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Clinical Chemistry
Stephen L. Upstone
in
Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.)
pp. 16991714
John Wiley & Sons Ltd, Chichester, 2000
Ultraviolet/Visible
Light Absorption
Spectrophotometry in
Clinical Chemistry
Acknowledgments
13
13
Related Articles
14
References
14
Stephen L. Upstone
PerkinElmer Ltd., Beaconsfield, UK
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
1
2
2
2
3
4
5
1 INTRODUCTION
6
6
6
6
6
8
8
8
9
9
9
10
11
11
12
12
13
CLINICAL CHEMISTRY
log T
.2/
.3/
where e D the absorptivity of the substance, c D concentration and l D path length. Provided that e and l
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
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.
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
(a)
(b)
230
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
1.4
Absorbance
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.
Point of inflection
Abs
Trough
D1
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
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
CLINICAL CHEMISTRY
Filter wheel
Entrance slit
single beam
double beam (single detector)
dual beam (dual detector)
photodiode array.
Mirror
Exit slit
Mirror
Sample
Detector
Tungsten lamp
Source mirror
Plane mirror
Deuterium
lamp
Light beam
Plane
mirror
Spherical
mirror
Holographic
grating
Open
Mirror
,
Q
Q,Q,
Slit
Chopper
assembly
Spherical
mirror
Spherical
mirror
Reference
cell
Optical
chopper
Collimating
mirror
Plane
mirror
Spherical
mirror
Spherical
mirror
Sample cell
Photomultiplier
detector
Mirror
Mirror
Deuterium
lamp
Filter
wheel
Entrance slit
Mirror
Reference
Mirror
Beam splitter
(half-silvered
mirror)
Exit slit
Mirror
Sample
Monochromator
Photodiode
detectors
CLINICAL CHEMISTRY
Diode array
Polychromator
Sample
Source
Dispersion
device
Entrance
slit
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.
Test strip
Blood
Membrane
LED
Detector
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
instruments 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).
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 405 (Soret peak)
340
520
340
PNP, p-nitrophenyl phosphate; GGT, g-glutamyl transferase; LDH, lactate dehydrogenase; GPO, glycerol-3-phosphate oxidase.
11
Absorbance
380
405
430
Wavelength (nm)
Figure 11 Porphyrin analysis using three wavelengths.
12
CLINICAL CHEMISTRY
.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 g 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
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.
13
Principle
Lowry.23/
Bradford.24/
Biuret.25/
BCA.26/
Absorbance at 280 nm.23/
Warburg Christian.27/
Range
(g mL 1 )
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
200 1200
>50
50 3000 purity check
Phenols, aromatics
ACKNOWLEDGMENTS
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
RELATED ARTICLES
Biomedical Spectroscopy (Volume 1)
Biomedical Spectroscopy: Introduction Glucose, In
Vivo Assay of Infrared Spectroscopy in Clinical
and Diagnostic Analysis Infrared Spectroscopy in
Microbiology Near-infrared Spectroscopy, In Vivo
Tissue Analysis by
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