THE FUNDAMENTALS OF...

Spectrophotometers
Robert M. Dondelinger

A
ll spectrophotometers
rely on the 150-year-old
observation that a mate-
rial, like an invisible gas or any
liquid, will absorb some amount
of specific wavelengths of light
while passing some amount of
other wavelengths. One might
think that this observation is,
at best, an interesting phenom-
enon, but developing this tech-
nology has given the research
and clinical laboratories one of
the most important and versa-
tile laboratory instruments ever
devised. Between the 1950s
and the 1970s, spectropho-
tometers were a fundamental
instrument found in every
A healthcare worker operates a spectrophotometer.
hospital-based clinical labora-
tory in the country.
Today, they are found pri-
marily in research facilities and reference laboratories. utilize these same optical principles.
This technology did not disappear from clinical labo- Before describing the spectrophotometer, some
ratories, however, since nearly every hospital laboratory knowledge of its use in performing basic laboratory tests
contains automated analyzers employing the same basic must be attained to understand how this instrument is
principles embodied in these legacy manual instruments. used and what it does. As the spectrophotometer evolved,
At the heart of many of these modern instruments lies aside from merely being able to measure absorbance (the
a spectrophotometer, albeit enveloped by some complex amount of light absorbed by the sample expressed as a
automated sample preparation hardware, sample flow percentage) or transmittance (how much light is passed,
plumbing, and, often, computer-based flow and output the mathematic reciprocal of absorbance), two important
controls. Likewise, dry chemistry analyzers utilizing light observations were made about the sample. First, the
reflectance technology, a cousin of spectrophotometry, amount of light absorbed or transmitted at a particular
wavelength is proportional to the concentration of a
Robert Dondelinger, CBET-E, MS, is the material. That is, the more of a particular material that
senior medical logistician at the U.S. exists in the sample, the greater the light absorbance. If
Military Entrance Processing Command
in North Chicago, IL. E-mail: robert. the material does not absorb light itself, one or more re-
dondelinger@mepcom.army.mil agents can be mixed into the solution and its absorbance
or transmittance will be proportional to the concentra-
tion of the material.

Biomedical Instrumentation & Technology 139

 THE FUNDAMENTALS OF...
Spectrophotometers
Figure 1. In this diagram of a basic spectrophotometer, light passes through the
monochromator, the sample in the cuvette, and finally strikes the detector, in
this case a photomultiplier tube. Its output can be used directly, amplified, or
passed to some other device like a computer.
Second, through prior testing, using titrated aliquots
of a material, a correlation can be drawn between the
absorbance or transmittance and the concentration of
the material. What is “the material”? It can be a plethora
of things from the amount of iron in the blood, alkaline
phosphatase in the serum, or the amount of copper in
tissue. The “secret,” if there is one, is in knowing which
reagent or reagents (when necessary) to use, the process
to employ (employing agitation, heat, cold, or simply
allowing time to pass until the reaction occurs), the
wavelength of light that yields the best results, and the
conversion from the absorbance or transmittance read-
ing to the known value of the material.
For manual lab tests performed with the spectropho-
tometer, this conversion information could be a graph
in a textbook containing a smooth curve showing the
instrument reading on one axis and the copper value, for
example, on the other axis. If the lab technician is using
a pre-packaged test kit, it will contain the reagents and a
packaging insert containing step-by-step instructions to
perform the test and the aforementioned graph.
Current Technology
To measure this absorbance or transmittance light by a
material and quantify the results, basic single-beam spec-
trophotometers rely on a collection of basic components:
a light source, light filter, a focusing device or devices, an
absorption cell or sample cuvette, a photodetector, and a
display device.
The first component is the light source, which must
provide both the correct wavelengths of light and a con-
stant intensity. Typically, a tungsten-filament bulb is used
because it provides light in the wavelength of 330 to about
140 March/April 2011
900 nanometers (nm), covering the visible region quite
well. Hydrogen or deuterium lamps are frequently used
for ultraviolet light, producing wavelengths from about
200 to 450 nm. Between the two lamps, the wavelengths
of interest are covered.
The full spectrum light from the bulb passes through a
light filter, called a “monochromator,” which allows only a
narrow slice (typically 0.05–20 nm wide) of the overall light
spectrum, centered on the wavelength of interest, to pass
through to the sample. The filter typically uses colored fil-
ters or a variable-width narrow slit to filter the light. The
filter can be adjusted manually by the lab technician or, on
highly automated spectrophotometer models, scanned or
automatically swept through a range of wavelengths while
documenting continuous successive readings.
To ensure the light rays are parallel as they go through
the sample, which both reduces scattering in and around
the sample, most spectrophotometers place a combina-
tion of lenses, slits, and/or mirrors in the light path.
These focusing devices may include components of the
monochromator as a first stage in the focusing process.
Next, the light passes through an absorption cell, or
cuvette, containing the sample being analyzed. Typically,
cuvettes are round or rectangular and are constructed of
glass, quartz, fused silica, or plastic. The key factor about
the cuvette is that the material of which it is made should
be completely transparent to the light (wavelength) being
used for the analysis. This is not easy to do since optical
glass absorbs light below 350 nm. Quartz cuvettes do not
possess this characteristic, but they are both more expen-
sive and more fragile than optical glass cuvettes. Cuvettes
are always placed in a dark analysis chamber while being
read; however, some designs employ a rotating cham-
ber so that a sample-filed cuvette can be changed while
another is being read. One variant of the basic design
uses a special flow-through cuvette design to aspirate the
sample into the cuvette, measure the absorbance, then
dispose of the sample and rinse the cuvette in preparation
for the next sample. A similar variant uses a continuous-
flow cuvette to provide constant absorbance readings of
a liquid stream.
When the light passes through the cuvette and the
sample contained therein, some of the spectrum has been
absorbed by the sample. The remaining light exits the
cuvette and strikes a photodetector (a photomultiplier
tube, photocell, or phototransistor) which provides an
electronic output proportional to the intensity of the
light leaving the sample. The simplest legacy units, still

found in daily use in developing countries, pass the elec-
tronic output to a sensitive galvanometer—a device simi-
lar to an analog meter movement—containing a mirror
in place of the customary needle or pen. Light bouncing
off the mirror illuminates a spot on a translucent scale
calibrated to provide a direct reading of the absorbance
or conductance. More sophisticated models amplify and
process the photodetector’s electronic output to an in-
tegral calculator or computer electronics to provide this
and other operator-selected information via a digital
readout on the unit. High-end units provide their output
to a personal computer that provides this and other valu-
able functionality.
Single-beam spectrophotometers must first be set to
zero absorbance with a reagent blank inserted into the
cuvette; that is, the reagent blank is read and the instru-
ment adjusted for 0.00% absorbance (100% transmit-
tance). Then the lab technician replaces the blank with
the sample and the absorbance of the sample is deter-
mined and read. To eliminate this wasteful step and to
increase lab throughput, a number of variant designs are
available in the marketplace. One design uses a special
mirror to split the light beam, passing one part through
the cuvette to the sample detector and the other part to a
reference detector. Another variant uses one or more ro-
The Origin and Evolution of the Device
I n 1859, a scientist studying the properties of various
gasses constructed the first spectrophotometer to
measure the absorptive powers of gasses such as carbon
dioxide, ozone, various hydrocarbons, and water vapor.
Surprisingly, to the scientist that is, these “perfectly col-
orless and invisible gasses and vapors” absorbed some
wavelengths of light while passing others. On Jan. 8,
1935, Professor Arthur C. Hardy of the Massachusetts
Institute of Technology was granted a patent for the
spectrophotometer. Five months later, General Electric
introduced the first commercial recording spectropho-
tometer based on his work. In 1940, Arnold O. Beck-
man, the founder of Beckman Instruments, assembled
a spectrophotometer using a glass prism, a vacuum
tube photocell, and an amplifier from his company’s
pH meter. The following year, Beckman introduced
an improved version which increased throughput and
raised accuracy from about 25% to around 99.99%,
setting a new standard in chemical analysis. This
Biomedical Instrumentation & Technology 141
THE FUNDAMENTALS OF...
Robert M. Dondelinger
Figure 2. In this diagram showing the basic components of a diode-array
spectrophotometer, light passes through the sample, the monochromator, and
then strikes the diode array. The output from the diode array is connected to a
computer. It records the output from each of the diodes in the array, providing
an absorbance profile—the absorbance at each wavelength in a spectrum—of
the sample, often in graphic form.
tating mirrors, each driven by a high-speed stepper mo-
tor, to provide an alternate light path around the sample
cuvette. This provides near simultaneous alternating
readings both through and around the sample cuvette,
interpreted by the electronics as nearly simultaneous
zero absorbance and sample absorbance readings. The
control electronics both synchronizes the stepper mo-
tors and mathematically determines sample absorbance.
This design automatically accounts for lamp aging and
momentary changes in lamp luminescence caused by
version was marketed, essentially unchanged, until it
was discontinued in 1976. After some 35 years of sales,
more than 30,000 of these instruments are used in
chemistry, biochemistry, and both clinical and indus-
trial laboratories.
Bausch & Lomb introduced the first highly accu-
rate, low-cost spectrophotometer in 1954, dubbing it
the “Spectronic 20.” Due to both features, it quickly
became an industry standard instrument and probably
the world’s most widely used spectrophotometer. The
early 1980s saw the first spectrophotometers coupled
with microprocessor controls, both automating the
analysis and reducing analysis time, again increas-
ing throughput. Incremental improvements continue
today, especially in the area of computer technology.
Bruce Merrifield, a Nobel laureate and author, called
the spectrophotometer “probably the most important
instrument ever developed towards the advancement of
bioscience.”
 THE FUNDAMENTALS OF...
Spectrophotometers
voltage changes, transient dust particles, etc. It also
automatically corrects for the change in light output as
the spectrophotometer automatically sweeps through a
range of wavelengths.
A contemporary alternate design (Figure 2) em-
ploys most of the same basic components, but passes
full-spectrum light through the sample and then filters
the light through a monochromator before it reaches a
diode array instead of a photodetector. This design has
the advantage of eliminating the moving parts associated
with the monochromator, but the disadvantage of higher
cost.
How to Manage the Device
The College of American Pathologists (CAP) has
established standards for clinical laboratories and the
maintenance of their equipment. Although not a regula-
tory body, following these standards is a requirement for
laboratories desiring CAP accreditation. Federal agen-
cies, such as the U.S. Centers for Medicare and Medicaid
Services (CMS) have established standards, through the
Clinical Laboratory Improvement Amendments (CLIA),
that must be followed to qualify the hospital, medical
center, or stand-alone laboratory for Medicare and Med-
icaid payments.
To comply with these standards, which do not spe-
cifically address maintenance management, the manager
should schedule recurring services, such as preventive
maintenance and electrical safety testing, uniquely to
each spectrophotometer in their area of responsibility.
Additionally, the manager should maintain a detailed
history covering initial installation, scheduled services,
remedial maintenance, and any modifications made to
the spectrophotometer.
Regulations
There is no specific regulation of spectrophotometers,
but a number of organizations have established standards
and guidance for the use and maintenance of this and
other laboratory instruments. If the laboratory seeks
certification by one of these organizations, its standards
and guidance must be followed as if they were regulatory
in nature.
Risk Management Issues
Spectophotometry presents two major risk management
issues. The first is the inability to perform certain specific
critical laboratory tests should the spectrophotometer
142 March/April 2011
“ Spectrophotometers have been common
in hospital laboratories since the early
1950s, so the basic technology
is well established.”
succumb to a hard failure. The second deals with inaccu-
rate results. Obviously whenever test results drive treat-
ments and diagnoses, a great potential exists for medical
misadventures.
If a test result incorrectly indicates the presence of a
particular treatable condition, the patient will experience
undue stress and anxiety as well as face unnecessary treat-
ment costs. In some cases, the physician may inadvertently
harm the patient by treating for a nonexistent condition.
The other extreme, not confirming a problem that does
in fact exist, can be equally or even more devastating—the
disease will progress untreated while physicians search
elsewhere for the cause of the presenting symptoms. In
either case, inaccurate lab results are detrimental for both
the patient and the healthcare facility.
Troubleshooting
Tungsten vapor, deposited on the interior of old lamps,
or dust and deposits on the exterior can cause inaccurate
results. Under these circumstances, the lamp must be
replaced. It will not be burnt out or appear to be “bad”
in the conventional sense, but the lamp is no longer us-
able. Single-beam spectrophotometers are particularly
prone to inaccurate results because of old lamps and the
accumulation of dust on the interior optics. Dual-beam
instruments automatically compensate for some optical
deterioration. Otherwise, due to the simplicity of their
design, spectrophotometers are quite failure free and not
maintenance intensive.
One oddity about the basic legacy spectrophotometer,
which a biomed may still find in use in developing nations,
is a problem rarely observed in modern equipment. Since
these units bounce a beam of light off the galvanometer-
connected mirror to an analog scale on the face of the in-
strument, a good hard shock to the instrument will cause
the galvanometer-connected mirror to oscillate back and
forth. While it is oscillating, the laboratory technician
will not see the light on the analog scale, leading the
technician to believe the bulb is burnt out, the instru-

ment cannot be zeroed, the galvanometer is not working,
or some other perceived problem. It can take as long as
an hour—more commonly one-half hour—for the galva-
nometer to settle down and again reflect the light onto
the translucent analog scale. This phenomenon does not
occur in modern spectrophotometers.
Training and Equipment
Again, due to their simple design, legacy manual spec-
trophotometers can be maintained with nothing more
than a voltmeter and common hand tools. As the design
becomes more complex, additional service aids, such
as manufacturer’s literature, oscilloscope, etc., become
essential items for the biomedical technician and the
maintenance of spectrophotometers.
Future Development
Spectrophotometers have been common in hospital
laboratories since the early 1950s, so the basic technol-
ogy is well established. The recent advances in computer
technology coupled to spectrophotometers, together
with improved detectors, diffraction gratings, and better
Biomedical Instrumentation & Technology 143
THE FUNDAMENTALS OF...
Robert M. Dondelinger
lamps have allowed industry to produce instruments of
unimagined accuracy, stability, higher resolution, and
improved reproducibility. Additionally, the integration
of spectrophotometry with computer technology has also
provided features such as automatic baseline correction,
improved monochromator performance, system control-
lers, and data analysis and storage. Additional improve-
ments and further instrument refinements are expected
as the level of computer technology increases. n
References
1.  Simoni R, Hill R, Vaughan M, Tabor H. A Classic Instrument:
The Beckman DU Spectrophotometer and Its Inventor, Arnold
O. Beckman. JBC. Available at: www.jbc.org/content/278/49/
e1.full. Accessed July 2010.
2.  Google Timeline Spectrophotometer History. Available at:
www.google.com/search?q=timeline+spectrophotometer+
history&hl=en&rls=com.microsoft:en-us:IE-SearchBox&rlz=
1I7HPIB_en&sa=X&ei=dCdzTMLMJYeOnweOsJTgDg&ved=
0CG4QpQI&tbs=tl:1,tlul:0,tluh:2010. Accessed July 2010.
3.  The History of Spectrophotometry. Available at: www.ehow.
com/about_6595173_history-spectrophotometry.html.
Accessed July 2010.
4.  BrainyHistory. Available at: www.brainyhistory.com/events/
1935/january_8_1935_93512.html. Accessed July 2010.