UNIT: Electromagnetic Radiation and

Photometric Equipment 3photo.wpd

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. State the wave and particle theory of electromagnetic radiation.

2. Know what colors correspond to visible wavelengths.
3. Identify the regions of the electromagnetic spectrum occupied by gamma, x-rays, UV, visible,
IR, and microwaves indicating relative wavelength, frequency, and energy.
4. Know basic principles of how instruments determine results.
5. 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.

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UNIT: Electromagnetic Radiation and Photometric Equipment (continued)

Wavelength Type of Radiant

Energy Frequency Reflects Absorbs
(nm) Energy

>* >* <0.1 gamma non-visible

* *
* * 0.1 - 10 x-rays non-visible
* *
* * <380 ultra-violet UV
* *
* * 380-435 green-yellow violet
* * 435-500 yellow blue
* * 500-570 red green
visible
* * 570-600 blue yellow
* * 600-630 green-blue orange
* * 630-700 green red

>750 infra-red (heat) non-visible IR

continuous
>25 x 107 radiowaves (uwaves)
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

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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.

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 UNIT: Electromagnetic Radiation and Photometric Equipment (continued)

Energy Mono- Sample

Equipment Clinical Applications Principle
Source chromater Holder
Colorimetry/ any procedure absorption/transmission tungsten filter
photometry measuring color of light through a colored (deuterium, (interferenc
development or loss solution providing a Hg, He) e filter)
means of measuring light
absorbing particles cuvette
slit
Spectrophotometry visible – change in Abs. tungsten/UV prism or
between 400-700 nm (deuterium, diffraction
UV – change in Abs. at Hg, He) grating
340nm of NAD : NADH
Reflectance Same as above Filtered light is focused on tungsten filter
Spectrophotometry a flat test surface. Some
of the light is absorbed, varies
the remainder is reflected paper,
onto a photodetector. The plastic
absorption of light by strip, dry
chromophores (reflection film or
density) is inversely slide
related to the intensity of
reflected light.
Turbidimetry lipids, CSF protein, light transmission through tungsten/UV filter
------------------- serum proteins, a turbid solution cuvette
Nephelometry amylase degree of light scattered UV filter slit
fluorometer
Fluorometry drugs, hormones, absorbs high intensity UV UV source 2 cuvette
intermediary and emits longer filters/prism slit
metabolites wavelength visible light Primary
(lower energy) exciter filter
Secondary
fluorescenc
e filter
Emission Flame electrolytes, trace The energy absorbed as hot flame specific burner-
Photometry minerals (Na, K, Li) heat is released in the filter for Na, aspirator
form of light energy of a K, Li slit
wavelength specific for
the element proportional
to its concentration
Atomic Absorption Ca, Mg, Cu, Mn, Fe, A hot flame releases hollow prism or burner-
Spectrometry Pb, Hg, etc. metallic atoms from mole- cathode tube diffraction aspirator
cules. The ground state of measured grating slit
metallic atoms absorb metal
monochromatic light,
generated by a hollow
cathode tube, proportional
to their concentration
Densitometry Hb, protein electro- photometer scans a tungsten/UV filter stage slit
(very basic phoresis, CPK, LDH colored strip
photometer) isoenzymes
Refractometry TP, specific gravity light bends with tungsten none stage slit
concentration; refractive
index
Scintillation drug assays, EM radiation – counts atomic none cuvette
Counters hormones directly proportional to nucleus slit
a. gamma immunoassays (TSH, concentration. Sample in
b. beta (liquid) T3, T4, B12 , Folate) liquid phosphor.
Fluorescence converted
into longer wavelength

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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.

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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 -

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