman)

Vapour phase

condensation reactions-

recombination of free

atoms in vapour phase to

molecular species

Wetting and spreading of

sample in the furnace


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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 (volatileanalyte less volatile) and Magnesium Nitrate which acts asoxidant to remove organic matter.


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STRENGTHS ~

Better detection limits

Small sample size

Few spectralinterferences

WEAKNESSES ~

Slower analysis time

Major chemical

interferences Element limitations

Limited dynamic range


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ICP-AES

Inductively coupled plasma atomic emission spectrometry.

Multi-element technique that uses an inductively coupledplasma 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, excitingthem to a level where they emit light of a characteristicwavelength.

A detector measures the light emitted specific for atom andintensity indicates concentration.


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ICP-AES


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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.


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STRENGTHS ~

Easy to use

Multi-elements in 1 sample in 1min and no compromise onprecision or detection limit

Few chemical interferences Excellent screening abilities, high

productivity

Solid and organic samples

WEAKNESSES ~ Moderate to low detection

Spectral interferences


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ICP-MS

Inductively coupled plasma mass spectrometry.

Multi-element technique

Uses a plasma source to dissociate the sample into itsconstituent positively charged ions.

The ions themselves are detected whereby they are passedthrough a mass spectrometer and are separated by their massto charge ratio.

Utilize mostly quadrupole mass spectrometers.

Isotope detection of elements is possible e.g. Cu (63 and 65).


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ICP-MS SETUP


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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 breakmolecules.

CHEMICAL: presence of argon gas and also solvents

used.

PHYSICAL: viscosity and surface tension.


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STRENGTHS ~

Excellent detectionlimit.

Multi-element High productivity

Wide dynamic range

Isotopic measurements

WEAKNESSES ~

High cost

Some spectral

interferences Method development

skill


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SUMMARY OF ELEMENTAL ANALYSIS

TECHNIQUES

FAAS GFAAS ICP ICPMS

DETECTION LIMIT SUB PPM SUB-PPB 1-10PPB 1-10PPT

SAMPLE

THROUGHPUT10-15s per

element

3-4min per

element

1-60

elements permin

All elements

per min

DYNAMIC RANGE 103 102 106 108

PRECISION(SHORT

AND LONG TERM)0.1-10% 0.5-10% 0.1-5% 0.5-4%

INTERFERENCES Manychemical

Manychemical

Manyspectral

Few spectral,some matrix

SAMPLE VOLUMES

REQUIREDLarge Small Medium small

CAPITAL AND

RUNNING COSTSLow Medium-

high

High High


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CALIBRATION

Signal caused by the detection of the element in a sample iscompared with a set of calibration samples of known content.

External calibration is utilized with suitable matrix matchingprecautions taken and the range of concern taken intoaccount.

Quality Assurance: procedures covering the sampling andstorage; contamination possibilities and treatment of samplesduring analytical phase.

Sources of Error: unsuitable methodology, contamination,interferences, calibration errors, losses and degradation,

incompetence and lack of care.

Biological Metals

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