Chapter 7
Spectrophotometry
Lorin M. Bachmann and W. Greg Miller
Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States
Learning objectives
After reviewing this chapter, the reader will be able to:
G Explain the principles of spectrophotometric measurement.
G Describe the sources of error in spectrophotometric
measurement.
G Discuss the calibration of spectrophotometric methods.
G Explain the principles of related optical measurement tech-
nologies such as fluorescence, chemiluminescence, and
atomic emission.
Principles of light absorption and
emission
Spectrophotometry is the measurement of the absorption
of electromagnetic radiation, in the wavelength range of
ultraviolet (UV), visible, and near-infrared (IR) light, by
an atom or a molecule to determine the quantity or iden-
tity of the substance present. In clinical chemistry, this
analytical technique is used to measure the quantity of an
analyte present in a body fluid sample. In most cases, an
analyte of clinical interest participates in a chemical reac-
tion resulting in the formation of a product, called a chro-
mogen (or chromophore), which absorbs light at a
specific measuring wavelength. The measuring wave-
length is selected based on the wavelength, or wavelength
range, at which the chromogen exhibits peak absorbance.
The quantity of chromogen produced in the reaction is
proportional to the quantity of analyte, and therefore the
amount of light absorbed by the chromogen is propor-
tional to analyte concentration in the clinical sample.
Spectrophotometry measures the amount of light absorbed
to quantitate the chromogen. A related analytical tech-
nique measures the light emitted as fluorescence. In fluo-
rescent measurement techniques, a specific wavelength of
light is absorbed by a chromogen, called a fluorophore,
and the subsequent emission of a longer wavelength of
light is measured. Other complimentary photometric tech-
niques measure light emitted from atoms when stimulated
by nonelectromagnetic energy sources (e.g., a gas flame
Contemporary Practice in Clinical Chemistry. DOI: https://doi.org/10.1016/B978-0-12-815499-1.00007-7
© 2020 Elsevier Inc. All rights reserved.
or plasma) or from molecules that emit light when stimu-
lated by a chemical reaction (chemiluminescence).
The energy state of the molecular orbitals of the
organic molecules can be represented as illustrated in
Fig. 7.1. At typical laboratory temperatures, the electrons
are in the ground (lowest energy) state, but the energy is
distributed among several vibrational and rotational
energy levels. A molecule can transition to an excited-
state electron orbital distribution if sufficient energy is
available for the transition. For molecules with an appro-
priate molecular orbital electron distribution, absorption
of radiation in the 200
1000-nm wavelength range can
cause a transition to a stable higher energy orbital. Such a
transition is illustrated in Fig. 7.1. The arrows represent
electrons going from ground-state energy levels to
excited-state levels. Absorption of radiation is quantized
between specific electronic, vibrational, and rotational
energy levels, which means that only the exact quantity of
energy required for a transition to a given higher energy
level will be absorbed. Because of quantized absorption, a
molecule absorbs specific wavelengths corresponding to
the energy required for a transition. The individual quan-
tized transitions are not resolved at typical laboratory
temperatures, because there are many transitions with
similar energy because of multiple vibrational and rota-
tional states. For this reason, it is typical to observe rela-
tively broad chromogen absorption bands (absorbance of
multiple wavelengths within a given wavelength range) in
liquids at ambient temperatures.
After absorption of radiation, the excited-state mole-
cule can dissipate the energy in several ways. One is for
the excited state to undergo a photochemical reaction
with another molecule to produce a new compound; this
process is not discussed in this chapter. In spectrophoto-
metric applications, thermal transfer primarily dissipates
the energy through collision with other molecules in the
liquid solution. Therefore no energy in the form of
photons is emitted. In fluorescence applications, the
excited state emits radiation at a longer wavelength
119
120 Contemporary Practice in Clinical Chemistry
FIGURE 7.1 Diagram showing relative energy distribution of electrons
among rotational and vibrational levels in a ground state and a first
excited state of an orbital, and quantized energy absorption or loss
(arrows) when an electron transitions between the states.
FIGURE 7.2 Absorption spectrum of the NAD1/NADH chromogens.
(lower energy), as it relaxes to the ground state. Not
shown in Fig. 7.1, and infrequently used in clinical chemistry,
is the possibility for singlet excited-state electrons to undergo
further electronic orbital transitions to a triplet excited state
and emit radiation at a longer wavelength, called phosphores-
cence, which persists for a longer time interval.
Typical chromogens that are absorbed in the
UV
visible
IR wavelength range are organic molecules
with highly conjugated C 5 C, C 5 O, C 5 N, and other
double bonds and/or aromatic rings that have molecular
orbital electron distributions that can transition to an excited
state at the energies available in these wavelength ranges.
Metallic atoms can also absorb energy in this wavelength
range to produce excited-state electronic orbitals.
The absorption spectrum describes chromogen absor-
bance vs. the wavelength range of light passing through a
cuvette that contains a solution of the chromogen. Fig. 7.2
shows an absorption spectrum for the oxidized and reduced
forms of nicotinamide adenine dinucleotide (NAD1 and
NADH, respectively). Many spectrophotometric assay reac-
tion mechanisms utilize the absorptive properties of the
NAD1/NADH redox pair. In this reaction scheme, an
analyte-specific chemical reaction results in product forma-
tion that is coupled to the reduction of NAD1 to NADH or
oxidation of NADH to NAD1. As shown in Fig. 7.2,
NAD1 and NADH absorb light at 250
280 nm because of
absorption by the adenine base. However, an absorbance
specific for NADH also occurs at approximately 340 nm
because of the orbital distribution of the electrons in the
dihydronicotinamide ring of NADH. In contrast, NAD1
exhibits minimal absorbance at 340 nm. An example of an
analyte reaction with the NAD1/NADH redox pair is the
hexokinase method for measuring glucose.
Hexokinase:
Glucose 1 ATP-glucose-6-phosphate 1 ADP (7.i)
Glucose-6-phosphate dehydrogenase:
Glucose-6-phosphate 1 NAD1 -6-phosphogluconate
1 NADH ðabsorbance at 340 nmÞ
(7.ii)
During the second step of the reaction, enzymatic con-
version of glucose-6-phosphate to 6-phosphogluconate is
coupled to the reduction of NAD1 to NADH, and absor-
bance is measured at 340 nm. The resulting increase in
absorbance due to NADH formation is directly proportional
to the concentration of glucose in the sample. It is important
to note that consumption of a chromogen is also utilized in
some spectrophotometric reactions. If the reaction mecha-
nism results in the oxidation of NADH to NAD1, the chro-
mogen (NADH) is converted into a nonchromogenic
product at 340 nm (NAD1), as shown in a method for mea-
suring lactate dehydrogenase (LDH) activity.
LDH:
Pyruvate 1 NADH ðabsorbance at 340 nmÞ-lactate
1 NAD1
(7.iii)
In this case, absorbance decreases in proportion to
pyruvate concentration conversion to lactate by LDH.
Other methods of chromogen production include dye-
binding reactions, chemical conversion of an analyte to a
chromogen, and enzyme-mediated conversion of an analyte
to a chromogen. In the dye-binding methods, direct binding
of an analyte to a precursor compound in the reagent leads
to the formation of a colored complex that absorbs light at a
specific wavelength. An example of a dye-binding reaction
is a commonly used method for measuring total calcium.
Ca21 1 arsenazo III-Ca:arsenazo III complex
ðabsorbance at 660 nmÞ
(7.iv)
Dye-binding reactions are used for many analytes,
including orthocresolphthalein complexone for calcium,

pyrogallol red and Coumassie brilliant blue for total
serum protein, and bromcresol green and bromcresol pur-
ple for serum albumin.
For quantitation of analytes using spectrophotometric
procedures, it is typical to select a single wavelength to
measure the amount of light energy absorbed by the chro-
mogen. The wavelength corresponding to the maximum
absorbance is usually used to optimize the analytical sen-
sitivity of the measurement. However, the influence of
interfering substances is a consideration when selecting a
measuring wavelength as discussed in a later section.
Principles of spectrophotometric
measurement
Fig. 7.3 illustrates a spectrophotometric measurement sys-
tem. Light of the desired wavelength impinges on a pho-
todetector that converts the light energy into an electrical
signal. A container, called a cuvette, is placed in the light
path of the spectrophotometer. The cuvette is filled with a
blank solution that, ideally, contains all reaction compo-
nents except the chromogen, and the amount of light hit-
ting the photodetector is measured (called I0). The cuvette
is then filled with a solution that contains all reaction
components, including the chromogen, and the light hit-
ting the photodetector is measured again (called I). The
amount of light hitting the photodetector is reduced by
the absorbance of light by the chromogen. The amount of
chromogen produced by the reaction and the correspond-
ing magnitude of absorbance are proportional to the
amount of analyte present in the cuvette. A spectrophoto-
metric measurement is the ratio of I to I0 and is called the
transmittance (T) or the percent transmittance (%T) when
the ratio is multiplied by 100. The formula is
T 5 I=I0 (7.1)
The transmittance ratio has an inverse, semilogarith-
mic relationship to the concentration of the chromogen.
The transmittance is more commonly converted into
absorbance (A), which has a linear relationship to concen-
tration to make it more convenient to use. It should be
noted that transmittance is the parameter actually being
measured and that the corresponding absorbance is a cal-
culated value. The formula is
FIGURE 7.3 Diagram showing major components and configuration of
a typical single-beam spectrophotometric system.
Spectrophotometry Chapter | 7 121


A 5 log10 1=T 5
log10 T (7.2)
Beer’s law describes the linear relationship between
absorbance and concentration.
A 5 ðεÞ ðbÞ ðCÞ (7.3)
where A is absorbance, ε is molar absorptivity
[L/(molGcm)] of the chromogen, b is path length (cm) of
light through the cuvette, and C is concentration (mol/L)
of the chromogen in the solution in the cuvette. The molar
absorptivity is a physical property of the chromogen. This
property is defined as the amount of light, at a specified
wavelength, that a 1 mol/L concentration of the chromo-
gen absorbs assuming the cuvette path length is 1 cm.
The molar absorptivity can be affected by the tempera-
ture, pH, and matrix of the solution containing the chro-
mogen. Variations in temperature, pH, and matrix (e.g.,
ionic strength, protein content, and surfactants), as well as
errors in wavelength and bandpass, can cause an error
when calculating concentration using Beer’s law. For
these reasons, many methods correct for such variations
by calibration, which will be discussed in more detail
later in the chapter.
It is important to remember that a spectrophotometric
measurement is a ratio of I to I0, because it is the intensity
of light hitting the photodetector that limits the measuring
range for a chromogen concentration. An absorbance of
0.0 is 100%T; an absorbance of 1.0 corresponds to 10%T,
which means the chromogen has absorbed 90% of the
incident light energy. Similarly, an absorbance of 2.0 cor-
responds to 1%T, which means the chromogen has
absorbed 99% of the incident light energy. Consequently,
the concentration change that causes an absorbance
change from 0 to 1 will use 90% of the measurement
range of the spectrophotometer; doubling the concentra-
tion will cause the absorbance to change from 1 to 2 but
will only use 9% of the measurement range of the spec-
trophotometer. The progressively smaller change in the
ratio of I to I0 as concentration increases and the corre-
sponding increasing contribution of noise sources to the
signal become a limiting factor in the ability of a spectro-
photometer to measure accurately the concentration of a
chromogen. For this reason, the optimal measurement
range for most spectrophotometers is between 0 and 1.0A.
Some instruments can make acceptable measurements at
larger A values, but there will be a progressive decrease
in precision and accuracy at higher values.
Configuration of spectrophotometers
Figs. 7.3 and 7.4 show two common configurations for a
single-beam spectrophotometric measurement system. A
wavelength selection device can be placed before the
cuvette as in Fig. 7.3 or after the cuvette as in Fig. 7.4,
122 Contemporary Practice in Clinical Chemistry
depending on the design of the spectrophotometer. In
Fig. 7.3, the specific wavelength of interest is isolated,
passes through the cuvette, and hits the detector. This
configuration can use a monochromator or an interference
filter to isolate the wavelength of interest. In Fig. 7.4,
the broad spectrum of wavelengths passes through the
cuvette, is diffracted by a grating monochromator, and
hits a series of individual diode detectors (called a diode
array configuration). Each diode in the array is impinged
by a very narrow band of wavelengths such that many dif-
ferent wavelengths can be measured simultaneously. The
diode array configuration has no moving parts to go out
of alignment. Diode array configurations are common in
automated analyzers because of their stability and the
ability to measure simultaneously multiple wavelengths.
Research-grade diode array spectrophotometers can mea-
sure in wavelength increments of 1 nm. However, many
clinical laboratory instruments are restricted to specific
intervals of wavelengths that may limit flexibility for
development of new assays.
Absorbance is measured by placing a cuvette contain-
ing the solution without chromogen in the light path mea-
suring I0. The solution in the cuvette is then replaced with
a solution containing the chromogen and I is measured.
Alternatively, the chromogen-containing solution could
be in a separate cuvette with a path length identical to
that of the one used for I0 measurement. This configura-
tion can easily be incorporated into a typical random
access automated analyzer in which a single reference
cuvette is used for the I0 measurement. A series of cuv-
ettes that contain the analyte-specific chromogens is mea-
sured to obtain I for a series of clinical samples. The ratio
of I/I0 for each cuvette/reference combination is then used
to determine the absorbance for each sample. In addition
to providing an I0 value for a specific set of measure-
ments, the reference channel can also be used to monitor
the stability of the spectrophotometric system over time
and to correct for optical or electronic signal drift.
Another common configuration for a spectrophotome-
ter is called a double-beam system, in which the light
path is split with mirrors and is directed alternately
through each of two cuvettes, one used for the I0 and one
for the I measurements. The frequency of the light
FIGURE 7.4 Diagram showing the configuration of a diode array
spectrophotometric system.
alternating between the two cuvettes is typically fast
enough to permit the measurement of transmittance over
a range of wavelengths to obtain an absorption spectrum.
Critical operating parameters: accuracy
Bandpass
Spectral bandpass (bandwidth) refers to the range of
wavelengths that pass through the cuvette when the
monochromator precedes the cuvette or can be isolated to
impinge on an individual detector in a diode array config-
uration. The intensity of light isolated at a specific wave-
length will be most intense at the nominal selected
wavelength, but a range of wavelengths with progres-
sively decreasing intensity will also be transmitted. The
bandpass is defined as the range of wavelengths at the
point of half-intensity of transmitted light. Consequently,
a range of wavelengths is actually measured by the detec-
tor. If the molar absorptivity of the chromogen changes
over the bandpass of the instrument, which is common,
there will be less absorbance observed than expected for
the concentration of chromogen present. This condition
results in a lower than expected absorbance, because the
observed absorbance is the integral of absorbance over
the band of wavelengths passing through the cuvette and
hitting the detector. If the conversion of absorbance to
concentration is based on the molar absorptivity (e.g., in
methods that measure enzyme activity), there will be an
error in the concentration measured unless the molar
absorptivity is corrected for the actual bandwidth of a spe-
cific spectrophotometer.
For example, the NADH chromogen shown in Fig. 7.2
exhibits a change in absorbance vs. wavelength. If a spec-
trophotometer with a wavelength bandpass of 1 nm was
used to measure NADH, there would be little absorbance
error, because 1 nm is small enough to isolate functionally
the peak absorbance at which the molar absorptivity was
determined. However, if a spectrophotometer with 10-nm
bandpass was used, some of the wavelengths would not
be measuring the peak absorbance, the integrated absor-
bance over the bandpass would be less than that expected,
and the concentration calculated from the molar

absorptivity would be erroneously low. For NADH, a
10-nm bandpass would produce an absorbance that is
approximately 1% lower than the correct value and a 20-
nm bandpass would produce an absorbance that is approx-
imately 3% lower than the correct value. Bandpass has a
greater influence for chromogens with a narrower absorp-
tion spectrum and a smaller influence for those with a
broader absorption spectrum. A bandpass of , 8 nm has
been recommended when measuring NADH in enzyme
assays that determine activity on the basis of molar
absorptivity [1].
When a relatively wide bandpass instrument is used, it
is necessary to correct for the condition by determining a
functional molar absorptivity value specific for that
instrument. Instrument manufacturers have various names
(e.g., filter factor) for such instrument-specific molar
absorptivity values. When interference filters are used,
the instrument-specific molar absorptivity values must be
verified periodically, because the devices are subject to
deterioration over time. Alternatively, an instrument-
specific calibration can be established using a calibrator
with known concentration (or activity) to establish the
relationship between absorbance and concentration (or
activity) for that instrument.
Stray light
Stray light is the light at wavelengths other than the mea-
suring wavelength that reaches the detector and is not
absorbed by the chromogen. Stray light causes the mea-
sured absorbance to be lower than it would be in the
absence of the stray light. Consider the equation for trans-
mittance when a stray light component (IS) is present.
I 1 IS
T5 (7.4)
I0 1 IS
The intensity of light hitting the detector has the stray
light added to it. IS is usually an insignificant fraction of
I0, but as I gets small due to large absorbance by the chro-
mogen, IS becomes a significant fraction of I, which
causes T to be larger (and A to be lower) than what would
be expected based on the concentration of chromogen.
Consequently, the relationship between absorbance and
concentration becomes nonlinear, as stray light becomes a
greater percentage of the total light hitting the detector.
For example, if stray light is 0.1% of the total hitting the
detector, the absorbance will have a 1% error at approxi-
mately 1.5A, and if stray light is 0.5%, the absorbance
will have a 1% error at approximately 0.6A.
Stray light can be caused by light leaks in the instru-
ment, degradation of the source lamp, and, rarely, fluores-
cence or phosphorescence from endogenous substances or
drug metabolites in the sample. However, the most common
source of stray light is undesired wavelengths originating
Spectrophotometry Chapter | 7 123
from the monochromator or wavelength isolation device
because of optical defects. Contemporary holographic grat-
ing monochromators have reduced stray light to insignifi-
cant levels for many applications, but this source of error
can be an issue when interference filters are used. Some
spectrophotometric systems have incorporated automated
multiwavelength stray light correction algorithms, which
are effective for some types of optical defects.
Stray light can be measured as residual absorbance
when measuring a solution or a filter that absorbs . 4A
(0.01 %T) at the wavelength of interest but passes most
other wavelengths. A signal observed in these conditions
is due to stray light reaching the detector. Note that only
potential stray light at wavelengths that are not blocked
by the filter or solution used to detect stray light will be
identified by this procedure. Two or more test filters or
solutions are typically needed to cover a range of wave-
lengths. Therefore stray light checks should be performed
as part of routine instrument performance assessment.
Use of a pulsed-frequency xenon lamp as the spectropho-
tometer source lamp is a common technique used to
reduce the effects of light leaks. The light from the xenon
lamp is pulsed at a frequency much faster (e.g., 1000 Hz)
than that of the overhead fluorescent lights (60 Hz) and is
synchronized with the photodetector. This approach
reduces the error contribution of minor amounts of envi-
ronmental light contamination.
Wavelength accuracy
The wavelength selected by the monochromator or other
device must be verified to be correct as indicated. If the
wavelength is not as indicated, the molar absorptivity used
to calculate concentration will be incorrect for the actual
wavelength in use, corrections for interfering substances
may not be accurate, or the analytical sensitivity of the
measurement may not be as expected. Wavelength accuracy
can be verified by scanning and identifying the peak absor-
bances for known substances such as holmium oxide in
solution [National Institute of Standards and Technology
Standard Reference Material (NIST SRM) 2034] or in
glass, or by removing wavelength-isolating filters from an
instrument and scanning them in a calibrated spectropho-
tometer. Some spectrophotometers have a provision to use
sharp line emissions from deuterium or mercury lamps used
as UV light sources. Verifying wavelength accuracy is the
responsibility of the clinical chemist for a stand-alone spec-
trophotometer, but is the responsibility of the manufacturer
for a spectrophotometer built into a diagnostic instrument.
Absorbance accuracy
The absorbance measured by the spectrophotometer must
be verified to be correct whenever results are based on
124 Contemporary Practice in Clinical Chemistry
molar absorptivity. Verification of absorbance accuracy
can be accomplished by measuring a set of certified
absorbance standards. Liquid solutions with a known con-
centration of chromogen with a known molar absorptivity
can be used. A set of liquid solutions for the range
302
678 nm (SRM 931h) and solid potassium dichro-
mate (SRM 136f) can be obtained from NIST to create
liquid solutions for use at wavelengths 235
350 nm.
Another approach (useful for automated analyzers for
which it is difficult to add solutions directly into cuvettes)
is to generate quantitatively NADH, for which the molar
absorptivity is known at 340 nm, from pure glucose
(NIST SRM 917c) using the hexokinase reaction.
Verifying absorbance accuracy is the responsibility of the
clinical chemist for a stand-alone spectrophotometer but
is the responsibility of the manufacturer for a spectropho-
tometer built into a diagnostic instrument.
Chromogen limitations
The measured chromogen is usually generated by a chem-
ical reaction that converts the amount of analyte present
in a biological sample into a proportional amount of the
chromogen. The proportionality of the chemical conver-
sion can be affected by defects in the reagents or by an
inadequate amount of one or more reagents that may limit
the completeness of the chemical reaction(s). The chro-
mogen, at higher concentrations, may form complexes or
adducts with itself or with other components of the reac-
tion that can change the molar absorptivity and thus the
ability to transform accurately the absorbance to the con-
centration of the analyte.
Calibration of spectrophotometric measurements
Calibration establishes the mathematical relationship
between the analytical response and the chromogen con-
centration in the solution in the cuvette. The concentration
of the analyte in the biological sample is typically directly
proportional to the analytical response of the chromogen.
Spectrophotometric measurements are based on the molar
absorptivity of the chromogen or on a calibration relation-
ship established by a calibrator that compensates for the
unique conditions of an instrument. It is a common prac-
tice to calibrate a spectrophotometric assay using the cali-
bration solutions of known analyte concentration, which
are assayed in the same manner as the clinical samples, in
which case the calculated concentration of an analyte in
an unknown sample is not reliant on knowing the molar
absorptivity. However, spectrophotometric assays for
enzyme activity typically use the molar absorptivity of a
chromogen to calculate the activity as the rate of substrate
consumption. Errors related to the molar absorptivity are
important to control when Beer’s law is used to calculate
enzyme activity or analyte concentration.
Molar absorptivity
When concentration is calculated from the molar absorp-
tivity, it is imperative that the pipette, absorbance, wave-
length and temperature accuracy, bandpass, and stray
light have been verified. Each of these variables can
affect the chemical reaction(s) and the observed molar
absorptivity of the chromogen in the cuvette. Any vari-
able that can influence the matrix of the solution in the
cuvette (e.g., pH, ionic strength, surfactants, and tempera-
ture) must be controlled to ensure the chromogen is in the
same matrix conditions as when the molar absorptivity
was determined. Note that it is acceptable and recom-
mended to establish the molar absorptivity of the chromo-
gen for the unique conditions of a specific method and
spectrophotometric system. Note that some automated
analyzers may not permit the user to make changes to
molar absorptivity settings. A measurement system-
specific molar absorptivity can be established by prepar-
ing a series of solutions containing various amounts of
pure chromogen dissolved in the reaction buffer for the
method. The molar absorptivity is calculated as the absor-
bance per mole per liter of chromogen in the cuvette for a
range of concentrations.
Calibration relationship
A calibration relationship can be established by measuring
a series of concentrations of calibrators (also called stan-
dard solutions) and establishing the mathematical relation-
ship between the absorbance and concentration of analyte
in the calibrator solutions. The calibrator solutions are
measured in the same manner as the clinical samples and
must have a matrix that is appropriate for the clinical
samples that will be quantitated using the calibration rela-
tionship. The term “standard curve” or “calibration curve”
is used to refer to a calibration relationship even when the
relationship is a straight line. For most spectrophotometric
assays, the relationship between absorbance and concen-
tration is linear, although the relationship between trans-
mittance (the actual measured quantity) and concentration
is semilogarithmic. If the absorbance relationship is not
linear, several concentrations of the calibrator are required
to describe accurately the calibration relationship over the
analytical measurement range.
Establishing a calibration relationship eliminates or
substantially reduces the influences of biases in pipette,
wavelength, and absorbance accuracy, bandpass, and stray
light, because these variables are compensated for by hav-
ing the calibrators measured at the same spectrophotomet-
ric conditions as the unknown samples. These variables
remain important for the measurement and must be at

settings appropriate for the reactions involved, but nomi-
nal differences from specifications can be tolerated as
long as they remain constant for calibrators and unknown
samples. The need for temperature accuracy is eliminated
as far as its effect on the molar absorptivity of the chro-
mogen but the role of temperature on the chemical reac-
tion that generates the chromogen may still be important.
Precision of the various steps in the assay remains an
important variable to control. The matrix of the calibrator
solutions must be appropriate for the clinical samples to
be assayed to ensure there is no bias introduced by a dif-
ference in the extent of the chemical reactions involved in
generating the chromogen or in the final matrix of the
solution in the cuvette that might differently affect the
molar absorptivity of the chromogen, depending on
whether it originated from the calibrator or from the clini-
cal sample. The matrix should also be taken into consider-
ation when diluting a sample. The selected diluent should
possess matrix characteristics similar to the sample to
avoid measurement biases due to matrix differences.
One of the critical factors affecting measurement accu-
racy is the target value assignment of the calibrator materi-
als. The calibrator value assignment should be traceable to
a higher order reference measurement procedure, if one is
available, or to a commutable certified reference material
when available and no reference measurement procedure
exists. Reference measurement procedures and reference
materials can be found in the database of the Joint
Committee on Traceability in Laboratory Medicine
(JCTLM) [2]. Many of the reference materials listed by
JCTLM are intended for use as trueness controls with refer-
ence measurement procedures and are not commutable with
clinical samples when used with routine clinical laboratory
methods. Use of noncommutable reference materials as
calibrators for routine clinical laboratory methods can cause
miscalibration and erroneous results for clinical samples
[3,4]. See Chapter 17, Harmonization of results among lab-
oratories, for additional information on calibration
traceability.
Critical operating parameters: precision
The precision of a spectrophotometric measurement varies
with design of the instrument system. At low absorbance
(high %T), the precision is limited by mechanical, optical,
and electronic stabilities. At high absorbance (low %T),
the precision is also limited by stray light and detector
background electronic noise, as these sources of error
become a larger fraction of the measured signal. Maximum
precision is generally obtained when the absorbance is
0.1
1.0A. Contemporary automated spectrophotometers
typically make multiple independent absorbance measure-
ments and average the results to reduce the effects of
imprecision. Precision improves proportional to the square
Spectrophotometry Chapter | 7 125
root of the number of independent measurements (e.g., the
mean of nine measurements reduces imprecision by a
factor of 3; 100 measurements reduces imprecision by a
factor of 10).
The precision of a spectrophotometric measurement
can be validated in several ways depending on the acces-
sibility of the cuvette. A dye can be measured in replicate
in a single cuvette or in multiple cuvettes. When multiple
cuvettes are used, minor differences in path length among
cuvettes will be included in the imprecision value. For
automated systems, it is frequently adequate to measure
the combined effects of pipette and absorbance variation
on the precision using a dye (which removes any influ-
ence of a chemical reaction) or with a chemical reaction
to generate a chromogen that may also include reagent
formulation variability and temperature effects.
Interferences
The absorbance of a substance other than the chromogen
derived from the analyte of interest will cause interfer-
ence in the measurement, unless the signal from the inter-
fering substance is removed from the total absorbance.
Absorbance from the reagents used in a reaction can be
measured before adding the sample and subtracted from
the total absorbance of the reagent plus chromogen. This
process is called a reagent blank correction. Clinical sam-
ples such as serum or urine frequently contain substances
that may absorb light at the wavelength used to measure
the analyte-specific chromogen and cause spectral inter-
ference. The effects of spectral interferences can be
reduced in several ways, including the use of a sample
blank, use of a kinetic reaction-rate method, and correc-
tion using wavelengths at which the interfering substances
absorb.
Measurement of a sample blank can correct for spec-
tral interferences in spectrophotometric end-point meth-
ods. In an end-point reaction (Fig. 7.5), the absorbance
value is obtained at a time point after the reaction has
proceeded to completion (when all of the analyte has
reacted with the reagents to produce a chromogen). To
correct for spectral interferences, a “sample blank” absor-
bance reading at the measuring wavelength is taken after
mixing the sample with all of the reagents, except those
that cause chromogen production. The reagent that trig-
gers production of chromogen is added, and the reaction
is allowed to proceed to completion. The total absorbance
is measured at the completion of the reaction. The absor-
bance of the sample blank, corrected for the volume of
the added second reagent, is then subtracted from the total
absorbance to yield the corrected chromogen-specific
absorbance. This method of correction is subject to error
if the absorbance of the spectral interferant is of such a
large magnitude that the chromogen-specific contribution
126 Contemporary Practice in Clinical Chemistry
FIGURE 7.5 Spectrophotometric end-point and rate reactions. The first
inflection on each graph represents the addition of reagents required for
chromogen production. Note: Equations are simplified examples; auto-
mated instruments typically include polychromatic corrections for inter-
fering substances, sample volume corrections, and averaging of many
measurements to improve precision.
to the total absorbance is small. It also is important to
note that sample or reagent blanks can only correct for
interfering absorbances that remain constant during the
reaction. The magnitude of the correction will be errone-
ous if an absorbing interferant reacts with any component
of the reaction mixture.
Another procedure to correct for spectral interferences
is by utilization of a kinetic (rate) reaction (Fig. 7.5).
Rather than calculating the analyte concentration on
the basis of total absorbance at the end of a reaction, the
absorbance is measured at multiple time points during the
reaction. The change in absorbance vs. time is then used
to calculate analyte concentration. Measurement of the
change in absorbance over time corrects for the absor-
bance of interfering substances that do not change during
the course of the reaction. As is the case with sample
blanking, this correction method will not work if the
interfering substance causes a large absorbance, such that
the change in absorbance due to the analyte-specific chro-
mogen cannot be accurately measured.
An important limitation to the preceding correction
techniques occurs when the absorbance from an interfer-
ing substance changes during the time of the reaction.
Examples of dynamic absorbance interference include
clearing of turbidity due to dispersion of triglyceride by
surfactants and reaction of an interfering chromogen such
as bilirubin in peroxide-coupled reactions as shown in the
following example:
Glucose oxidase:
Glucose 1 O2 -H2 O2 1 products (7.v)
H2 O2 1 dye-chromogen ðanalyte-specific absorbanceÞ
(7.vi)
H2 O2 1 bilirubin-nonchromogen
(7.vii)
ðdecrease in total absorbanceÞ
Note that the interference from bilirubin has two
mechanisms. First, some H2O2 that would otherwise be
available to react with the dye to produce chromogen
instead reacts with bilirubin and is not converted into the
analyte-specific chromogen, thus resulting in a falsely
low absorbance. Second, bilirubin itself can absorb light
and may contribute to the total absorbance. The interfer-
ing absorbance from bilirubin decreases in magnitude
over the time course of the reaction. As the bilirubin
reacts with H2O2, the absorbance from bilirubin no longer
contributes to the total absorbance. Dynamic interferences
must be controlled by modifying the reaction system to
remove the interfering substance independently from the
reaction that generates the analyte-specific chromogen.
For example, inactivating agents such as sodium dodecyl
sulfate (SDS) or potassium ferricyanide can be added to
the reagent to reduce the interference of bilirubin for reac-
tion methods that use peroxide to produce the chromogen
such as those based on the Trinder reaction. In some
instances, the interfering substance exhibits different reac-
tion kinetics than the reaction of the analyte. In these
cases, the time points at which absorbance readings are
obtained can be modified to reduce the effect of the
interference.
Another limitation in the spectrophotometric methods,
as in all methods, is caused by the nonspecificity of the
reaction for the analyte of interest. In this case, interfering
substances react to produce the same chromogen that
is produced from the analyte of interest. For example,
acetoacetic acid can react with alkaline picrate used in
some creatinine methods to produce a chromogen that
cannot be discriminated from that produced from creati-
nine. Method nonspecificity must be corrected by modify-
ing the method to include additional separation steps to
remove interfering substances or to change the chemical
mechanism to be more specific for the analyte. When
interferences exist in a method, they must be documented
as limitations in method specificity.
Bichromatic and polychromatic measurements
Measuring absorbance at a second wavelength at which
no change in absorbance occurs during the reaction can
be used as a reference point to correct the spectrophoto-
metric system for drift in the measurement signal. Two or

more wavelengths can also be used to correct for some
types of absorbance interferences. A simple case occurs
when an interfering substance absorbs light at the same
wavelength as the analyte-specific chromogen (primary
wavelength) but also absorbs light at a second wavelength
at which the analyte-specific chromogen does not absorb.
The ratio of molar absorptivities of the interfering sub-
stance at the two wavelengths can be used to predict the
absorbance due to the interfering substance at the primary
wavelength from its absorbance at the second wavelength.
This value can then be subtracted from the total absor-
bance at the primary wavelength to obtain the net absor-
bance due to the analyte-specific chromogen. This
correction approach has been referred to as the Allen
correction.
A more complex situation occurs when the analyte-
specific chromogen and an interfering substance both
absorb light at all wavelengths available for measurement.
This situation is shown in Fig. 7.6. When the molar
absorptivities of the analyte-specific chromogen and the
interfering substance(s) are different and are known at
two wavelengths, two equations with two unknowns can
be written to describe the total absorbance at each of the
two wavelengths. This correction method has been
referred to as bichromatic correction. A is the absorbance;
λ1 and λ2 are the two respective wavelengths; ε1 and ε2
are the molar absorptivities of substances 1 and 2, respec-
tively, at each of the two wavelengths; and C1 and C2 are
the concentrations of substances 1 and 2, respectively.
At wavelength 1:



Aðλ1Þ 5 ε1ðλ1Þ 3 C1 1 ε2ðλ1Þ 3 C2 (7.5)
At wavelength 2:



Aðλ2Þ 5 ε1ðλ2Þ 3 C1 1 ε2ðλ2Þ 3 C2 (7.6)
All arguments in the two equations are known or can
be measured except C1 and C2. Thus the two equations
FIGURE 7.6 Absorption spectra illustrating bichromatic measurement
to correct for an interfering substance that has absorption at all wave-
lengths available to measure a chromogen.
Spectrophotometry Chapter | 7 127
can be simultaneously solved for C1 and C2, one of which
is the concentration of the analyte of interest.
Note that more than one interfering substance can be
corrected using a bichromatic (two wavelengths) tech-
nique as long as the substances have similar molar
absorptivities at the two wavelengths used. For example,
bilirubin and hemoglobin have very similar absorbance
spectra at several wavelengths and can frequently be ade-
quately corrected using bichromatic measurements.
However, interfering substances that have different molar
absorptivity ratios at the two wavelengths cannot be cor-
rected using bichromatic measurements. For example, tur-
bidity from triglyceride-rich lipoproteins has a very
different absorption spectrum than bilirubin and hemoglo-
bin; thus a bichromatic technique cannot simultaneously
correct for each of these sources of spectrophotometric
interference. Three (trichromatic) or more wavelengths
can be used to correct for more complex interferences or
to measure simultaneously multiple analytes, such as in
cooximetry (COOX) determination of hemoglobin spe-
cies. Imprecision of a final result will increase as more
individual absorbance measurements are used and must
be accommodated in the total error budget of the
measurement.
Other applications of spectrophotometric
or light emission measurements
Cooximetry
COOX is the measurement of blood total hemoglobin and
the relative percentages of hemoglobin derivatives includ-
ing oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb),
carboxyhemoglobin (COHb), and methemoglobin
(MetHb). COOX instrumentation may be integrated with
routine blood gas analyzers or function as stand-alone
analyzers. O2Hb, HHb, COHb, and MetHb each exhibit
characteristic spectrophotometric absorbance profiles over
the wavelength range of 422
695 nm. The absorbances at
the measuring wavelengths are obtained for venous or
arterial whole blood samples, and a series of algorithms
based on the absorbance spectrum for each molecule is
used to identify and quantify each hemoglobin derivative.
Total hemoglobin is quantified using conductivity or the
sum of the spectrophotometric measurements of O2Hb,
HHb, COHb, and MetHb. Due to similar absorbance spec-
tra, sulfhemoglobinemia from medications containing
sulfonamides can cause interference with MetHb mea-
surements and fetal hemoglobin can cause interference
with COHb measurements. Therefore COOX instruments
employ additional algorithms for qualitative spectrophoto-
metric detection of sulfhemoglobin and fetal hemoglobin
to alert the user of elevated concentrations of these deri-
vatives. Although different combinations of instrument
128 Contemporary Practice in Clinical Chemistry
optics and measuring wavelengths are used to reduce the
effects of interferences, high concentrations of bilirubin,
intralipid, or methylene blue can also cause errors in
COOX measurements. More information on the use and
interpretation of COOX measurements can be found in
Chapter 35, Contemporary practice in clinical chemistry:
blood gas and critical care testing.
Reflectance spectrophotometry
Reflectance refers to a configuration in which the incident
light impinges on the chromogen contained in a thin coat-
ing on a surface, and the light is reflected from the rear
surface of the coating support to make a second pass
through the chromogen matrix. Examples include dry
slide chemistry analyzers and urinalysis dipstick readers.
In these applications, the path length is small and mea-
surement precision is influenced by the uniformity of the
coating containing reagent(s) and chromogen. Reflectance
is characterized by increased influence of scatter at each
reagent layer surface that has a change in the index of
refraction. Consequently, a diffuse reflectance measure-
ment typically has a nonlinear relationship with concen-
tration and appropriate calibration algorithms are used.
Densitometry
Densitometry measures absorbance by passing light
through a solid support material on which the chromogen
is contained by absorption or adsorption to the solid (or
gel) layer. Thin-layer chromatography and electrophoresis
are typical examples. Accuracy and precision are limited
by variations in path length due to inconsistent thickness
of the medium, scatter from components in the solid
medium, and exceed the limited dynamic range of the
detector caused by too high of a concentration of the
chromogen in the medium.
Turbidimetry and nephelometry
Turbidimetry and nephelometry are the techniques to
measure concentration on the basis of light scatter, as
illustrated in Fig. 7.7. Conventional spectrophotometers
measure light transmitted at 180 degrees from (or in line
with) the incident light source. Turbidity is measured in
FIGURE 7.7 Configuration used to measure light scatter by turbidime-
try or by nephelometry.
this configuration as absorbance, because scattered light
does not reach the photodetector and causes a decrease in
I0 (increase in absorbance), just as does absorbance by a
chromogen. This mechanism is the reason for which tur-
bidity causes interference in spectrophotometric measure-
ments. Nephelometry measures the intensity of light
scattered at an angle away from the incident light path.
Nephelometry is not a measurement of the ratio of light
intensity in the absence and presence of scattering mole-
cules but is a measure of absolute intensity of light caused
by scattering molecules. Nephelometry is capable of mea-
suring orders of magnitude lower concentrations of scat-
tering molecules than turbidity. Nephelometers frequently
use laser light sources, because the increased intensity can
produce increased intensity of scattered light and lower
detection limits. In addition, optics and optical filters are
used to focus the scattered light and reduce the contribu-
tion of stray light to improve the analytical sensitivity of
the measurement.
Light is scattered by molecules large enough that their
hydrated diameter is of similar magnitude to the wave-
length of the incident light [5]. Light scatter is typically
used to quantitate antibody complexes of protein analytes,
because these complexes are large enough to scatter light.
Light scatter is a complex process that depends on the
size and index of refraction of the scattering molecular
complex. Light scatter is typically greater at shorter wave-
lengths, with the intensity being approximately propor-
tional to the inverse of the wavelength to the fourth
power (1/λ4). Light scatter is not symmetrical; the inten-
sity of scatter at a given angle from incident varies with
the size of the complex. As the scattering complex gets
larger, a greater proportion of light is scattered at angles
.90 degrees from the incident (called forward scatter).
Nephelometers typically measure scatter at forward angles
from the incident to improve sensitivity for immune com-
plexes and similar sized molecular aggregates. It is com-
mon to control the size of immune complexes by the use
of surfactants and polymers (e.g., polyethylene glycol) to
optimize conditions for quantitation of complexes of dif-
ferent sizes that may be produced by analytes at different
concentrations. Measurement of light scatter can be used
to measure amylase and lipase hydrolysis of starch or tri-
glycerides, respectively, and for assays based on the
aggregation of latex particles coated with various sub-
stances, including antibodies. Flow cytometry measures
scatter of light by cells (typically larger than the molecu-
lar aggregates) at small forward angles to determine the
size and concentration of the cells.
The response of turbidimetry and nephelometry is not
linear with the concentration of analyte because of the
variable size of scattering complexes formed at different
analyte concentrations. When the scattering complexes
are formed by the classical antibody
antigen reaction,

the complex size is susceptible to antigen-excess condi-
tions. Antigen excess may cause falsely low results at
very high concentrations of analyte (called the “high-dose
hook effect” or “prozone effect”). To detect a high-dose
hook effect, the absorbance of the undiluted sample is
compared with the absorbance of a sample that has been
diluted. If the absorbance of the diluted sample is greater
than the undiluted sample, a prozone effect is present.
Most modern spectrophotometers and nephelometers
automatically analyze the kinetics of antigen
antibody
complex formation to increase the probability of prozone
effect detection and alert the user.
Nephelometry and turbidimetry are sensitive to inter-
ferences from light scatter due to triglyceride-rich lipopro-
teins (lipemia). The effects of interfering lipoproteins may
be partially reduced by ultracentrifugation and removal of
the lipid particles. Because turbidimetric methods are
based on the measurement of transmitted light, they are
also sensitive to the absorbance of colored substances
such as hemoglobin and bilirubin in the sample.
Antibodies are often utilized as detection reagents, and
therefore the presence of cross-reacting substances or
interfering antibodies (e.g., rheumatoid factor or hetero-
phile antibodies) can be a source of measurement error.
Atomic absorption
Atomic absorption is a special application in which
ground-state atoms of metals absorb light at very specific
wavelengths corresponding to the energy needed to cause
electronic transitions in their electron orbitals. Fig. 7.8
shows the configuration of an atomic absorption spectro-
photometer. The atoms are created by heating the sample
in an acetylene gas flame or a cylindrical graphite tube
furnace to a temperature that burns off most of the
organic matrix and produces ground-state atoms of
the metal to be measured. The “cuvette” is the flame or
the center of the graphite tube, and the atoms are in the
gas state. The population of atoms is relatively short-lived
in the light path, and rapid measurements are necessary.
The thermal energy must be carefully controlled and
FIGURE 7.8 Diagram showing major components and configuration of
an atomic absorption spectrophotometer.
Spectrophotometry Chapter | 7 129
ionization of metals must be avoided maintaining the
atoms in the ground state.
Atomic electron transitions have very narrow band-
widths that require specialized light sources with very nar-
row bandpass (a few angstroms or tenths of nanometers).
Hollow cathode lamps have an electrode coated with the
metal to be measured and use an electronic discharge to
generate an emission wavelength from the metal that is at
the correct wavelength to be absorbed by the ground-state
atoms in the cuvette. A high intensity of stray light is pro-
duced by incandescence of sample materials in the flame
or furnace that necessitates placing the monochromator
after the cuvette in the light path to the detector. A very
narrow bandpass monochromator is used to isolate the
wavelength of interest for the metal from the stray
light. Because of the stray light, linearity of atomic absorp-
tion is frequently limited to a small range of absorbance
(e.g., 0
0.3). Background absorbance and light scatter
from sample components can also interfere and can be cor-
rected by specialized bichromatic (Zeeman effect) or poly-
chromatic (deuterium lamp or Hieftje) techniques. Atomic
absorption can be used for accurate assay of many metals
in body fluid and tissue samples, because most sample
interferences can be eliminated by correct choice of instru-
ment and thermal conditions.
Atomic emission
Atomic emission is a photometric technique in which
thermal energy is used similarly to atomic absorption,
except that, in this technique, the thermal energy is ade-
quate to create excited-state atoms, which then emit light
as they relax back to the ground state. Atomic emission
offers the possibility to measure simultaneously several
different atomic species and can have lower limits of
quantitation than atomic absorption. The emitted light is
at wavelengths specific to the metals present. Flame pho-
tometry using propane gas provides the reference mea-
surement procedures for sodium, potassium, and lithium
in clinical samples. An inductively coupled argon ion
plasma (ICP) can be used to create excited-state atoms for
many metals. The ICP is much hotter than propane or
acetylene flames and thus produces more complete com-
bustion of potential interfering organic substances. The
hotter plasma also produces more emission lines from
metals and requires a more sophisticated optical system to
isolate wavelengths of interest. ICP methods are used for
toxic and trace metal measurements from body fluid and
tissue samples.
Fluorescence
Fluorescence is the light emitted from an excited-state
molecular orbital as it relaxes to the ground state by
130 Contemporary Practice in Clinical Chemistry
FIGURE 7.9 Diagram showing major components and typical configu-
ration to measure fluorescence.
emitting a photon of light. Fluorescent molecules, called
fluorophores, are characterized by the extended conjuga-
tion of double bonds with aromatic resonance that allows
the lifetime of the excited state to persist long enough for
relaxation to occur by the emission of light. The excited
state is produced by the absorption of light that has
greater energy (shorter wavelength) than that of the emit-
ted light. The emitted light is always at a longer wave-
length (lower energy), because the excited-state electrons
rapidly lose thermal energy to reach a minimal energy
excited state before the emission of light to relax to the
ground state. The difference in wavelength between the
excitation and emission wavelengths is called the Stokes
shift. Fig. 7.9 shows a fluorescence measurement configu-
ration. Fluorescent light is emitted with equal intensity in
all directions, which allows for various optical configura-
tions. Fluorescence intensity is measured at an angle and
using a wavelength selection device that excludes the
excitation light. The fluorescence intensity is directly pro-
portional to the concentration of a fluorophore in the
cuvette. The quantum yield is the fraction of absorbed
energy that is emitted by the fluorophore and is a measure
of the efficiency of molecular fluorescence.
Fluorescence typically provides a 100-fold to 1000-
fold increase in concentration sensitivity compared with
absorbance measurements. For maximum sensitivity, the
detection system must be able to measure the fluorescence
emission without any background signal from the excita-
tion light (i.e., measure the signal against a zero back-
ground). Potential interferences are from substances in the
sample that can quench the excited-state molecule before
fluorescent light is emitted or that can absorb the emitted
light in the cuvette before it reaches the detector (called
an inner filter effect).
Fluorescence lifetime
Fluorescence emission typically persists for 10
100 ns
after excitation to the excited state, depending on the
fluorophore, until all of the excited-state molecules have
FIGURE 7.10 Time course of fluorescence emission after a pulse of
excitation light.
returned to the ground state. When longer lifetime fluoro-
phores are used, this characteristic can be exploited to
eliminate interferences from substances that contribute
background fluorescence emission and to improve the
limit of quantitation. Fig. 7.10 illustrates a time-resolved
fluorescence measurement when a relatively long-lifetime
fluorophore is used. A pulse of light is used for excitation;
when the pulse ends, the background fluorescence decays
to 0 in 30 ns, but the fluorescence from the long-lifetime
fluorophore persists and can be measured against a zero-
signal background at 40 ns.
Fluorescence polarization (depolarization)
If a fluorescent molecule absorbs polarized light (i.e., all
electromagnetic waves are oriented in the same direction)
to produce the excited state, then the fluorescence emis-
sion will have the same polarization as long as the fluoro-
phore does not rotate during the lifetime of the excited
state. If the fluorophore rotates before the fluorescence
emission takes place, the emission will be depolarized
proportional to the degree of rotation before the light
emission. This property has been the basis for fluores-
cence polarization (more correctly depolarization) immu-
noassay for low-molecular-weight haptens. In this
application, a fluorescent analog of the analyte and the
analyte from the sample are mixed and compete for
analyte-binding sites on a fixed number of antibodies. If
analyte is absent, the fluorescent analog binds to the anti-
bodies and rotates in solution at the rate of the antibodies.
Because an antibody is a large protein, it rotates relatively
slowly compared with the low-molecular-weight fluores-
cent analyte analog, such that there is little change in
position during the lifetime of the excited state.
Consequently, the fluorescence polarization of the emitted
light is not changed from that of the excitation light. If
analyte is present, it binds to some of the antibody mole-
cules, which leaves some of the fluorescent analog
unbound to antibodies and free in solution. Because the

fluorescent analog is small, it rotates relatively quickly,
such that its orientation changes during the lifetime of the
excited state before fluorescent light is emitted.
Consequently, the fluorescence polarization of the emitted
light is randomized or depolarized. The amount of depo-
larization or the ratio of polarized to depolarized signal is
the basis to quantitate the analyte. Historically, the fluo-
rescence polarization approach was used in several immu-
noassay instruments but is not used in current practice.
Chemiluminescence
Chemiluminescence is related to fluorescence in that light
emitted from a molecular orbital excited state is measured
and is proportional to the concentration of the chemilumi-
nescent substance. In this technique, the excited state is
produced by a chemical reaction rather than by absorption
of light, as in fluorescence excitation. The excited chemi-
luminescent compound emits light as it relaxes to a
ground electronic energy state. Many precursor com-
pounds used in chemiluminescent reactions (e.g., acridi-
nium ester, luminol, and luciferin analogs) are activated
to emit light upon oxidation. In the presence of an oxidiz-
ing agent, metal ions or enzymes (e.g., horseradish perox-
idase or alkaline phosphatase) mediate oxidation of the
precursor compounds. Chemiluminescent reactions often
have a wide dynamic range and can have greater sensitiv-
ity to low concentrations than absorption or fluorescence,
because the measurements are made against a zero-signal
background.
The most common applications for chemiluminescent
measurement are immunoassay methods, in which the
chemiluminescent compound or enzyme mediator func-
tions as part of the detection label. Light emission is
detected by a luminometer, in which emitted photons are
detected to produce a measurement signal. The analytical
Spectrophotometry Chapter | 7 131
sensitivity of chemiluminescent reactions is improved by
the inclusion of a second molecule (sometimes called an
intensifier agent) that can amplify the intensity of emitted
light by a nonradiative energy transfer mechanism or
stabilize the excited state to optimize light emission.
Common sources of error in clinical practice include the
development of light leaks in the luminometer and insuffi-
cient removal of unbound chemiluminescent label because
of defects in the wash system. Like fluorescence, potential
interferences include molecules that can quench the
excited state before light emission and molecules that can
absorb the emitted light in the cuvette before it reaches
the detector.
References
[1] Guidelines for photometric instruments for measuring enzyme reac-
tion rates. Clin. Chem.23 (1977) 2160
2162.
[2] Joint Commission on Traceability in Laboratory Medicine
Database. ,https://www.bipm.org/jctlm/. (accessed 13.11.18).
[3] W.G. Miller, G.L. Myers, R. Rej, Why commutability matters,
Clin. Chem. 52 (2006) 553
554.
[4] H.W. Vesper, W.G. Miller, G.L. Myers, Reference materials and
commutability, Clin. Biochem. Rev. 28 (2007) 139
147.
[5] G. Sittampalam, G.S. Wilson, Theory of light scattering measure-
ments as applied to immunoprecipitation reactions, Anal. Chem. 56
(1984) 2176
2180.
Suggested reading
J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New
York, 2006. 954pp.
H.A. Strobel, W.R. Heineman, Chemical Instrumentation: A Systematic
Approach, third ed., Wiley, New York, 1989. 1210 pp.
H.H. Willard, L.L. Merritt, J.A. Dean, F.A. Settle, Instrumental Methods
of Analysis, seventh ed., Wadsworth Publishing, Belmont, CA,
1988. 895 pp.
132 Contemporary Practice in Clinical Chemistry
Self-assessment questions
(More than one answer may be correct for each question.)
1. What is actually measured to determine the absor-
bance of a chromogen?
a. light energy hitting a detector
b. decrease in light intensity hitting a detector
c. increase in light intensity hitting a detector
d. ratio of light intensity hitting a detector between
the chromogen and the blank
2. Which of the following reaction types can be used to
produce chromogens that absorb light?
a. redox reactions
b. chemiluminescent reactions
c. dye-binding reactions
d. enzyme-mediated reactions
3. What can cause the relationship between absorbance
and concentration to be nonlinear?
a. stray light
b. too large of a bandpass
c. chromogen complexation
d. wavelength calibration error
4. Why is 0.1
1.0 the most precise measurement range
for absorbance?
a. Effects of stray light are minimized.
b. The proportion of light intensity change with con-
centration decreases at larger A values.
c. The noise of the detector becomes dominant at
low A values.
d. The noise of the detector becomes dominant at
high A values.
5. What can cause the apparent molar absorptivity to be
different from theoretical?
a. too large of a bandpass
b. stray light
c. inner filter effect
d. miscalibrated wavelength
6. What type of calibration can make molar absorptivity
unimportant?
a. calibration relationship using calibrator solutions
b. correct wavelength calibration
c. bracketing with standards
d. use of a filter factor
7. What sources of bias or error can be corrected for by
calibration?
a. wavelength inaccuracy
b. autopipettor imprecision
c. absorbance inaccuracy
d. spectrophotometric interferences
8. How can the absorbance from an interfering sub-
stance be corrected?
a. sample blank before final reagent addition
b. kinetic measurement
c. bichromatic measurement for a sample with
hemoglobin and turbidity
d. rapid absorbance measurement
9. What type of interference cannot be corrected with a
bichromatic measurement?
a. bilirubin
b. turbidity
c. bilirubin and turbidity in the same sample
d. nonchromogenic drugs
10. What is the relationship between turbidimetric and
nephelometric measurements?
a. Nephelometry is the inverse of turbidimetry.
b. Turbidimetry is more sensitive.
c. Nephelometry can be measured with a conven-
tional spectrophotometer.
d. Both measure light scatter.
11. How can the presence of a prozone effect in an
immunoturbidimetric assay be detected?
a. measuring the absorbance after ultracentrifugation
b. measuring the absorbance after concentrating
sample
c. measuring the absorbance after diluting the
sample
d. measuring the absorbance after treating the sam-
ple with SDS
12. How does atomic absorption spectrophotometry dif-
fer from solution-phase measurements?
a. The chromogen has broad absorbance bands in
atomic absorption.
b. Stray light is more significant in atomic absorption.
c. The chromogen is short-lived in atomic absorption.
d. The flame or furnace in atomic absorption pro-
duces a more stable chromogen.
13. How does fluorescence polarization immunoassay
discriminate between free and bound fluorescent
analogs?
a. The bound fluorescent analog emits polarized
light in all directions.
b. The bound fluorescent analog emits polarized
light with the same orientation as that of the light
absorbed.
c. Free fluorescent analog rotates during the lifetime
of fluorescence and depolarizes the emitted light.
d. Fluorescent-labeled analog and native analyte
compete for antibody-binding sites.
14. Which of the following are sources of error in
chemiluminescent assays?
a. insufficient wash
b. presence of substances that quench emission
c. presence of light leaks
d. lamp deterioration
 Spectrophotometry Chapter | 7 133

Answers

1. d
2. a, c, d
3. a, c
4. a, b, d
5. a, b, d
6. a, c
7. a, c
8. a, b, d
9. c
10. d
11. c
12. b, c
13. b, c
14. a, b, c