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?
 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”

 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”

 pH Meters / Ion Selective Electrodes (ISE)

Electrically charged ions effect potentials of electrochemical circuits

 Electrophoresis
Charged molecules move at different rates when “pulled” through an electrical field

 Osmometers
Dissolved molecules & ions are measured by freezing point depression and vapor pressure

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Electromagnetic Radiation:
Properties of light and radiant energy

 Electromagnetic radiation is described as photons of energy traveling

in waves

 There is a relationship between energy and the length of the wave

(wavelength)

 The more energy contained, the more frequent the wave and
therefore, the shorter the wavelength

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Electromagnetic Radiation:
Properties of light and radiant energy

 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

 The formula shows that the higher the frequency; the higher the energy
or the lower the frequency, the lower the energy

 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

 White light
 Combination of all wavelengths of light

 Diffract (bend) white light and all the colors

become visible

 The color you see depends on the wavelength of

color(s) that are not being absorbed

 Light that is not being absorbed is being

transmitted

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Electromagnetic Radiation:
Properties of light and radiant energy

 Wavelength
 Measured in nanometers (nm) or 10-9 meters.

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Electromagnetic Radiation
Properties of light and radiant energy

 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

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Electromagnetic Radiation (Properties of
light and radiant energy)

 In order for a ray of radiation to be absorbed it

must:
1. Have the same frequency of the rotational or
vibrational frequency in the molecules it strikes, and;
2. Be able to give up energy to the molecule it strikes.

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Electromagnetic Radiation

 Many lab chemistry instruments measure either the

absorption or emission of radiant energy /light.

 Spectroscopy is based on the mathematical relationship

between solute concentration & light absorbance
 Beer’s law

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Electromagnetic Radiation

 Beer's Law

 States the relationship between the absorption of light

by a solution and the concentration of the material in
the solution.

 The absorption and/or transmission of light through a

specimen is used to determine molar concentration of a
substance.

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Beer-Lambert law (Beer’s Law)

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Beer-Lambert law (Beer’s Law)

 A = 2 – log%T

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Requirements for Beer’s Law
 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

 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.

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Photometry/Spectrophotometry

 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

 Spectrophotometers differ from photometers in

that they use prisms or diffraction gratings to form
monochromatic light.

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Spectrophotometer: Components

 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

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Spectrophotometer: Components

 Monochromators
 Promote spectral isolation
 Operator selects specific wavelength
 Isolate a single wavelength of light
 Provides increased sensitivity & specificity

 Types
 Glass filters
 Prisms
 Diffraction gratings

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Spectrophotometer: Components

 Monochromator characteristic:
 Bandpass/bandwidth –
 Measures the success of the monochromator
 Defines the width of the segment of the spectrum that will be isolated
by the monochromator

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 Spectrophotometer: Components

 Cuvet
 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

 Photodetectors

 Purpose – to convert the transmitted light into an

equivalent amount of electrical energy
 Most common is the photomultiplier tube

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Spectrophotometer: Component

 Readout devices

 Purpose – to convert the electrical

signal from the detector to a
usable form

 Types
 Meters/Galvanometers
 Recorders
 Digital Readout

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Spectrophotometer:
Quality Assurance

 Wavelength calibration or accuracy is checked by

using special filters with known peak transmission
 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.

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Spectrophotometer: QA

 Stray light
 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

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Spectrophotometer: QA

 Linearity Check
 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.

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Spectrophotometer:
Sources of Error

 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

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Spectrophotometer:
Sources of Error

 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.

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Nephelometer
 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 Most of the component parts are similar
to those of the spectrophotometer.
Scatter” Major differences:
•The position of the detector and
reduces stray light
 Clinical Applications •Light source/beam= LASER light
 Protein measurements in
serum, CSF, immunoglobulins,
etc.
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References
 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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