1

Dr Chris Harrington

SAS Trace Element Centre

Surrey Research Park

Guildford

TEQAS

External Quality

Assessment at

Surrey

Trace Elements in Clinical

Science

2

Outline of Todays Lecture

Setting the Scene

Why trace elements?

Clinical applications

Analytical Methods

Atomic absorption spectroscopy

• AAS

Atomic emission spectroscopy

• Flame photometry

• ICP-OES

Atomic mass spectrometry

• ICP-MS

3

Why Trace Elements ?

Elemental Classification

4

5

Trace Elements

Definition:

Trace elements have a concentration of less

than 0.0001 g/g or 0.1 g/L.

Significance:

14 elements are essential for bacteria, plants

and animals (including humans);

• the transition elements V, Cr, Mn, Fe, Co, Ni, Cu, Mo

and Zn;

• the metalloids B, Si and Se;

• the halogen elements F and I.

Why?:

There are two main reasons for their

measurement in a clinical-nutritional setting:

• to determine deficiency or toxicity.

6

Overload and Deficiency

Metals act as nutrients:

Macronutrients (K) and Micronutrients (Mo, Zn etc).

Metals act as toxic agents:

(As, Pb, Hg, U, Tl etc).

• The toxicity depends on the concentration and

the target organism.

• Toxicity also depends on the chemical form of

e.g. Cr (III) essential, Cr (VI) toxic.

7

Trace Element Examples

8

Lead: Still a Pernicious Problem

Lead use dates back to the Romans, but still

used in construction industry

Tetraethyllead now banned, but may

have left lasting scars…crime!

Blood lead > 0.5umol/L in children requires

intervention

Mostly lead in old paint

9

Arsenic – the silent killer…wallpaper

Associated with murder throughout time, which

lead to the 1st analytical method, the Marsh test

in 1832

Lewisite was 1st developed in

1904 in USA.

Rather horrible blistering

agent, which put the PhD

student who synthesised it in

hospital.

Not used in WWI because of

British Anti-lewisite

In 1893 Gosio produced

gas from green wall paper

Trimethylarsine produced

by action of mould

10

Why Doesn’t a Prawn Sarnie Kill you?

• Shellfish contains up to 70 mg/kg As,

so a sandwich has about 3500 ug As

• The LD50 for AsO3 in mice is 35 mg/kg

body weight

• So 10 prawn sandwiches contain

enough As to kill one mouse/person!!

• The LD50 for AsB is 1200 ug/g

• Arsenobetaine is non-toxic

• Normal < 10 ug As/g Creatinine

Arsenobetaine

11

Chronic Exposure

• Long term exposure to arsenic is very problematic

• Ground water contamination in

Bangladesh and SE Asia affects a

huge number of people

12

The First “magic bullet”

ca1786….cures agues, remitting fevers and periodic

headaches, for bowel problems use with laudanum.

• Paul Ehrlich 1854

• The Nobel Prize in Physiology or

Medicine 1908

• Produced Salvarsan in 1909 the

first treatment for syphilis

Salvarsan

• Trisenox - arsenic

trioxide approved

by FDA in 2000 for

the treatment of

Acute

Promyelocytic

Leukemia

13

Clinical Applications

14

The SAS Handbook

15

Applications in Clinical Biochemistry 1

Nutritional status

Cu, Zn, Se etc

Total parenteral nutrition; assessment after GI

surgery etc

Toxicology

High/acute Hg, Cd, Tl, Pu

Medium/chronic iAs (speciation required), Pb

Low/narrow essentiality Se etc

Metal-on-Metal Hip Replacement Patients

Co, Cr mainly, also Ti, Mo, Ni

Indicative of poorly performing joints that may need

replacement

16

Clinical Applications 2

Genetic disorders of trace element metabolism:Haemochromatosis - Fe accumulation

Acrodermatitis enteropathica - Zn deficiency

Wilsons disease - Cu accumulation

Menkes disease - Cu deficiency

Issues to address:Sample types:

hair, urine, nails, blood, serum.

Contamination:

Al from dust, Zn/Sb in plastics, Cr/Mn/Al

secondary tubes, also primary tubes eg Mn

Reference Ranges

Can be age related; for Co/Cr use MHRA action level

17

Analytical Methods

Analytical Chemistry to the Rescue

• Qualitative - identification of trace metals

• Quantitative – concentration (amount) present

eg ug/L, mol/L, %

• Requirements of method –

• Liquid form required so solid samples have to be

dissolved or digested using an acid;

• Specific for the trace metal of interest;

• Accurate, not affected by other components, in

agreement with other methods;

• Precise, stable over short and long term;

• Able to detect low concentrations as well as high;

• Automated and multi-elemental.

19

Instrument Detection Limits

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Molecular and Atomic Spectroscopy

0

200

400

600

800

1000

1200

1400

250 350 450 550 650 750

/nm

Mo

lar

ab

so

rptv

ity

0

200

400

600

800

1000

1200

1400

Flu

ore

sc

en

ce

In

ten

sit

y (

arb

itra

ry)

0

200

400

600

800

1000

1200

1400

250 350 450 550 650 750

Wavelength /nm

Mo

lar

ab

so

rptv

ity

0

200

400

600

800

1000

1200

1400

Flu

ore

sc

en

ce

In

ten

sit

y (

arb

itra

ry)

21

Similarities & Differences?

Major Differences:

Very narrow

"lines!„

No molecular

vibrations

No chemical

information

Similarities:

– Light mediates the

transitions from ground to

excited states.

– The difference in energy

between the states is the

same as the energy of the

photon.

Atomic lines are typically 10-2 – 10-3 nm

Molecular "bands" are typically ~50 nm wide or more.

22

Consequences of Atomic "lines"

We need a different type of spectrophotometer.

Need narrow line light source

• Otherwise stray light is a problem.

Need to "atomize"

• All chemical information must be destroyed or we will be doing molecular spectroscopy NOT atomic spectroscopy.

23

Absorption or Emission ?

All spectroscopic techniques depend on the absorption

or emission of electromagnetic radiation.

Absorption or emission arise from quantized energy

changes within the atom or molecule.

E = h x f = h x c/l (h Planck constant and c speed of light)

Atomic Absorption

Ground State

High Energy State

Atomic Emission

Absorption of

thermal, radiation

or electrical energy.

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Atomic Absorption Spectroscopy

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Atomic Absorption Spectroscopy

Radiation

source

(HCL)

Atomisation cell

(flame) Monochromator

Detector

(photomultiplier

tube)SAMPLE

Aspiration

via nebuliser

(I0) (I)

Double Beam

Instrument

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Atomic Absorption Spectroscopy

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Absorption of Radiation

The beam of light from the radiation source passes

through the atom cell and some of the radiation is

absorbed.

The absorbance follows the Beer-Lambert Law:-

A = e.l.c

Where e is the absorptivity (constant), l is the path

length (constant) and c is the concentration.

28

Atomic Absorption Spectroscopy

The radiation source _ narrow-line emission

The atomisation cell produces ground state

atoms via thermal energy

Flame - the liquid sample enters via a nebuliser

Only 10% reaches the flame, the rest goes to waste

Wavelength selector (monochromator) isolates

the line of interest from other emission lines

Detection is by a photomultiplier tube which

converts light to electric current

29

Radiation Sources: HCL

Silica

window

•The hollow cathode lamp (HCL) is an emission

source i.e. it emits radiation characteristic of the metal

from which the cathode is made.

Hollow cathode

Connecting

pinsAnode

(tungsten)Neon or Argon

Glass envelopeUnder vacuum

1-5 torr

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Atomic Absorption Spectroscopy

burner

Wavelength

Selection

detector

holllow

cathode

lamp

Hollow cathode lamp – special light source.

Fe coated

~300 V potential in the lamp causes ionization of the fill gas

Ne Ne + + e -

The Ne+ hits the surface (cathodes attract positive charge )

Fe 0* (Excited state Fe 0) is ejected .

Fe 0* Fe 0 + hn.

Ne +

Fe 0*

Fe coated

~300 V potential in the lamp causes ionization of the fill gas

Ne Ne + + e -

The Ne+ hits the surface (cathodes attract positive charge )

Fe 0* (Excited state Fe 0) is ejected .

Fe 0* Fe 0 + hn.

Ne +

Fe 0*

Hollow cathode

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“Lock & Key Effect”

The absorption line of the metal of interest is broadened in

the atomisation cell

The narrow emission line from the HCL coincides with this

The monochromator therefore only has to isolate the

emission line from others generated by the HCL.

Unique features gives high degree of selectivity.

Spectral bandpass of monochromator (0.1nm)

Absorption

in flame

HCL

emissionl

Lock & Key Effect

32

Atomisation Cells

Burner and flame – atomization method.

Flames Temperature (C) Burn velocity (cm/s)

Air/Acetylene 2100-2400 160-270

N20/Acetylene 2600-2800 290

O2/Acetylene 3050-3150 1100-2500

The nitrous flame is useful for elements that form "refractory oxides" like titanium.

33

Flame AAS

Commonly used method for analysis of

Group I and II, also transition metals

Detection limits approx. 100 ug L-1

Limitations include:

Sample introduction system is inefficient (10%)

Residence time of the atom in the flame/light from

HCL leads to poor detection limits

Inability to analyse solid samples without pre-

treatment

Number of important interferences

Single element

34

Physical Interferences

Physical interferences affect the transportation of the

sample to the FLAME and conversion to an aerosol.

Related to viscosity of sample. 2 solutions are possible:

Matrix match standards and samples.

Use standard additions for quantitation.

In standard additions add same volume of sample to

each standard.

Run samples recording response.

Plot response against conc of standard.

Graph does not pass through zero.

Conc in sample given by x-intercept.Conc

Response

x-conc

35

Chemical Interferences

Chemical interferences arise when the metal of interest

forms a thermally stable complex with molecular or ionic

species in the FLAME

phosphate, silicate or aluminate suppress the alkaline

earth metals:

Ca2+ (aq)

3 solutions to the problem:

(a) Addition of a releasing agent eg. lanthanum or

strontium salt to mop up phosphate

(b) Use a hotter flame

(c) Add protective chelating agent e.g. EDTA to form

thermally unstable complex

PO43-

Ca3(PO4)2

36

Ionisation Effects

Ionisation effects are most severe for Group I and II

metals because low ionisation potentials lead to

ionisation in the FLAME.

This means that only ions are present so no

absorption occurs eg:

Na absorbs at a different wavelength to Na+.

One solution is to add a more easily ionisable

element (EIE) e.g. Cs.

This is ionised in preference to Na:

Na+ + e-Na

Cs+ + e-Cs (large excess)

Na+ + e-Na

37

Spectral Interferences

Spectral overlap is not common in AAS because of

the lock and key effect

Does occur for some elements Cu 324.754nm and

europium 324.753nm

Calcium hydroxide (molecular) and barium 553.55nm

corrected by background correction.

Alternatively use a different absorption line which will

affect sensitivity.

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Atomic Emission Spectroscopy

39

Flame emission is the oldest spectroscopic technique

In contrast to AAS where specificity is generated by

HCL this is not the case in FES

Spectral interferences cannot be resolved by monochromator

Flame photometry only applicable to specific elements

in simple matrices. Group I and Group II

Uses cooler flame (air-propane/butane/natural gas)

No other metals are excited so monochromator is not

needed. An Interference filter is used

Cheap

Atomic Emission Spectroscopy

40

Energy Level Diagram for Sodium

Ground

State

Excited

States

Several types of

transition:

Excited states to

other excited

states (emission).

Excited to ground

states (emission).

Ground to excited

states

(absorption).

41

Flame (Emission) Photometry

Corning 410

42

Inductively Coupled Plasma

ICP-OES Plasma Cross Section of Plasma

• Use a hotter flame eg ICP Temperature is between

7000 and 10 000 K (same temp. as surface of sun).

• Can do multi-elemental analysis

43

ICP-OES or ICP-AES

44

Inductively Coupled Plasma

Cool and Plasma

tangential gas

flows

Sample

aerosol

Load Coil

Quartz glass tubes

Magnetic field

Annular Plasma

45

A plasma is a dense ball of highly excited electrons,

ions, & neutral species formed from an inert gas (Ar,

He, N2).

A stream of argon (15 - 20 l/min) flows through three

concentric quartz tubes (the torch).

The torch is encircled by a copper induction coil, which

is water cooled.

This is connected to a radio-frequency (RF) generator

giving an output of 1-2 kW.

The magnetic field generated by the RF wave through

the load coil induces a current in the argon gas.

The plasma is formed by seeding the gas with high

energy electrons.

Formation of an ICP

46

Sample Introduction

Liquid sample introduction involves the use of a

nebuliser.

The nebuliser converts the liquid sample into an aerosol.

In this way the plasma is not extinguished.

Transport efficiency is the amount of the original sample

solution that is converted to an aerosol and reaches the

plasma. Typically 1-2%.

The aerosol passes through a spraychamber where

collisions and condensation reduce the particle size to

the ideal 10 mm.

Nebulisation is affected by sample viscosity and surface

tension - match standard and sample matrices.

47

ICP Characteristics

Why argon?

It is mono-isotopic so the spectrum is simple

compared to a flame which contains many

molecular species.

Average energy of plasma is determined by

1st ionisation potential of Ar (15.8 eV)

Produces singly charged ions for most

elements. Exceptions include Ba & Sr which

have 2nd ionisation potential below that of Ar.

48

Spectrometer separates emitted light into its

component wavelengths - majority of

wavelengths lie between 160 to 860nm

Oxygen absorbs at wavelengths below

200nm so flush system with N2, Ar or under

vacuum

Separation of light achieved using a

diffraction grating

Light striking the grating will be diffracted to

a degree depending on wavelength

Plasma Based Spectrometers

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ICP Torch Lens Pinhole

Focusing

Mirror

Prism

Echelle

Grating Focusing

Mirror

Solid State

Detector

50

ICP Torch Lens Pinhole

Focusing

Mirror

Prism

Echelle

Grating Focusing

Mirror

Solid State

Detector

Ord

er

(gra

ting

)

Wavelength

(prism)

51

ICP-OES vs ICP-MS

Comparison of

(a) ICP-optical

spectrum for 100

ppm cerium and

(b) ICP-MS

spectrum for

10ppm cerium.

52

Atomic Mass Spectrometry

53

Detection Limits

54

ICP-MS Instrument

18

18

Detector

Mass analyser

(quadrupole)

Ion lenses

Sampler

Skimmer

Turbomolecular

pumps

Rotary pumps

ICP

Intermediate stage

(<1 x 10-7 bar)

Analyser stage

(<5 x 10-9 bar)

Expansion Chamber

(1x10-3 bar)

55

Quadrupole Mass Analyzers

56

Mass Spectrometer

The mass spectrometer acts as a filtertransmitting ions with a pre-selected mass/charge ratio.

All mass analysers perform two functions:

they separate ions according to their m/z ratio;

they measure the relative abundance of isotopes at each mass.

For successful operation there must be a collision free path for the ions to follow.

57

Mass Spectra

Typical ICP mass

spectrometers have a

mass range of 3-300

daltons, and provide

unit mass resolution.

Over 90% of the

elements have been

determined by ICP-MS.

58

Mass Spectra

The spectra produced by ICP-MS are remarkably simple

compared with ICP optical spectra (ie. ICP-AES).

They consist of a single peak for each element present and a

simple series of their isotopes.

• ICP-MS mass

spectrum of lead.

• Lead is not stable

so abundance is

source dependent

59

Interferences

•There are two types; spectral and non-spectral

•Spectral:

• Sub-divided into 'polyatomic' (e.g. 40Ar2+ on 80Se+) and

'isobaric' (e.g. 64Ni+ on 64Zn+)

• Polyatomic interferences formed from combination of

plasma gases and sample matrix constituents

• In many cases, can avoid interference by using

another isotope (e.g. 82Se+ in place of 80Se+, 66Zn+ in

place of 64Zn+)

• For interferences on mono-isotopic elements (e.g. 40Ar35Cl+ on 75As+), other strategies are required

60

Interferences

•There are two types; spectral and non-spectral

•Non-spectral:

• Signal suppression and enhancement effects

• Can overcome using internal standards and sample -

standard matrix matching approaches

61

Limit of Detection

The limit of detection is the smallest amount of a

substance that can be detected but not necessarily

quantified

Has to be significantly different to the blank

Recent guidelines (IUPAC) suggest the criteria should

be:

LOD = 3 x std. dev. of the blank concentration

Limit of determination is the lower limit for precise

quantitative measurement

LOQ = 10 x std. dev. of the blank concentration

62

Background Literature

Atomic Spectroscopy.

Harris, Quantitative Chemical Analysis

Chapter 21.

J.R. Dean, Atomic Absorption and Plasma

Spectroscopy, ACOL, John Wiley, ISBN 0471

972541

Web resource:

http://www.spectroscopynow.com

Food Aspects

ASU Review

http://www.asureviews.org/clinabstr.php

63

• At the end of this lecture you should:

• Appreciate the function and effect of

trace elements

• Understand the basic concepts behind

their measurement

• Appreciate the different types of atomic

spectrocopy: AAS, FES, ICP-OES and

ICP-MS

• Understand the main interferences

associated with each technique

Learning Outcomes

64

• Thursday 20th November 2014

• Meet in SAS Trace Element Lab, 15 Frederick

Sanger Road, Surrey Research Park

• Directions on the research park website

http://www.surrey-research-park.com/location/

• Practical sheets will be handed out

• The practical involves a tour of the lab, a

demonstration of ICP-MS and ICP-OES,

followed by some problems to be solved.

Practical Session