Download UV/Vis Light Absorption in Clinical Chemistry

Ultraviolet/Visible Light Absorption Spectrophotometry in
Clinical Chemistry
Stephen L. Upstone
in
Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.)
pp. 1699–1714
 John Wiley & Sons Ltd, Chichester, 2000
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
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Ultraviolet/Visible
Light Absorption
Spectrophotometry in
Clinical Chemistry
Acknowledgments
13
Abbreviations and Acronyms
13
Related Articles
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References
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Stephen L. Upstone
PerkinElmer Ltd., Beaconsfield, UK
Ultraviolet/visible (UV/VIS) absorption spectroscopy has
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
analysis systems, absorption spectroscopy still has a place
in most clinical laboratories.
This article discusses the range of application types for
which absorption spectroscopy can be used and some
examples of common analyses are given. The article
also discusses the merits of various instrument types and
discusses some more advanced spectroscopic techniques,
such as derivative spectroscopy, to enhance the data
measured by the spectrophotometer. Brief reference is
also made to the use of reflectance in clinical analysis.
1 Introduction
2 Principles of Analysis and Instrument
Parameters
2.1 Fundamentals of Ultraviolet/Visible
Spectroscopy and the Beer – Lambert
Law
2.2 Linearity and Deviations from the
Beer – Lambert Law
2.3 Cuvettes and Solvents
2.4 Resolution (Band-pass) and Slit
Width
2.5 First- and Second-derivative
Spectroscopy
2.6 Verification of Spectrophotometer
Performance
3 Overview of Instrument Designs and
Their Advantages and Disadvantages
3.1 Dispersive and Diode-array Systems
3.2 Dispersive Ultraviolet/Visible
Spectrophotometers
3.3 Single-beam Spectrophotometers
3.4 Double-beam Spectrophotometers
3.5 Dual-beam Spectrophotometers
3.6 Photodiode-array Spectrophotometers
3.7 Microplate Reader Spectrophotometers
3.8 Reflectance-based Analyzers
4 Common Clinical Applications Using
Ultraviolet/Visible Absorption Spectroscopy
4.1 Enzyme Rate Assays
4.2 Colorimetric and End-point Assays
4.3 Immunoassays, Enzyme-linked
Immunosorbent Assays and
Microplate Assays
4.4 Porphyrin Analysis
4.5 Hemoglobin Analysis
4.6 Protein Assays
4.7 Molecular Biology
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1 INTRODUCTION
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Ultraviolet/visible (UV/VIS) absorption spectroscopy has
been used in the clinical laboratory for many years. The
technique has appeal, as it is almost universal in its
application. The reliance on UV/VIS spectrophotometers (and other spectroscopic techniques) has diminished
over the years, however, as dedicated multichannel clinical analyzers and readers have appeared on the market.
These analyzers offer high sample throughput and clinically validated proprietary chemistries with ready-mixed
reagents for greater speed and convenience. These analyzers normally utilize a range of analytical techniques such
as ion-selective electrodes, turbidometry and UV/VIS
absorption in one integrated instrument. Reagent manufacturers are also trying to simplify the analysis even
further so that much simpler instrumentation is required.
There is some work on solid-phase chemistry systems in
which the completed reaction on the solid surface can
be measured on a simple reader which uses a simple
light-emitting diode (LED) source and measures light
reflectance from the active face.
Spectroscopic analysis, in general, is in a slow decline
in the clinical laboratory as the increasing adoption of
molecular biological techniques such as the polymerase
chain reaction (PCR) and DNA sequencing are now
being used to measure (for example) genetic abnormalities rather than the more indirect biochemistry-based
approach.
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Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.) Copyright  John Wiley & Sons Ltd
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CLINICAL CHEMISTRY
I have spent some length explaining the fundamentals
of the technique and about instrument design. This is
deliberate – too often people treat UV/VIS spectrophotometers as a simple ‘‘meter’’ without understanding
their limitations or suitability for a particular analysis.
This article should also be useful for understanding spectrophotometric assays on dedicated analyzers.
2 PRINCIPLES OF ANALYSIS AND
INSTRUMENT PARAMETERS
2.1 Fundamentals of Ultraviolet/Visible Spectroscopy
and the Beer – Lambert Law
UV/VIS spectroscopy is the study of how a sample
responds to light. When a beam of light passes through
a substance or a solution, some of the light may be
absorbed and the remainder transmitted through the
sample. The ratio of the intensity of the light entering the
sample (I0 ) to that exiting the sample (It ) at a particular
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):
I0
%T D
ð 100
.1/
It
The absorbance (A) of a sample is the negative logarithm
of the transmittance (Equation 2):
AD
log T
.2/
The UV/VIS range of the electromagnetic spectrum
covers the range 190 – 700 nm (most instruments are
capable of measuring at longer wavelengths than this,
depending on their detector type). For clinical analysis
this is useful, as water (most assays are in aqueous
solution) is almost completely transparent in this region.
Most clinical assays are concerned with quantitation
rather than identification (there are more powerful
techniques available to perform the latter) as absorption
spectra tend to be featureless – they lack the fine structure
which is found, for example, in an infrared (IR) spectrum.
Some techniques to enhance resolution and qualitative
information will be discussed later.
The most important principle in absorption analysis is
the Beer – Lambert law. This law states that, for a given
ideal solution, there is a linear relationship between concentration and absorbance provided that the path length
is kept constant; the absorptivity (e) is a constant for each
molecule for each wavelength (Equation 3):
A D ecl
.3/
where e D the absorptivity of the substance, c D concentration and l D path length. Provided that e and l
are kept constant for a given set of experiments, a
plot of the sample absorbance against the concentration
of the absorbing substance should give a straight line.
In practice, a calibration curve is prepared by plotting
the absorbance of a series of standard samples as a
function of their concentration. If the absorbance of an
unknown sample is then measured, the concentration
of the absorbing component can be assessed from this
graph. Another consequence of the Beer – Lambert law
is that it is possible to change the path length to affect
the absorbance. This can be useful where lower detection
limits are required as the path length can be increased
(longer path length cuvettes are available) or, where
the absorbance is too high to be measured on the
instrument, the path length can be reduced. Alternatively,
it is possible to reduce the absorbance by diluting the
sample, but one has to take care when dealing with
biologically active samples, particularly enzyme-based
solutions, as this may have a profound effect on the
activity.
2.2 Linearity and Deviations from the Beer – Lambert
Law
Above, the Beer – Lambert Law has been shown to be
a linear relationship between sample concentration and
absorbance. If this relationship is tested experimentally
by measuring samples of increasing concentration and
the results are plotted, the relationship will be seen
to break down with increasing absorbance. With general laboratory UV/VIS spectrophotometers of recent
design, this will probably be around 3 absorbance units
(AU).
What is observed here is not a breakdown in the
Beer – Lambert law (although some assumptions about
the sample being infinitely dilute are made in the law) but
a limitation in the instrument’s performance.
The greatest contributor to this nonlinearity is the
instrument’s stray light. Stray light can be loosely defined
as the amount of light present in the instrument at
nonanalytical wavelengths or, put another way, the
amount of unwanted light present in the system. This
unwanted light can come from several sources. First, there
may be light from outside entering the optical system.
Manufacturers usually go to some lengths to exclude this
light and so it ought to be negligible in a well-designed
instrument. The main source of stray light comes from
the monochromator itself.
Monochromators (in dispersive instruments) are usually diffraction gratings. These gratings, although highly
efficient producers of monochromatic radiation, are not
entirely efficient. They will also allow small amounts of
light to pass through at other discrete wavelengths, in
addition to small amounts of white light. We have seen
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
Table 1 Observed absorbance readings for three
different instrument stray light specifications
True
absorbance
1.0
2.0
3.0
4.0
5.0
a
b
c
1% T
stray lighta
0.01% T
stray lightb
0.0001% T
stray lightc
0.9788
1.8239
2.2218
2.2924
2.3009
0.9996
1.9957
2.9586
3.6999
3.9586
1.0000
2.0000
3.0000
3.9957
4.9586
Low-cost or old instrument.
High-performance double-beam instrument.
‘‘Top of the range’’ double-monochromator, double-beam
instrument.
earlier that absorbance is a logarithmic scale and so 1 AU
corresponds to 10% T, 2 AU corresponds to 1% T, 3 AU
to 0.1% T and so on.
If the light throughput is expressed in percentage
terms (%T) and a stray light value (from the instrument
manufacturer’s data) of 0.05% transmittance is quoted,
this will represent a third of the total light seen by
the detector at 3 AU (i.e. 0.1% T). This will cause
an underreporting of the true absorbance. By 4 AU
(0.01% T), there will be more stray light than sample
signal, rendering the instrument unusable at this value.
Table 1 shows the anticipated effect on the observed
absorbance for a range of stray light values which may
be encountered on various instruments found within the
clinical laboratory.
2.2.1 Other Causes of Non-linearity
Occasionally, there are other reasons why there may be
unexpected deviations from linearity.
One major cause is sample fluorescence. If a fluorophore is excited at the same wavelength at which one
is trying to measure absorption (the chromaphore), the
detector will see a combination of absorbed light and
the fluorescence emission. This emission will occur at a
longer wavelength than the absorption. It should be borne
in mind that there is no monochromator (on a dispersive
instrument) between the sample and detector, so the
emission will be detected. The observed result will be a
lower absorbance reading than expected (as the detector
is receiving light from two phenomena instead of one).
If sample fluorescence is severe, it can be removed
by inserting a cutoff filter between the sample and the
detector. This filter should allow light of the absorbance
wavelength to pass through but block higher wavelengths.
Sometimes the linearity can be affected by changes
in pH. Some chromophores (the functional group in the
molecule responsible for the light absorption) will shift
wavelength with a change in pH. If the absorption peak
is particularly sharp, it may mean that measurement
3
is no longer at the actual peak but at its side. More
sophisticated instruments can detect the actual peak
for every measurement or measure areas under the
absorption curve between two user-defined points.
Another possible reason for nonlinearity is sample
scatter. The instrument expects light to travel through the
sample in a straight line (unless there is special provision
in the instrument design). If the sample is turbid, this
light will be scattered and the light will be splayed out
in a cone-like fashion. As a result, less light will fall on
the detector and a falsely high absorbance reading will be
observed. Turbidity is relatively easy to observe as it is
not wavelength specific. A scattering sample, therefore,
will have a raised baseline. In bad cases, the baseline may
be elevated over 1 AU.
2.3 Cuvettes and Solvents
Apart from ensuring that the instrument design is
sufficient for the required analysis, the user must also
check that the other components of the analytical
system – the cuvettes and the solvents – are applicable
for the required task. Cuvettes are offered in either
optical glass or quartz. Optical glass will transmit light
above 320 nm whereas quartz will transmit light well
below 190 nm (the usual low-wavelength limit for UV/VIS
spectrophotometers).
As an alternative to glass and quartz cuvettes, several
manufacturers offer disposable cuvettes. These are made
from various plastics including polystyrene and acrylic.
These cuvettes offer the main advantage that they do not
need cleaning between samples, which is very useful when
handling biohazardous samples. The disadvantages are
that they have a restricted wavelength range (one should
carefully check the manufacturer’s stated wavelength
specification before using disposable cuvettes) and they
have a lower optical performance. In some cases striations
across the face of the cuvette might be observed. These
are due to the molding process in the cell manufacture.
There will also be a molding mark at the bottom of
the cuvette (from the extrusion process) and this can
make stirring with a small magnetic flea difficult (with
instruments which provide a stirring facility). Another
disadvantage with disposable cuvettes is that the cell path
length (an important consideration for the Beer – Lambert
law) will not be as accurate as with glass or quartz cells
and this may have quantitative accuracy implications.
Figure 1 shows the light transmission charateristics of
some common cuvette materials.
As regards solvents, water has the widest range
and the lowest cutoff wavelength. Cutoff wavelengths
are somewhat arbitrary and there will be variations
in the stated values from different literature sources.
I have used the definition as being the wavelength
4
CLINICAL CHEMISTRY
100
60
40
Quartz
Plastic
(acrylic)
Glass
20
0
190
300
400
500
600
700
Wavelength (nm)
Figure 1 Light transmission characteristics of various cuvette
materials.
Table 2 Cutoffs for common solvents
Solvent
Cutoff (transmittance of
10% in a 1-cm cell) (nm)
Water
2-Propanol (isopropyl alcohol)
Ethanol
Methanol
Acetonitrile
Dichloromethane
Toluene
Acetone
Chloroform
190
210
210
210
210
235
270
330
250
Absorbance
%T
80
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
in clinical analysis, such as porphyrins and the various
types of hemoglobin (oxy-, met- and carboxy-) give
sharp structures and the position of the peak has to
be accurately measured or a misdiagnosis may otherwise
result.
Figure 2 shows the effect on a sample with fine
structure, i.e. benzene vapor in a sealed cuvette. This
is a highly artificial situation, as typical samples in the
clinical laboratory do not exhibit such sharp features.
Figure 3 shows the effect of variable resolution on a
more typical sample with a single broad peak. In this
instance there is little or no difference between a highand low-resolution (band-pass) scan.
(a)
(b)
230
at which a 1-cm cuvette filled with pure solvent has
a transmittance of 10% (equivalent to 1 AU). The
cutoffs for common solvents are given in Table 2.
Some buffers [e.g. tris(hydroxymethyl)aminomethane
(TRIS)] have organic components and so it should not
be assumed that the cutoff is identical with that for
water.
235
240
245
250
255
260
265
270
Wavelength (nm)
Figure 2 Effect of changing the instrument resolution on a
sample with sharp peaks (benzene vapor) showing the effect
on band shapes and illustrating sharper discrimination using a
0.5-nm slit (b) compared with a 2-nm slit (a).
1.8
1.6
UV/VIS instruments have a specified band-pass. This
term relates to the instrument’s ability to resolve peaks
which are very close together or ‘‘shoulders’’ (small
features on the side of a larger peak). The band-pass
will either be fixed, usually between 1 and 2 nm, or,
in the case of more expensive instruments, variable
(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
software may be able to simulate other slit conditions);
the resolution is defined by the light dispersion, the
polychromator and the number of elements in the
array.
The band-pass will affect the instrument’s ability to
discriminate between sharp features. Most molecules
1.4
Absorbance
2.4 Resolution (Band-pass) and Slit Width
4 nm
1.2
1.0
0.5 nm
0.8
0.6
0.4
0.2
500
550
600
650
700
750
Wavelength (nm)
Figure 3 Effect of using various slit settings on a typical
spectrum with broad bands (spectra offset for clarity) showing
that the slit setting is largely irrelevant for broad peaks.
5
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
2.5 First- and Second-derivative Spectroscopy
UV/VIS spectra are rather featureless at room temperature, which is why the technique is primarily used for
quantitative analysis rather than for sample identification
purposes. A broad ultraviolet (UV) spectrum is composed
of many separate electronic transitions which, at room
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
of these two conditions is applicable in routine clinical
analysis.
One major advantage that UV/VIS analysis possesses
for biological measurements is that water (which is
the main component of all living systems) is virtually
transparent across the entire range (there is some very
weak activity around 860 nm) and so any technique to
improve the qualitative data is welcome.
Derivative spectroscopy is a useful tool to give some
improvement. As the name suggests, the technique
consists of plotting the rate of change of the absorbance
spectrum versus wavelength. This will give a plot (shown
in Figure 4 with the original spectrum). At peak maxima
and minima (and also points of inflection), the graph
is seen to pass through zero on the ordinate. As a
result, the technique can be used to identify peak
maxima and minima. This is termed a first-derivative
spectrum.
First-derivative spectra have their uses but they are
difficult to interpret (as the brain often sees the ‘peaks’
rather than the true information). If we take the secondderivative spectrum (by derivatizing the absorbance
spectrum twice), we obtain a plot such as that in
Figure 5.
The second-derivative spectrum can be thought of
as an inverted spectrum. Sharp peaks will be made
even sharper. Broad peaks (and also broad background
Peak
Point of inflection
Abs
Trough
D1
0
200 210 220 230 240 250 260 270 280 290 300 310
Wavelength (nm)
Figure 4 Explanation of the first-derivative (D1) spectrum,
and its relationship to the absorption spectrum (Abs).
D2
Broad peak almost
lacking in D2 spectrum
Some peak
shoulders
Note sharper
peak
Sharp peak
Broad peak
Abs
200
250
300
350
400
450
500
550
600
650
Wavelength (nm)
Figure 5 Second-derivative (D2) spectrum showing sharp band
discrimination.
features) will become flattened and so the technique can be used both as a peak-enhancement and
background-rejection tool. The second-derivative spectrum, like its absorbance counterpart, still contains
quantitative information. If two points are consistently
used on the spectrum and the difference in their values on the ordinate is taken, this can be plotted against
concentration and a linear relationship established.
Derivative spectra represent an alternative presentation of the original data. Information cannot be created
and so there is a cost involved. This cost is at the expense
of a much poorer signal-to-noise ratio (as the noise is
also being derivatized) and so if a requirement for derivative spectroscopy is anticipated, an instrument with a good
noise specification is highly desirable. Single-beam instruments should not be considered for serious derivative
spectroscopy.
Higher order derivatives also are possible, and usually
up to the fourth derivative is offered on current
instrumentation, but the increased noise makes their
general analytical use questionable.
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
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
of the instrument and to use the correct operating and
sampling procedures.
Instruments should be checked regularly, using a
certified reference material (CRM). As a minimum, the
user should possess a sample for checking wavelength
6
CLINICAL CHEMISTRY
accuracy and a set of absorbance filters. Wavelength
standards should never be used to check absorbance
accuracy and vice versa. Wavelength standards (such
as holmium oxide glass filters) have very sharp peaks
whereas absorbance standards such as the National
Institute of Standards and Technology (NIST) 930D
filters are of neutral density.
Additional checks such as for stray light and noise may
also be advantageous. Most instrument companies will
be able to give advice on instrument performance checks
and routine maintenance.
Microplate reader users are more limited in the
choice of calibration aids. For filter-based instruments
(the majority at the time of writing), there is no way
to check wavelength accuracy (other than to scan the
filter on a spectrophotometer) and there are few CRMs
available for all formats of microplates. If one is using a
microplate reader, the manufacturer should be contacted
for advice.
3 OVERVIEW OF INSTRUMENT DESIGNS
AND THEIR ADVANTAGES AND
DISADVANTAGES
3.1 Dispersive and Diode-array Systems
Before going into details of analysis, it is important to
discuss the design of a UV/VIS specrophotometer and the
implications of the various design types for a particular
analysis, and to understand the limiting factors in making
that measurement.
UV/VIS instruments are available in two main types,
dispersive and photodiode array (usually shortened to
diode array). The dispersive design uses a monochromator before the sample to convert the white light produced
by the light source into a single pure wavelength of light.
This single wavelength is then passed through the sample and detected. The monochromator can be either a
fixed-wavelength filter or a variable-wavelength design
such as a prism (rare nowadays) or a diffraction grating.
On higher specification instruments, there may be two
monochromators linked in series to permit higher performance – particularly at high absorbance values (e.g. over
3.0 AU).
3.3 Single-beam Spectrophotometers
The single-beam spectrophotometer is the simplest
optical configuration. It consists of a light source,
monochromator (either a grating or a filter), the sample
area and the detector, as shown in Figure 6. In the singlebeam design, it is necessary first to zero the instrument (to
establish the I0 value) and then to measure the sample.
Single-beam instruments are used mainly on grounds
of cost. They have poorer noise specifications compared
with their dual- and double-beam counterparts and are
prone to drifting with time. This makes accurate kinetics
and applications involving repetitive sampling over time
more difficult. The cheaper models often lack the spectral
resolution demanded by some clinical applications (e.g.
porphyrin analysis).
3.4 Double-beam Spectrophotometers
In the double-beam design as shown in Figure 7, there
is still a single detector – usually a photomultiplier (a
phototube). The beam is then sent alternately through
sample and reference positions using a chopper wheel
or vibrating mirror. The electronics in the instrument
are also able to synchronize the beam switching with the
detector so that it can distinguish whether the detector is
measuring the sample or reference beam at any one time.
Even in complete darkness, a photomultiplier will
output a signal. This is known as the dark current. As
a result, the beam shuttling device will also have some
means of temporarily blocking the light from both beams
so that this residual signal (the dark current) can be
measured and subtracted from each sample and reference
measurement.
Tungsten lamp
(for visible region)
Mirror
Deuterium
lamp (UV
models only)
Mirror
(for
source
change)
Filter wheel
Entrance slit
3.2 Dispersive Ultraviolet/Visible Spectrophotometers
ž
ž
ž
ž
single beam
double beam (single detector)
dual beam (dual detector)
photodiode array.
Mirror
Exit slit
Dispersive UV/VIS spectrophotometers have four main
optical configurations:
Diffraction grating
(monochromator)
Mirror
Figure 6 Single-beam optical configuration.
Sample
Detector
7
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
Tungsten lamp
Source mirror
Plane mirror
Deuterium
lamp
Light beam
Plane
mirror
Spherical
mirror
Holographic
grating
Open
Mirror
S
R
¢
,
Q
¢Q,¢Q,
Slit
Chopper
assembly
Blank portions for zeroing
detector dark current
Spherical
mirror
Spherical
mirror
Reference
cell
Optical
chopper
Collimating
mirror
Plane
mirror
Spherical
mirror
Spherical
mirror
Sample cell
Photomultiplier
detector
Figure 7 Double-beam (chopper wheel) optical layout.
Tungsten
lamp
Mirror
Mirror
Deuterium
lamp
Filter
wheel
Entrance slit
Mirror
Reference
Mirror
Beam splitter
(half-silvered
mirror)
Exit slit
Mirror
Sample
Monochromator
Figure 8 Dual-beam spectrophotometer optical layout.
Photodiode
detectors
8
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3.5 Dual-beam Spectrophotometers
An alternative to the double-beam design is the dualbeam layout (Figure 8). In this design, the beam is
split using a half-silvered mirror (beam splitter) into
its sample and reference components. Each beam has its
own detector.
This design is only practical with solid-state detectors
(which have fairly constant dark current) and gives
equivalent results compared with a double-beam system
for most analyses. The design removes the need for a
mechanical beam shuttling device (and hence improves
reliability) and, as solid-state detectors are used, there
is no need for a high-tension power supply to provide
the high voltages (around 1000 V) which are required to
set the photomultiplier gain. The design also produces
very good baseline noise characteristics as, again, there
is no contribution from the mechanical beam shuttle. If
the design has a weakness, it is at very high absorbance
(over 3 AU) as the photomultiplier is able to detect
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
the upper absorbance limit of a spectrophotometer, they
can either be diluted or a shorter path length cell can be
used.
3.6 Photodiode-array Spectrophotometers
Photodiode-array spectrophotometers have been available since the early 1980s with the advent of the early
designs from Hewlett-Packard (now Agilent) (Figure 9).
The design is basically a single-beam instrument but
the sample is irradiated with white light (as opposed to
monochromatic light in a dispersive instrument). This
light, after it has passed through the sample, is then dispersed by a dispersion monochromator (often referred
Diode array
Polychromator
Sample
Source
Dispersion
device
Entrance
slit
Figure 9 Diode-array spectrophotomer optical layout.
to as a polychromator) on to a special solid-state detector with individual segments, one for each wavelength.
The main appeal of these instruments is that the measurement of a spectrum takes only a few seconds. The
system has some disadvantages, however. A diode-array
spectrophotometer is a single-beam instrument (although
a nonanalytical wavelength may be used as a pseudoreference to overcome nonwavelength-specific drift). It
is also less suitable for some single-wavelength measurements as a whole spectrum has to be collected irrespective
of whether the data points are required or not. The design
is also more prone to errors from sample fluorescence
(as the sample irradiation is at all wavelengths and so
any fluorophore present will be also excited) and any
nonparallel surfaces in the sample (as this will affect
the beam dispersion on to the individual elements in
the array). Nevertheless, the diode-array system offers
a high throughput of scanned data and the ability also
to visualize whole spectrum for single-wavelength analyses so that any unexpected results can be investigated
further.
3.7 Microplate Reader Spectrophotometers
Over the past 20 years, many clinical analyses have
been transferred from the traditional, cuvette-based
spectrophotometer to a microwell (normally 96-well)
format. The microwell started life in the early 1960s
for microbiological culture. Later, it was realized that
the format could be applied to bulk analysis using clear
microplates and a dedicated reader.
Microplates are manufactured from plastic (usually
polystyrene or acrylic, depending on the required wavelength range). They are cheap, disposable and provide a
universal format. The plates also have the advantage that
they are compatible with liquid handling devices such as
plate fillers and washers.
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
384 wells per plate.
The dedicated reader is really a spectrophotometer
in an applied form. Most plate readers work in the
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
bioluminescence in addition to absorbance. Many modern
readers can also be used in conjunction with a robotic
system.
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
greater convenience which this format offers.
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
3.8 Reflectance-based Analyzers
In recent years, reagent manufacturers have investigated
ways of making tests even more simple to perform
and less dependent on expensive analytical equipment.
An example of this is the portable glucose monitoring
systems which diabetics use to check their blood sugar
levels.
The color reaction is performed on a solid support
which is coated with the required reagents. This support
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
opaque it is not possible to pass light through them).
When light falls on a surface it can be reflected in
two main ways, as either specular or diffuse reflectance.
Specular reflectance (from the Latin word speculum
for mirror) is the study of mirror-like reflectance. The
path of the reflected ray of light is entirely predictable
as it should be at the same angle as the angle of
light incidence. In diffuse reflectance, the surface has
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
exhibit both types of reflectance (e.g. a coating with a
glossy surface) and so the geometry of the collection
sphere can be adjusted either to include or exclude the
specular component. Reflectance is normally expressed
as a percentage compared with a standard – normally a
white surface such as Teflon . The relationship between
reflectance and concentration is much less clearly defined
when compared with absorbance as there are physical
factors to consider (e.g. particle size, layer thickness).
A fairly accurate quantitative result can be obtained
by taking the logarithm of reflectance or by using the
Kubelka – Munk equation. Manufacturers of reflectancebased devices need to spend considerable development
work in providing an accurate calibration which can be
stored inside the instrument to produce the correct results.
These results will need to be verified by regulatory bodies
Test strip
Blood
Membrane
LED
Detector
Figure 10 Principle of reflectance-based hand-held analyzer
for diabetes monitoring.
9
such as the US Food and Drug Administration (FDA)
before such a device can be marketed.
As many of these devices are designed for use in the
home or general practitioner’s surgery, they must be
manufactured as cheaply as possible. As a result, an LED
can be used in place of a light source and monochromator.
Detection is by means of a silicon photodiode or other
solid-state detector. A diagram of a reflectance-based
analyser for the measurement of blood sugar for diabetes
monitoring is shown in Figure 10.
4 COMMON CLINICAL APPLICATIONS
USING ULTRAVIOLET/VISIBLE
ABSORPTION SPECTROSCOPY
There are many clinical tests which employ UV and visible
spectrophotometry. For readers who want a detailed and
exhaustive list, there are many textbooks on the subject..1/
In this section I have highlighted a range of these tests
and detailed how they work.
4.1 Enzyme Rate Assays
Enyzmes are biocatalysts, which are extremely efficient
in converting their chosen substrates into product. Apart
from their efficiency, they are also highly specific and
often will not work with a slightly different substrate.
Often it is useful to study the rate of this catalysis
by measuring either the rate of depletion of substrate
or the formation of products. This may be a matter
of simply measuring the absorbance of one of the
reaction components directly or by forming an absorbing
conjugate with another molecule.
Enzyme kinetics are usually zero order. This means
that, after an initial lag phase, there should be a linear
relationship between substrate (or product) concentration with time until one of these components becomes
limiting. It is therefore useful if the instrument software
is able to allow the user to choose where this linear
portion is (either prior to or, better, after data collection) and to use this portion to calculate the rate. The
enzyme activity is normally expressed in International
Units (IU) by applying a simple factor to the measured
slope (absorbance/time).
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
thermostated water circulator). Some cell holders use
thermoelectric (or Peltier effect) cell holders. These cell
10
CLINICAL CHEMISTRY
holders have the advantage of much more precise temperature control and the ability to work below ambient
temperatures (Peltier cells can cool by reversing the electric current flow). Some Peltier designs, particularly those
requiring high or low temperatures, will still require a
flow of cold water in order to operate correctly whereas
those covering a more restricted range do not.
Most enzyme reactions are fairly slow, taking place for
5 min or more. In order to increase productivity, most
UV/VIS instruments offer a cell changer as an accessory.
This is a shuttle device, which can hold six or more
cuvettes at once. The instrument then cycles through
each of the cell positions taking a reading on each cell
every 30 s during the course of the reaction.
The collected data can be analyzed either using the
instrument’s own kinetic software or externally, using
either a computer or manual calculation. Some statistical
data on the quality of the curve fit are also useful.
An example of a clinical rate assay is the determination
of butyrylcholinesterase (BchE). Certain individuals
express a mutant form of the BchE gene. This then
encodes for a defective form of the enzyme, which lacks
the ability to hydrolyze succinylcholine. In some rare
cases, the complete BchE gene is missing. A defective or
missing gene will not, normally, be of any consequence.
If, however, succinylcholine is used during tracheal
intubation in the administration of inhalation anesthetics,
this will then cause the patient to undergo complete
paralysis. The test for this enzyme.2/ is commonly
performed using a UV/VIS spectrophotometer with a
temperature-controlled cell holder (most tests will be
performed at 37 ° C).
One important class of enzyme-catalyzed reactions
involves the oxidation and reduction of pyridine
nucleotides [nicotinamide adenine dinucleotide (NADC )
and nicotinamide adenine dinucleotide (reduced form)
(NADH), respectively]. If the reaction is followed at
340 nm, NADC does not absorb whereas NADH shows
a strong absorbance. Examples of NADC /NADH kinetic
reactions include glucose dehydrogenase,.3/ aspartate
aminotransferase.4/ and urea..5/
Other enzymatic tests often include a colorless
compound, which is added to the reaction. This is
then enzymatically converted to a colored product (e.g.
p-nitrophenol). Examples of this type of test include alkaline phosphatase,.6 – 8/ acid phosphatase.4/ and amylase..1/
These tests are included in the summary of common
clinical tests in Table 3.
4.2 Colorimetric and End-point Assays
It is also possible to perform a reaction to completion
and then to calculate the quantity of initial substrate
by extrapolation. This type of assay is termed an endpoint assay. There are a vast number of such tests and,
if an exhaustive list is sought, the reader is directed to
a compendium of methods such as Tietz..1/ A summary
of some common clinical end-point analyses is given in
the summary of common clinical tests in Table 3. The
methods will prescribe a time after which the reading can
be taken. In the case of enzyme-catalyzed reactions, the
incubation time will need to be at constant temperature.
Most diagnostics companies provide premixed reagents
to perform these tests together with a detailed protocol
describing their use. Different protocols may use slightly
Table 3 Some common clinical tests
Analyte
.4/
Acid phosphatase
Alanine aminotransferase.9/
Alkaline phosphatase.6 – 8/
a-Amylase.1/
Aspartate
aminotransferase.10/
Bilirubin.11/
Cholesterol.12/
Creatinine.13/
GGT.14/
Glucose.15/
LDH.16/
Porphyrins (total).17/
Pseudocholinesterase.28/
Triglycerides.18,19/
Urea.20/
Method
UV wavelength (nm)
Kinetic: PNP
Kinetic: NADC /NADH
Kinetic: PNP
Kinetic:
2-chloro-4-nitrophenylmaltoheptaoside
2-chloro-PNP
Kinetic: NADC /NADH
405
340
405
405
Evelyn – Molloy
Kinetic: cholesterol oxidase
Jaffe
Kinetic: carboxy substrate
Kinetic: hexokinase (NADC /NADH)
Kinetic: lactate/pyruvate
Acidification using HCl
NADC /NADH
Kinetic: GPO colorimetric
Kinetic: NADC /NADH
340
555
500
510
405
340
340
Absorbance ¾ 405 (Soret peak)
340
520
340
PNP, p-nitrophenyl phosphate; GGT, g-glutamyl transferase; LDH, lactate dehydrogenase; GPO, glycerol-3-phosphate oxidase.
11
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
4.3 Immunoassays, Enzyme-linked Immunosorbent
Assays and Microplate Assays
Immunoassays rely on the very strong affinity between
an antibody and its target molecule (antigen). This strong
affinity has been used to design a wide range of assays
for a variety of targets. These include pathogens (where
a chemical associated with the organism or virus will be
targeted), tumor markers and drug monitoring (either
therapeutic or drugs of abuse). In fact, anything which
can elicit an antibody response can be developed into
an immunoassay. These assays are ideal where high
specificity is required. They are not good at dealing with
a range of possible compounds. For example, it is difficult
to design an immunoassay to detect all abused drugs, only
specific types. If a ‘‘designer’’ drug is synthesized with an
additional functional group, this may adversely affect the
assay.
Immunoassays are competitive assays. The reaction
mixture will contain antigens labeled with some kind
of tag (this is either a radiolabel, a fluorophore or
a site which will bind to a chromophore). When the
(unlabeled) sample antigens are introduced, there will
be competition between the labeled and unlabeled
antigens and this can be calibrated against a binding
curve (which will be sigmoidal in nature). Prior to
measurement, it will be necessary to separate the free
and bound antigens and so a separation step will be
required (heterogeneous assay). Much effort has been
spent in previous years trying to simplify this process
(e.g. using magnetized, latex-covered beads) or using
a technique which inherently separates bound from
unbound antigens (e.g. fluorescence polarization) to
produce a homogeneous assay. For the highest sensitivity
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 immunosorbent 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 antibody is washed away. The blocking agent is to help
prevent unbound antigen from adhering directly to the
plate, thus affecting the final result.
The analyte and enzyme conjugate [e.g. horseradish
peroxidase (HRP)] is added. The analyte will bind to
the antibody, which has previously been coated on the
microplate. The enzyme conjugate also binds to the
analyte. This will be used later in conjunction with a
specific dye to produce the color, which will then be
measured on the reader.
4.4 Porphyrin Analysis
Porphyria is the name given to a number of related
conditions, some genetic, which result from an over
production of porphyrins, which are precursors in the
production of hemoglobin. Sufferers are prone to bouts
of severe abdominal pain, vomiting, severe personality
changes and sensitivity to light. Some types of porphyria
produce characteristic dark-colored (‘‘port wine’’) urine
and this is a good first indication that porphyria is present.
One of the main types of porphyria (a generic term for
porphyrin-related disease) is variegate porphyria. In this
case a mitochondrial enzyme called protoporphyrinogen
oxidase is defective (owing to incorrect genetic coding)
and as a result excess protoporphyrin (one of the
porphyrin types) is produced. The protoporphyrinogen
reacts with oxygen to produce protoporphyrin in an
uncontrolled reaction. Other porphyria types include
acute intermittent porphyria. Accurate diagnosis of the
exact type of porphyria is vital as incorrect treatment
could have very serious consequences.
Porphyrins.17/ have a characteristic UV/VIS absorption
peak (Soret peak) in the region 400 – 410 nm depending
on the type of porphyrin present (coproporphyrin has a
peak between 402 and 403 nm whereas the uroporphyrin
peak lies between 406 and 407 nm).
The urine sample is filtered, diluted with distilled
water and acidified with hydrochloric acid. The sample
is scanned between 300 and 500 nm and the spectrum
peak positions are noted. The measurement is taken by
first constructing a baseline at points at either side of
the main peak (usually around 380 and 430 nm) and then
measuring the height of the peak down to this baseline
(Figure 11).
Absorbance
altered wavelengths for each analyte. As with rate assays,
the test may be based on reading the analyte directly or
an additional color reaction may be required to develop
the test.
380
405
430
Wavelength (nm)
Figure 11 Porphyrin analysis using three wavelengths.
12
CLINICAL CHEMISTRY
total porphyrin.µg L 1 / D 2A.lmax /
.A380 C A430 /e
.4/
where A is absorbance, lmax is the wavelength at
maximum absorption and e is the molar absorptivity
(a constant) for the analyte; for porphyrins (in a 1-cm
cell) e D 4740 µg L 1 .
Alternatively, second-derivative spectroscopy has been
used.1/ (as this reduces background effects and produces
sharper peaks). It should be borne in mind that, for a
full and correct diagnosis, the type of porphyrin must be
accurately identified. This can only be done using a good
UV/VIS spectrophotometer offering narrow slits and a
skilled user, as interpretation of the corrected spectrum or
the second-derivative spectrum may be involved. UV/VIS
absorption spectroscopy is generally used for screening
and other techniques such as high-performance liquid
chromatography (HPLC) or fluorescence spectroscopy
(which gives much better selectivity as each porphyrin has
a different excitation and emission wavelength) are often
used to make the final, confirmatory, diagnosis. These
techniques are usually offered by porphyria reference
centers.
4.5 Hemoglobin Analysis
Hemoglobin is a protein with a nonprotein core consisting of an iron atom surrounded by heme groups. It
has remarkable oxygen transportation properties where
it can change its conformation to accept oxygen (oxyhemoglobin). This process can be inhibited by carbon
monoxide, which has a 200 times stronger affinity for
hemoglobin (carboxyhemoglobin) than oxygen, resulting
in severe respiratory problems and death in cases of carbon monoxide poisoning. Hemoglobin possesses an iron
atom core in its ferrous (Fe2C ) state. If the iron is oxidized
to its ferric (Fe3C ) state, its oxygen transport capabilities
are diminished and the molecule is called methemoglobin
(metHb).
Total hemoglobin can be measured by performing a
reaction of the total hemoglobin present with potassium cyanide to form a hemoglobin– cyanide complex
(Figure 12).
This is the basis of the method of Zijlstra et al..20/
In this method, hemoglobin is reacted with potassium
cyanide (taking great care with pipetting and certainly
never by mouth!) and sodium (or potassium) hexacyanoferrate(III) to produce a hemoglobin– cyanide complex
(Equation 5):
hemoglobin C CN C Fe(CN)6 3
! hemoglobin– cyanide
.5/
Absorbance
The total porphyrin concentration is given by
Equation (4):
0.300
0.280
0.260
0.240
0.220
0.200
0.180
0.160
0.140
0.120
0.100
0.080
0.060
0.040
0.020
0.000
470
500 520 540 560 580 600 620 640 660 680 700 720
750
Wavelength (nm)
Figure 12 Absorption spectrum of hemoglobin – cyanide
complex.
This complex has a peak around 546 nm, which can be
measured and quantitated.
Franzini et al..21/ have described a method based on
second-derivative spectroscopy to measure hemoglobin
and its homologs. This method helps to overcome the
interference from bilirubin, which is also often present in
these analyses.
Shih et al..22/ also used multicomponent analysis in
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
with a calibration set of known references – either
mixtures or single components – and attempts to calculate
the relative proportions of each in the unknown spectrum.
This approach has its attractions as it is potentially
able to measure each component without the need for
a separation step. The main drawback is when there
is significant spectral overlap or in situations where
the concentrations of the various components differ
widely. Problems may also occur if there is a strong
background matrix which is not constant. More powerful
techniques exist, such as principle component regression
or partial least-squares fitting. These techniques are a
science in themselves (chemometrics) and go beyond this
article.
4.6 Protein Assays
Proteins are composed of amino acid building blocks.
Protein has some intrinsic absorbance at approximately
280 nm (from the aromatic amino acids – tyrosine and
tryptophan) but it is more common to perform a reaction
to produce a colored complex which can be assayed in the
visible region. The three most common procedures for
protein analysis are biuret, Bradford and bicinchoninic
acid (BCA) assays.
Table 4 lists the various protein methods. These are all
simple colorimetric determinations.
13
UV/VIS LIGHT ABSORPTION SPECTROPHOTOMETRY IN CLINICAL CHEMISTRY
Table 4 Summary of protein assays
Assay method
and reference
Principle
Lowry.23/
Bradford.24/
Biuret.25/
BCA.26/
Absorbance at 280 nm.23/
Warburg – Christian.27/
Range
(µg mL 1 )
As biuret plus determination of
aromatic amino acids
Dye binding with coomassie
brilliant blue
Determination of number of
peptide bonds
Reduction of copper by protein
and formation of Cu(I) – BCA
complex
Determination of tyrosine and
tryptophan in protein
Determination of aromatic amino
acids with compensation for
nucleic acids
Absorbance
1.10
1.00
0.80
0.60
0.40
0.20
0.00
220
240
260
280
300
320
340
Wavelength (nm)
Figure 13 Absorption spectrum of DNA.
4.7 Molecular Biology
The use of UV/VIS spectroscopy for molecular biology
strictly falls outside the scope of clinical analysis. On
the other hand, more routine clinical laboratories are
using molecular biology techniques (such as the PCR and
automated dideoxy sequencing) as these give more direct
diagnosis for genetically based disorders.
As a result of these techniques, UV/VIS spectroscopy is
useful for assessing the purity of the starting template in
either a sequencing or PCR reaction and, as a result,
saves time in optimizing the reaction and helps to
reduce reagent costs. Pure DNA and RNA absorb at
260 nm (Figure 13). Protein (which is the main source
of contamination) absorbs at around 280 nm (this is
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
adopted in molecular biology laboratories as a quick
method for assessing purity. This method measures the
absorbance at 260 and 280 nm and the ratio (A260 /A280 )
5 – 200
10 – 200
Interferences
Phenols, aromatic amino acids
Detergents
200 – 5000
Amines, ammonium salts
200 – 1200
High concentrations of metals,
strong reducing agents,
chelating agents
Nucleic acids, phenols, aromatics
>50
50 – 3000 purity check
Phenols, aromatics
is calculated. Optionally, a third reference point can be
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
ratio. If the ratio of the two absorbances is between 1.7 and
2.0, then the DNA preparation is considered to be pure.
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.
It is not uncommon to have volumes of 10 µL and so
special low-volume cells are available. These special
cuvettes are made from quartz (as plastic and glass
cuvettes generally absorb below 300 nm). Dedicated lowcost instruments are available for the assessment of
nucleic acid purity.
ACKNOWLEDGMENTS
I thank Hanswilly Müller at PerkinElmer, Überlingen,
Germany, for providing some of the spectra shown here,
Jackie Woolf at the Porphyria Reference Centre, Heath
Park Hospital, Cardiff, and Ipswich Hospital, UK, for
some information and spectra of porphyrins and Chris
Royle at the Brompton Hospital, UK, for keeping me up
to date with the latest developments in clinical analysis.
I also thank Agilent Technologies (formerly HewlettPackard Instruments) and Hypoguard for permission to
use their diagrams.
ABBREVIATIONS AND ACRONYMS
BchE
BSA
Butyrylcholinesterase
Bovine Serum Albumin
14
CRM
ELISA
FDA
GGT
GPO
HPLC
HRP
IR
LDH
LED
metHb
NADC
NADH
NIST
PCR
PNP
TRIS
UV
UV/VIS
CLINICAL CHEMISTRY
Certified Reference Material
Enzyme-linked Immunosorbent
Assay
Food and Drug Administration
g-Glutamyl Transferase
Glycerol-3-phosphate Oxidase
High-performance Liquid
Chromatography
Horseradish Peroxidase
Infrared
Lactate Dehydrogenase
Light-emitting Diode
Methemoglobin
Nicotinamide Adenine
Dinucleotide
Nicotinamide Adenine
Dinucleotide (Reduced Form)
National Institute of
Standards and Technology
Polymerase Chain Reaction
p-Nitrophenyl Phosphate
Tris(hydroxymethyl)aminomethane
Ultraviolet
Ultraviolet/Visible
RELATED ARTICLES
Biomedical Spectroscopy (Volume 1)
Biomedical Spectroscopy: Introduction ž Glucose, In
Vivo Assay of ž Infrared Spectroscopy in Clinical
and Diagnostic Analysis ž Infrared Spectroscopy in
Microbiology ž Near-infrared Spectroscopy, In Vivo
Tissue Analysis by
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