LECTURE 2.7-2.
8
Lecturer: Samantha Brown
ICP-AES Instrumentation
Sample introduction system
and the ICP torch
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NR149.htm /refiningandassaying.htm
Detectors in ICP- AES
• Photomultiplier Tubes
• Charge Transfer Devices (CCDs, CIDs)
• PMTs: Single channel devices
(one wavelength measured at a time)
• CTDs: Multichannel devices
(multiple wavelengths measured simultaneously)
Semiconductor based charge storage and readout
ICP-AES
Offer several advantages over flame:
Lower inter-element interference (higher temperatures)
With a single set of conditions signals for dozens of elements can be
recorded simultaneously
Lower LOD for elements resistant to decomposition
Permit determination of non-metals (Cl, Br, I, S)
Can analyse concentration ranges over several decades (vs 1 or 2
decades for other methods)
Disadvantages:
More complicated and expensive to run
Require higher degree of operator skill
Elements by ICP-AES
core
electrons
Different elements have different emission intensities.
Alkalis (Na, K, Rb, Cs) are weakly emitting. Alkaline
Earths (Be, Mg, Ca, Sr, Ba ) are strongly emitting.
Performance characteristics
• Elements determined
• ~ 60 elements
• Line selection
• Most elements have several lines that can be selected
• Linear range
• Better than AAS
• Interferences
• Chemical interferences (lowered)
• Spectral interferences (still a problem)
• Detection limits
• Comparable or better than other atomic spectral
techniques
Modern ICP-OES spectrometer
• Over 70 elements (in principle simultaneously)
• Including non-metals such as sulfur, phosphorus,
and halogens (not possible with AAS)
• ppm to ppb range
Applications
ICP-OES used for quantitative analysis of:
Soil, sediment, rocks, minerals, air
Geochemistry
Mineralogy
Agriculture
Forestry
Forensics
Environmental sciences
Food industry
Elements not accessible using AAS
Sulfur, Boron, Phosphorus, Titanium, and Zirconium
ICP-MS
• The ICP is also a rich source of singly charged
positive ions
• Mass spectrometry method: detects ions
distinguished by their mass-to-charge ratio (m/z
value)
• Based on ions moving under influence of electrical or
magnetic field
• Mass analysers generally require operation under
vacuum, to avoid ions colliding with other particles
• Recommended series of short articles: Robert
Thomas: A beginner’s guide to ICP-MS
ICP-MS instrumentation and principle
Plasma generates Under vacuum
positive ions
Detector (e.g.
electron
multiplier)
nebuliser
Interface
Sorted by mass analyser,
Spray chamber e.g. quadrupole, magnetic
sector, according to m/z
ratio
sample
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Instrumentation
low vacuum intermediate vacuum high vacuum
~3x10-3 bar ~1x10-7 bar ~5x10-10 bar
ion beam
14 ICP-MS Training - Cees-Jan De Hoog
Possible factors that can affect the
performance of ICP-MS
• Variations in plasma ionization efficiency
• Possible clogging or corrosion of cone apertures
• Differing concentrations of other components in
matrix (e.g. acid, bulk elements) in samples could
result in matrix suppression
• Ion current influenced by matrix composition
• Temperature and humidity fluctuations in the
laboratory environment
• Isobaric elemental and polyatomic interferences:
Used to be greatest limitation for applicability
Comparison: AAS, ICP-AES, and ICP-MS
• AAS: Single element, ppm/ppb range
• Cheap, simple
• Small dynamic range
• GFAAS about 100 times more sensitive than FAAS, but also
more challenging
• ICP-AES: Multi-element, ppb range
• Limited spectral interferences, good stability, low matrix
effects
• ICP-MS: Multi-element, possible to reach ppt (or
even ppq)
• Most complex, most expensive, lowest detection limits,
isotope analysis possible
Typical detection limits of ICP-MS instrument
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Comparison: Detection limits and
working ranges
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How much
money (US$) do
you have?
Flame AAS:
$25k
GFAAS:
$50k
ICP-AES :
$100k
ICP-MS:
$200k
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bro_atomicspectroscopytechniqueguide.pdf
X rays
PANalytical (Netherlands) http://www.panalytical.com/
XRF
• XRF (X-ray fluorescence spectrometry) is a non-
destructive analytical technique used to identify and
determine the concentrations of elements present in
solid, powdered and liquid samples.
XRF Principles
• When x-rays interact with matter there are two
possible outcomes; photoelectric absorption or
scattering of the incident photons.
• Photoelectric absorption results in
characteristic peaks in an XRF spectrum
• Scattering results in increased background
Sample preparation for XRF
• SOLIDS (uniform particle size)
• Metals (polished)
• Loose powders (grind)
• Pressed (powder) pellets
• Fused beads
• LIQUIDS
• Filter media
Quantification
XRF is a reference method,
standards are required for
quantitative results.
Standards are analysed,
Concentration
intensities obtained, and a
calibration plot is generated
(intensities vs. concentration).
Intensity
XRF instruments compare the
spectral intensities of unknown
samples to those of known
standards.
Applications of XRF
• Very useful for non-destructive analysis of valuable artifacts
and discovery of forgeries
• Also used for on-site detection of Pb in paint and children’s
toys
• Geochemical Mapping
• Essential and Trace elements in the soil-plant chain
• Heavy Metal Pollution
• Biomonitoring of Air pollution
• Trace Elements in Animal/Human Tissue
Immunoassay
Immunoassay is used to screen a biological sample for the presence of
an antigen (drug)
Definitions
• Immunoassay :is a scientific test which uses antibodies to identify and
measure the amounts of a chemical substance.
• Antibody : Proteins produced by animal lymphocytes in response to
the presence of a foreign substance.
• Antigen: antigen -- any agent (molecule) that binds to components of
the immune response.
• Immunogen : any agent capable of inducing an immune response
Using Antibodies
• Antibodies are used to impart specificity.
• Inject the compound into animal – produce antiserum
for that specific compound
• Anti serum is collected and mixed with labeled drug
and with the drug itself.
• Labeled and unlabeled drug compete for the antibody
binding sites.
• Concentration of drug can be determined using the
Law of Mass action or by comparing the amount of
labeled drug bound to antibody in a sample to that of
reference standards containing known concentration of
antigen .
Antibody formation
• Binding site recognizes the antigen
by molecular structure and spatial
arrangement.
• Antibodies reject unrelated
molecules
• But may bind compounds that are
structurally related ie cross-
reactivity.
Cross Reactivity vs Specificity
• Specificity -Extent to which a an assay can
correctly identify only the compound of interest.
• In IA antiserum specificity.
• Cross reactivity - Extent to which an assay
responds to compounds other than that which it
was designed to detect.
• Cross reactivity can give “false positive” results.
Need for confirmation by another method e.g. GCMS
• “cutoff” concentration of drug below which all
specimen results are considered negative.
Cross Reactivity vs Specificity
barbital pentobarbital phenobarbital
Linking barbituate at substituent group - antibodies
recognize the barbituate ring
Link barbituate at nitrogen - then substituent groups are
recognized and a more selective assay for the particular
assay is obtained.
Commercial Immunoassays for drug
detection
• Principle of ‘competitive binding’. An antigen similar to the target
compound is conjugated to a signaling molecule and competes with
target drug in a sample for antibody binding.
• Immunoassays are also classified as homogeneous and
heterogeneous.
• Homogeneous assays do not separate the original sample from the
final detection sample. They must use a signal that changes when
antibody is bound.
• Heterogenous assays –signaling molecules are removed from the
original sample before measurement
Characteristics of labelled compounds
• Use labelled compounds which
• Should be Immunologically similar to substance being tested
i.e. Able to compete for antibody binding
• Should be readily detected and not affected by interference
from matrix components.
• Label can be
• Radioactive
• Fluorescent
• Enzyme
• Microparticle
• Add labelled molecule to reaction mixture
• Assay detects changes associated with the label when
bound vs. when free
Homogeneous Immunoassays
Homogeneous immunoassays include :
• FPIA - Fluorescent Polarization Immunoassay -
(emission in a polar field increases when bound)
• KIMS - Kinetic Interaction of Microparticles in
Solution immunoassay (lattice formation inhibited
when bound).
• EMIT – Enzyme Multiplied Immunoassy Technique
Example of Homogenous (no separation) IA -
EMIT
Uses enzyme linked antigen
• Components of the EMIT Assay Method
– Drug
– Antibody
– Substrate
– Enzyme bound to drug
• Enzyme reacts with substrate to give product NADH.
NADH production monitored spectrophotometrically
• Adding drug (sample) reduces antibody available and
increases the rate of NADH production .
• Change in absorbance at is directly proportional to the
concentration of drug in the sample.
EMIT Assay Components in Action
Redrawn from: Pieper and Rutledge, Laboratory Techniques for Pharmacists, Upjohn 1989.
Heterogeneous (separation) Immunoassays
Include:
• RIA - radio immunoassay (measure unbound
radiolabeled antigen)
• ELISA - enzyme-linked immunosorbent assay
(monitor enzyme conjugated antigen or antibody)
RIA
1.Known quantity of antigen labelled with Iodine 125
2.Mix with known amount of antibody
3.Sample with unknown amount of antigen is added which displaces
I125 labelled bound antigen
4.Bound separated from free (supernatant)
5.Radioactivity of free measured using gamma counter.
Example insulin.
Strengths
•Sensitive
•Free from matrix effects therefore can be used for post mortem
samples
Limitations
1.Disposing of radioactive waste
2.Shelf life is limited (radioactive decay) less than 60days
3.Heterogeneous assays cannot be adapted to automated analysis
RIA
Antigen- labelled Competitive ELISA
Competitive, solid-phase, heterogeneous, enzyme
immunoassay
• Antibodies immobilized
• Free drug and drugs conjugated to an enzyme compete for
binding to the antibody.
• Incubated and washed.
• Bound enzyme conjugate and bound drug are left on plate .
• Substrate solution added – gives colored product in the
presence of enzyme.
• Measure OD – Readings are inversely proportional to the
amount of free drug present in the original sample.
Antigen- labelled Competitive ELISA
Redrawn from: Pieper and Rutledge, Laboratory Techniques for Pharmacists, Upjohn 1989.
Antibody- labelled Competitive ELISA
• Antibody is directly coupled to the enzyme
• Antigen is bound to the microplate.
• Competition between the analyte in the sample and the
immobilized analyte for binding to the enzyme labelled antibody.
• Incubation then wash to separates bound and free fractions
• Leave enzyme- labelled antibody bound to the analyte derivative in
the well.
• Add substrate solution to get colour reaction.
• Inverse relationship . Calibration curve has negative slope.
Sandwich ELISA
• Sandwich or indirect ELISA uses two or more
antibodies.
• Analyte must have more than one binding site.
• Excess primary antibody is bound to the microplate
and incubated with the sample.
• After washing a second enzyme-labelled antibody is
added and allowed to incubate.
• After a second wash to remove excess label a solution
is added to develop the colour.
• Advantage : calibration curve has a positive slope,
more sensitive than competitive ELISA, equilibrium
times are short .
ELISA vs. RIA
• ELISA uses an enzyme for labelling rather than a
radioisotope.
• ELISA measures bound enzyme activity rather than
bound counts per minutes.
• Quantification requires measuring the colour intensity of
the colored products generated by the enzyme and
added substrate.
• The intensity of the colour is equivalent to the amount of
labeled antibody bound to antigen.
• ELISA has replaced RIA in many clinical and basic
science laboratories.
Various formats of ELISA
Sandwich ELISA
Micro plate reader
An instrument for optical reading of microplates.
An instrument that enables optical reading and determination of the components
displayed on microplates as a result of a specific assay.
ELISA
ANALYZER
Analyzers for photometric immunoassays.
Immunoassay analyzers using photometric radiation restriction methods.
Many of these analyzers use a visible or UV light source.
Immunoassays are optically read from within the microplate sample tray.
Results are presented and printed.
Immunoassay Analyzer
Analyzers for enzymes using fluorescence and chemiluminescence
immunoassay techniques.
FIA, EIA, and CIA analyzers are instruments that use reagents
to analyze drugs and endogenous substances in biological fluids.
May be used to determine drug concentrations
( such as antiarrhythmics, antibiotics, anticonvulsants, or cardiac glycosides)
as well as testing for endocrines, proteins, and bacterial toxins.
