Trace Elements in Clinical Science

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

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.

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

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

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

20
Molecular and Atomic Spectroscopy
0
200
400
600
800
1000
1200
1400
250 350 450 550 650 750
/nm
Molarabsorptvity
0
200
400
600
800
1000
1200
1400
Fluorescence
Intensity(arbitrary)
0
200
400
600
800
1000
1200
1400
250 350 450 550 650 750
Wavelength/nm
Molarabsorptvity
0
200
400
600
800
1000
1200
1400
Fluorescence
Intensity(arbitrary)

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.

24
Atomic Absorption Spectroscopy

25
Radiation
source
(HCL)
Atomisation cell
(flame) Monochromator
Detector
(photomultiplier
tube)
SAMPLE
Aspiration
via nebuliser
(I0) (I)
Double Beam
Instrument

26

27
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
 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
pins
Anode
(tungsten)
Neon or Argon
Glass envelope
Under vacuum
1-5 torr

30
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 )
Fe0
* (Excited state Fe0
) is ejected .
Fe0
*  Fe0
+ hn.
Ne+
Fe0
*
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 )
Fe0
* (Excited state Fe0
) is ejected .
Fe0
*  Fe0
+ hn.
Ne+
Fe0
*
Hollow cathode

31
“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
emission
l
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
PO4
3-
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.

38
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

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

49
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
Order
(grating)
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.

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)

56
Mass Spectrometer
 The mass spectrometer acts as a filter
transmitting 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

Trace Elements in Clinical Science

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (20)

Similar to Trace Elements in Clinical Science

Similar to Trace Elements in Clinical Science (20)

Recently uploaded

Recently uploaded (20)

Trace Elements in Clinical Science