MLAB 2401:
Clinical Chemistry
Keri Brophy-Martinez
Analytical Techniques and Instrumentation
Electromagnetic Radiation &
Spectrophotometry
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Introduction
How do we actually measure the concentrations of molecules that are
dissolved in the blood?
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Spectrophotometry
Mix chemicals together to produce colored products , shine a specific wavelength of light
thru the solution and measure how much of the light gets “absorbed”
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Nephelometry and Turbidimetry
Mix chemicals together to produce cloudy or particulate matter , shine a light thru the
suspension and measure how much light gets “ absorbed” or “refracted”
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pH Meters / Ion Selective Electrodes (ISE)
Electrically charged ions effect potentials of electrochemical circuits
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Electrophoresis
Charged molecules move at different rates when “pulled” through an electrical field
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Osmometers
Dissolved molecules & ions are measured by freezing point depression and vapor pressure
Electromagnetic Radiation:
Properties of light and radiant energy
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Electromagnetic radiation is described as photons of energy traveling
in waves
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There is a relationship between energy and the length of the wave
(wavelength)
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The more energy contained, the more frequent the wave and
therefore, the shorter the wavelength
Electromagnetic Radiation:
Properties of light and radiant energy
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This relationship between energy and light is expressed by Planck's
formula:
E = hf
Where:
E= energy of a photon
h = a constant
f = frequency
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The formula shows that the higher the frequency; the higher the energy
or the lower the frequency, the lower the energy
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We do not use this to perform any calculations. You only need to
recognize Planck’s formula and its components
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Electromagnetic Spectra
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Electromagnetic Radiation:
Properties of light and radiant energy
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White light
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Combination of all wavelengths of light
Diffract (bend) white light and all the colors
become visible
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The color you see depends on the wavelength of
color(s) that are not being absorbed
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Light that is not being absorbed is being
transmitted
Electromagnetic Radiation:
Properties of light and radiant energy
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Wavelength
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Measured in nanometers (nm) or 10-9 meters.
Electromagnetic Radiation
Properties of light and radiant energy
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Interactions of light and matter
 When an atom, ion, or molecule absorbs a photon, the
additional energy results in an alteration of state (it
becomes excited). Depending on the individual
“species,” this may mean that a valence electron has
been put into a higher energy level, or that the vibration
or rotation of covalent bonds of the molecule have
been changed.
 Ultimately, as energy is released, an emission spectra is
formed
Electromagnetic Radiation (Properties of
light and radiant energy)
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In order for a ray of radiation to be absorbed it
must:
1.
2.
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Have the same frequency of the rotational or
vibrational frequency in the molecules it strikes, and;
Be able to give up energy to the molecule it strikes.
Electromagnetic Radiation
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Many lab chemistry instruments measure either the
absorption or emission of radiant energy /light.
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Spectroscopy is based on the mathematical relationship
between solute concentration & light absorbance
 Beer’s law
Electromagnetic Radiation
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Beer's Law
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States the relationship between the absorption of light
by a solution and the concentration of the material in
the solution.
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The absorption and/or transmission of light through a
specimen is used to determine molar concentration of a
substance.
Beer-Lambert law (Beer’s Law)
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Beer-Lambert law (Beer’s Law)
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A = 2 – log%T
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Requirements for Beer’s Law
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Keep light path constant by using matching sample cuvettes
standardized for diameter and thickness
Solution demonstrates a straight line or linear relationship between two
quantities in which the change in one (absorption) produces a
proportional change in the other (concentration).
Not all solutions demonstrate a straight line graph at all concentrations.
If these rules are followed, we can calculate / determine an
unknown’s concentration, by comparing a characteristic (its
absorbance) to the same characteristic of the standard (whose
concentration is known – by definition)
Concentration unk = (Aunk /Astd) * Concentration std
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Percent transmittance
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Photometry/Spectrophotometry
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In photometry we measure the amount of light
transmitted through a solution in order to determine
the concentration of the light absorbing molecules
present within.
Photometry/Spectrophotometry
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Types -Simple photometers and colorimeters use a
filter to produce light of one wavelength
(monochromatic light).
Major components of a simple photometer.
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Spectrophotometer /
Spectrophotometry
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Spectrophotometers differ from photometers in
that they use prisms or diffraction gratings to form
monochromatic light.
Spectrophotometer: Components
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Light source/lamps
 Vary according to need, but must be a constant beam,
cool and orderly
 Types
 Tungsten or tungsten iodide lamps for visible and near
infrared
 Incandescent light (400 nm - 700 nm)
 Deuterium or mercury-arc lamps required for work
in U.V. range
 Range 160-375 nm
Spectrophotometer: Components
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Monochromators
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Promote spectral isolation
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Isolate a single wavelength of light
Provides increased sensitivity & specificity
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Types
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Operator selects specific wavelength
Glass filters
Prisms
Diffraction gratings
Spectrophotometer: Components
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Monochromator characteristic:
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Bandpass/bandwidth –
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Measures the success of the monochromator
Defines the width of the segment of the spectrum that will be isolated
by the monochromator
Spectrophotometer: Components
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Cuvet
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Made of high quality glass or quartz
 Glass – for work in the visible light range
 Quartz or fused silica – for work in the UV range
Shape
 Round cuvets are cheaper but light refraction and distortion
occur
 Square cuvets have less light refraction but usually more costly
Optically clean
 No inconsistencies in composition
 No marks, scratches, or fingerprints
Positioning
 Orientation and placement into the instrument important.
Each time must be the same so light passes through the cuvet
at the same place.
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Spectrophotometer: Component
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Photodetectors
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Purpose – to convert the transmitted light into an
equivalent amount of electrical energy
Most common is the photomultiplier tube
Spectrophotometer: Component
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Readout devices
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Purpose – to convert the electrical
signal from the detector to a
usable form
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Types
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Meters/Galvanometers
Recorders
Digital Readout
Spectrophotometer:
Quality Assurance
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Wavelength calibration or accuracy is checked by
using special filters with known peak transmission
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Should be done periodically
Must be done if a parameter, such as a change in light /
lamp has taken place.
Must be done if the instrument has been bumped or
traumatized.
Wavelength calibration verifies that the wavelength
indicated on the dial is what is being passed through the
monochromator.
Spectrophotometer: QA
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Stray light
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any wavelength of light reaching the detector, outside
the range of wavelengths being transmitted by the
monochromator.
Spectrophotometers must be periodically checked for
Stray Light
Causes insensitivity and linearity issues
Resolve by cleaning optical system
Spectrophotometer: QA
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Linearity Check
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A linearity check is made by reading the absorbance of a
set of standard solutions (obtained commercially) at
specified wavelength(s), or by using neutral density filters
Produces a graph similar in appearance to standard curve.
Spectrophotometer:
Sources of Error
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Lamp burnout – most frequent source of error
 Hours of use can be logged by system
 Watch for lamp to turn dark or smoky in color
Monochromator error
 Poor resolution due to wide bandpass
 Results in decreased linearity and sensitivity
Cuvet errors
 Dirt, scratches, loose cuvet holder - all cause stray light
Air bubbles in specimen
Spectrophotometer:
Sources of Error
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Reagent make-up
 some test procedures make a product that easily foams
Volume too low for light path
Electrical static (noise)
Dark current - from the detector. Leakage of electrons
when no light passing through.
Nephelometer
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Principle
 Measures scattered light
 Light “bounces” off insoluble
complexes and hits a
photodetector
 The photodetector is at an
angle off from the initial
direction of the light.
 This is a measure of ‘Light
Scatter”
Clinical Applications
 Protein measurements in
serum, CSF, immunoglobulins,
etc.
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Most of the component parts are similar
to those of the spectrophotometer.
Major differences:
•The position of the detector and
reduces stray light
•Light source/beam= LASER light
References
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Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry:Techniques,
principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams &
Wilkins.
Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper
Saddle River: Pearson .
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