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Ultraviolet/Visible Light Absorption Spectrophotometry in

Clinical Chemistry

Stephen L. Upstone

Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.)
pp. 16991714
John Wiley & Sons Ltd, Chichester, 2000

Ultraviolet/Visible Acknowledgments 13
Abbreviations and Acronyms 13
Light Absorption Related Articles 14
Spectrophotometry in References 14
Clinical Chemistry
Stephen L. Upstone Ultraviolet/visible (UV/VIS) absorption spectroscopy has
PerkinElmer Ltd., Beaconsfield, UK been used in the clinical laboratory for many years.
The technique has appeal, as it is almost universal in
its application. Although much of the routine work
is performed using high-throughput dedicated clinical
1 Introduction 1 analysis systems, absorption spectroscopy still has a place
2 Principles of Analysis and Instrument in most clinical laboratories.
Parameters 2 This article discusses the range of application types for
2.1 Fundamentals of Ultraviolet/Visible which absorption spectroscopy can be used and some
Spectroscopy and the Beer Lambert examples of common analyses are given. The article
Law 2 also discusses the merits of various instrument types and
2.2 Linearity and Deviations from the discusses some more advanced spectroscopic techniques,
Beer Lambert Law 2 such as derivative spectroscopy, to enhance the data
2.3 Cuvettes and Solvents 3 measured by the spectrophotometer. Brief reference is
2.4 Resolution (Band-pass) and Slit also made to the use of reflectance in clinical analysis.
Width 4
2.5 First- and Second-derivative
Spectroscopy 5 1 INTRODUCTION
2.6 Verification of Spectrophotometer
Performance 5 Ultraviolet/visible (UV/VIS) absorption spectroscopy has
been used in the clinical laboratory for many years. The
3 Overview of Instrument Designs and technique has appeal, as it is almost universal in its
Their Advantages and Disadvantages 6 application. The reliance on UV/VIS spectrophotome-
3.1 Dispersive and Diode-array Systems 6 ters (and other spectroscopic techniques) has diminished
3.2 Dispersive Ultraviolet/Visible over the years, however, as dedicated multichannel clin-
Spectrophotometers 6 ical analyzers and readers have appeared on the market.
3.3 Single-beam Spectrophotometers 6 These analyzers offer high sample throughput and clini-
3.4 Double-beam Spectrophotometers 6 cally validated proprietary chemistries with ready-mixed
3.5 Dual-beam Spectrophotometers 8 reagents for greater speed and convenience. These analyz-
3.6 Photodiode-array Spectrophoto- ers normally utilize a range of analytical techniques such
meters 8 as ion-selective electrodes, turbidometry and UV/VIS
3.7 Microplate Reader Spectrophoto- absorption in one integrated instrument. Reagent man-
meters 8 ufacturers are also trying to simplify the analysis even
3.8 Reflectance-based Analyzers 9 further so that much simpler instrumentation is required.
4 Common Clinical Applications Using There is some work on solid-phase chemistry systems in
Ultraviolet/Visible Absorption Spectro- which the completed reaction on the solid surface can
scopy 9 be measured on a simple reader which uses a simple
4.1 Enzyme Rate Assays 9 light-emitting diode (LED) source and measures light
4.2 Colorimetric and End-point Assays 10 reflectance from the active face.
4.3 Immunoassays, Enzyme-linked Spectroscopic analysis, in general, is in a slow decline
Immunosorbent Assays and in the clinical laboratory as the increasing adoption of
Microplate Assays 11 molecular biological techniques such as the polymerase
4.4 Porphyrin Analysis 11 chain reaction (PCR) and DNA sequencing are now
4.5 Hemoglobin Analysis 12 being used to measure (for example) genetic abnormal-
4.6 Protein Assays 12 ities rather than the more indirect biochemistry-based
4.7 Molecular Biology 13 approach.

Encyclopedia of Analytical Chemistry

R.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd

I have spent some length explaining the fundamentals are kept constant for a given set of experiments, a
of the technique and about instrument design. This is plot of the sample absorbance against the concentration
deliberate too often people treat UV/VIS spectropho- of the absorbing substance should give a straight line.
tometers as a simple meter without understanding In practice, a calibration curve is prepared by plotting
their limitations or suitability for a particular analysis. the absorbance of a series of standard samples as a
This article should also be useful for understanding spec- function of their concentration. If the absorbance of an
trophotometric assays on dedicated analyzers. unknown sample is then measured, the concentration
of the absorbing component can be assessed from this
graph. Another consequence of the Beer Lambert law
2 PRINCIPLES OF ANALYSIS AND is that it is possible to change the path length to affect
INSTRUMENT PARAMETERS the absorbance. This can be useful where lower detection
2.1 Fundamentals of Ultraviolet/Visible Spectroscopy limits are required as the path length can be increased
and the Beer Lambert Law (longer path length cuvettes are available) or, where
the absorbance is too high to be measured on the
UV/VIS spectroscopy is the study of how a sample instrument, the path length can be reduced. Alternatively,
responds to light. When a beam of light passes through it is possible to reduce the absorbance by diluting the
a substance or a solution, some of the light may be sample, but one has to take care when dealing with
absorbed and the remainder transmitted through the biologically active samples, particularly enzyme-based
sample. The ratio of the intensity of the light entering the solutions, as this may have a profound effect on the
sample (I0 ) to that exiting the sample (It ) at a particular activity.
wavelength is defined as the transmittance (T). This is
often expressed as the percentage transmittance (%T),
which is the transmittance multiplied by 100 (Equation 1): 2.2 Linearity and Deviations from the Beer Lambert
%T D 100 .1/ Above, the Beer Lambert Law has been shown to be
a linear relationship between sample concentration and
The absorbance (A) of a sample is the negative logarithm absorbance. If this relationship is tested experimentally
of the transmittance (Equation 2): by measuring samples of increasing concentration and
the results are plotted, the relationship will be seen
AD log T .2/ to break down with increasing absorbance. With gen-
eral laboratory UV/VIS spectrophotometers of recent
The UV/VIS range of the electromagnetic spectrum design, this will probably be around 3 absorbance units
covers the range 190 700 nm (most instruments are (AU).
capable of measuring at longer wavelengths than this, What is observed here is not a breakdown in the
depending on their detector type). For clinical analysis
Beer Lambert law (although some assumptions about
this is useful, as water (most assays are in aqueous
the sample being infinitely dilute are made in the law) but
solution) is almost completely transparent in this region.
a limitation in the instruments performance.
Most clinical assays are concerned with quantitation
The greatest contributor to this nonlinearity is the
rather than identification (there are more powerful
instruments stray light. Stray light can be loosely defined
techniques available to perform the latter) as absorption
as the amount of light present in the instrument at
spectra tend to be featureless they lack the fine structure
nonanalytical wavelengths or, put another way, the
which is found, for example, in an infrared (IR) spectrum.
amount of unwanted light present in the system. This
Some techniques to enhance resolution and qualitative
unwanted light can come from several sources. First, there
information will be discussed later.
may be light from outside entering the optical system.
The most important principle in absorption analysis is
Manufacturers usually go to some lengths to exclude this
the Beer Lambert law. This law states that, for a given
light and so it ought to be negligible in a well-designed
ideal solution, there is a linear relationship between con-
instrument. The main source of stray light comes from
centration and absorbance provided that the path length
the monochromator itself.
is kept constant; the absorptivity (e) is a constant for each
Monochromators (in dispersive instruments) are usu-
molecule for each wavelength (Equation 3):
ally diffraction gratings. These gratings, although highly
A D ecl .3/ efficient producers of monochromatic radiation, are not
entirely efficient. They will also allow small amounts of
where e D the absorptivity of the substance, c D con- light to pass through at other discrete wavelengths, in
centration and l D path length. Provided that e and l addition to small amounts of white light. We have seen

Table 1 Observed absorbance readings for three is no longer at the actual peak but at its side. More
different instrument stray light specifications sophisticated instruments can detect the actual peak
for every measurement or measure areas under the
True 1% T 0.01% T 0.0001% T
absorbance stray lighta stray lightb stray lightc
absorption curve between two user-defined points.
Another possible reason for nonlinearity is sample
1.0 0.9788 0.9996 1.0000 scatter. The instrument expects light to travel through the
2.0 1.8239 1.9957 2.0000 sample in a straight line (unless there is special provision
3.0 2.2218 2.9586 3.0000
4.0 2.2924 3.6999 3.9957 in the instrument design). If the sample is turbid, this
5.0 2.3009 3.9586 4.9586 light will be scattered and the light will be splayed out
in a cone-like fashion. As a result, less light will fall on
a Low-cost or old instrument.
b the detector and a falsely high absorbance reading will be
High-performance double-beam instrument.
c Top of the range double-monochromator, double-beam observed. Turbidity is relatively easy to observe as it is
instrument. not wavelength specific. A scattering sample, therefore,
will have a raised baseline. In bad cases, the baseline may
earlier that absorbance is a logarithmic scale and so 1 AU be elevated over 1 AU.
corresponds to 10% T, 2 AU corresponds to 1% T, 3 AU
to 0.1% T and so on. 2.3 Cuvettes and Solvents
If the light throughput is expressed in percentage
terms (%T) and a stray light value (from the instrument Apart from ensuring that the instrument design is
manufacturers data) of 0.05% transmittance is quoted, sufficient for the required analysis, the user must also
this will represent a third of the total light seen by check that the other components of the analytical
the detector at 3 AU (i.e. 0.1% T). This will cause system the cuvettes and the solvents are applicable
an underreporting of the true absorbance. By 4 AU for the required task. Cuvettes are offered in either
(0.01% T), there will be more stray light than sample optical glass or quartz. Optical glass will transmit light
signal, rendering the instrument unusable at this value. above 320 nm whereas quartz will transmit light well
Table 1 shows the anticipated effect on the observed below 190 nm (the usual low-wavelength limit for UV/VIS
absorbance for a range of stray light values which may spectrophotometers).
be encountered on various instruments found within the As an alternative to glass and quartz cuvettes, several
clinical laboratory. manufacturers offer disposable cuvettes. These are made
from various plastics including polystyrene and acrylic.
These cuvettes offer the main advantage that they do not
2.2.1 Other Causes of Non-linearity
need cleaning between samples, which is very useful when
Occasionally, there are other reasons why there may be handling biohazardous samples. The disadvantages are
unexpected deviations from linearity. that they have a restricted wavelength range (one should
One major cause is sample fluorescence. If a fluo- carefully check the manufacturers stated wavelength
rophore is excited at the same wavelength at which one specification before using disposable cuvettes) and they
is trying to measure absorption (the chromaphore), the have a lower optical performance. In some cases striations
detector will see a combination of absorbed light and across the face of the cuvette might be observed. These
the fluorescence emission. This emission will occur at a are due to the molding process in the cell manufacture.
longer wavelength than the absorption. It should be borne There will also be a molding mark at the bottom of
in mind that there is no monochromator (on a dispersive the cuvette (from the extrusion process) and this can
instrument) between the sample and detector, so the make stirring with a small magnetic flea difficult (with
emission will be detected. The observed result will be a instruments which provide a stirring facility). Another
lower absorbance reading than expected (as the detector disadvantage with disposable cuvettes is that the cell path
is receiving light from two phenomena instead of one). length (an important consideration for the Beer Lambert
If sample fluorescence is severe, it can be removed law) will not be as accurate as with glass or quartz cells
by inserting a cutoff filter between the sample and the and this may have quantitative accuracy implications.
detector. This filter should allow light of the absorbance Figure 1 shows the light transmission charateristics of
wavelength to pass through but block higher wavelengths. some common cuvette materials.
Sometimes the linearity can be affected by changes As regards solvents, water has the widest range
in pH. Some chromophores (the functional group in the and the lowest cutoff wavelength. Cutoff wavelengths
molecule responsible for the light absorption) will shift are somewhat arbitrary and there will be variations
wavelength with a change in pH. If the absorption peak in the stated values from different literature sources.
is particularly sharp, it may mean that measurement I have used the definition as being the wavelength

100 possess a broad spectrum, so a small band-pass will

not change the appearance of the spectrum (apart from
increased noise). Some molecules commonly encountered
Quartz in clinical analysis, such as porphyrins and the various
types of hemoglobin (oxy-, met- and carboxy-) give

(acrylic) sharp structures and the position of the peak has to
Glass be accurately measured or a misdiagnosis may otherwise
20 result.
Figure 2 shows the effect on a sample with fine
0 structure, i.e. benzene vapor in a sealed cuvette. This
190 300 400 500 600 700
is a highly artificial situation, as typical samples in the
Wavelength (nm) clinical laboratory do not exhibit such sharp features.
Figure 3 shows the effect of variable resolution on a
Figure 1 Light transmission characteristics of various cuvette more typical sample with a single broad peak. In this
instance there is little or no difference between a high-
and low-resolution (band-pass) scan.
Table 2 Cutoffs for common solvents

Solvent Cutoff (transmittance of

10% in a 1-cm cell) (nm)

Water 190
2-Propanol (isopropyl alcohol) 210
Ethanol 210 (a)
Methanol 210
Acetonitrile 210
Dichloromethane 235
Toluene 270
Acetone 330
Chloroform 250 (b)

230 235 240 245 250 255 260 265 270

at which a 1-cm cuvette filled with pure solvent has Wavelength (nm)
a transmittance of 10% (equivalent to 1 AU). The
cutoffs for common solvents are given in Table 2. Figure 2 Effect of changing the instrument resolution on a
Some buffers [e.g. tris(hydroxymethyl)aminomethane sample with sharp peaks (benzene vapor) showing the effect
on band shapes and illustrating sharper discrimination using a
(TRIS)] have organic components and so it should not 0.5-nm slit (b) compared with a 2-nm slit (a).
be assumed that the cutoff is identical with that for
2.4 Resolution (Band-pass) and Slit Width 1.6

UV/VIS instruments have a specified band-pass. This 1.4

4 nm

term relates to the instruments ability to resolve peaks 1.2

which are very close together or shoulders (small
features on the side of a larger peak). The band-pass 0.5 nm
will either be fixed, usually between 1 and 2 nm, or, 0.8
in the case of more expensive instruments, variable 0.6
(either continuously or in finite steps). Photodiode-array
instruments have a fixed optical band-pass as there is no
physical slit aperture on these instruments (although the 0.2
software may be able to simulate other slit conditions); 500 550 600 650 700 750
the resolution is defined by the light dispersion, the
Wavelength (nm)
polychromator and the number of elements in the
array. Figure 3 Effect of using various slit settings on a typical
The band-pass will affect the instruments ability to spectrum with broad bands (spectra offset for clarity) showing
discriminate between sharp features. Most molecules that the slit setting is largely irrelevant for broad peaks.

2.5 First- and Second-derivative Spectroscopy Broad peak almost

lacking in D2 spectrum
UV/VIS spectra are rather featureless at room temper- D2
ature, which is why the technique is primarily used for
Some peak
quantitative analysis rather than for sample identification shoulders
purposes. A broad ultraviolet (UV) spectrum is composed Note sharper
of many separate electronic transitions which, at room peak
temperature, become blurred to give the appearance
of a single entity. Spectra only become more defined
at very low temperatures and in the gas phase. Neither Sharp peak
of these two conditions is applicable in routine clinical Broad peak
analysis. Abs
One major advantage that UV/VIS analysis possesses
for biological measurements is that water (which is 200 250 300 350 400 450 500 550 600 650
the main component of all living systems) is virtually
Wavelength (nm)
transparent across the entire range (there is some very
weak activity around 860 nm) and so any technique to Figure 5 Second-derivative (D2) spectrum showing sharp band
improve the qualitative data is welcome. discrimination.
Derivative spectroscopy is a useful tool to give some
improvement. As the name suggests, the technique
features) will become flattened and so the tech-
consists of plotting the rate of change of the absorbance
nique can be used both as a peak-enhancement and
spectrum versus wavelength. This will give a plot (shown
background-rejection tool. The second-derivative spec-
in Figure 4 with the original spectrum). At peak maxima
trum, like its absorbance counterpart, still contains
and minima (and also points of inflection), the graph
quantitative information. If two points are consistently
is seen to pass through zero on the ordinate. As a
used on the spectrum and the difference in their val-
result, the technique can be used to identify peak
ues on the ordinate is taken, this can be plotted against
maxima and minima. This is termed a first-derivative
concentration and a linear relationship established.
Derivative spectra represent an alternative presenta-
First-derivative spectra have their uses but they are
tion of the original data. Information cannot be created
difficult to interpret (as the brain often sees the peaks
and so there is a cost involved. This cost is at the expense
rather than the true information). If we take the second-
of a much poorer signal-to-noise ratio (as the noise is
derivative spectrum (by derivatizing the absorbance
also being derivatized) and so if a requirement for deriva-
spectrum twice), we obtain a plot such as that in
tive spectroscopy is anticipated, an instrument with a good
Figure 5.
noise specification is highly desirable. Single-beam instru-
The second-derivative spectrum can be thought of
ments should not be considered for serious derivative
as an inverted spectrum. Sharp peaks will be made
even sharper. Broad peaks (and also broad background
Higher order derivatives also are possible, and usually
up to the fourth derivative is offered on current
Peak instrumentation, but the increased noise makes their
general analytical use questionable.

Point of inflection
Abs 2.6 Verification of Spectrophotometer Performance

Clinical analysis is concerned with producing an accurate
result to enable the clinician to make an accurate
diagnosis and to provide the correct treatment. It is
D1 important, therefore, that all equipment used to make
the diagnosis is well maintained and that the operators
have a sufficient skill level to understand the limitations
200 210 220 230 240 250 260 270 280 290 300 310 of the instrument and to use the correct operating and
Wavelength (nm) sampling procedures.
Instruments should be checked regularly, using a
Figure 4 Explanation of the first-derivative (D1) spectrum, certified reference material (CRM). As a minimum, the
and its relationship to the absorption spectrum (Abs). user should possess a sample for checking wavelength

accuracy and a set of absorbance filters. Wavelength 3.3 Single-beam Spectrophotometers

standards should never be used to check absorbance
accuracy and vice versa. Wavelength standards (such The single-beam spectrophotometer is the simplest
as holmium oxide glass filters) have very sharp peaks optical configuration. It consists of a light source,
whereas absorbance standards such as the National monochromator (either a grating or a filter), the sample
Institute of Standards and Technology (NIST) 930D area and the detector, as shown in Figure 6. In the single-
filters are of neutral density. beam design, it is necessary first to zero the instrument (to
Additional checks such as for stray light and noise may establish the I0 value) and then to measure the sample.
also be advantageous. Most instrument companies will Single-beam instruments are used mainly on grounds
be able to give advice on instrument performance checks of cost. They have poorer noise specifications compared
and routine maintenance. with their dual- and double-beam counterparts and are
Microplate reader users are more limited in the prone to drifting with time. This makes accurate kinetics
choice of calibration aids. For filter-based instruments and applications involving repetitive sampling over time
(the majority at the time of writing), there is no way more difficult. The cheaper models often lack the spectral
to check wavelength accuracy (other than to scan the resolution demanded by some clinical applications (e.g.
filter on a spectrophotometer) and there are few CRMs porphyrin analysis).
available for all formats of microplates. If one is using a
microplate reader, the manufacturer should be contacted 3.4 Double-beam Spectrophotometers
for advice.
In the double-beam design as shown in Figure 7, there
is still a single detector usually a photomultiplier (a
3 OVERVIEW OF INSTRUMENT DESIGNS phototube). The beam is then sent alternately through
AND THEIR ADVANTAGES AND sample and reference positions using a chopper wheel
DISADVANTAGES or vibrating mirror. The electronics in the instrument
are also able to synchronize the beam switching with the
3.1 Dispersive and Diode-array Systems detector so that it can distinguish whether the detector is
measuring the sample or reference beam at any one time.
Before going into details of analysis, it is important to Even in complete darkness, a photomultiplier will
discuss the design of a UV/VIS specrophotometer and the output a signal. This is known as the dark current. As
implications of the various design types for a particular a result, the beam shuttling device will also have some
analysis, and to understand the limiting factors in making means of temporarily blocking the light from both beams
that measurement. so that this residual signal (the dark current) can be
UV/VIS instruments are available in two main types, measured and subtracted from each sample and reference
dispersive and photodiode array (usually shortened to measurement.
diode array). The dispersive design uses a monochroma-
tor before the sample to convert the white light produced
Tungsten lamp
by the light source into a single pure wavelength of light. (for visible region)
This single wavelength is then passed through the sam-
ple and detected. The monochromator can be either a Mirror
fixed-wavelength filter or a variable-wavelength design lamp (UV
such as a prism (rare nowadays) or a diffraction grating. models only)
On higher specification instruments, there may be two Mirror Filter wheel
monochromators linked in series to permit higher perfor- (for
mance particularly at high absorbance values (e.g. over change)
Entrance slit
3.0 AU).

3.2 Dispersive Ultraviolet/Visible Spectrophotometers Mirror

Exit slit
Dispersive UV/VIS spectrophotometers have four main
optical configurations:

single beam
Diffraction grating Mirror Sample Detector
double beam (single detector) (monochromator)
dual beam (dual detector)
photodiode array. Figure 6 Single-beam optical configuration.
Tungsten lamp
Source mirror

Plane mirror

lamp Light beam

Open Mirror

Slit Chopper
mirror S R assembly
Blank portions for zeroing
detector dark current

Optical cell
Collimating Plane
mirror mirror

Spherical Spherical
mirror Sample cell mirror


Figure 7 Double-beam (chopper wheel) optical layout.

lamp Mirror



Entrance slit


Mirror Reference
Exit slit Beam splitter detectors



Figure 8 Dual-beam spectrophotometer optical layout.


3.5 Dual-beam Spectrophotometers to as a polychromator) on to a special solid-state detec-

tor with individual segments, one for each wavelength.
An alternative to the double-beam design is the dual-
The main appeal of these instruments is that the mea-
beam layout (Figure 8). In this design, the beam is
surement of a spectrum takes only a few seconds. The
split using a half-silvered mirror (beam splitter) into
system has some disadvantages, however. A diode-array
its sample and reference components. Each beam has its
spectrophotometer is a single-beam instrument (although
own detector.
a nonanalytical wavelength may be used as a pseudo-
This design is only practical with solid-state detectors
reference to overcome nonwavelength-specific drift). It
(which have fairly constant dark current) and gives
is also less suitable for some single-wavelength measure-
equivalent results compared with a double-beam system
ments as a whole spectrum has to be collected irrespective
for most analyses. The design removes the need for a
of whether the data points are required or not. The design
mechanical beam shuttling device (and hence improves
is also more prone to errors from sample fluorescence
reliability) and, as solid-state detectors are used, there
(as the sample irradiation is at all wavelengths and so
is no need for a high-tension power supply to provide
any fluorophore present will be also excited) and any
the high voltages (around 1000 V) which are required to
nonparallel surfaces in the sample (as this will affect
set the photomultiplier gain. The design also produces
the beam dispersion on to the individual elements in
very good baseline noise characteristics as, again, there
the array). Nevertheless, the diode-array system offers
is no contribution from the mechanical beam shuttle. If
a high throughput of scanned data and the ability also
the design has a weakness, it is at very high absorbance
to visualize whole spectrum for single-wavelength anal-
(over 3 AU) as the photomultiplier is able to detect
yses so that any unexpected results can be investigated
lower light levels than solid-state detectors. Routine
clinical analyses rarely exceed these absorbance values
and, in any case, a single monochromator instrument is
limited by its stray light (see later). If samples exceed 3.7 Microplate Reader Spectrophotometers
the upper absorbance limit of a spectrophotometer, they Over the past 20 years, many clinical analyses have
can either be diluted or a shorter path length cell can be been transferred from the traditional, cuvette-based
used. spectrophotometer to a microwell (normally 96-well)
format. The microwell started life in the early 1960s
3.6 Photodiode-array Spectrophotometers for microbiological culture. Later, it was realized that
the format could be applied to bulk analysis using clear
Photodiode-array spectrophotometers have been avail-
microplates and a dedicated reader.
able since the early 1980s with the advent of the early
Microplates are manufactured from plastic (usually
designs from Hewlett-Packard (now Agilent) (Figure 9).
polystyrene or acrylic, depending on the required wave-
The design is basically a single-beam instrument but
length range). They are cheap, disposable and provide a
the sample is irradiated with white light (as opposed to
universal format. The plates also have the advantage that
monochromatic light in a dispersive instrument). This
they are compatible with liquid handling devices such as
light, after it has passed through the sample, is then dis-
plate fillers and washers.
persed by a dispersion monochromator (often referred
Although 96 wells is the most common format, the
requirement for greater speed and throughput has seen
the introduction of even higher density formats such as
Diode array 384 wells per plate.
The dedicated reader is really a spectrophotometer
in an applied form. Most plate readers work in the
Polychromator visible region only (as the polystyrene microplates absorb
in the UV region) and use optical filters rather than
monochromators. More sophisticated readers may also
offer multiple reading modes such as fluorescence and
Dispersion bioluminescence in addition to absorbance. Many modern
device readers can also be used in conjunction with a robotic
Entrance system.
Source slit
In terms of performance, there is a compromise
compared with using a spectrophotometer, but for many
assays this is far outweighed by the reduced costs and
Figure 9 Diode-array spectrophotomer optical layout. greater convenience which this format offers.

3.8 Reflectance-based Analyzers such as the US Food and Drug Administration (FDA)
before such a device can be marketed.
In recent years, reagent manufacturers have investigated As many of these devices are designed for use in the
ways of making tests even more simple to perform home or general practitioners surgery, they must be
and less dependent on expensive analytical equipment. manufactured as cheaply as possible. As a result, an LED
An example of this is the portable glucose monitoring can be used in place of a light source and monochromator.
systems which diabetics use to check their blood sugar Detection is by means of a silicon photodiode or other
levels. solid-state detector. A diagram of a reflectance-based
The color reaction is performed on a solid support analyser for the measurement of blood sugar for diabetes
which is coated with the required reagents. This support monitoring is shown in Figure 10.
is usually a small stick or strip. After exposure to a blood
or urine sample, this support is inserted in the analytical
device and the reading displayed. These devices are based
on reflectance rather than absorbance (as the supports are 4 COMMON CLINICAL APPLICATIONS
opaque it is not possible to pass light through them). USING ULTRAVIOLET/VISIBLE
When light falls on a surface it can be reflected in ABSORPTION SPECTROSCOPY
two main ways, as either specular or diffuse reflectance.
Specular reflectance (from the Latin word speculum There are many clinical tests which employ UV and visible
for mirror) is the study of mirror-like reflectance. The spectrophotometry. For readers who want a detailed and
path of the reflected ray of light is entirely predictable exhaustive list, there are many textbooks on the subject..1/
as it should be at the same angle as the angle of In this section I have highlighted a range of these tests
light incidence. In diffuse reflectance, the surface has and detailed how they work.
a matt surface which scatters the reflected light in
all directions and this scattered reflectance can be
collected by a detector. In practice, a sample may 4.1 Enzyme Rate Assays
exhibit both types of reflectance (e.g. a coating with a Enyzmes are biocatalysts, which are extremely efficient
glossy surface) and so the geometry of the collection in converting their chosen substrates into product. Apart
sphere can be adjusted either to include or exclude the from their efficiency, they are also highly specific and
specular component. Reflectance is normally expressed often will not work with a slightly different substrate.
as a percentage compared with a standard normally a Often it is useful to study the rate of this catalysis
white surface such as Teflon . The relationship between by measuring either the rate of depletion of substrate
reflectance and concentration is much less clearly defined or the formation of products. This may be a matter
when compared with absorbance as there are physical of simply measuring the absorbance of one of the
factors to consider (e.g. particle size, layer thickness). reaction components directly or by forming an absorbing
A fairly accurate quantitative result can be obtained conjugate with another molecule.
by taking the logarithm of reflectance or by using the Enzyme kinetics are usually zero order. This means
Kubelka Munk equation. Manufacturers of reflectance- that, after an initial lag phase, there should be a linear
based devices need to spend considerable development relationship between substrate (or product) concentra-
work in providing an accurate calibration which can be tion with time until one of these components becomes
stored inside the instrument to produce the correct results. limiting. It is therefore useful if the instrument software
These results will need to be verified by regulatory bodies is able to allow the user to choose where this linear
portion is (either prior to or, better, after data collec-
tion) and to use this portion to calculate the rate. The
Test strip
enzyme activity is normally expressed in International
Units (IU) by applying a simple factor to the measured
Blood slope (absorbance/time).
Membrane Enzyme rate assays are nearly always performed at a
single wavelength (340 and 405 nm are commonly used)
and require a temperature-controlled environment. This
may take the form of a simple water-jacketed cell holder
where water (or other liquid) is passed through the
water jacket at a constant temperature (supplied by a
Figure 10 Principle of reflectance-based hand-held analyzer thermostated water circulator). Some cell holders use
for diabetes monitoring. thermoelectric (or Peltier effect) cell holders. These cell

holders have the advantage of much more precise tem- One important class of enzyme-catalyzed reactions
perature control and the ability to work below ambient involves the oxidation and reduction of pyridine
temperatures (Peltier cells can cool by reversing the elec- nucleotides [nicotinamide adenine dinucleotide (NADC )
tric current flow). Some Peltier designs, particularly those and nicotinamide adenine dinucleotide (reduced form)
requiring high or low temperatures, will still require a (NADH), respectively]. If the reaction is followed at
flow of cold water in order to operate correctly whereas 340 nm, NADC does not absorb whereas NADH shows
those covering a more restricted range do not. a strong absorbance. Examples of NADC /NADH kinetic
Most enzyme reactions are fairly slow, taking place for reactions include glucose dehydrogenase,.3/ aspartate
5 min or more. In order to increase productivity, most aminotransferase.4/ and urea..5/
UV/VIS instruments offer a cell changer as an accessory. Other enzymatic tests often include a colorless
This is a shuttle device, which can hold six or more compound, which is added to the reaction. This is
cuvettes at once. The instrument then cycles through then enzymatically converted to a colored product (e.g.
each of the cell positions taking a reading on each cell p-nitrophenol). Examples of this type of test include alka-
every 30 s during the course of the reaction. line phosphatase,.6 8/ acid phosphatase.4/ and amylase..1/
The collected data can be analyzed either using the These tests are included in the summary of common
instruments own kinetic software or externally, using clinical tests in Table 3.
either a computer or manual calculation. Some statistical
data on the quality of the curve fit are also useful. 4.2 Colorimetric and End-point Assays
An example of a clinical rate assay is the determination It is also possible to perform a reaction to completion
of butyrylcholinesterase (BchE). Certain individuals and then to calculate the quantity of initial substrate
express a mutant form of the BchE gene. This then by extrapolation. This type of assay is termed an end-
encodes for a defective form of the enzyme, which lacks point assay. There are a vast number of such tests and,
the ability to hydrolyze succinylcholine. In some rare if an exhaustive list is sought, the reader is directed to
cases, the complete BchE gene is missing. A defective or a compendium of methods such as Tietz..1/ A summary
missing gene will not, normally, be of any consequence. of some common clinical end-point analyses is given in
If, however, succinylcholine is used during tracheal the summary of common clinical tests in Table 3. The
intubation in the administration of inhalation anesthetics, methods will prescribe a time after which the reading can
this will then cause the patient to undergo complete be taken. In the case of enzyme-catalyzed reactions, the
paralysis. The test for this enzyme.2/ is commonly incubation time will need to be at constant temperature.
performed using a UV/VIS spectrophotometer with a Most diagnostics companies provide premixed reagents
temperature-controlled cell holder (most tests will be to perform these tests together with a detailed protocol
performed at 37 C). describing their use. Different protocols may use slightly

Table 3 Some common clinical tests

Analyte Method UV wavelength (nm)

Acid phosphatase Kinetic: PNP 405
Alanine aminotransferase.9/ Kinetic: NADC /NADH 340
Alkaline phosphatase.6 8/ Kinetic: PNP 405
a-Amylase.1/ Kinetic: 405
Aspartate Kinetic: NADC /NADH 340
Bilirubin.11/ Evelyn Molloy 555
Cholesterol.12/ Kinetic: cholesterol oxidase 500
Creatinine.13/ Jaffe 510
GGT.14/ Kinetic: carboxy substrate 405
Glucose.15/ Kinetic: hexokinase (NADC /NADH) 340
LDH.16/ Kinetic: lactate/pyruvate 340
Porphyrins (total).17/ Acidification using HCl Absorbance 405 (Soret peak)
Pseudocholinesterase.28/ NADC /NADH 340
Triglycerides.18,19/ Kinetic: GPO colorimetric 520
Urea.20/ Kinetic: NADC /NADH 340

PNP, p-nitrophenyl phosphate; GGT, g-glutamyl transferase; LDH, lactate dehydrogenase; GPO, glycerol-3-phosphate oxidase.

altered wavelengths for each analyte. As with rate assays, the antibody, which has previously been coated on the
the test may be based on reading the analyte directly or microplate. The enzyme conjugate also binds to the
an additional color reaction may be required to develop analyte. This will be used later in conjunction with a
the test. specific dye to produce the color, which will then be
measured on the reader.
4.3 Immunoassays, Enzyme-linked Immunosorbent
Assays and Microplate Assays 4.4 Porphyrin Analysis
Porphyria is the name given to a number of related
Immunoassays rely on the very strong affinity between
conditions, some genetic, which result from an over
an antibody and its target molecule (antigen). This strong
production of porphyrins, which are precursors in the
affinity has been used to design a wide range of assays
production of hemoglobin. Sufferers are prone to bouts
for a variety of targets. These include pathogens (where
of severe abdominal pain, vomiting, severe personality
a chemical associated with the organism or virus will be
changes and sensitivity to light. Some types of porphyria
targeted), tumor markers and drug monitoring (either
produce characteristic dark-colored (port wine) urine
therapeutic or drugs of abuse). In fact, anything which
and this is a good first indication that porphyria is present.
can elicit an antibody response can be developed into
One of the main types of porphyria (a generic term for
an immunoassay. These assays are ideal where high
porphyrin-related disease) is variegate porphyria. In this
specificity is required. They are not good at dealing with
case a mitochondrial enzyme called protoporphyrinogen
a range of possible compounds. For example, it is difficult
oxidase is defective (owing to incorrect genetic coding)
to design an immunoassay to detect all abused drugs, only
and as a result excess protoporphyrin (one of the
specific types. If a designer drug is synthesized with an
porphyrin types) is produced. The protoporphyrinogen
additional functional group, this may adversely affect the
reacts with oxygen to produce protoporphyrin in an
uncontrolled reaction. Other porphyria types include
Immunoassays are competitive assays. The reaction
acute intermittent porphyria. Accurate diagnosis of the
mixture will contain antigens labeled with some kind
exact type of porphyria is vital as incorrect treatment
of tag (this is either a radiolabel, a fluorophore or
could have very serious consequences.
a site which will bind to a chromophore). When the
Porphyrins.17/ have a characteristic UV/VIS absorption
(unlabeled) sample antigens are introduced, there will
peak (Soret peak) in the region 400 410 nm depending
be competition between the labeled and unlabeled on the type of porphyrin present (coproporphyrin has a
antigens and this can be calibrated against a binding peak between 402 and 403 nm whereas the uroporphyrin
curve (which will be sigmoidal in nature). Prior to peak lies between 406 and 407 nm).
measurement, it will be necessary to separate the free The urine sample is filtered, diluted with distilled
and bound antigens and so a separation step will be water and acidified with hydrochloric acid. The sample
required (heterogeneous assay). Much effort has been is scanned between 300 and 500 nm and the spectrum
spent in previous years trying to simplify this process peak positions are noted. The measurement is taken by
(e.g. using magnetized, latex-covered beads) or using first constructing a baseline at points at either side of
a technique which inherently separates bound from the main peak (usually around 380 and 430 nm) and then
unbound antigens (e.g. fluorescence polarization) to measuring the height of the peak down to this baseline
produce a homogeneous assay. For the highest sensitivity (Figure 11).
immunoassays, either radioactivity or fluorescence (or
time-resolved fluorescence) has to be used. In situations
where lower sensitivity is acceptable, an absorption-based
assay can be employed.

A popular assay type is the enzyme-linked immunosor-

bent assay (ELISA). In this assay, an antibody (specific to
the analyte) is coated on the bottom of the microplate. In
some cases, any exposed area on the microplate (i.e. any
area where the antibody has not been coated) is blocked
using bovine serum albumin (BSA) and the excess anti-
body is washed away. The blocking agent is to help
prevent unbound antigen from adhering directly to the 380 405 430
plate, thus affecting the final result. Wavelength (nm)
The analyte and enzyme conjugate [e.g. horseradish
peroxidase (HRP)] is added. The analyte will bind to Figure 11 Porphyrin analysis using three wavelengths.

The total porphyrin concentration is given by 0.300

Equation (4): 0.260

total porphyrin.g L 1 / D 2A.lmax / .A380 C A430 /e 0.200
.4/ 0.160
where A is absorbance, lmax is the wavelength at 0.140
maximum absorption and e is the molar absorptivity 0.100
(a constant) for the analyte; for porphyrins (in a 1-cm 0.080
cell) e D 4740 g L 1 . 0.040
Alternatively, second-derivative spectroscopy has been 0.020
used.1/ (as this reduces background effects and produces 470 500 520 540 560 580 600 620 640 660 680 700 720 750
sharper peaks). It should be borne in mind that, for a Wavelength (nm)
full and correct diagnosis, the type of porphyrin must be
accurately identified. This can only be done using a good Figure 12 Absorption spectrum of hemoglobin cyanide
UV/VIS spectrophotometer offering narrow slits and a complex.
skilled user, as interpretation of the corrected spectrum or
the second-derivative spectrum may be involved. UV/VIS This complex has a peak around 546 nm, which can be
absorption spectroscopy is generally used for screening measured and quantitated.
and other techniques such as high-performance liquid Franzini et al..21/ have described a method based on
chromatography (HPLC) or fluorescence spectroscopy second-derivative spectroscopy to measure hemoglobin
(which gives much better selectivity as each porphyrin has and its homologs. This method helps to overcome the
a different excitation and emission wavelength) are often interference from bilirubin, which is also often present in
used to make the final, confirmatory, diagnosis. These these analyses.
techniques are usually offered by porphyria reference Shih et al..22/ also used multicomponent analysis in
centers. order to quantitate carboxy-, met- and oxyhemoglobin
independently from a single scan. This is a statistical
approach which compares each data point in a spectrum
4.5 Hemoglobin Analysis
with a calibration set of known references either
Hemoglobin is a protein with a nonprotein core consis- mixtures or single components and attempts to calculate
ting of an iron atom surrounded by heme groups. It the relative proportions of each in the unknown spectrum.
has remarkable oxygen transportation properties where This approach has its attractions as it is potentially
it can change its conformation to accept oxygen (oxy- able to measure each component without the need for
hemoglobin). This process can be inhibited by carbon a separation step. The main drawback is when there
monoxide, which has a 200 times stronger affinity for is significant spectral overlap or in situations where
hemoglobin (carboxyhemoglobin) than oxygen, resulting the concentrations of the various components differ
in severe respiratory problems and death in cases of car- widely. Problems may also occur if there is a strong
bon monoxide poisoning. Hemoglobin possesses an iron background matrix which is not constant. More powerful
atom core in its ferrous (Fe2C ) state. If the iron is oxidized techniques exist, such as principle component regression
to its ferric (Fe3C ) state, its oxygen transport capabilities or partial least-squares fitting. These techniques are a
are diminished and the molecule is called methemoglobin science in themselves (chemometrics) and go beyond this
(metHb). article.
Total hemoglobin can be measured by performing a
reaction of the total hemoglobin present with potas- 4.6 Protein Assays
sium cyanide to form a hemoglobin cyanide complex
(Figure 12). Proteins are composed of amino acid building blocks.
This is the basis of the method of Zijlstra et al..20/ Protein has some intrinsic absorbance at approximately
In this method, hemoglobin is reacted with potassium 280 nm (from the aromatic amino acids tyrosine and
cyanide (taking great care with pipetting and certainly tryptophan) but it is more common to perform a reaction
never by mouth!) and sodium (or potassium) hexacyano- to produce a colored complex which can be assayed in the
ferrate(III) to produce a hemoglobin cyanide complex visible region. The three most common procedures for
(Equation 5): protein analysis are biuret, Bradford and bicinchoninic
acid (BCA) assays.
hemoglobin C CN C Fe(CN)6 3 Table 4 lists the various protein methods. These are all
! hemoglobin cyanide .5/ simple colorimetric determinations.

Table 4 Summary of protein assays

Assay method Principle Range Interferences

and reference (g mL 1 )
Lowry.23/ As biuret plus determination of 5 200 Phenols, aromatic amino acids
aromatic amino acids
Bradford.24/ Dye binding with coomassie 10 200 Detergents
brilliant blue
Biuret.25/ Determination of number of 200 5000 Amines, ammonium salts
peptide bonds
BCA.26/ Reduction of copper by protein 200 1200 High concentrations of metals,
and formation of Cu(I) BCA strong reducing agents,
complex chelating agents
Absorbance at 280 nm.23/ Determination of tyrosine and >50 Nucleic acids, phenols, aromatics
tryptophan in protein
Warburg Christian.27/ Determination of aromatic amino 50 3000 purity check Phenols, aromatics
acids with compensation for
nucleic acids

1.10 is calculated. Optionally, a third reference point can be

1.00 taken at 320 nm (to assess the amount of turbidity and
scatter) and this absorbance value can be subtracted from

either of the two absorbance values prior to calculating the
0.60 ratio. If the ratio of the two absorbances is between 1.7 and
2.0, then the DNA preparation is considered to be pure.
0.40 If it is >2, then there is probably a high RNA content.
A lower ratio would indicate a high protein or phenol
content (a reagent commonly used in DNA extraction).
One major issue in molecular biology is sample volume.
220 240 260 280 300 320 340 It is not uncommon to have volumes of 10 L and so
Wavelength (nm) special low-volume cells are available. These special
cuvettes are made from quartz (as plastic and glass
Figure 13 Absorption spectrum of DNA. cuvettes generally absorb below 300 nm). Dedicated low-
cost instruments are available for the assessment of
nucleic acid purity.
4.7 Molecular Biology
The use of UV/VIS spectroscopy for molecular biology
strictly falls outside the scope of clinical analysis. On ACKNOWLEDGMENTS
the other hand, more routine clinical laboratories are
using molecular biology techniques (such as the PCR and I thank Hanswilly Muller at PerkinElmer, Uberlingen,
automated dideoxy sequencing) as these give more direct Germany, for providing some of the spectra shown here,
diagnosis for genetically based disorders. Jackie Woolf at the Porphyria Reference Centre, Heath
As a result of these techniques, UV/VIS spectroscopy is Park Hospital, Cardiff, and Ipswich Hospital, UK, for
useful for assessing the purity of the starting template in some information and spectra of porphyrins and Chris
either a sequencing or PCR reaction and, as a result, Royle at the Brompton Hospital, UK, for keeping me up
saves time in optimizing the reaction and helps to to date with the latest developments in clinical analysis.
reduce reagent costs. Pure DNA and RNA absorb at I also thank Agilent Technologies (formerly Hewlett-
260 nm (Figure 13). Protein (which is the main source Packard Instruments) and Hypoguard for permission to
of contamination) absorbs at around 280 nm (this is use their diagrams.
dependent on the exact amino acid composition as
individual amino acids have slightly different absorption
maxima). There is a rule of thumb which is now widely ABBREVIATIONS AND ACRONYMS
adopted in molecular biology laboratories as a quick
method for assessing purity. This method measures the BchE Butyrylcholinesterase
absorbance at 260 and 280 nm and the ratio (A260 /A280 ) BSA Bovine Serum Albumin

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