UNIT: Electromagnetic Radiation and
Photometric Equipment
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Instrumentation I
Task
To review the theory of electromagnetic radiation and the principle and use of common laboratory
instruments using electromagnetic radiation.
Objectives
Upon completion of this exercise, the student will be able to:
1.
2.
3.
4.
5.
State the wave and particle theory of electromagnetic radiation.
Know what colors correspond to visible wavelengths.
Identify the regions of the electromagnetic spectrum occupied by gamma, x-rays, UV, visible,
IR, and microwaves indicating relative wavelength, frequency, and energy.
Know basic principles of how instruments determine results.
List and compare the principles/applications of different photometric equipment.
Principle
1.
Properties of light and radiant energy.
Radiant energy behaves as if it has electric and magnetic fields and is found as discrete
bundles of energy (photons) traveling in wave form (particle and wave theories). The more
energy contained, the more frequent the wave and therefore, the shorter the wavelength.
Wavelengths are measured in nanometers (nm) or 10-9 meters.
2.
Interactions of light and matter.
A.
Absorption – (absorption spectroscopy – UV, visible, infra-red, atomic)
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.
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.
MLAB 2401 - Clinical Chemistry Lab Manual CB 15
UNIT: Electromagnetic Radiation and Photometric Equipment (continued)
Energy
Frequency
>*
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Wavelength
(nm)
Type of Radiant
Energy
<0.1
gamma
non-visible
0.1 - 10
x-rays
non-visible
<380
ultra-violet
380-435
435-500
500-570
570-600
600-630
630-700
visible
>750
>25 x 107
Reflects
Absorbs
UV
green-yellow
yellow
red
blue
green-blue
green
violet
blue
green
yellow
orange
red
infra-red (heat)
non-visible
IR
radiowaves (uwaves)
continuous
non-visible
The energy absorbed is distributed throughout the structure. The absorption pattern of
complex organic molecules will be the cumulative sum of the absorption of all of the
individual covalent bonds as a result of the energy's distribution throughout the molecule.
As the energy is released from the atoms, molecules, or incandescent solids, an emission
spectra is formed.
B.
Emission – spectra are of three types.
1.
Line spectrum is light of one wavelength formed as the result of excited electrons
of atoms returning to their ground state. The atom emits the same wavelengths
as those absorbed by the atom.
2.
Band spectrum is a group(s) of wavelengths formed from the return of excited
electrons in molecules returning to their ground state. An array of wavelengths are
emitted as the process is complicated by the internal energy of vibration and
rotation (all substances except rarefied gas and atoms).
3.
Continuous spectrum is a very broad and continuous group of wavelengths
emitted by incandescent solids (tungsten lamps).
Electromagnetic radiation in its various forms is measured in common laboratory instruments:
A.
Absorption of EM radiation
1. atomic absorption
2. spectrophotometry
B.
Emission of EM radiation
1. flame photometer
2. fluorometer
3. scintillation counter
B 16 C MLAB 2401 - Clinical Chemistry Lab Manual
UNIT: Electromagnetic Radiation and Photometric Equipment (continued)
Transmission and
Absorbance Instruments
Fluorescence and
Instruments
Nephelometers
Reflectance Spectrophotometers (diagram above left), as found in many modern instruments
such as UA dipstick readers and Kodac’s Ectachem:
A, light source;
B, slit;
C, filter or wavelength selector;
D, collimating lens or slit;
E, test surface;
F, collimating lens or slit;
G, detector;
H, readout device.
MLAB 2401 - Clinical Chemistry Lab Manual CB 17
UNIT: Electromagnetic Radiation and Photometric Equipment (continued)
Energy
Source
Monochromater
absorption/transmission
of light through a colored
solution providing a
means of measuring light
absorbing particles
tungsten
(deuterium,
Hg, He)
filter
(interferenc
e filter)
visible – change in Abs.
between 400-700 nm
UV – change in Abs. at
340nm of NAD : NADH
tungsten/UV
(deuterium,
Hg, He)
prism or
diffraction
grating
Filtered light is focused on
a flat test surface. Some
of the light is absorbed,
the remainder is reflected
onto a photodetector. The
absorption of light by
chromophores (reflection
density) is inversely
related to the intensity of
reflected light.
tungsten
filter
lipids, CSF protein,
serum proteins,
amylase
light transmission through
a turbid solution
tungsten/UV
filter
UV
filter
fluorometer
Fluorometry
drugs, hormones,
intermediary
metabolites
absorbs high intensity UV
and emits longer
wavelength visible light
(lower energy)
UV source
2
filters/prism
Primary
exciter filter
Secondary
fluorescenc
e filter
cuvette
slit
Emission Flame
Photometry
electrolytes, trace
minerals (Na, K, Li)
The energy absorbed as
heat is released in the
form of light energy of a
wavelength specific for
the element proportional
to its concentration
hot flame
specific
filter for Na,
K, Li
burneraspirator
slit
Atomic Absorption
Spectrometry
Ca, Mg, Cu, Mn, Fe,
Pb, Hg, etc.
A hot flame releases
metallic atoms from molecules. The ground state
metallic atoms absorb
monochromatic light,
generated by a hollow
cathode tube, proportional
to their concentration
hollow
cathode tube
of measured
metal
prism or
diffraction
grating
burneraspirator
slit
Densitometry
(very basic
photometer)
Hb, protein electrophoresis, CPK, LDH
isoenzymes
photometer scans a
colored strip
tungsten/UV
filter
stage slit
Refractometry
TP, specific gravity
light bends with
concentration; refractive
index
tungsten
none
stage slit
Scintillation
Counters
a. gamma
b. beta (liquid)
drug assays,
hormones
immunoassays (TSH,
T3, T4, B12 , Folate)
EM radiation – counts
directly proportional to
concentration. Sample in
liquid phosphor.
Fluorescence converted
into longer wavelength
atomic
nucleus
none
cuvette
slit
Equipment
Colorimetry/
photometry
Clinical Applications
any procedure
measuring color
development or loss
Spectrophotometry
Reflectance
Spectrophotometry
Turbidimetry
------------------Nephelometry
Same as above
Principle
degree of light scattered
B 18 C MLAB 2401 - Clinical Chemistry Lab Manual
Sample
Holder
cuvette
slit
varies
paper,
plastic
strip, dry
film or
slide
cuvette
slit
UNIT: Electromagnetic Radiation and Photometric Equipment (continued)
Study Questions
Name
Instructions: Legibly write your answers in the space provided. Unless otherwise indicated, each
question is worth one point.
1.
Describe / define wavelength.
2.
In clinical laboratory work, wavelength is most commonly measured in what unit?
3.
The discrete bundles of energy emission in the electromagnetic spectrum are called
__________________________________________.
4.
Ultraviolet light has shorter / longer (circle one) wavelength than visible light and would
therefore have higher / lower (circle one) energy than visible light.
5.
Gamma rays have a very short wavelength. You would, therefore, expect them to have
higher / lower (circle one) frequency and energy.
6.
What type of emission spectra is expected from:
a.
atoms –
b.
molecules –
c.
solids –
7.
According to information presented in this lab, briefly summarize what happens when
electromagnetic radiation is absorbed by an atom or molecule. (2 points)
8.
A spectrophotometer and an atomic absorption spectrophotometer can be grouped together
as they measure light _________________________________________.
9.
A flame photometer and scintillation counter (gamma counter) measure light/radiation
_________________________________.
10.
From the Table of Photometric Equipment included in the lab, list two (2) types of chemistry
equipment that measure change in the electromagnetic radiation.
MLAB 2401 - Clinical Chemistry Lab Manual CB 19
UNIT: Electromagnetic Radiation and Photometric Equipment (continued)
After reviewing the lab, provide a brief explanation of the principles of the following in your own
words. (2 points each)
11.
photometry –
12.
emission flame photometry –
13.
atomic absorption spectrophotometry –
14.
reflectance photometry -
15.
fluorometry -
B 20 C MLAB 2401 - Clinical Chemistry Lab Manual