UVPRIMER.BK : uvprimer.

UVPRIMER.BK : uvprimer.

toc Page i Friday, December 20, 1996 4:12 PM
Contents
Chapter 1 Principles and applications of UV-visible
spectroscopy
Basic principles.................................................................................................... 10
The electromagnetic spectrum..................................................................... 10
Wavelength and frequency .......................................................................... 11
Origin of UV-visible spectra ....................................................................... 11
Transmittance and absorbance .................................................................... 14
Derivative spectra........................................................................................ 14
Obtaining derivative spectra ............................................................... 16
Applications ........................................................................................ 17
Signal-to-noise .................................................................................... 17
Instrumental considerations ................................................................ 18
Qualitative analysis.............................................................................................. 18
Identification—spectra and structure .......................................................... 18
Confirmation of identity .............................................................................. 19
Color............................................................................................................ 21
Other qualitative information ...................................................................... 22
Protein and nucleic acid melting temperature..................................... 22
Enzyme activity .................................................................................. 23
Instrumental considerations......................................................................... 24
Quantitative analysis............................................................................................ 24
Beer’s law.................................................................................................... 24
Sample requirements........................................................................... 28
Multicomponent analysis ............................................................................ 29
Principle of additivity ......................................................................... 29
Simple simultaneous equations method.............................................. 29
Least squares method.......................................................................... 32
Other methods..................................................................................... 34
Sample requirements........................................................................... 34
Instrumental requirements ........................................................................... 34
Indirect quantification .......................................................................................... 35
Chemical derivatization............................................................................... 35
Spectrophotometric titrations ...................................................................... 35
Enzyme kinetic assays................................................................................. 36
Chapter 2 Instrumentation
Instrumental design.............................................................................................. 38
Components................................................................................................. 38
Sources................................................................................................ 39
Dispersion devices .............................................................................. 40
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Contents
Detectors ............................................................................................. 41
Optics .................................................................................................. 43
The conventional spectrophotometer .......................................................... 44
The diode array spectrophotometer............................................................. 45
Configuration............................................................................................... 47
Single-beam design............................................................................. 47
Dual-beam design ............................................................................... 48
Split-beam design ............................................................................... 50
Dual-wavelength design ..................................................................... 51
Measuring a spectrum ................................................................................. 51
Key instrumental parameters ............................................................................... 52
Spectral resolution ....................................................................................... 52
Wavelength accuracy and precision ............................................................ 55
Photometric accuracy and precision............................................................ 57
Stray light............................................................................................ 57
Noise ................................................................................................... 58
Linear dynamic range .................................................................................. 59
Drift ............................................................................................................. 61
Chapter 3 Sample handling and measurement

Liquid samples ..................................................................................................... 64
Cells............................................................................................................. 64
Material ............................................................................................... 64
Cell types ............................................................................................ 65
Sources of error................................................................................... 66
Care of cells ........................................................................................ 67
Choice of solvent......................................................................................... 67
Effect of solvent, concentration, pH, and temperature................................ 68
Solid samples ....................................................................................................... 70
No reference ................................................................................................ 70
Refractive index .......................................................................................... 71
Sample geometry ......................................................................................... 71
Weak absorbance ................................................................................................. 72
Changing slit width ..................................................................................... 72
Time averaging............................................................................................ 73
Wavelength averaging ................................................................................. 73
Strong absorbance ................................................................................................ 74
Interference .......................................................................................................... 75
Types of interference................................................................................... 75
Other absorbing compounds ............................................................... 76
Scattering ............................................................................................ 76
Correction techniques.................................................................................. 77
Isoabsorbance...................................................................................... 78
Multicomponent analysis .................................................................... 78
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Contents
Background modeling......................................................................... 79
Internal referencing............................................................................. 80
Three-point correction ........................................................................ 80
Derivative spectroscopy...................................................................... 81
Photochemical problems...................................................................................... 85
Fluorescence ................................................................................................ 85
Sample decomposition ......................................................................................... 86
Chapter 4 Method development and validation

Method development ........................................................................................... 88
Linearity ...................................................................................................... 89
Accuracy...................................................................................................... 93
Precision ...................................................................................................... 94
Sensitivity.................................................................................................... 95
Range........................................................................................................... 97
Selectivity.................................................................................................... 97
Ruggedness.................................................................................................. 99
Instrumental requirements ......................................................................... 100
Method validation .............................................................................................. 100
Chapter 5 Routine operation

Instrument performance verification.................................................................. 102
Test parameters.......................................................................................... 102
Wavelength accuracy and precision ................................................. 103
Photometric accuracy and precision ................................................. 104
Stray light.......................................................................................... 104
Resolution ......................................................................................... 105
Noise ................................................................................................. 105
Baseline flatness ............................................................................... 106
Stability ............................................................................................. 106
Linearity............................................................................................ 106
Standards ................................................................................................... 107
Emission standards ........................................................................... 107
Solid absorption standards ................................................................ 107
Liquid absorption standards.............................................................. 108
Regulatory requirements ........................................................................... 110
GLP/GMP ......................................................................................... 110
European Pharmacopoeia ................................................................. 110
United States Pharmacopoeia ........................................................... 111
American Standard Testing Methods ............................................... 113
Recommendations ..................................................................................... 115
Instrument self-test ............................................................................................ 119
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System suitability............................................................................................... 120

Proper operation................................................................................................. 120
Electronic storage ...................................................................................... 121
Standard operating procedures .................................................................. 121
Collateral data .................................................................................................... 121
Confirmation wavelengths ........................................................................ 122
Full spectra ................................................................................................ 122
Statistics..................................................................................................... 124
Appendix A Accuracy and precision

Definition of terms............................................................................................. 126
Appendix B Characteristics of diode array

spectrophotometers
Advantages of diode array spectroscopy ........................................................... 128
Fast spectral acquisition ............................................................................ 128
Simultaneous multiwavelength measurement ........................................... 129
Wavelength resettability............................................................................ 130
Sensitivity.................................................................................................. 131
Measurement statistics .............................................................................. 131
Ruggedness and reliability ........................................................................ 131
Open sample area ...................................................................................... 132
Disadvantages of diode array spectroscopy ....................................................... 132
Resolution.................................................................................................. 132
Stray light .................................................................................................. 133
Sample decomposition .............................................................................. 133
Complexity ................................................................................................ 134
Errors in measuring fluorescent samples................................................... 134
References..................................................................135
Index...........................................................................139
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UVPRIMER.BK : front.fm5 Page v Friday, December 20, 1996 4:12 PM
Fundamentals of
UV-visible
spectroscopy
A Primer
Tony Owen
UVPRIMER.BK : front.fm5 Page vi Friday, December 20, 1996 4:12 PM
 Copyright Hewlett-Packard Company,

1996. All rights reserved. Reproduction,
adaption, or translation without prior written
permission is prohibited, except as allowed
under the copyright laws.
The information contained in this primer is
subject to change without notice.
Printed in Germany 09/96
Hewlett-Packard publication number
12-5965-5123E
UVPRIMER.BK : front.fm5 Page vii Friday, December 20, 1996 4:12 PM
Preface
Preface In 1988 we published a primer entitled “ The Diode-Array

Advantage in UV/Visible Spectroscopy”. At the time, although
diode array spectrophotometers had been on the market since
1979, their characteristics and their advantages compared with
conventional scanning spectrophotometers were not
well-understood. We sought to rectify the situation. The primer
was very well-received, and many thousands of copies have been
distributed.
Much has changed in the years since the first primer, and we felt
this was an appropriate time to produce a new primer. Computers
are used increasingly to evaluate data; Good Laboratory Practice
has grown in importance; and a new generation of diode array
spectrophotometers is characterized by much improved
performance. With this primer, our objective is to review all
aspects of UV-visible spectroscopy that play a role in obtaining
the best results. Microprocessor and/or computer control has
taken much of the drudgery out of data processing and has
improved productivity. As instrument manufacturers, we would
like to believe that analytical instruments are now easier to
operate. Despite these advances, a good knowledge of the basics
of UV-visible spectroscopy, of the instrumental limitations, and
of the pitfalls of sample handling and sample chemistry remains
essential for good results.
With this primer, we also want to show that the conventional
“single measurement at a single wavelength” approach to
obtaining results is insufficient for assuring optimum results.
Multiple measurements at multiple wavelengths or (preferably)
full spectra yield the best accuracy and precision of results and
provide the information necessary to detect erroneous results.
I would like to take this opportunity to thank my colleagues, too
numerous to mention by name, at Hewlett-Packard from whom I
have learned so much about UV-visible spectroscopy over the
years.
vii
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viii
UVPRIMER.BK : chap01.fm5 Page 9 Friday, December 20, 1996 4:12 PM
chapter 1
Chapter 1 Principles and

applications of
UV-visible
spectroscopy
9
Principles and applications of UV-visible spectroscopy
This chapter outlines the basic theories and principles of
UV-visible spectroscopy. These provide valuable insight into
the uses and limitations of this technique for chemical
analysis. The primary applications of UV-visible
spectroscopy are also briefly reviewed.
Basic principles
The electromagnetic Ultraviolet (UV) and visible radiation comprise only a small part
spectrum of the electromagnetic spectrum, which includes such other
forms of radiation as radio, infrared (IR), cosmic,
and X rays (see Figure 1).
Frequency [Hz]
Ultraviolet Visible Infrared

Gamma ray
Cosmic ray
Microwave
Ultraviolet
Ultrasonic
Infrasonic
Television
(audible)
Infrared
visible
Radar
Radio
X ray
NMR
Sonic
Wavelength [m]
Figure 1
The electromagnetic spectrum
10
The energy associated with electromagnetic radiation is defined

by the following equation:
E = hν
where E is energy (in joules), h is Planck’s constant

(6.62 × 10-34 Js), and ν is frequency (in seconds).
Wavelength and frequency Electromagnetic radiation can be considered a combination of

alternating electric and magnetic fields that travel through space
with a wave motion. Because radiation acts as a wave, it can be
classified in terms of either wavelength or frequency, which are
related by the following equation:
ν = c⁄λ
where ν is frequency (in seconds), c is the speed of light

(3 × 108 ms-1), and λ is wavelength (in meters). In UV-visible
spectroscopy, wavelength usually is expressed in nanometers
(1 nm = 10-9 m).
It follows from the above equations that radiation with shorter
wavelength has higher energy. In UV-visible spectroscopy, the
low-wavelength UV light has the highest energy. In some cases,
this energy is sufficient to cause unwanted photochemical
reactions when measuring sample spectra (remember, it is the
UV component of light that causes sunburn).
Origin of UV-visible When radiation interacts with matter, a number of processes can
spectra occur, including reflection, scattering, absorbance,
fluorescence/phosphorescence (absorption and reemission), and
photochemical reaction (absorbance and bond breaking). In
general, when measuring UV-visible spectra, we want only
absorbance to occur.
Because light is a form of energy, absorption of light by matter
causes the energy content of the molecules (or atoms) to increase.
The total potential energy of a molecule generally is represented
as the sum of its electronic, vibrational, and rotational energies:
11
Etotal = E electronic + E vibrational + E rotational
The amount of energy a molecule possesses in each form is not a

continuum but a series of discrete levels or states. The
differences in energy among the different states are in the order:
Eelectronic > E vibrational > E rotational
In some molecules and atoms, photons of UV and visible light

have enough energy to cause transitions between the different
electronic energy levels. The wavelength of light absorbed is that
having the energy required to move an electron from a lower
energy level to a higher energy level. Figure 2 shows an example
of electronic transitions in formaldehyde and the wavelengths of
light that cause them.
transition
transition
Figure 2
Electronic transitions in formaldehyde
These transitions should result in very narrow absorbance bands

at wavelengths highly characteristic of the difference in energy
levels of the absorbing species. This is true for atoms, as depicted
in Figure 3.
12
Figure 3
Electronic transitions and spectra of atoms
However, for molecules, vibrational and rotational energy levels

are superimposed on the electronic energy levels. Because many
transitions with different energies can occur, the bands are
broadened (see Figure 4). The broadening is even greater in
solutions owing to solvent-solute interactions.
electronic energy levels

vibrational energy levels
rotational energy levels
electronic transition
Figure 4
Electronic transitions and UV-visible spectra in molecules
13
Transmittance and When light passes through or is reflected from a sample, the
absorbance amount of light absorbed is the difference between the incident
radiation (Io) and the transmitted radiation (I). The amount of
light absorbed is expressed as either transmittance or absorbance.
Transmittance usually is given in terms of a fraction of 1 or as a
percentage and is defined as follows:
T = I ⁄ Io or % T = ( I ⁄ Io ) × 100
Absorbance is defined as follows:
A = – log T
For most applications, absorbance values are used since the
relationship between absorbance and both concentration and path
length normally is linear.
Derivative spectra If a spectrum is expressed as absorbance (A) as a function of

wavelength (λ), the derivative spectra are:
Zero order: A = f(λ)
dA
First order: ------- = f ′( λ )
dλ
2
d A
Second order: --------2- = f ″( λ )
dλ
Figure 5 on the next page shows the effects of derivatization on a

simple Gaussian absorbance band. The derivative spectra are
always more complex than the zero-order spectrum.
The first derivative is the rate of change of absorbance against
wavelength. It starts and finishes at zero, passing through zero at
the same wavelength as λmax of the absorbance band. This
14
derivative has a positive and a negative band with maximum and

minimum at the same wavelengths as the inflection points in the
absorbance band. This bipolar function is characteristic of all
odd-order derivatives.
The most distinctive feature of the second-order derivative is a
negative band with minimum at the same wavelength as the
maximum on the zero-order band. This derivative also shows two
positive satellite bands on either side of the main band. The
fourth derivative shows a positive band with a maximum at the
same wavelength as the maximum on the zero order band.
Even-order derivatives show a negative or positive band with
minimum or maximum at the same wavelength as λmax on the
absorbance band.
15
Absorbance Absorbance
1st derivative 2nd derivative
3rd derivative 4th derivative
Figure 5
Derivative spectra of a Gaussian absorbance band
Obtaining derivative spectra Optical, electronic, and mathematical methods all can be used to
generate derivative spectra. Although optical and electronic
techniques formed the basis of early UV-visible spectroscopy,
these have been largely superseded by mathematical methods.
To calculate the derivative at a particular wavelength (λ), a
window of ± n data points is selected, and a polynomial
Aλ = a 0 + a 1 λ + … + a l λ
is fitted by the least squares method. The coefficients a0 … al at

each wavelength are the derivative values, where a1 is the first
16
derivative, a2 is the second derivative, and so on. Savitzky and

Golay developed a highly efficient method to perform the
calculations that is the basis of the derivatization algorithm in
most commercial instruments. This method also smooths the
data. If the polynomial order (l) is less than the number of data
points (2n+1) in the window, the polynomial generally cannot
pass through all data points. Thus the least squares fit gives a
smoothed approximation to the original data points.
Although transforming a UV-visible spectrum to its first or a
higher derivative usually yields a more complex profile than the
zero-order spectrum (see Figure 5), the intrinsic information
content is not increased. In fact, it is decreased by the loss of
lower-order data such as constant offset factors.
Applications Derivative spectra can be used to enhance differences among

spectra, to resolve overlapping bands in qualitative analysis (see
“Confirmation of identity” on page 19) and, most importantly, to
reduce the effects of interference from scattering, matrix, or other
absorbing compounds in quantitative analysis (see “Derivative
spectroscopy” on page 81).
Signal-to-noise An unwanted effect of the derivatization process is the decrease

in S/N with higher orders of derivatives. This decrease follows
from the discrimination effect (see “Derivative spectroscopy” on
page 81) and from the fact that noise always contains the sharpest
features in the spectrum. Thus, if the spectral data used in the
derivative calculation are at 2-nm intervals, the noise has a 2-nm
bandwidth. If the analyte band has a bandwidth of 20 nm, the S/N
of the first derivative will be 10 times worse than with the
zero-order spectrum. The smoothing properties of the
Savitzky-Golay polynomial technique can be used to mitigate the
decrease in S/N, but care must be taken as too high a degree of
smoothing will distort the derivative spectrum.
Instrumental considerations The higher resolution of derivative spectra places increased

demands on the wavelength reproducibility of the
spectrophotometer. Small wavelength errors can result in much
17
larger signal errors in the derivative mode than in the absorbance

mode.
The negative effect of derivatization on S/N also places increased
demands on low-noise characteristics of the spectrophotometer.
If the spectrophotometer can scan and average multiple spectra,
S/N can be improved further prior to derivatization.
Qualitative analysis
Identification—spectra UV-visible spectra generally show only a few broad absorbance

and structure bands. Compared with techniques such as infrared spectroscopy,
which produces many narrow bands, UV-visible spectroscopy
provides a limited amount of qualitative information. Most
absorption by organic compounds results from the presence of π
(that is, unsaturated) bonds. A chromophore is a molecular group
usually containing a π bond. When inserted into a saturated
hydrocarbon (which exhibits no UV-visible absorbance
spectrum), it produces a compound with absorption between 185
and 1000 nm. Table 1 lists some chromophores and the
wavelengths of their absorbance maxima.
Table 1 Selected chromophores and their absorbance maxima
Chromophore Formula Example λmax (nm)
Carbonyl (ketone) RR’C=O Acetone 271
Carbonyl RHC=O Acetaldehyde 293

(aldehyde)
Carboxyl RCOOH Acetic acid 204
Amide RCONH2 Acetamide 208
Ethylene RCH=CHR Ethylene 193
Acetylene RC=CR Acetylene 173
18
Table 1 Selected chromophores and their absorbance maxima
Nitrile RC=N Acetonitrile < 160
Nitro RNO2 Nitromethane 271
The presence of an absorbance band at a particular wavelength

often is a good indicator of the presence of a chromophore.
However, the position of the absorbance maximum is not fixed
but depends partially on the molecular environment of the
chromophore and on the solvent in which the sample may be
dissolved. Other parameters, such as pH and temperature, also
may cause changes in both the intensity and the wavelength of
the absorbance maxima.
Conjugating the double bond with additional double bonds
increases both the intensity and the wavelength of the absorption
band. For some molecular systems, such as conjugated
hydrocarbons or carotenoids, the relationship between intensity
and wavelength has been systematically investigated.
Transition metal ions also have electronic energy levels that
cause absorption of 400–700 nm in the visible region.
Confirmation of identity Although UV-visible spectra do not enable absolute

identification of an unknown, they frequently are used to confirm
the identity of a substance through comparison of the measured
spectrum with a reference spectrum.
Where spectra are highly similar, derivative spectra may be used.
As shown in Figure 6, the number of bands increases with higher
orders of derivatives. This increase in complexity of the
derivative spectra can be useful in qualitative analysis, either for
characterizing materials or for identification purposes. For
example, the absorbance spectrum of the steroid testosterone
shows a single, broad, featureless band centered at around
330 nm, whereas the second derivative shows six distinct peaks.
The resolution enhancement effect may be of use as well in
identifying an unknown. Figure 6 shows a computer simulation.
19
When two Gaussian bands with a 40-nm natural spectral

bandwidth (NBW) separated by 30 nm are added in absorbance
mode, a single band with a maximum midway between the two
component bands results. The two components are not resolved.
In the fourth derivative, these two bands are clearly visible, with
maxima centered close to the λmax of the component bands.
Absorbance
4th derivative
Figure 6
Resolution enhancement
Color Color is an important property of a substance. The color of matter

is related to its absorptivity or reflectivity. The human eye sees
the complementary color to that which is absorbed, as shown in
Figure 7 and Figure 8.
20
Figure 7
Transmission and color
Wavelength [nm] Absorbed color Complementary color

red blue-green
orange greenish blue
yellow-green purple
green red-purple
bluish green red
greenish blue orange
blue yellow
violet yellow-green
Figure 8
Absorbance and complementary colors
In practice, both the generation and sensation of color are highly

complex and depend on many factors, including the spectrum of
the illuminant and, in the case of solids, the surface structure.
Specialized color measurement systems, such as the CIE L*a*b,
21
and instrumentation to measure color have been developed.

When equipped with the appropriate software, most
spectrophotometers can be used to measure color. An in-depth
discussion of color is beyond the scope of this primer. Several
well-written publications2,3 discuss color and the measurement
of color in detail.
Other qualitative UV-visible spectroscopy can be used to determine many

information physicochemical characteristics of compounds and thus can
provide information as to the identity of a particular compound.
Two examples follow.
Protein and nucleic acid melting The absorbance spectra of proteins result largely from the
temperature presence of the aromatic amino acids tryptophan, tyrosine, and
phenylalanine. A protein at room temperature has a specific
tertiary structure or conformation that in turn creates a specific
electronic environment for the aromatic amino acids. If the
protein is heated it will, at a certain temperature, unfold or melt
and lose its structure. In this process, the electronic environment
of the aromatic amino acids changes, which in turn results in
spectral changes or shifts.
Multicomponent analysis (see “Multicomponent analysis” on
page 27) can be used to determine how many of each aromatic
amino acid are present in an intact protein.4
Deoxyribonucleic acid (DNA) in its native state comprises two

strands of deoxyribose molecules helically wound around the
same axis. The strands are linked by hydrogen bonds between the
purine and pyrimidine bases—adenine is joined to thymine
(A-T), and guanine to cytosine (G-C). These bases are primarily
responsible for the UV absorbance of DNA, with a peak
maximum at 260 nm. As in any multicomponent system, the
observed absorption of any DNA molecule should equal the sum
of the individual absorbances:
ADNA = A adenine + Aguanine + Acyto sin e + A thymine
22
However, the observed absorbance is always significantly less

than expected because the hydrogen bonding between the bases
changes their electronic environment. When a molecule is
heated, the hydrogen bonds break, the double helix unwinds, and
the absorbance increases so that it approaches that expected from
the sum of all bases. This denaturation process also is known as
melting. In a DNA melt experiment, the temperature of a DNA
solution is increased in a stepwise fashion, and the absorbance at
260 nm at each temperature is measured and plotted as a melting
curve.
The midpoint of the temperature range over which the melting
occurs is the Tm value. The Tm value of a particular DNA sample
depends primarily on the percentage of G-C pairs in the sample,
each of which contains three hydrogen bonds (in contrast, each
A-T pair contains two hydrogen bonds). The higher the
percentage of G-C pairs in the sample, the higher the observed
Tm .
Enzyme activity The activity of an enzyme is a measure of its effectiveness as a

catalyst. The concentration of enzyme in an impure preparation
can be expressed in terms of units per milliliter, and the specific
activity of the preparation can be expressed as units per
milligram of protein. As an enzyme is purified, the specific
activity increases to a limit (that of the pure enzyme).
Because reaction rate varies with such factors as substrate
concentration, pH, ionic strength, and temperature, the conditions
under which the activity is determined must be defined precisely.
These conditions are usually the optimal assay conditions at a
fixed temperature (25, 30, or 37 °C), with all substrates present at
saturating conditions. To determine activity, a system is set up
with known concentrations of substrate and, if necessary,
coenzyme. A known weight of the enzyme is added and the rate
of reaction determined. Activity measurements are conducted
primarily in the research environment as enzymes are isolated
and purified, and in the manufacture of enzyme assay kits, in
which the enzyme activity must be consistent from batch to
batch.
23
Instrumental Absolute wavelength accuracy and absolute photometric

considerations accuracy are very important in qualitative analysis, particularly
for the identification and confirmation of unknowns. Often
spectra acquired on different instruments at different times are
compared. In this regard, spectra may have to be measured at a
defined instrumental resolution.
Quantitative analysis
Beer’s law If 100 photons of light enter a cell and only 50 emerge from the
other side, the transmittance is 0.5, or 50 %. If these 50 photons
then pass through an identical cell, only 25 will emerge, and so
forth. Figure 9 shows the plot of transmittance against
concentration.
Transmission
Path length
Figure 9
Transmittance and concentration—the Bouguer-Lambert law
Lambert (1760) generally is credited with the first mathematical

formulation of this effect, although it now appears that Bouguer
first stated it in 1729. The mathematical expression is:
– kb
T = I ⁄ Io = e
24
where Io is the incident intensity, I is the transmitted intensity, e

is the base of natural logarithms, k is a constant, and b is the path
length (usually in centimeters).
Beer’s law is identical to Bouguer’s law, except that it is stated in
terms of concentration. The amount of light absorbed is
proportional to the number of absorbing molecules through
which the light passes. Figure 10 shows a plot of transmittance
against path length.
Transmission
Path length
Figure 10
Transmittance and path length—Beer’s law
Combining the two laws gives the Beer-Bouguer-Lambert law:

– kbc
T = I ⁄ Io = e
where c is the concentration of the absorbing species (usually
expressed in grams per liter or milligrams per liter). This
equation can be transformed into a linear expression by taking
the logarithm and is usually expressed in the decadic form:
A = – log T = – log ( I ⁄ I o ) = log ( Io ⁄ I ) = εbc
25
where ε is the molar absorption or extinction coefficient. This

expression is commonly known as Beer’s law. Figure 11 shows a
plot of absorbance against concentration.
Absorbance [AU]
Concentration
Figure 11
The Beer–Bouguer-Lambert law
The extinction coefficient (ε) is characteristic of a given

substance under a precisely defined set of conditions, such as
wavelength, solvent, and temperature. In practice, the measured
extinction coefficient also depends partially on the characteristics
of the instrument used. For these reasons, predetermined values
for the extinction coefficient usually are not used for quantitative
analysis. Instead, a calibration or working curve for the substance
to be analyzed is constructed using one or more standard
solutions with known concentrations of the analyte.
For electronic transitions, the difference in energy between
ground and excited states is relatively large. Therefore, at room
temperature, it is highly likely that all molecules are in the
electronic ground state. Absorption and return to ground state are
fast processes, and equilibrium is reached very quickly. Thus,
absorption of UV-visible light is quantitatively highly accurate.
The simple linear relationship between absorbance and
concentration and the relative ease of measurement of UV-visible
26
light have made UV-visible spectroscopy the basis for thousands

of quantitative analytical methods.
Assuming Beer’s law is obeyed for the zero-order spectrum, a
similar linear relationship exists between concentration and
amplitude for all orders of derivative spectra:
Zero order: A = εbc
dA dε
First derivative: ------- = ------ bc
dλ dλ
n n
d A d ε
th
n derivative: --------- = -------- bc
dλ′ dλ′
at λ, where A is absorbance, ε is the extinction coefficient, b is

the sample path length, and c is the sample concentration.
For single-component quantification, the selection of
wavelengths is more difficult with derivative spectra than with
absorbance spectra since both positive and negative peaks are
present. The even-order derivatives have a peak maximum or
minimum at the same λmax as the absorbance spectrum, but for
the odd-order derivatives, this wavelength is a zero-crossing
point. Taking the difference between the highest maximum and
the lowest minimum gives the best S/N but may result in
increased sensitivity to interference from other components.
Sample requirements For accurate results, the sample to be analyzed must contain only
the absorbing component for which the calibration has been
performed. If the sample is a solution, a pure sample of the
solvent should be used as a blank. It may be possible to correct
for an interfering component with a second wavelength.
Multicomponent analysis Multicomponent analyses using UV-visible spectra have been

performed for almost as long as single-component analyses, but
because the techniques used in multicomponent analysis often
27
gave incorrect results (as detailed below), they were not widely
applied. However, modern instruments yield more precise data,
and modern curve-fitting techniques give more accurate results
and—perhaps more importantly—indicate when results are
incorrect. For these reasons, multicomponent UV-visible
analyses are becoming more popular.
Principle of additivity According to Beer’s law (see “Beer’s law” on page 24),
absorbance is proportional to the number of molecules that
absorb radiation at the specified wavelength. This principle is
true if more than one absorbing species is present. All
multicomponent quantitative methods are based on the principle
that the absorbance at any wavelength of a mixture is equal to the
sum of the absorbance of each component in the mixture at that
wavelength.
Simple simultaneous equations The simple approach to multicomponent analysis is based on

method measurements at a number of wavelengths equal to the number of
components in the mixture. The wavelengths chosen usually are
those of the absorbance maximum of each component. For
calibration, the absorbance of standards of known concentrations
of pure components is measured to determine the extinction
coefficient for each component at each wavelength selected.
The absorbance of the mixture at each wavelength is the sum of
the absorbance of each component at that wavelength, which in
turn depends on the extinction coefficient and the concentration
of each component. Thus for two components x and y, the
equations are:
A′ ( x + y ) = A′ x + A′ y = e′ x bc x + e′ y bc y
and
A″( x + y ) = A″ x + A″y = e″ x bc x + e″ y bc y
28
where A′ is absorbance at wavelength ′, A′′ is absorbance at

wavelength ′′, e′ is molar absorptivity at wavelength ′, e′′ is molar
absorptivity at wavelength ′′, c is concentration, and b is path
length.
These equations are easily solved to determine the concentration
of each component. If measurements were always perfect,
accurate results could be obtained even for complex mixtures of
components with very similar spectra. In practice, however,
measurement errors always occur. Such errors can affect
significantly the accuracy of results when spectra overlap
significantly. Figure 12 shows a simulated two-component
mixture with no overlap of the spectra at the absorbance maxima.
Absorbance [AU]
Wavelength [nm]
Figure 12
A two-component mixture with little spectral overlap
In contrast, Figure 13 shows a simulated two-component mixture

with significant overlap of the spectra at the absorbance maxima.
29
Absorbance [AU]
Wavelength [nm]
Figure 13
A two-component mixture with significant spectral overlap
For a mixture of x and y where cx = cy = 1, the measured

absorbances should be:
With little spectral overlap With substantial spectral overlap
A’ (x + y) = 1.1 + 0.0 = 1.1 A’(x + y) = 0.6 + 0.5 = 1.1
A’’(x + y) = 0.0 + 0.9 = 0.9 A’’(x + y) = 0.4 + 0.5 = 0.9
If a 10 % error occurs in the measurement of A′(x + y) and A′′(x +

y), that is, A′(x + y) = 0.99 (- 10 %) and A′′(x + y) = 0.99 (+ 10 %),
the quantitative calculation yields the results shown in Table 2:
30
Table 2 Comparison of multicomponent analysis results for examples with little and
substantial spectral overlap
Substantial spectral
Little spectral overlap overlap
Nominal Calculated Calculated

concentratio concentratio % concentratio %
Component n n error n error
x 1 0.9 - 10 % 0 - 100 %
y 1 1.1 + 10 % 1.98 + 98 %
Least squares method The effect of random noise can be reduced through the use of
additional spectral information, that is, a series of data points can
be used for quantification instead of only two. In this so-called
overdetermined system, a least squares fit of the standard spectra
to the spectrum of the measured sample yields quantitative
results.5,6 Figure 14 depicts a spectrum for the two-component
mixture shown in Figure 13 with a 10 % random error at each
measurement point.
True Measured
Absorbance [AU]
Wavelength [nm]
Figure 14
Mixture spectrum with 10 % random error at each wavelength
31
With 21 data points (2-nm intervals over 200–240 nm), the

quantitative results from the least squares method have an error
of < 1 % compared with an error of approximately 100 % from
the usual measurements at two wavelengths, as shown in Table 3.
Table 3 Comparison of multicomponent analysis results from simple simultaneous

equations and least squares methods
Using 210 and 230 nm

only Using 200–240 nm
Nominal Calculated Calculated

concentratio concentratio % concentratio %
Component n n error n error
x 1 0.0 - 10 % 1.003 + 0.3 %

y 1 1.98 + 10 % 0.995 - 0.5 %
This method enables the analysis of more complex mixtures and

of simple mixtures of components with similar spectra.
The residual from the least squares calculation is a good indicator
of how well the standard spectra fit the sample spectra and is
therefore a good indicator of the probable accuracy of the results.
An example of multicomponent analysis is the quantification of
five hemoglobins in blood with minimum sample preparation.7
Figure 15 shows the absorption spectra of hemoglobin
derivatives. This analysis was previously performed using
various analytical techniques, including spectroscopy and
titrations.
32
Sulfhemoglobin
Oxyhemoglobin
Carboxyhemoglobin
Absorbance [AU]
Hemoglobin (pH 7.0–7.4)
Deoxyhemoglobin
Wavelength [nm]
Figure 15
Absorption spectra of hemoglobin derivatives
Other methods Other statistical approaches to multicomponent analysis include

the partial least squares (PLS), principle component regression
(PCR), and multiple least squares (MLS) methods. In theory,
these methods offer some advantages over those described
above, but in practice the calibration process is much more
complex.
Sample requirements The simple simultaneous equations and least squares methods
yield accurate results only if calibration is performed using pure
standards or mixtures of standards for each component in the
sample that contributes to the UV spectrum. The unknown
sample must not have any additional absorbing capacity.
Instrumental requirements Single-component quantification is normally performed by

measuring with the same instrument a standard or series of
standards followed by an unknown. This calibration process
should eliminate instrumental bias, making absolute wavelength
accuracy and absolute photometric accuracy relatively
unimportant. On the other hand, photometric reproducibility is
essential for precise results. If measurements are performed only
33
at the absorbance maximum, wavelength reproducibility is also

of little importance because the rate of change of absorbance with
wavelength is low. However, if a wavelength on the side of the
band is used, wavelength reproducibility becomes very
important. Finally, the instrumental linear range is critical, as the
calibration process relies on a linear relationship.
Accurate multicomponent analyses require excellent S/N,
especially if the simple simultaneous equations method is used.
In the least squares method, data from the sides of absorbance
bands is incorporated into the calculation, making excellent
wavelength reproducibility essential as well. Moreover, because
more data is required, fast scanning is necessary for productivity.
Indirect quantification
Chemical derivatization Because many compounds exhibit either very weak or no

absorbance in the UV or visible regions, a number of methods
using chemical derivatization have been developed. Such
methods usually involve adding an organic reagent, which forms
a complex with strong absorptivity. The final stage of
measurement closely resembles that of the direct methods. With
this technique, the choice of an appropriate reagent can enhance
significantly both sensitivity and selectivity.
Spectrophotometric In volumetric analyses, the color changes that signify the end
titrations point of a titration are most often detected through visual
inspection. This process is inherently subjective and can be a
source of error. The use of a spectrophotometer for endpoint
detection introduces objectivity into the analysis and lends itself
to automation.
Enzyme kinetic assays Direct UV-visible analysis of one component in biological

matrices, for example blood or foodstuffs, is difficult.
Interference from other components often makes impossible
direct measurement of a specific property, such as absorbance.
34
Separation of the compound of interest may be costly and

time-consuming and thus impracticable for routine analysis.
Enzyme assays can be used in the indirect analysis of one
compound or a group of compounds in a complex matrix. If the
enzyme is carefully selected, any change in the sample following
addition of the enzyme will result only from the reaction of the
specific compound or compounds. This selectivity is the basis of
enzyme assays.
Enzyme assays can be divided broadly into two types: rate assays
and end point assays. The rate of an enzyme depends on many
factors, including temperature, pH, enzyme activity, enzyme
concentration, and substrate concentration. However, if all other
parameters are controlled at a constant level, the rate of reaction
is directly proportional to the substrate concentration. With end
point assays, the conditions are selected so that the conversion of
substrate to product is completed within a reasonable period of
time (5–20 min). The difference between initial absorbance and
final absorbance is directly proportional to the amount of
substrate.
35
36
chapter 2
Chapter 2 Instrumentation
37
Instrumentation
Ideally, analytical instruments always yield correct
measurments of a chemical or physicochemical parameter,
but in practice all instruments are subject to error. In this
chapter we review the basic components of a
spectrophotometer and the various instrument
configurations available. Key instrumental parameters and
their potential adverse effects on the measured values are
also discussed.
Instrumental design
Components A spectrophotometer is an instrument for measuring the

transmittance or absorbance of a sample as a function of the
wavelength of electromagnetic radiation. The key components of
a spectrophotometer are:8
• a source that generates a broad band of electromagnetic
radiation
• a dispersion device that selects from the broadband radiation
of the source a particular wavelength (or, more correctly, a
waveband)
• a sample area
• one or more detectors to measure the intensity of radiation
38
Instrumentation
Other optical components, such as lenses or mirrors, relay light

through the instrument.
Sources The ideal light source would yield a constant intensity over all
wavelengths with low noise and long-term stability.
Unfortunately, however, such a source does not exist. Two
sources are commonly used in UV-visible spectrophotometers.
The first source, the deuterium arc lamp, yields a good intensity
Spectral irradiance
continuum in the UV region and provides useful intensity in the

visible region (see Figure 16). Although modern deuterium arc
lamps have low noise, noise from the lamp is often the limiting
factor in overall instrument noise performance. Over time, the
intensity of light from a deuterium arc lamp decreases steadily.
Such a lamp typically has a half-life (the time required for the
Wavelength [nm]
intensity to fall to half of its initial value) of approximately
1,000 h.
Figure 16
Intensity spectrum of the deuterium arc
lamp
The second source, the tungsten-halogen lamp (see Figure 17),

Spectral irradiance
yields good intensity over part of the UV spectrum and over the
entire visible range. This type of lamp has very low noise and
low drift and typically has a useful life of 10,000 h.
Most spectrophotometers used to measure the UV-visible range
contain both types of lamps. In such instruments, either a source
selector is used to switch between the lamps as appropriate, or
Wavelength [nm] the light from the two sources is mixed to yield a single
Figure 17 broadband source.
Intensity spectrum of the
tungsten-halogen lamp
39
Instrumentation
An alternate light source is the xenon lamp (see Figure 18),

Spectral irradiance
which yields a good continuum over the entire UV and visible

regions. However, because the noise from currently available
xenon lamps is significantly worse than that from deuterium or
tungsten lamps, xenon lamps are used only for applications such
as diffuse reflectance measurements, in which high intensity is
the primary concern.
Wavelength [nm]
Figure 18
Intensity spectrum of the
xenon lamp
Dispersion devices
(a) Prism
Dispersion devices cause different wavelengths of light to be
dispersed at different angles. When combined with an
appropriate exit slit, these devices can be used to select a
particular wavelength (or, more precisely, a narrow waveband) of
light from a continuous source. Two types of dispersion devices,
prisms and holographic gratings, are commonly used in
UV-visible spectrophotometers.
A prism generates a rainbow from sunlight. This same principle
(b) Grating is used in spectrophotometers. Prisms are simple and
inexpensive, but the resulting dispersion is angularly nonlinear
(see Figure 19a). Moreover, the angle of dispersion is
temperature sensitive.
For these reasons, most modern spectrophotometers contain
holographic gratings instead of prisms. These devices are made
from glass blanks, onto which very narrow grooves are ruled.
First order
Second order Traditionally, this task was done mechanically, but modern
production methods use a holographic optical process. The
Figure 19 dimensions of the grooves are of the same order as the
Dispersion devices wavelength of light to be dispersed. Finally, an aluminum coating
is applied to create a reflecting source. Light falling on the
grating is reflected at different angles, depending on the
wavelength. Holographic gratings yield a linear angular
40
Instrumentation
dispersion with wavelength and are temperature insensitive.

However, they reflect light in different orders, which overlap (see
Figure 19b). As a result, filters must be used to ensure that only
the light from the desired reflection order reaches the detector. A
concave grating disperses and focuses light simultaneously.
A monochromator consists of an entrance slit, a dispersion
device, and an exit slit. Ideally, the output from a monochromator
is monochromatic light. In practice, however, the output is
always a band, optimally symmetrical in shape. The width of the
band at half its height is the instrumental bandwidth (IBW).
Detectors A detector converts a light signal into an electrical signal. Ideally,

it should give a linear response over a wide range with low noise
and high sensitivity. Spectrophotometers normally contain either
a photomultiplier tube detector or a photodiode detector.
The photomultiplier tube (see Figure 20) combines signal

conversion with several stages of amplification within the body
of the tube. The nature of the cathode material determines
spectral sensitivity. A single photomultiplier yields good
sensitivity over the entire UV-visible range. This type of detector
Cathode yields high sensitivity at low light levels. However, in analytical
Anode
spectroscopy applications, high sensitivity is associated with low
Figure 20 concentrations, which result in low absorbances, which in turn
The photomultiplier tube detector result in high intensity levels. To detect accurately small
differences between blank and sample measurements, the
detector must have low noise at high intensity levels.
Increasingly, photodiodes are used as detectors in
spectrophotometers (see Figure 21). Photodiode detectors have a
wider dynamic range and, as solid-state devices, are more robust
than photomultiplier tube detectors. In a photodiode, light falling
on the semiconductor material allows electrons to flow through
it, thereby depleting the charge in a capacitor connected across
the material. The amount of charge needed to recharge the
capacitor at regular intervals is proportional to the intensity of the
light. Earlier photodiodes had low sensitivity in the low UV
range, but this problem has been corrected in modern detectors.
41
Instrumentation
The limits of detection are approximately 170–1100 nm for

silicon-based detectors.
Metal contact Photon
p layer Intrinsic region

n layer Gold block
Figure 21
The photodiode detector
Some modern spectrophotometers contain an array of photodiode

detectors instead of a single detector. A diode array consists of a
series of photodiode detectors positioned side by side on a silicon
crystal. Each diode has a dedicated capacitor and is connected by
a solid-state switch to a common output line. The switches are
controlled by a shift register (see Figure 22). Initially, the
capacitors are charged to a specific level. When photons
penetrate the silicon, free electrical charge carriers are generated
that discharge the capacitors. The capacitors are recharged at
regular intervals that represent the measurement period for each
scanning cycle.
42
Instrumentation
Light
Photodiode
Capacitator
Shift
register
Transistor
switch
Video line
Readout cycle
Figure 22
Schematic diagram of a photodiode array
The amount of charge needed to recharge the capacitors is

proportional to the number of photons detected by each diode,
which in turn is proportional to the light intensity. The absorption
spectrum is obtained by measuring the variation in light intensity
over the entire wavelength range. The array typically comprises
between 200 and 1000 elements, depending on the instrument
and its intended application. For example, the diode array of the
HP 8453 spectrophotometer comprises 1024 detector elements,
and the photosensitive area measures approximately
25 × 0.5 mm. The readout cycle, which corresponds to the
illumination time, is 100 ms.
Photodiode array technology is similar to microprocessor
technology. Photodiode arrays are complex devices but, because
they are solid state, have high reliability.
Optics Either lenses or concave mirrors are used to relay and focus light
through the instrument. Simple lenses are inexpensive but suffer
from chromatic aberration, that is, light of different wavelengths
is not focused at exactly the same point in space. However, with
careful design, the chromatic aberrations of individual lenses in
43
Instrumentation
an optical system can be used to cancel each other out, and an

effective optical system can be constructed with these simple and
inexpensive components.
Achromatic lenses combine multiple lenses of different glass
with different refractive indices in a compound lens that is
largely free of chromatic aberration. Such lenses are used in
cameras. They offer good performance but at relatively high cost.
Concave mirrors are less expensive to manufacture than
achromatic lenses and are completely free of chromatic
aberration. However, the aluminum surface is easily corroded,
resulting in a loss of efficiency.
At each optical surface, including the interfaces between
components in an achromatic lens, 5–10 % of the light is lost
through absorbance or reflection. Thus spectrophotometers
ideally should be designed with a minimum number of optical
surfaces.
The conventional Figure 23 shows a schematic of a conventional single-beam

spectrophotometer spectrophotometer. Polychromatic light from the source is
focused on the entrance slit of a monochromator, which
selectively transmits a narrow band of light. This light then
passes through the sample area to the detector. The absorbance of
a sample is determined by measuring the intensity of light
reaching the detector without the sample (the blank) and
comparing it with the intensity of light reaching the detector after
passing through the sample. As discussed above, most
spectrophotometers contain two source lamps, a deuterium lamp
and a tungsten lamp, and use either photomultiplier tubes or,
more recently, photodiodes as detectors.
44
Instrumentation
Sample
Monochromator
Detector
Exit slit
Dispersion
device
Source Entrance
slit
Figure 23
Schematic of a conventional spectrophotometer
This design is well-suited for measuring absorbance at a single

point in the spectrum. It is less appropriate, however, for
measuring different compounds at different wavelengths or for
obtaining spectra of samples. To perform such tasks with a
conventional spectrophotometer, parts of the monochromator
must be rotated, which introduces the problem of mechanical
irreproducibility into the measurements. Moreover, serial data
acquisition is an inherently slow process.
The diode array Figure 24 shows a schematic diagram of a diode array

spectrophotometer spectrophotometer. Polychromatic light from a source is passed
through the sample area and focused on the entrance slit of the
polychromator. The polychromator disperses the light onto a
diode array, on which each diode measures a narrow band of the
spectrum. The bandwidth of light detected by a diode is related to
the size of the polychromator entrance slit and to the size of the
diode. Each diode in effect performs the same function as the exit
slit of a monochromator.
45
Instrumentation
Diode array
Polychromator
Sample Dispersion
device
Source Entrance
slit
Figure 24
Schematic of a diode array spectrophotometer
The polychromator (entrance slit plus dispersion device) and the

diode array are contained in a unit known as a spectrograph.
Because the relative positions of the sample and the dispersive
element are reversed compared with a conventional instrument,
this configuration often is referred to as reversed optics.
To minimize possible photochemical reactions, a shutter is used
to block light from the source until a measurement can be
performed. When the measurement is initiated, the shutter is
automatically opened, and light passes through the sample to the
array of diodes. The difference in the intensities of the light
reaching the detector with and without the sample is measured as
described in “The conventional spectrophotometer” on page 44.
A diode array spectrophotometer is inherently very fast owing to
its parallel data acquisition and electronic scanning capabilities,
has excellent wavelength reproducibility, and is highly reliable.
Configuration Various configurations of spectrophotometers are commercially

available. Each has its advantages and disadvantages.
46
Instrumentation
Single-beam design Both conventional and diode array spectrophotometers are single
beam. Single-beam instruments are low in cost, and the simple
optical system offers high throughput and hence high sensitivity.
The reference spectrophotometers used by national standards
institutions such as the National Institute of Standards and
Technology (NIST) in the United States and the National
Physical Laboratory (NPL) in the United Kingdom are single
beam.
Diode array spectrophotometers in particular are well-suited to
single-beam configuration because spectra are acquired very
quickly and because the time interval between blank and sample
measurements is minimized. In addition, internal referencing can
be used to reduce further the effects of lamp drift (see “Internal
referencing” on page 79).
Figure 25 shows the optical system of a modern diode array
spectrophotometer, the HP 8453. This single-beam configuration
has a minimum number of optical components for highest
throughput efficiency and contains a 1024-element diode array
for measuring the wavelength range from 190 to 1100 nm with
good resolution.
Shutter
Lens
Tungsten
lamp
Sample
Lens Deuterium
Slit lamp
Grating
1024-element
diode array
Figure 25
Optical diagram of the HP 8453 diode array spectrophotometer
47
Instrumentation
Dual-beam design In a conventional single-beam spectrophotometer, the blank and

the sample are measured consecutively, with an interval of
several seconds for a single wavelength measurement and up to
several minutes for a full spectrum measurement with a
conventional instrument. Lamp drift can result in significant
errors over long time intervals.
The dual-beam spectrophotometer was developed to compensate
for these changes in lamp intensity between measurements on
blank and sample cuvettes. In this configuration, a chopper is
placed in the optical path, near the light source. The chopper
switches the light path between a reference optical path and a
sample optical path to the detector. It rotates at a speed such that
that the alternate measurements of blank and sample occur
several times per second, thus correcting for medium- and
long-term changes in lamp intensity (drift).
Figure 26 shows a schematic of a dual-beam spectrophotometer.
Compared with single-beam designs, dual-beam instruments
contain more optical components, which reduces throughput and
sensitivity. For high sensitivity, long measurement times may be
required. In addition, the more complex mechanical design of the
dual-beam spectrophotometer may result in poorer reliability.
Monochromator
Exit
slit Reference
Dispersion
device
Entrance Chopper
Source slit Detector
Sample
Figure 26
Optical system of a dual-beam spectrophotometer
48
Instrumentation
Traditionally, the higher stability of dual-beam instruments has

been a major factor in the design of high-performance
spectrophotometers. However, recent advances in lamp and
electronics design have improved the stability of the single-beam
spectrophotometer and led to the resurgence of this
configuration. Single-beam instruments offer higher sensitivity
and greater ease of use, with drift typically only a factor of two
worse than that of dual-beam instruments.
The first commercially available diode array spectrophotometer,
the HP 8450A, was a multibeam design (see Figure 27). The
beam director is used to shift the beam alternately through the
reference position and as many as four sample positions (for
clarity only one is shown in the figure).
Visible lamp
Cube
Reference corner
UV lamp cell mirrors
Source
Source ellipse
mirror
Upper beam
Spectrograph director mirror
UV ellipse
Sample
cell
Visible
Cube
corner
Holographic Spectrograph
mirrors
slit and Lower beam
grating
detector arrays director mirror
Figure 27
Optical system of the HP 8450A diode array spectrophotometer
Split-beam design The split-beam spectrophotometer (see Figure 28) resembles the
dual-beam spectrophotometer but uses a beam splitter instead of
a chopper to send light along the blank and sample paths
49
Instrumentation
simultaneously to two separate but identical detectors. This

configuration enables the blank and the sample to be measured at
the same time. Although the split-beam design is mechanically
simpler than the true dual-beam instrument and requires fewer
optical elements, the use of two independent detectors introduces
another potential source of drift.
Monochromator
Exit
slit
Dispersion Reference Detector
device
Entrance
Source slit
Sample Detector
Figure 28
Optical system of a split-beam spectrophotometer
This design provides high stability, although not as high as a

dual-beam instrument since two detectors can drift
independently, and good noise, although not as good as a
single-beam instrument since the light is split so that less than
100 % passes through the sample.
Dual-wavelength design With a dual-wavelength spectrophotometer, two wavelengths can

be measured simultaneously for special applications, such as the
study of two concurrent reactions in a sample. The
monochromator contains two dispersion devices (in effect
transforming it into a duochromator), with the output combined
into a single beam. These complex instruments typically are
significantly more expensive than conventional
spectrophotometers and have been largely replaced by diode
50
Instrumentation
array spectrophotometers, which are multiwavelength

instruments.
Measuring a spectrum The degree of interaction of the sample with radiation

(transmittance or absorbance) is determined by measuring both
the intensity of the incident radiation (without the sample) and
the transmitted intensity (with the sample). These intensities are
denoted Io and I, respectively, in the equations in “Transmittance
and absorbance” on page 14.
Because most samples measured with UV-visible spectroscopy
are in solution, the blank should be measured on a cuvette
containing the pure solvent used to prepare the sample. This
process eliminates from the sample measurement any absorbance
due to the solvent.
With a single-beam instrument, the cuvette containing the
solvent is placed in the spectrophotometer, and the blank is
measured. The sample solution is then measured in the same
cuvette. All modern instruments automatically store the reference
Io values, which are used to calculate absorbance values for the
sample.
With a dual- or split-beam instrument, two cuvettes are required.
Both cuvettes are initially filled with pure solvent, and a
so-called balance measurement is performed. This measurement
reflects the difference in absorbance between the two optical
paths in use. The sample cuvette is then filled with the sample
solution for measurement, and Io and I are measured virtually
simultaneously. The resulting spectrum is corrected by
subtracting the balance spectrum.
Key instrumental In this section we discuss some instrumental parameters that may
parameters affect the accuracy and precision of measured absorbance values
(see Appendix A for a detailed definition of the terms accuracy
and precision). Sources of error in measurements related to
sample handling are described in Chapter 3 “Sample handling
and measurement”.
51
Instrumentation
Spectral resolution Spectral resolution is a measure of the ability of an instrument to

differentiate between two adjacent wavelengths. Two
wavelengths usually are considered resolved if the minimum
between the two peaks of the detector output signal is lower than
80 % of the maximum. This condition is known as the Rayleigh
criterion. Figure 29 shows schematically a case for two closely
adjacent emission lines (the input to the instrument) and the
actual signal that is output from the detector.
Detector
Intensity output signal
Wavelength Wavelength
Figure 29
Definition of resolution
52
Instrumentation
Absorbance Resolution is closely related to instrumental spectral bandwidth

(SBW). The SBW is defined as the width, at half the maximum
I intensity, of the band of light leaving the monochromator (see
Figure 30).
0.5 I
SBW Wavelength
Figure 30
Instrumental spectral bandwidth
Absorbance The accuracy of any measured absorbance depends on the ratio

of the SBW to the natural bandwidth (NBW) of the absorbing
substance. The NBW is the width of the sample absorption band
at half the absorption maximum (see Figure 31).
Wavelength
NBW
Figure 31
Natural spectral bandwidth
53
Instrumentation
An SBW/NBW ratio of 0.1 or less will yield an absorbance

measurement with accuracy of 99.5 % or better.8,9 At an
SBW/NBW ratio of higher than 0.1, the measured spectrum
becomes progressively more distorted, as shown in Figure 32.
Bands may not be resolved correctly, and significant errors in
Absorbance
absorbance values will occur at most wavelengths. SBW is

primarily a function of the entrance and exit slit widths of the
monochromator, and of the dispersion generated by the grating.
Resolutions of 0.5, 0.2, and 0.1 nm are not unusual, but higher
resolutions cause considerable deterioration in S/N.10
Wavelength
Figure 32
Effect of varying SBW on measured
band shape
In modern spectrophotometers, the sampling interval used to

digitize the spectrum for computer evaluation and storage also
Absorbance
Original
spectrum affects resolution (in a diode array spectrophotometer,
digitization occurs on the array itself). Figure 33 shows this
effect. If the sampling interval is large relative to the SBW, the
resolution of the instrument will be degraded. A smaller
Sampling sampling interval improves resolution but results in much larger
process spectral files, which may be difficult to manage. In practice, the
sampling interval is best set at equal to or slightly smaller than
Absorbance
Digitized
the SBW.
spectrum
When considering instrumental requirements, it is important to
determine what resolution is required. As discussed in Chapter 1
“Principles and applications of UV-visible spectroscopy”,
Wavelength absorption bands in the UV-visible region are normally rather
Figure 33 broad, particularly for samples in solution. For approximately
Effect of digital sampling 99 % of routine measurements, an SBW of 2 nm is more than
adequate to yield accurate absorbance measurements of bands
with an NBW of 20 nm or greater.
54
Instrumentation
If an instrument with an SBW of 2 nm is used to measure

samples with an NBW narrower than 20 nm (for example,
benzene), an error in absolute absorbance measurements will
result. This error increases as the NBW decreases (see Figure
32). For absolute absorbance measurements, an instrument with a
narrower SBW is necessary. However, most UV-visible
measurements are used for quantification, which normally
requires only relative measurements (for example, the
absorbance of an unknown concentration relative to the
absorbance of a standard). A calibration performed using
standards, which bracket the concentration of the unknown
sample, will yield accurate quantitative results even for very
narrow bands.
Wavelength accuracy and The difference between wavelength accuracy and wavelength
precision precision usually is not well understood (see Appendix A for an
explanation of the difference between accuracy and precision).
Wavelength accuracy is important for the comparison of
measurements made on different instruments. In most UV-visible
analyses, however, measurements are made on the same
instrument relative to a standard, and wavelength precision (that
is, resettability) is most important.
Figure 34 shows the effect of poor wavelength resettability. If a
wavelength at the absorption maximum is selected for
quantitative measurements, the small wavelength errors that
occur in resetting the spectrophotometer to that wavelength will
have a minimal effect on the measured absorbance. This method
yields the most reproducible quantitative results. On the other
hand, the choice of a wavelength on the side of the absorption
band, with the same wavelength resetting error, will result in
significant errors in measured absorbance. In this case, the
quantitative results are unreliable.
55
Instrumentation
Error = 0.0 AU
Absorbance [AU]
Error = 0.1 AU (10 %)
Wavelength [nm]
Figure 34
Effect of poor wavelength resettability
All standard texts on UV-visible spectroscopy emphasize that,

for accurate quantification, the analytical wavelength must be at
the absorption maximum, even though other wavelengths may
yield better selectivity. However, these texts are based on
conventional mechanical scanning techniques and do not apply to
diode array instruments, which have virtually absolute
wavelength resettability.
Photometric accuracy and Assuming good optical and electronic design, only two factors
precision influence photometric accuracy and precision: stray light and
noise.
Stray light Stray light is defined as detected light of any wavelength that lies
outside the bandwidth of the selected wavelength. The equation
used to calculate transmittance and thereby absorbance is:
T = ( I + Is ) ⁄ ( Io + I s )
where T is transmittance, Io is the intensity of incident light, I is

the intensity of transmitted light, and Is is the intensity of stray
light.
56
Instrumentation
The intensity of stray light normally does not depend on that of

transmitted light. If Is remains near constant, it becomes the
dominant term at low levels of I. At high absorption, stray light
causes a negative bias in instrument response and eventually is
the limiting factor for the absorbance, and thereby concentration,
that can be measured. The photometric accuracy of the
instrument is thus compromised. Figure 35 shows the effect of
various levels of stray light on measured absorbance compared
with actual absorbance.
0.00 % Stray light

Measured absorbance [AU]
0.01 % Stray light

0.10 % Stray light
1.00 % Stray light
True absorbance [AU]
Figure 35
Effect of stray light on measured sample absorbance
Noise A spectrophotometer typically has two noise elements. The first

element (photon or Schott noise) results from the statistical
distribution of the photons emitted by a light source. It is
proportional to the square root of the intensity of light. When low
concentration samples with low absorbances are measured, this
element may prevent an accurate measurement of the small
difference between two high light levels. The second element is
inherent to the instrument electronics (detector amplifier,
analog-to-digital converter, and so forth) and is independent of
the intensity measured. This element becomes significant at high
57
Instrumentation
absorbance levels, where the sample signal is very small. It can

be minimized through good design.
Noise negatively affects the precision of measurements and, for
any single measurement, may introduce errors in accuracy as
well. However, noise can be reduced by increasing measurement
time (see “Time averaging” on page 73).
Linear dynamic range A frequently quoted and often misunderstood specification is

instrument range. In most cases, the instrument range is simply
the numerical range an instrument can display. A more useful
specification is linear dynamic range, which specifies for a given
acceptable deviation from linearity (as a percentage of
absorbance) the minimum and maximum absorbance values.
Potential errors at different absorbances can be calculated from
stray light and noise.
Thus, error due to stray light (%) is equal to
( A t – A m ) ⁄ ( At × 100 )
where A t = – log ( l ⁄ l o )
and A m = – log [ ( l + I s ) ⁄ ( l o + Is ) ]
= log [ ( l + Is ⁄ l o ) ⁄ ( l ⁄ l o + I s ⁄ l o ) ]
where lo is incident light intensity, l is transmitted light intensity,

Is is stray light intensity, lf is stray light as a percentage of lo, At is
true absorbance, and Am is measured absorbance. Figure 35
shows the error curve due only to stray light.
In addition, the measured absorbance may be in error because of
the instrument noise.
Thus, error due to noise (%) is equal to
( A t – A m ) ⁄ ( At × 100 )
where A t = – log ( l ⁄ l o )
58
Instrumentation
and Am = At + T ⁄ 100 × A pn + A en
or Am = T ⁄ 100 × Apn – Aen
where Apn is photon noise in AU, Aen is electronic noise in AU,
and T is transmittance as a percentage.
The total error at any absorbance is the sum of the errors due to
stray light and noise. Figure 36 depicts the total error for an
example with photon noise of ± 0.0004 AU and electron noise of
± 0.0001 AU. The plot shows that absorbance measurements
made from approximately 0.3 to 1.0 AU have the highest
accuracy and precision. The instrumental dynamic range can be
determined from the acceptable measurement error.
% Error
Stray light error curve

Stray light + noise error curve
Stray light - noise error curve
Absorbance [AU]
Figure 36
Theoretical absorbance error versus absorbance
59
Instrumentation
Drift Another potential cause of photometric error is drift. Drift

normally results from variations in lamp intensity between the
measurement of Io and the measurement of I. Changes in the
instrument electronics also can cause drift. Good instrumental
design can minimize drift, but this effect can reduce the accuracy
of results, especially over a long period of time (see Figure 37).
With mulitwavelength data, the problem of drift can be
minimized using techniques such as internal referencing (see
“Internal referencing” on page 79).
Error
Absorbance [AU]
Wavelength [nm]
Figure 37
Effect of drift on measured absorbance values
60
Instrumentation
61
Instrumentation
62
chapter 3
Chapter 3 Sample handling

and
measurement
63
Sample handling and measurement
Assuming the instrumental limitations are understood and
the spectrophotometer is correctly operated, the largest
sources of error in UV-visible spectroscopy are related to
sample handling and sample chemistry. In this chapter we
discuss potential sources of error and steps to avoid them.
Liquid samples
Cells UV-visible spectroscopy is used primarily to measure liquids or

solutions. This mode is simpler and allows more accurate
quantitative analysis than do reflectance measurements on solids.
With this technique, a cell must contain the liquid or solution in
the spectrophotometer sample area.
Material Ideally, cells would be completely transparent at all wavelengths

since any absorbance from the cell itself reduces the effective
linear dynamic range for the sample. For example, if the upper
limit of the instrumental linear range is 2.5 AU but the cell
absorbs 1 AU, the remaining usable range for the sample is
between 1 and 2.5 AU, that is, only 1.5 AU.
The cells lowest in cost are made of plastic, usually an acrylic.
These cells are not resistant to all solvents and absorb strongly
below 300 nm, making them unsuitable for measurements in this
region. Consistency (absorbance and path length) can vary from
cell to cell, depending on the manufacturer.
Glass cells are slightly more expensive than plastic cells but are
more durable and, with proper care, can provide years of use.
64
Glass absorbs strongly below 320 nm and thus is not suitable for
measurements in this area.
Fused quartz cells are reasonably transparent down to 210 nm.
The best cells are made of high-purity fused synthetic silica and
are reasonably transparent down to 190 nm.
Figure 38 shows the absorption characteristics of cells of
different materials. Note that all materials exhibit at least an
approximately 10 % loss in transmission at all wavelengths.
Fused
silica
Transmittance [%]
Fused
quartz
Acrylic Glass
plastic
Optical
glass
Wavelength [nm]
Figure 38
Optical transmission characteristics of some cell materials
Cell types A wide range of cells are available, and only the most important
are described here. The most frequently used cell is the
open-topped rectangular cell (see Figure 39a). These cells are
available in path lengths from 1 to 100 mm, but the most popular
path length by far is 10 mm. Almost all rectangular cells have an
external width of 12.5 mm. When sample volume is limited,
apertured cells are often used. (see Figure 39b).
65
When sample volume is extremely limited, microcells can be

used that reduce the aperture of the sample area to a very small
cross section (typically 2 × 2.5 mm), as shown in Figure 40a.
Only approximately 60 µl of sample are required for
measurement. With special ultramicrocells, sample volumes
down to 5 µl can be measured. For automated application,
flow-through cells (see Figure 40b) are used. Modern cells are
connected to sample transfer tubing with screw fittings. Flow
cells of various aperture sizes and geometries are available in a
wide range of path lengths.
Figure 39
With apertured cells and microcells, part of the light beam is
Standard (a) and apertured (b) cells
blocked, throughput is reduced, and sensitivity is to some extent
compromised. The loss of sensitivity depends on the degree of
aperturing and on the optical geometry.
Sources of error
If cells are properly designed and used, their contribution to

errors in absorbance measurements should be minimal. However,
the analyst should be aware of a few potential sources of error.
Because measured absorbance depends on path length, the
precision of the path length is important in absolute
measurements. Cell path length tolerances for cells of good
quality are ± 0.01 mm for path lengths from 0.5 to 100 nm.11 For
maximum quantitative accuracy, the same cell should be used for
both standard and sample measurements.
When placed in the sample beam, a cell becomes an active
Figure 40 optical component. Nonflat or nonparallel optical surfaces can
Micro (a) and flow-through (b) cells deviate the optical beam and cause apparent absorbance errors
(see “Sample geometry” on page 71). The best cells have very
flat and very parallel optical surfaces that minimize their
influence as optical components. The cell always should face the
same direction in a cell holder to ensure that any cell optical
effects are identical for both blank and sample measurements.
66
With apertured cells, the parts of the cell that do not contain the
sample must be properly masked (as shown in the figures) to
avoid unwanted transmissions and reflections through the side
walls. If unmasked cells are used, the measurements will be in
error. The degree of error depends on the optical geometry in the
sample area. If the optical beam is highly focused so that a large
proportion of the light passes through a very small opening in the
cell, results with unmasked cells will be reasonably accurate
because the optics are in effect self-masking. On the other hand,
if the optical beam is broad and collimated (parallel), much of the
light will pass through the walls of the cell instead of through the
sample, and measurments will be inaccurate.
Care of cells Cells should be handled carefully to prevent scratching. Care

should be taken to avoid touching the optical surfaces with the
fingers, as oil from fingerprints can cause significant absorbance.
If the optical surfaces of the cell become mildly contaminated,
the cell can be wiped carefully with photographic tissue. If a cell
becomes seriously contaminated, it may be cleaned with a mild
sulfonic detergent or with special cleaning fluid available from
cell manufacturers. In severe cases, treatment with hydrochloric
or nitric acid can be used.
Choice of solvent The ideal solvent for the preparation of sample solutions would
dissolve all types of compounds, would be nonflammable and
nontoxic, and would be completely transparent at all
wavelengths. Distilled water approaches the ideal but is not
suitable for many nonpolar organic compounds. Table 1 lists
some of the most commonly used solvents. With the exception of
water, these solvents all exhibit a cut-off wavelength in the UV
range below which they absorb too strongly for sample
measurements to be performed.
67
Table 4 Properties of some common solvents
Cut-off
wavelength
Solvent Polarity* (nm)** Hazard***
Distilled water 78.5 < 195 none
Hexane 1.9 199 F
Ethanol (absolute) 24.3 207 F
Methanol 32.6 210 F
Cyclohexane 2.0 211 F
Chloroform 4.8 246 F/T
Dimethylsulfoxide none 270 H
Acetone 20.7 331 F
*
Dielectric constant at ambient temperature
**
Wavelength at which transmittance of 10-mm path length is < 25 %
*** F = flammable; T = toxic; H = health hazard
With volatile organic solvents, such as acetone or methylene

chloride, it is advisable to use a stoppered cell to eliminate
evaporation, which can result very quickly in changes in
concentration.
Effect of solvent, A number of factors, including the solvent used as well as the
concentration, pH, and concentration, pH, and temperature of the sample, can affect the
temperature position and intensity of absorption bands of molecules. These
parameters should be controlled to ensure maximum precision
and when comparing spectra measured under different
conditions.
The polarity of a solvent can modify the electronic environment
of the absorbing chromophore. In general, the magnitude of the
shift can be correlated with solvent polarity. Thus, for example,
the absorption maximum of acetone can vary from 259 to
68
279 nm, depending on the solvent used. For comparative

analysis, a single solvent should be used for all measurements.
Concentration normally affects only the intensity of bands. At
higher concentrations, however, molecular interactions (for
example, dimerization) may cause changes in the shape and
position of the absorbance band. These changes in turn affect the
linearity of the concentration versus absorbance relationship and
may lead to inaccurate quantitative results.
The effects of pH on absorbance spectra can be very large and
result primarily from the shifting of equilibrium between two
different forms. For example, pH indicators visually change color
at different pH values. If the spectrum of the sample under study
is found to be affected by pH, a buffer should be used to control
this parameter. Note, however, that most buffers themselves
exhibit significant absorbance, which may affect the wavelength
range over which measurements can be performed.
Temperature also can affect UV-visible measurements. Simple
expansion of the solvent, especially for some organic solvents,
may be sufficient to change the apparent absorbance and thereby
the accuracy of quantitative results. In addition, temperature may
affect equilibria, which can be either chemical or physical. A
good example of a physical equilibrium is the denaturation of
nucleic acids as temperature is increased, which changes
absorptivity. Finally, notably for organic solvents, changes in
refractive index with temperature can be significant. When
convection currents cause different temperatures to occur in
different parts of the cell, the resulting Schlieren effect can
change the apparent absorbance. If temperature is found to have a
significant effect on the measurements, the sample temperature
should be controlled using a thermostatted cell holder. This cell
holder may be a simple water-jacketed holder used in
conjunction with a circulating water bath, or a more sophisticated
Peltier temperature controller. With organic solvents, it is
advisable to use a stoppered cell to minimize the Schlieren effect.
69
Solid samples A number of factors can interfere with the accurate and precise
measurement of transparent solid samples such as glasses or
crystals.
No reference Often solid samples are measured in order to determine the

spectrum of, or to quantitate, one component in the solid matrix.
However, a sample of the matrix that does not contain the analyte
is not always available. In this case, the blank measurement must
be performed on air since a true blank measurement (matrix
without analyte) is not possible. This process at best will result in
a constant offset at all wavelengths and at worst in a measured
spectrum that is a composite of the analyte and the matrix.
Accurate quantitative analysis is possible if two or more
standards are available and if a calibration curve that is not forced
through zero is used. The intercept on the y-axis then represents
the absorbance due to the matrix. If multiple standards are not
available, internal referencing using a reference wavelength at
which no analyte absorbance occurs is often a viable alternative.
70
Refractive index
Collimated beam The lack of a reference sample also causes a significant change in
the refractive index between the blank (air) and the solid sample.
If the optical beam is collimated perpendicular to the sample, this
effect is unimportant. If, however, the beam is focused, the solid
sample becomes an active optical component and alters the
optical path length. This change in path length may in turn cause
Sample Detector a significant change in the degree of illumination of the detector
between the blank and the sample (see Figure 41), resulting in an
Focused beam apparent absorbance error. Such a problem is very difficult to
detect and, if the instrument is a focused-beam design, has no
simple solution. However, this effect can be minimized by
placing the sample as close to the detector as possible.
Light not
detected
Figure 41
Effect of refractive index
Sample geometry Solid samples often may be glass or molded plastic filters or
lenses (for example, sunglass lenses). Such samples are active
optical components in the system and will deviate or change the
focal length of the light beam. As a result, the detector fails to
detect some of the light (see Figure 42), which is then measured
as an apparent absorbance. This effect can be tested for by
rotating or reversing the sample in its holder and can be
minimized by placing the sample as close to the detector as
possible.
71
Photosensitive area
Detector Detector
Planar sample “Wedge” sample
Figure 42
Effect of nonplanar sample geometry
Weak absorbance A recurring problem in analytical chemistry is low sensitivity,

which may be due to low concentrations of the sample or to very
weak absorptivity of the analyte. At low absorbances, noise in the
measurement results in a loss of precision such that any single
measurement may be inaccurate. Reducing the noise level
directly improves the precision of results. Noise specification
should be a key parameter in selecting a spectrophotometer, but
note that varying the measurement parameters will reduce noise
level.
Changing slit width If the spectrophotometer has a variable slit width, the noise level
can be reduced by increasing the slit width to allow more light
through the optics. This increase in width yields better S/N but
reduces instrument resolution.
72
Time averaging
Taking the average of the data points reduces noise by the square
root of the number of points averaged. Figure 43 shows the
Absorbance [AU]
S/N = 5.5
improvement in S/N with increasing integration time for a
dyestuff at very low concentration. The measurements were
performed using a diode array spectrophotometer with
0.1 sec integration times of 0.1 and 1.6 s. Actual improvement is close to
the expected theoretical improvement of four. Note that
extending the integration time will improve sensitivity only until
other effects, such as drift, become dominant.
Absorbance [AU]
S/N = 18
1.6 sec
Wavelength [nm]
Figure 43
Effect of integration time on S/N
Wavelength averaging Another averaging technique is wavelength averaging. Figure 44

shows a broad absorption band. Conventional quantitative
measurements would be performed at the absorption maximum.
If data at all wavelengths is available, additional values on either
side of the maximum can be averaged. The reduction in noise is
equivalent to the square root of the number of data points.
However, as more data points are added, the average absorbance
is reduced, which has a negative effect on the signal. For a given
absorption bandwidth, a certain number of data points will yield
optimum S/N (see Figure 44).
73
S/N
Signal
Noise
Absorbance
Absorbance
Wavelength # of data points
NBW Optimum
range
Figure 44
Effect of wavelength averaging on S/N
Drift also affects the accuracy of absorbance measurements.

Internal referencing (see “Internal referencing” on page 79) can
be used to improve precision and accuracy at low absorbance
levels.
Strong absorbance When samples absorb too strongly, the linear dynamic range of
the instrument is exceeded, and the relationship between
absorbance and concentration becomes nonlinear (see “Linear
dynamic range” on page 59). The easiest solution to this problem
is to dilute the sample to an absorbance level within the linear
dynamic range. With solid samples, however, this is not possible.
Moreover, any sample handling step, even dilution, introduces
error and thus may be better avoided. An alternative is to select
one or more wavelengths on the side of the absorbance band,
where absorptivity is lower (see Figure 45). When the
wavelength of the absorbance maximum (241 nm) is used for
calibration, a maximum concentration of approximately
0.26 mg/ml can be measured with reasonable accuracy.
However, switching to the absorbance on the side of the band
(266 nm) enables concentrations of up to 5 mg/ml to be measured
74
with equal accuracy. A prerequisite for use of this technique is

excellent wavelength reproducibility (see “Wavelength accuracy
and precision” on page 55).
Measured absorbance [AU]

0.1428 mg/ml
spironoloactone
Absorbance [AU]
Wavelength [nm] Concentration [mg/ml]
Figure 45
The use of wavelength switching to increase dynamic range
Interference
Types of interference Ideally, the absorbance that occurs during UV-visible

measurements should be due only to the target analyte. In
practice, however, absorbances that interfere with the
measurements often occur for chemical or physical reasons.
Other absorbing compounds The presence of any other compound that absorbs in the same
region as the target compound will result in an error in the
absorbance measurement. In some cases, only a single known
75
interfering compound, for example a by-product from the

synthesis of a pharmaceutical product, is present. In other cases,
multiple absorbing species, many of them unknown (for
example, biological samples such as blood), may cause a broad
matrix absorption.
Scattering A recurring problem in pharmaceutical and biological analyses is

scattering caused by particles suspended in solution. In
pharmaceutical analyses, these particles are usually the
excipients or fillers used in tablet or capsule formulations. The
scattering of radiation results in an apparent background
absorbance that interferes with absorbance measurements.
Filtering samples prior to measurement eliminates scattering but
may not always be practicable, and the analyst often must work
with spectra that include a scattering component.
Scattering causes an apparent absorbance because light, instead

Detector of passing through the solution to the detector, is scattered at an
angle. Therefore, even if no absorption occurs, less light reaches
the detector, as shown in Figure 46.
In the UV-visible part of the spectrum, two types of scattering
Transparent sample can be observed. Rayleigh scattering occurs when the particles
are small relative to the wavelength of light and is inversely
proportional to the fourth power of the wavelength. Tyndall
scattering occurs when the particles are large relative to the
Detector
wavelength of light and is inversely proportional to the square of
the wavelength. In chemical systems, the exponent of the
wavelength may range from - 4 to - 2, depending on the
distribution of particle sizes:
Scattering sample n
A scatter ∝ 1 ⁄ λ
Figure 46
Scattering where A is absorbance due to scatter, λ is wavelength, and n is
the order of scattering. This relationship causes the error due to
scattering to increase at lower wavelengths, as shown in Figure
47.
76
Absorbance [AU]
Tyndall scattering
Rayleigh scattering
Wavelength [nm]
Figure 47
Scatter spectra
The magnitude of the scattering effect can be reduced by placing

the sample as close as possible to the detector. However, with
significant scattering, light is lost and the sensitivity and
accuracy of quantitative analysis are seriously impaired.
Correction techniques A number of correction techniques can be used to eliminate or

reduce interference errors. In general, if the source of the error is
known and is consistent from sample to sample, the error can be
eliminated. On the other hand, if the source is unknown and
varies from sample to sample, the error can be reduced but not
eliminated. Correction techniques always require data from at
least two wavelengths. The more sophisticated correction
techniques require multiwavelength or spectral data.
Isoabsorbance When a known interfering component with a known spectrum is

present, the error introduced by this component at the analytical
wavelength for the target analyte can be eliminated by selecting a
reference wavelength at which the interfering compound exhibits
the same absorbance as it does at the analytical wavelength. The
absorbance at this reference wavelength is subtracted from the
77
absorbance at the analytical wavelength, as shown in Figure 48.

The residual absorbance is the true absorbance of the analyte.
Analyte spectrum
Interferent spectrum
Absorbance [AU] Measured spectrum
Wavelength [nm]
Figure 48
Isoabsorbance correction
This technique is less reliable when the spectra of the analyte and
of the interferent are highly similar. Moreover, it can correct for
only one interferent.
Multicomponent analysis An extension of isoabsorbance is multicomponent analysis, as

described in “Multicomponent analysis” on page 27. Here,
spectra of the interferent or interferents must be measured as
standards. This technique can be applied successfully when the
spectra overlap considerably and when more than one interferent
is present.
Background modeling Background modeling is appropriate when the interference is due

to a physical process (most often scattering) in which the
interference at the analytical wavelength can be estimated by
extrapolation from modeling at another wavelength range. A
portion of the spectrum where the apparent absorbance is due
only to interference is selected. A polynomial is then fitted to this
78
part of the spectrum using a least squares fit to the logarithm of

the absorbance:
n
A = aλ
log ( A ) = log ( a ) + n log ( λ )
where A is absorbance, λ is wavelength, n is the order of the

relationship between absorbance and wavelength, and a is a
constant. The background absorbance at all other wavelengths
can be estimated using the coefficients determined from the fit.
These values are then subtracted from the measured values to
give the absorbances due to the analyte, as shown in Figure 49.
Measured
spectrum
Absorbance [AU]
Extrapolated
scatter spectrum
Wavelength [nm]
Figure 49
Background modeling
Internal referencing One of the simplest correction techniques is internal referencing,

which uses a single reference wavelength. This method is most
often used when a baseline shift occurs between measurements.
In general, it is best to choose a reference wavelength as close as
possible to the analytical wavelength but with no significant
absorbance from the analyte. The absorbance at the reference
wavelength is subtracted from the absorbance at the analytical
79
wavelength. Any interference that is constant at all wavelengths

is corrected for, as shown in Figure 50. Although in practice
interference usually is not constant at all wavelengths, this simple
technique often yields surprisingly good improvements in
accuracy.
Spectrum a
Spectrum b
Absorbance [AU]
Wavelength [nm]
Figure 50
Internal referencing
Three-point correction The three-point, or Morton-Stubbs, correction uses two reference

wavelengths, usually those on either side of the analytical
wavelength. The background interfering absorbance at the
analytical wavelength is then estimated using linear interpolation
(see Figure 51). This method represents an improvement over the
single-wavelength reference technique because it corrects for any
background absorbance that exhibits a linear relationship to the
wavelength. In many cases, if the wavelength range is narrow, it
will be a reasonable correction for nonlinear background
absorbances such as that resulting from scattering or from a
complex matrix.
80
Measured
spectrum
Absorbance [AU]
Wavelength [nm]
Figure 51
Three-point (Morton-Stubbs) correction
Derivative spectroscopy Owing to two of its properties, derivative spectroscopy can be

used to reduce or eliminate background interference from a wide
range of sources. First, any interference components that exhibit
a directly proportional relationship to different orders of
wavelength with the general form
1 n
A = a 0 + a1 λ + … + a n λ
are eliminated through the use of increasingly higher orders of

derivatives. Thus a constant baseline offset (a0) is eliminated by
the first derivative, a background absorbance that increases
linearly with wavelength is eliminated by the second derivative,
and so on. Unfortunately, only the constant baseline offset is
common in UV-visible spectroscopy.
Second, and perhaps more important than the first property,
derivatives discriminate against broad absorbance bands relative
to narrow absorbance bands. This discrimination results from the
fact that the amplitude (Dn) of a Gaussian band in the nth
derivative is inversely proportional to the original bandwidth (W)
raised to the nth degree:
81
n 1
D ∝ -------n.
W
Thus for two coincident bands of equal intensity but different

bandwidth in the zero order, the nth derivative amplitude of the
sharper band (X) is greater than that of the broader band (Y) by a
factor that depends on the relative bandwidth and on the
derivative order:
n n
DX WY
---------
n
= ---------
n
-
DY DX
Figure 52 shows the effect of taking derivatives of two bands

with NBWs of 160 and 40 nm, respectively. In absorbance mode,
these bands have equal amplitude. In the first derivative, the
narrower band has 4 times greater amplitude, and in the second
derivative it has 16 times greater amplitude. This property
improves the accuracy of quantification of any narrow band
component in the presence of any broadband component. The
latter may be an interfering component, as is shown in Figure 52.
82
Absorbance
First derivative
Second derivative
Wavelength [nm]
Figure 52
Discrimination against broad bands by derivative spectroscopy
The spectrum resulting from scattering is also broadband, and the

use of derivatives can reduce its contribution as well. For
example, Figure 53 shows an absorbance band with an NBW of
40 nm and the same band in the presence of a scattering
background. Without any correction, the amplitude at 500 nm is
1.0920 A instead of 1.0 A because of the scattering contribution.
Quantification at this wavelength results in an error of + 9.2 %.
Taking the first derivative reduces the contribution from the
scattering component such that, using peak maximum to
minimum, the signal in the presence of scattering is 0.02992 A
instead of 0.03024—a quantification error of only - 1.1 %.
83
Absorbance
Measured spectrum
Actual spectrum
First derivative
Wavelength [nm]
Figure 53
Scatter correction by derivative spectroscopy
Usually, however, the analytical problem cannot be defined

simply as scattering, baseline shift, or unwanted broad absorbing
components. Rather, a combination of two or more of these
effects results in a broad absorbing background matrix.
Derivative spectroscopy is an excellent tool for reducing or
eliminating these difficult-to-
characterize sources of error.
84
Photochemical
problems
Fluorescence Some samples fluoresce, that is, they emit light over a
wavelength range when irradiated with light of a shorter (more
energetic) wavelength. This emitted light results in an error in the
absorbance measurement. The position and magnitude of the
error depend on whether the instrument has conventional forward
optics or reversed optics.
In a conventional forward optics instrument, the sample is
illuminated with light of varying wavelengths over time. As the
excitation wavelength range is scanned, absorption occurs,
initiating the fluorescence process that emits light at longer
wavelengths. Because the detector cannot differentiate among
the individual wavelengths, the absorbance measured at the
excitation wavelength is too low (see Figure 54). As the emission
wavelength range is scanned, no fluorescence occurs, and the
absorption measurements are thus accurate.
True absorbance spectrum

Excitation
Spectrum measured
wavelength with forward optics
Spectrum measured
Absorbance [AU]
with reversed optics
Emission
wavelength
Wavelength [nm]
Figure 54
Effect of fluorescence on the measured absorbance spectrum
85
In a reversed optics instrument, the sample is illuminated with all

wavelengths of light simultaneously, including light at the
excitation wavelength. The sample therefore fluoresces and emits
light but, because the wavelength selection occurs after the light
has passed through the sample, the emitted light is directed to the
correct wavelength. The absorbance measured at the emission
wavelength is therefore too low (see Figure 54), but the
absorbance measured at the excitation wavelength is correct.
Forward optics An additional factor that affects the magnitude of the error is the
so-called acceptance angle of the detector, as shown in Figure 55.
Detector
The fluorescent light is emitted in all directions. If the acceptance
angle is wide, a significant portion of the fluorescent light will
reach the detector. Conversely, if the detector acceptance angle is
narrow, only a small amount of the fluorescent light will reach
Reversed optics the detector, and the absorbance error will be correspondingly
Slit Detector small. Reversed optics instruments have a narrow detector
acceptance angle and thus are less susceptible to fluorescence
error.
Placing a filter in the light beam can eliminate the error due to
fluorescence. In a conventional instrument, the filter is placed
Figure 55 between the sample and the detector to filter out the emission
Acceptance angles and magnitude of wavelength or wavelengths, whereas in a reversed optics
fluorescence error instrument, the filter is placed between the source and the sample
to eliminate excitation wavelengths.
Sample decomposition Some samples are sensitive to photochemical reaction, especially

when exposed to low-wavelength UV light. In extreme cases, a
filter may be necessary to eliminate this light.
86
chapter 4
Chapter 4 Method
development
and validation
87
Method development and validation
An awareness of the errors that can be introduced by the
instrument, by sample handling and by the sample itself
enables you to develop analytical methods that minimize
their effect on results. In this chapter we review criteria for
defining a good method and strategies for finding optimum
method parameters.
Method development Because most UV-visible applications are single-component

quantitative analyses, in this chapter we focus on the
optimization of such methods. Method development involves
selecting the wavelength or wavelengths that yield the best
results for a particular analysis on a particular instrument. Here,
best is defined by such parameters as accuracy, precision,
sensitivity, linearity, range, selectivity, and ruggedness. Because
all of these parameters cannot be optimized at the same time, the
parameter or parameters for optimization must be determined and
the requirements defined prior to analysis. Until recently,
instrumentation limited the choice of wavelength (owing mainly
to problems of wavelength reproducibility) and/or suitable tools
for the comparison of choices were not available. These
limitations have been eliminated in modern instruments.
NOTE: Method development involves the use of various
statistical tools. An in-depth discussion of how these tools are
calculated and of their relative advantages and disadvantages is
beyond the scope of this primer, but detailed information may be
found in the references.12–15
88
Linearity Linearity is the ability of the method to produce test results that
are proportional, either directly or by a well-defined
mathematical transformation, to the concentration of analyte in
samples within a given range.16
For UV-visible measurements, the usual linear relationship is
Beer’s law, which states that the absorbance of a solute is directly
proportional to its concentration (see “Beer’s law” on page 24).
A linear calibration curve relating absorbance to concentration
should have the form:
A = kc
where A is absorbance, c is concentration, and k is the calibration

factor (the slope of the calibration curve). Thus testing for
linearity in effect tests how well our theoretical model (Beer’s
law) fits the actual measurements.
Theoretically, an absorbance measurement of only one standard
of known concentration is required in order to calibrate for
quantification. The measured absorbance value divided by the
concentration gives the slope. A number of instrumental and
sample parameters (see Chapter 2 “Instrumentation” and
Chapter 3 “Sample handling and measurement”) can cause
deviations from Beer’s law, and significant quantitative errors
can result if the calibration curve is not accurately characterized
(see Figure 56). However, because these deviations are
wavelength dependent, selection of the appropriate wavelength
can minimize their influence on results.
89
e
r ve Possible true
cu
rv
cu
on calibration curves on
a ti ati
i br l i br
Absorbance [AU]
ca l Measured sample ca
ica
l al
tic
o ret absorbance
e ore
Measured standard
T he Th
absorbance
Possible
Known concentration quantification results
of standard
Concentration Concentration
Figure 56
Potential errors resulting from inadequate calibration
To construct a calibration curve, the spectra of a set of at least

three standard analyte solutions should be measured. The
Absorbance [AU]
concentrations of the standard solutions should bracket the

expected concentration range of the samples for analysis. Ideally,
all measured standard values would lie on a straight line, but in
practice the values always exhibit some scatter (see Figure 57). A
statistical method must be applied to find the best fit of the
calibration curve to the data and, in a second step, to determine
Concentration
which type of calibration curve gives the best fit. The statistical
method most often used is linear regression, which is also known
as the least squares method.
To compare two calibration curves, a measure of the goodness of
Absorbance [AU]
the fit of the standards to the line is required. Several statistical

values, including correlation coefficient, standard error of
regression, and uncertainty can be used to obtain this
measurement. Of these, the correlation coefficient is the most
popular. This value always lies between + 1 and - 1. A value of
+ 1 indicates a perfect linear relationship between absorbance
Concentration and concentration, with A increasing. A value of - 1 also
Figure 57 indicates a perfect linear relationship, but with A decreasing
Calibration data sets (which can occur if derivative data is used). A value of 0
90
indicates that there is no correlation between absorbance and

concentration.
To determine the best wavelength or combination of
wavelengths, the spectra of a set of pure standards with a wide
range of concentrations are measured. A linear calibration curve
is applied to each wavelength, and the chosen statistic for the
assessment of linearity is calculated. For quickest evaluation, a
graphical plot of the linearity statistic versus wavelength is
useful.
Figure 58 depicts the spectra of four yellow dye standards and the
corresponding correlation coefficient spectrum for a simple
linear calibration curve. The best calibration in terms of linearity
is achieved at the point at which the correlation coefficient
approaches unity. In this example, the wavelength of maximum
absorbance (414 nm) is not identical to the wavelength (402 nm)
that gives the best linearity. Typically, correlation coefficient
values of better than 0.999 can be expected.
Absorbance [AU]
Correlation coefficient
Optimum
range
Wavelength [nm]
Figure 58
Selecting the wavelength or wavelengths for best linearity
If an individual wavelength does not give the required degree of

linearity, a combination of wavelengths may be used. For
91
example, the use of an internal reference wavelength to eliminate

baseline shift errors in measurements will improve results.
If the desired degree of linearity cannot be achieved with a
simple linear calibration curve, a different type of calibration
curve can be applied to minimize the influence of the nonideality
on the results. Typically, equations of the form:
A = a + kc
2
A = kc + k′c
and
2
A = a + kc + k′c
are used. Again, the correlation coefficient or another statistical

tool should be used to assess the relative quality of the calibration
curves. Note that when calibration curve types with higher
degrees of freedom are used, more standards are required in order
to characterize properly the curve.
For multicomponent analyses, Beer’s law is also assumed. Here,
however, the calculation model includes the law of additivity to
explain total absorbances at each wavelength. The simple
linearity test described above cannot be used. Instead, the
standard deviation of the residual can be used to test how well
our standard set fits the measured spectrum. This value is the sum
of the squares of the differences at each wavelength between the
measured spectrum and the calculated spectrum. It may be
assumed that if the residual is zero—that is, if the standards can
be fitted perfectly to the measured spectrum—Beer’s law and the
law of additivity hold for the system under study. If the standard
deviation of the residual is not zero, the theoretical model does
not reflect the system under study because one of the laws is not
obeyed or because an additional component is present for which
there is no calibration standard. In practice, however, the residual
is never zero, and the acceptable value for an analysis must be
92
determined empirically using analytical results that meet the

requirements for accuracy and precision.
Accuracy Accuracy of a method is the degree of agreement between an

individual test result generated by the method and the true
value.16 (See Appendix A for an explanation of the difference
between accuracy and precision.)
To determine which wavelength or wavelengths give the best
accuracy, the spectra of a set of standards are measured and
calibration curves constructed at all wavelengths, as described
above. A sample of known concentration is then required. This
sample is ideally one for which the concentration of the analyte
has been determined using a different technique. However, if
such a sample is not available, a synthetic sample containing a
known weight of the sample is prepared. The spectrum of the
sample is measured, and quantification is performed at all
wavelengths over the measured wavelength range. The
quantitative results at each wavelength are then compared with
the known value. A graphical plot of the quantitative results
versus wavelength enables quick evaluation.
Figure 59 shows the results for a yellow dye sample. Although
the analytical wavelength would have been set at 414 nm using
traditional methods, the wavelength or wavelengths that give the
best accuracy lie in the region of 400 nm.
93
Absorbance [AU]
Concentration
Measured concentration Optimum

True concentration range
Wavelength [nm]
Figure 59
Selecting the wavelength(s) for best accuracy
Because noise may bias the accuracy of any individual

measurement, it is preferable to perform a series of
measurements on the sample and then calculate the average. This
method reduces the contribution of noise to errors in accuracy.
In multicomponent analyses with spectral ranges, however, the
optimum wavelength range cannot be derived using such a
simple technique. Instead, the best range must be determined
through trial-and-error variation of the wavelength range and
through comparison of the calculated results with actual values.
Precision Precision of a method is the degree of agreement among

individual test results when the procedure is applied repeatedly
to multiple samplings.16 (See Appendix A for an explanation of
the difference between accuracy and precision.)
A statistical value is required in order to determine precision.
Standard deviation, percent relative standard deviation (obtained
by dividing the standard deviation by the average value and
multiplying by 100), and confidence interval are the most
94
popular tools for assessing the precision or repeatability of a set

of values.
To determine the precision of a method, a set of typically 10–20
samples with the same concentration are prepared. These
samples are then measured, and the amount of analyte is
calculated. The standard deviation of the results is a measure of
the precision. Figure 60 depicts an example of the scatter of
results and the standard deviation that may be expected for a
good analysis.
Value [mg/l]
Average = 31.41 mg/l

Standard deviation = 0.022 mg/l
Measurement number
Figure 60
Determining the precision of an analysis
If the desired level of precision is not achieved, noise reduction

techniques such as wavelength averaging, time averaging, and
internal referencing should be used to improve the values. The
same techniques can be applied in multicomponent analyses.
Sensitivity Sensitivity refers to the response obtained for a given amount of

analyte and is often denoted by two analytical factors: the limit of
detection (LOD) and the limit of quantification (LOQ).16
The LOD is the lowest concentration of analyte that can be
detected but not necessarily quantified in sample matrices. In
general, the LOD is the point at which the signal from the analyte
95
is equal to three times the noise in the measurement.

Measurement results from some spectrophotometers list standard
deviations based on the noise in the measurement. The LOD is
approximately three times the standard deviation.
The LOQ is the lowest concentration of analyte that can be
determined with acceptable precision and accuracy in sample
matrices. To calculate the LOQ, the acceptable limits of precision
and accuracy (which depend on the objectives for the analysis)
must be defined. The tools described above then can be used to
determine the acceptable limits.
It is often assumed that the wavelength with maximum
absorbance will give the best sensitivity. However, because
instrumental noise can vary significantly with wavelength, this is
not necessarily the case. A better way to determine the
wavelength or wavelengths of optimum sensitivity is to measure
the spectrum of a sample of low concentration several times. The
average and percent relative standard deviation of the measured
values at each wavelength are then calculated. The wavelength
with the lowest percent relative standard deviation likely will
yield the best sensitivity. Figure 61 illustrates this technique for a
yellow dye sample. Although wavelengths between 400 and
450 nm give excellent sensitivity, the best sensitivity is obtained
at 220 nm.
96
Absorbance [AU]
% RSD
Optimum
range
Wavelength [nm]
Figure 61
Selecting the wavelength or wavelengths for best sensitivity
For multicomponent analysis, this technique can be used to

identify the optimum wavelength or wavelengths for each
component. These wavelengths can be applied directly in the
simple simultaneous equations method or as a guide for the
optimum wavelength range in the least squares method.
Range Range is the interval between (and including) the upper and
lower levels of analyte that have been calculated with the
required precision, accuracy, and linearity.16
The range is determined by first analyzing samples that contain
varying concentrations of the analyte and then using the tools
described above to calculate the linearity, precision, and accuracy
of the results.
Selectivity Selectivity is the ability of a method to quantify accurately and

specifically the analyte or analytes in the presence of other
compounds.16
The presence of any other compound that absorbs at the
wavelength used to quantify the analyte will result in quantitative
97
error. These other compounds may be synthesis precursors,

known impurities, excipients, or degradation products in the
sample matrix. If the type of interferent is known, the method
developer can examine the spectra of the analyte and of the
interferent to select a wavelength at which the analyte has
significant absorbance but the interferent has little absorbance. If
the choice of wavelength does not suppress sufficiently the effect
of the interferent, an appropriate correction technique such as use
of an isoabsorbance wavelength (see “Isoabsorbance” on page
77) may be applied.
If the identity and/or spectra of possible interferents are
unknown, an empirical approach almost identical to that
described above may be used to determine which wavelength or
wavelengths give the best accuracy. In this case, the test samples
must contain the interferent. If the concentration of the analyte in
the sample is known, the wavelength that gives the result closest
to the known value yields the best selectivity. If the concentration
of the analyte is unknown, the wavelength that gives the lowest
concentration usually yields the best selectivity (impurities
always add absorbance, causing erroneously high results).
In the example in Figure 62, the yellow dye sample has been
contaminated by a red dye. The plot of concentration versus
wavelength shows that wavelengths between 390 and 420 nm
yield the best selectivity. Note that, owing to the contaminant, the
accuracy error range is much wider than it is for pure yellow dye
(see Figure 59).
98
Absorbance [AU]
Concentration [mg/l]
Measured concentration Optimum

True concentration range
Wavelength [nm]
Figure 62
Selecting the wavelength or wavelengths for best selectivity
Ruggedness Ruggedness is the degree of reproducibility of test results

obtained by analyzing the same samples under a variety of
normal test conditions.16
The method should not be affected by changes in time or place.
The reproducibility of the method should be established under
various conditions, for example with different reagents batches,
at different assay temperatures, and at different elapsed assay
times.
The ruggedness of an analytical method is determined by
analyzing subsamples of a homogeneous sample in different
laboratories and on different instruments. These tests should be
performed by different analysts under operational and
environmental conditions that may vary but that fall within the
specified parameters for the method. The degree of
reproducibility of the results is then calculated as a function of
the assay variables. This value can be compared with the
precision of the method under normal conditions to obtain a
measure of its ruggedness.
99
Instrumental requirements In developing an analytical method, full spectral data is valuable,

if not essential, for the evaluation of all possible choices. The
wavelength reproducibility of the instrument also must be
excellent so as not to restrict the choice of wavelength or
wavelengths. A spectrophotometer that automatically measures
multiple spectra and calculates average and standard deviation
values for each absorbance value can greatly improve
productivity as well. Finally, the instrumental software must be
able to process a large amount of data and use appropriate
statistical tools. Calculation by hand or the transferring of data
from the spectrophotometer to other applications for evaluation
seriously limits productivity.
Method validation Any analytical method designed for use in a regulated

environment, such as a pharmaceutical quality control laboratory,
must be validated. Method validation is the process for
determining whether performance characteristics of the method
are suitable for the intended purpose.
The United States Pharmacopoeia (USP) contains specific
guidelines for method validation.17 Method validation is similar
to method development in that it includes studies on specificity,
linearity, accuracy or recovery, sensitivity, and precision or
reproducibility. However, method validation must be kept
separate from method development and selection.16 Whereas
method development involves the selection of specific
parameters and conditions, method validation is used to confirm
that the performance characteristics of the method are in keeping
with the intended application in laboratory studies.
100
chapter 5
Chapter 5 Routine
operation
101
Routine operation
UV-visible spectroscopy is considered a routine technique
and is used extensively in QA/QC and similar laboratories.
Yet neither the considerations discussed in previous chapters
nor the best method development guarantees good results.
This chapter reviews steps to ensure accurate and precise
results on a regular basis and, perhaps more importantly, to
detect erroneous results.
Instrument In recent years, quality requirements as outlined by ISO 9000

performance (BS 5750), Good Laboratory Practice (GLP), Good
Manufacturing Practice (GMP), and the United Kingdom
verification National Measurement Accreditation Service (NAMAS) have
assumed increasing importance. Consequently, in the
pharmaceutical industry, the recommendations contained in
pharmacopoeias also have become more influential. Verification
of the continued proper performance of UV-visible
spectrophotometers is a key element of these quality
requirements.
Test parameters Although there is no clear definition as to which parameters

should be tested to verify instrument performance, manufacturers
and users generally agree that the criteria discussed below merit
inclusion.
Wavelength accuracy and Wavelength accuracy is the most important performance

precision criterion for differentiating spectra when the spectra are
measured on different instruments. It also plays a role in
102
Routine operation
quantitative analysis when extinction coefficients or factors are

used. Because these factors are wavelength dependent,
measurements should be performed at exactly the same
wavelength at which the factors were originally determined.
When comparing spectra or absorbance values measured on the
same instrument (as in most quantitative analyses in which a
standard is measured), however, wavelength precision is the
critical parameter (see “Wavelength accuracy and precision” on
page 55).
To measure wavelength accuracy and precision, a reference
standard is required. Ideally, this standard would have very
narrow, well-defined peaks at a series of wavelengths throughout
the UV and visible ranges, as shown in Figure 63. This ideal
standard does not exist, although some standards approach the
ideal. For actual wavelength accuracy standards, because the
peaks are not ideally symmetrical, changes in the slit width of the
instrument will affect slightly the measured position of the peaks.
Absorbance standard
Wavelength
Absorbance [AU]
standard
Wavelength [nm]
Figure 63
Ideal spectra of absorbance and wavelength standards
Photometric accuracy and Photometric accuracy is the most important criterion for
precision quantitative analysis when extinction coefficients or factors are
used. For comparative measurements (as above), however,
photometric precision is the critical parameter (see “Photometric
accuracy and precision” on page 56).
103
Routine operation
A reference standard is necessary in order to measure

photometric accuracy and precision. Ideally, this standard would
have constant absorbance at all wavelengths throughout the UV
and visible ranges (see Figure 63) so that any errors in
wavelength accuracy do not affect results. In practice, no such
standard exists, although neutral density glass approaches the
ideal in the visible wavelength range. Standards used typically
have broad peaks and valleys.
Stray light Stray light is the factor that affects most strongly the linear
relationship between absorbance and concentration at high
absorbances. It introduces a systematic bias to lower absorbances
at increasing concentrations. Stray light is also the primary
influence on the upper limit of the linear dynamic range for an
analysis (see “Stray light” on page 56).
To measure stray light, a filter is needed. Ideally, this filter would
absorb all light of the wavelength at which the measurement is to
be performed and transmit higher and lower wavelengths (the
sources of the stray light, as shown in Figure 64). In practice,
however, such a filter does not exist. Instead, cut-off filters that
transmit all light above or below a certain wavelength and that
block all light in the wavelength range are used.
Transmittance [%]
Wavelength [nm]
Figure 64
Ideal spectrum of a stray light filter
104
Routine operation
Resolution Resolution is a critical factor in determining the shape of

measured peaks. In general, the instrumental resolution should be
approximately 10 times better than the bandwidth of the peak
measured. If the instrumental resolution is not sufficient, the
absorbance measured at the wavelength maximum will be too
low. Thus photometric accuracy has a systematic bias. This bias
plays a key role if absolute accuracy is essential but, as discussed
in “Spectral resolution” on page 52, it is less important for
relative measurements such as quantification.
Measuring resolution is difficult, and empirical approaches, such
as measuring the relative heights of peaks and valleys for a
sample with a narrow band, are often used (see “Resolution” on
page 118).
Noise Noise is the major factor affecting the precision of absorbance

measurements. It is especially significant at low absorbances, at
which it is used determine the detection limit (see “Noise” on
page 57).
Noise typically is measured at zero absorbance, that is, with no
sample in the light path.
Baseline flatness To measure noise at all wavelengths usually is not practicable.

However, baseline flatness indicates relative noise at all
wavelengths and reveals wavelengths with instrumental
problems resulting from the switching of filters or source
exchange, for example.
Baseline flatness typically is measured at zero absorbance.
Stability Stability affects the accuracy of absorbance measurements as a

function of time. Drift in absorbance measurements introduces
systematic errors in photometric accuracy (see “Drift” on page
60).
Stability typically is measured at zero absorbance.
Linearity Linearity often is considered an important parameter for

performance verification. However, because linearity is strongly
105
Routine operation
sample dependent, we feel it is best measured during a system

suitability test, as described below.
The main problem in performance verification is the fact that all
of the above-listed parameters are wavelength dependent and, in
the case of stray light, sample dependent. Because it is not
practicable to perform all tests at all wavelengths, a few
wavelengths representative of the intended purpose should be
selected. The results of these tests should be compared with
absolute performance specifications for the methods in use.
Performance verification then will demonstrate effectively
whether the performance characteristics have changed in a way
that could affect the goodness of the analytical results.
Compliance with the above criteria (as determined using an
appropriate set of reference standards) does not guarantee that a
particular analysis can be performed with the required accuracy
and linearity, however. Because many parameters are sample
dependent, the desired accuracy and linearity can be achieved
only with an appropriate system suitability test performed on the
sample itself.
Standards A detailed discussion of all standards and their relative

advantages and disadvantages is beyond the scope of this primer.
Several excellent publications cover these topics in depth.18,19
Some general comments on the relative merits of the three main
types of standards follow.
Emission standards Certain emission sources, such as mercury and deuterium arc
lamps, exhibit sharp lines at specific wavelengths that are ideal
for wavelength accuracy and precision testing. In fact, in many
modern spectrophotometers, deuterium lines at 486.0 and
656.1 nm of the built-in source are used to check and recalibrate
for wavelength accuracy. However, emission sources require
electrical power supplies, and if the instrument has been
self-calibrated using its built-in deuterium lamp, another standard
should be used for verification. Moreover, because emission
sources are not typical samples, they do not test the full system.
106
Routine operation
Solid absorption standards Solid standards do not require any preparation, are easy to use
and maintain, are relatively insensitive to temperature, and have
good stability over time. However, with solid standards,
homogeneity cannot be ensured from one standard to another or
from one batch of material to another. Each standard therefore
must be calibrated individually on a reference
spectrophotometer. Because this process is time-consuming,
solid standards tend to be expensive. Moreover, since solid
standards are not absolutely stable, they must be returned for
recalibration at regular intervals. Finally, these samples must be
kept scrupulously clean and cannot be used for testing flow
systems.
Table 5 summarizes some of the best-known solid standards,
their uses, and their advantages and disadvantages.
Table 5 Some solid standards
Standard Use Advantages Disadvantages
Holmium oxide glass Wavelength standard Peaks at many wavelengths from Position of peaks varies from batch to
280 to 2000 nm batch
Didymium oxide Wavelength standard Peaks at many wavelengths from Position of peaks varies from batch to
glass 400 to 1920 nm batch
No peaks below 400 nm
Neodymium yttrium Wavelength standard Peaks at many wavelengths from No peaks below 350 nm
aluminum garnet 350 to 1000 nm
Neutral density glass Photometric standard Very flat absorbance profile in Not suitable for UV range
visible wavelength range Not absolutely stable; must be
recalibrated at intervals
Metal on quartz Photometric standard Very flat absorbance profile in UV Frequent interreflection
and visible wavelength ranges error problems
Temperature sensitive
Not very stable; must be recalibrated at
intervals
Liquid absorption standards Liquid standards normally are absolute physical standards. In
other words, if liquid standards are prepared using appropriately
pure materials, they possess inherently the properties of
107
Routine operation
absorptivity, peak maxima, and so on required for accuracy

checks. Because calibration of the solutions is unnecessary,
liquid standards can be relatively inexpensive. These standards
also can be used to check flow systems. Moreover, the process of
using a solution as a standard closely resembles that for a normal
sample.
The main disadvantage of liquid standards is that, in general, they
must be freshly prepared. This preparation is time-consuming
and requires a reasonable degree of skill on the part of the
operator. Some suppliers have attempted to overcome this
problem by supplying standards sealed in cuvettes. However,
because the cuvette itself contributes some absorbance, these
standards must be calibrated and recalibrated individually, which
increases costs. Furthermore, because liquid standards are less
stable than solid standards, they must be recalibrated more often.
Prepared solutions sealed in ampoules for use with normal
cuvettes are now available that are both easy to use and
cost-effective.20
Table 6 summarizes some of the best-known liquid standards,
their uses, and their advantages and disadvantages.
Table 6 Some liquid standards
Holmium oxide in perchloric Wavelength standard Peaks at many wavelengths from No usable peaks above 650 nm
acid 240 to 650 nm
Samarium oxide in perchloric Wavelength standard Peaks at many wavelengths from No usable peaks above 500 nm
acid 300 to 500 nm
Benzene (vapor) Wavelength standard Very narrow peaks from 230 to Very limited wavelength range
260 nm
Potassium dichromate in Photometric standard Broad peaks at 257 and 350 nm No absorbance in visible range
perchloric or sulfuric acid and broad valleys at 235 and Very pH sensitive and, as a
313 nm powerful oxidizing agent, can be
unstable
108
Routine operation
Table 6 Some liquid standards
Mixture of cobalt and nickel Photometric standard Peaks at 302, 395, 512, and Because bands are rather narrow,
salts 678 nm cover both UV and wavelength accuracy can affect
visible regions results
Sodium nitrite solution (50 g/l) Stray light Cuts off at ca. 390 nm None
Used to measure stray light at
340 nm or below
Potassium or sodium iodide Stray light Cuts off at ca. 260 nm Tendency to decompose
solution (10 g/l) Used to measure stray light at
200 nm or below
Potassium chloride solution Stray light Cuts off at ca. 200 nm Because the measurement is
(12 g/l) Used to measure stray light at performed on the side of the cut-off
220 nm or below slope, wavelength accuracy errors
can affect the measured stray light
Toluene in hexane Resolution Easy empirical approach using Can only be used to determine
peak (269 nm) and valley resolution at one point in the
(266 nm) in toluene spectrum spectrum May vary with
wavelength
Regulatory requirements In the following section we review some of the more important
regulatory requirements governing the use of UV-visible
spectrophotometers.
GLP/GMP GLP and GMP requirements for the validation of instruments can
be summarized as follows:
Documented verification that the system or subsystem performs
as intended throughout representative or anticipated operating
ranges.21
For spectroscopy, the following guideline is given:
Where appropriate, periodic performance checks should be
carried out (for example, ... the resolution, alignment and
wavelength accuracy of spectrophotometers etc.).22
109
Routine operation
European Pharmacopoeia The requirements governing UV-visible spectrophotometers used

for pharmaceutical analyses in Europe are contained in the
European Pharmacopoeia (EP).23 These requirements are based
on—and virtually identical to—those listed in national
pharmacopoeia such as the British Pharmacopoeia (BP) in Great
Britain and the Deutsche Arzneimittelbuch (DAB) in Germany:
Control of wavelengths—Verify the wavelength scale using the
absorption maxima of holmium perchlorate solution, the line of a
hydrogen or deuterium discharge lamp or the lines of a mercury
vapor arc shown below. The permitted tolerance is ± 1 nm for the
ultraviolet range and ± 3 nm for the visible range.
241.15 nm (Ho) 404.66 nm (Hg)
253.70 nm (Hg) 435.83 nm (Hg)
287.15 nm (Ho) 486.00 nm (Dβ)
302.25 nm (Hg) 486.10 nm (Hβ)
313.16 nm (Hg) 536.30 nm (Ho)
334.15 nm (Hg) 546.07 nm (Hg)
361.50 nm (Ho) 576.96 nm (Hg)
365.48 nm (Hg) 579.07 nm (Hg)
Control of absorbance—Check the absorbance using potassium
dichromate solution R at the wavelengths indicated in the
following table, which gives for each wavelength the exact value
of A (1 per cent, 1 cm) and the permitted limits.
Wavelength (nm) A (1 per cent, 1 cm) Maximum tolerance
235 124.5 122.9 to 126.2
257 144.0 142.4 to 145.7
313 48.6 47.0 to 50.3
350 106.6 104.9 to 108.2
Limit of stray light—Stray light may be detected at a given

wavelength with suitable filters or solutions: for example the
110
Routine operation
absorbance of a 1.2 per cent m/V solution of potassium chloride

R in a 1 cm cell should be greater than 2 at 200 nm when
compared with water as compensation liquid.
Resolution power—When prescribed in a monograph, measure
the resolution of the apparatus as follows: record the spectrum of
a 0.02 % V/V solution of toluene R in hexane R. The minimum
ratio of the absorbance at the maximum at 269 nm to that at the
minimum at 266 nm is stated in the monograph.
NOTE: The BP states that the ratio should “... be not less than
1.5 unless otherwise specified in the monograph.”
United States Pharmacopoeia In the United States, the regulatory requirements for UV-visible
spectrophotometers are not as clearly defined as in Europe. The
United States Pharmacopoeia24 (USP) XII, Section 831
(“Spectrophotometry and light scattering”) states:
Check the instrument for accuracy of calibration. ... The
wavelength scale may be calibrated also by means of suitable
glass filters, which have useful absorption bands through the
visible and ultraviolet regions. Standard glasses containing
didymium (a mixture of praseodymium and neodymium) have
been widely used. Glass containing holmium is considered
superior.
For checking the photometric scale, a number of standard
inorganic glass filters as well as standard solutions of known
transmittance such as potassium chromate or potassium
dichromate are available.
The latter contains a cross reference:
For further details regarding checks on both wavelength and
photometric scales of a spectrophotometer, reference may be
made to the following publications of the National Institute of
Standards and Technology ...
The National Institute of Standards and Technology (NIST) gives
a range of solid and liquid standards for determining wavelength
accuracy, photometric accuracy, and stray light.25 Table 7
summarizes the most important of these.
111
Routine operation
Table 7 NIST standards
SRM # Type NIST description
930 Neutral density glass filters This SRM is for the verification and calibration of the transmittance and absorbance
scales of visible absorption spectrometers.
931 Cobalt and nickel solution in The SRM is for the verification and calibration of the absorbance scales of ultraviolet
nitric/perchloric acid mixture and visible absoprtion spectrometers having narrow bandpasses.
935 Potassium dichromate solid This SRM is for the verification and calibration of the absorbance scales of ultraviolet
for preparation of test absorption spectrometers.
solution
2031 Metal on quartz This SRM is for the verification and calibration of the transmittance and absorbance
scales of ultraviolet and visible absorption spectrometers.
2034 Holmium oxide solution in This SRM is for use in the verification and calibration of the wavelength scale of
perchloric acid ultraviolet and visible absorption spectrometers having nominal spectral bandwidths
not exceeding 3 nm.
2032 Potassium iodide solid for This SRM is for use in the assessment of heterochromic stray radiant energy (stray
preparation of test solution light) in ultraviolet absorption spectrometers.
American Standard Testing The American Standard Testing Methods (ASTM)26 publishes
Methods test methods for measuring the key performance characteristics
of UV-visible spectrophotometers:
Wavelength accuracy and precision: determined using a
mercury vapor discharge lamp (UV region), a deuterium or
hydrogen arc lamp (visible region), benzene vapor (UV region),
or holmium oxide glass or holmium perchlorate solution (UV and
visible regions). The ASTM recommends that the calibration
wavelengths used bracket the analytical wavelength.
Linearity: determined by preparing an analytical working curve
with the target analyte (see “System suitability” on page 120).
Photometric accuracy: determined using NIST 930, 2031, or
935 standards.
Photometric precision: determined using a metallic screen or a
suitable glass filter.
112
Routine operation
The importance of slit width and resolution are mentioned in

ASTM documentation, but no practical method for measuring
these parameters is given. An additional ASTM publication27
describes standards and procedures for measuring stray light.
Table 8 summarizes the requirements and recommendations of
the various regulatory bodies.
Table 8 Test parameters and standards used by the main regulatory agencies
NIST AST
Test type Standard USP SRM EP M
Wavelength Holmium perchlorate solution — 2034 S X

accuracy
holmium oxide glass X — — X
Deuterium arc lamp X — S X
Mercury arc lamp X — S X
Benzene vapor — — — X
Photometric Potassium dichromate solution X 935 S X

accuracy and
Neutral density glass — 930 — X
linearity
Cobalt and nickel salts — 931 — X
solution
Metal on quartz — 2031 — X
Stray light Potassium chloride solution — — S X
Potassium iodide solution — 2032 — —
Sodium iodide solution — — — X
Sodium nitrite solution — — — X
Resolution Toluene in hexane solution — — S —
USP: Test method only

NIST SRM: Standard material and test method
EP: Test method and minimum performance specification
ASTM: Test method only
113
Routine operation
The choice of standards depends on the analyses to be performed

on the instrument. This condition is highlighted by GLP
requirements that verification should be performed for the
intended and anticipated operating ranges, by ASTM
recommendations that wavelengths used for calibration bracket
the analytical wavelength, and by NIST regulations. Thus, for
example, if the intended purpose is to measure absorbance in the
UV region (as in most pharmaceutical analyses), photometric
accuracy should be verified not in the visible range but in the UV
range, preferably at several wavelengths. Similarly, it is not
appropriate to verify wavelength accuracy only at the 656.1-nm
deuterium line since this is not a reliable indicator of wavelength
accuracy in the UV region.
Recommendations Although there is no definite set of standards for verifying the

performance of a spectrophotometer, the following tests are
recommended for those users who analyze primarily liquid
samples and who need to comply with general regulatory
requirements.
Wavelength accuracy: holmium perchlorate solution (40 g/l
holmium oxide in 10 % v/v perchloric acid). Figure 65 shows the
spectrum of this solution as measured on an HP 8453
spectrophotometer.
114
Routine operation
Absorbance [AU]
Wavelength [nm]
Figure 65
Spectrum of holmium perchlorate solution
Table 9 shows the values specified by NIST for the usable

reference peaks for three different instrumental spectral
bandwidths (SBWs). These values are preferable to EP values
because the EP does not specify the SBW or temperature used
and because the EP and NIST values exhibit significant
discrepancy.
115
Routine operation
Table 9 NIST values for holmium perchlorate solution peaks28
SBW
0.5 nm 1.0 nm 2.0 nm
241.01 241.08 240.90
249.79 249.87 249.98
278.13 278.10 278.03
287.01 287.18 287.47
333.43 333.44 333.40
345.52 345.47 345.49
361.33 361.31 361.16
385.50 385.66 385.86

416.09 416.28 416.62
— 451.30 451.30
467.80 467.83 467.94
485.27 485.29 485.33
536.54 536.64 536.97
640.49 640.52 640.48
Photometric accuracy: potassium dichromate solution

(approximately 60 mg/l in 0.01N sulfuric acid). Figure 66 shows
the spectrum of potassium dichromate. The EP values for this
standard are given on page 112.
116
Routine operation
Absorbance [AU]
Wavelength [nm]
Figure 66
Spectrum of potassium dichromate
If measurements are to be performed in the visible region,

additional testing with neutral density glass filters such as the
NIST SRM 930 is advisable.
Stray light: solutions of sodium nitrite (50 g/l), sodium iodide
(10 g/l), and potassium chloride (12 g/l) in water. These three
solutions enable the measurement of stray light at three different
wavelengths. Figure 67 shows the spectra of these solutions. In
general, sodium nitrite and sodium or potassium iodide is
recommended but, for those users who need to comply with EP
requirements, potassium chloride also may be necessary.
117
Routine operation
Transmittance [%]
Wavelength [nm]
Figure 67
Spectra of stray light standard solutions
Resolution: solution of toluene in hexane (0.02 % v/v), as

specified in the EP. Figure 68 depicts the spectrum of this
solution.
Absorbance [AU]
Wavelength [nm]
Figure 68
Spectrum of toluene in hexane
The resolution is estimated by taking the ratio of the absorbance

of the maximum near 269 nm to that of the minimum near
118
Routine operation
266 nm. This ratio is empirically related to the SBW, as shown in

Table 10.29
Table 10 SBW and 269/266 nm ratio (toluene)
Ratio of absorbance at
269 nm to absorbance at
SBW (nm) 266 nm
0.25 2.30
0.50 2.20
1.00 2.00
2.00 1.40
3.00 1.10
4.00 1.00
Noise, baseline flatness, and drift also should be measured with a

clear sample area. No standard is required.
Instrument self-test Full performance verification of a spectrophotometer is

time-consuming and thus normally done only at periodic
intervals. These intervals are determined according to the
stability of the instrument. To ensure that any deviations from
performance occurring between verifications are detected in a
timely fashion, the instrument should be equipped with self-test
routines that can be run on a daily basis. These routines should
include a check of the electronic and optical operation of the
spectrophotometer as well as wavelength accuracy checks with
one or both lines from the deuterium lamp.
119
Routine operation
System suitability System suitability is designed to evaluate the components of the

analytical system in order to verify that system performance
meets the standards required by the method. System suitability
should not be confused with method validation. Whereas method
validation is performed once at the end of method development,
system suitability tests are performed on a given system
periodically to determine its adequacy or effectiveness. System
suitability requirements are well-defined for chromatography
systems but not for UV-visible spectroscopy systems.
In practice, analysts have developed their own strategies for
performing system suitability tests on UV-visible instruments.
Two examples follow:
A. Measure and calibrate using one standard with a concentration
equal to 100 % of the expected component concentration. Then
measure and quantify the standard and the standard diluted by a
factor of two. The results of both samples should fall within a
specified percentage of the known concentration. Remeasuring
the standard confirms the accuracy of the initial measurement.
B. Measure first the standard and then a series of dilutions of the
standard and calculate the extinction coefficient (absorbance
divided by concentration) for each concentration. The values of
the extinction coefficients should not vary by more than a
specified percentage.
Proper operation However good quality control measures such as instrument

performance verification tests and method validation, accurate
and precise results remain highly dependent on the human factor.
Although this factor can never be eliminated, steps nonetheless
can be taken to reduce human error.
Electronic storage Modern spectrophotometers are almost invariably

microprocessor or computer controlled and should provide for
electronic storage of method parameters. Ideally, all relevant
120
Routine operation
parameters should be stored in a single method file under a

unique name. When the operator enters the name of the method,
all parameters should set automatically, thus eliminating
potential error. Regulated environments in particular would
benefit from a system that prevents operators from changing
method parameters or that keeps track of and reports any method
changes.
Standard operating Although spectrophotometers can store and automatically set

procedures most method parameters, some procedures—for example,
emptying, rinsing, and filling cuvettes with samples—must be
performed by the operator and can introduce substantial errors.
Operators should be properly trained in these procedures, and all
steps should be clearly documented in working instructions. In a
regulated environment, these steps are known as standard
operating procedures (SOPs).
Collateral data Errors or entirely incorrect results can occur despite the best
instrumentation, method development and validation, and
operator training. For example, the sample may be contaminated.
Whereas an incorrect result in itself is not necessarily
problematic, failing to recognize that a result is incorrect can
have serious consequences. Most analytical results obtained with
UV-visible spectroscopy are based on a single measurement at a
single wavelength. With a single data point, however, there is
virtually no way to detect whether a result is suspect unless a
typical result is known and the actual result deviates significantly
from the known value. Collateral data and multiple
measurements at a single wavelength or (preferably) at multiple
wavelengths can help ensure that a result is correct. The data also
can be used to investigate the reason for an incorrect result.
Confirmation wavelengths A simple way to verify quantitative analytical results is through

confirmation analysis. In confirmation analysis, the absorbance
at one or more wavelengths in addition to the analytical
wavelength is measured both on standards and on unknown
121
Routine operation
samples. Quantification is performed at all measured

wavelengths. If the sample is pure, the results at the analytical
wavelength and at the confirmation wavelength or wavelengths
will be identical. If the sample is contaminated, it is highly
probable that the contaminant will contribute a different
absorbance value at the analytical and conformation wavelength
or wavelengths, and results will differ. An example is shown in
Figure 69. Confirmation analysis also can be used to detect
whether the correct sample has been measured and whether
measurements lie outside the linear dynamic range of the
instrument.
Caffeine
Absorbance [AU]
Salicylic acid
Wavelength [nm]
Figure 69
Confirmation analysis
Full spectra For optimum results, the full spectra of the sample for analysis
should be acquired. The sample spectra can be overlayed onto the
standard spectra to check for any differences, or mathematical
methods can be used to obtain a match factor, which indicates the
similarity of samples to standards. The match factor is obtained
by plotting the absorbance values at each wavelength of the
sample against the standard. If the spectra are identical, the
plotted points will fall on a straight line. The slope of this line is
122
Routine operation
the ratio of the concentration of the sample to the standard. If the

spectra are not identical, the points will not fall on a straight line
but exhibit scatter. Figure 70 shows plots of both similar and
dissimilar spectra. In both cases, linear regression can be used to
fit the best straight line, and the correlation coefficient can be
calculated. The correlation coefficient is a measure of the
similarity of the sample spectra to the standard spectra. If spectra
are perfectly similar, the correlation coefficient will be 1,
whereas for completely dissimilar spectra, it will be close to 0.
Correlation = 0.999
Normalized
Standard
Unknown
Wavelength [nm] Unknown [mAU]
Unknown
Correlation = 0.056
Normalized
Standard
Wavelength [nm] Unknown [AU]
Figure 70
Comparative plots of similar and dissimilar spectra
If full spectra are used, all analytical information is available for

reevaluation of results and for determining why a result was
incorrect.
Statistics A single measurement does not have a high degree of validity.

All measurements include some degree of bias and random
variation due to noise. Although bias can be detected using
123
Routine operation
techniques such as those described above, noise also can result in

significant errors. The precision of results always should be
estimated by performing multiple measurements and then
calculating the average and the standard deviation of the average.
The standard deviation is an indicator of the validity of a
measurement. Monitoring of changes in the standard deviation of
results over time can be a useful diagnostic for many
measurement problems.
124
UVPRIMER.BK : appdxa.fm5 Page 125 Friday, December 20, 1996 4:12 PM
Appendix A
125
UVPRIMER.BK : appdxa.fm5 Page 126 Friday, December 20, 1996 4:12 PM
Definition of terms The terms accuracy and precision are used throughout this
primer, but not interchangeably. It is therefore important to
clearly understand the difference between them. As an analogy,
Figure 71 shows the performance of a marksman on a rifle range.
Figure 71
Example of precision and accuracy
In (a), the shots are neither accurate nor precise. In (b), the shots
are precise but inaccurate; the marksman is peforming well, but a
consistent bias is evident. In (c), the shots are accurate but
imprecise: the average of the shots would lie in the center of the
target, but the individual shots deviate significantly. In (d), the
shots are both accurate and precise.
126
UVPRIMER.BK : appdxb.fm5 Page 127 Friday, December 20, 1996 4:12 PM
Appendix B
Appendix A Characteristics of
diode array
spectrophotometers
127
Characteristics of diode array spectrophotometers
Advantages of diode As the leading manufacturer of diode array spectrophotometers,

array spectroscopy we feel this technology offers considerable advantages over
conventional scanning spectrophotometers. In the following
sections we review the advantages of diode array technology and
comment on some of the real or perceived disadvantages.
Fast spectral acquisition Fast spectral acquisition makes diode array spectrophotometers
the first choice for measurement of dynamic systems such as
flow injection analysis detection, process control, and kinetic
measurements. For example, Figure 72 shows spectra measured
at intervals of 1 s during a hydrolysis reaction of sultone. With
this data, the disappearance of the reactant and the appearance of
the product can be monitored simultaneously.
Absorbance [AU]
Wavelength [nm]
Figure 72
Spectra of hydrolysis of sultone
For most UV-visible spectrophotometer users who are not

performing measurements on dynamic systems, fast full spectral
acquisition still offers significant advantages. Because it is so
fast, this method enables the acquisition of full spectra even
when the analysis requires only a single wavelength. The full
spectral data can be used for error correction (see “Background
modeling” on page 78 and “Derivative spectroscopy” on page
128
81) and as collateral data for confirming data quality (“Full

spectra” on page 122).
Finally, full spectra can be obtained with high productivity for
use in method development (see “Method development” on page
88) and for multicomponent analysis (see “Multicomponent
analysis” on page 27).
Simultaneous Most conventional spectrophotometers can perform

multiwavelength multiwavelength measurements but must physically move from
measurement one point in the spectrum to another to do so, which takes time.
With a diode array spectrophotometer, all points in the spectrum
can be measured simultaneously. Multiple wavelength
measurements thus are completed in the time a conventional
instrument requires to perform a single wavelength
measurement. This technique facilitates the use of various
techniques for eliminating errors (see “Isoabsorbance” on page
77, “Internal referencing” on page 79, and “Three-point
correction” on page 80). Moreover, it provides collateral data for
confirming data quality (see “Confirmation wavelengths” on
page 121) and enhances productivity when the simple
simultaneous equations method is used (see “Multicomponent
analysis” on page 27). With multiwavelength measurement,
smaller quantities of data need to be stored than with full spectral
measurement. However, error-eliminating techniques are
somewhat less effective than those that can be used with full
spectra.
Through the use of alternative wavelengths for high and low
concentration ranges, dynamic range can be maximized with
diode array spectrophotometers. The absorption maxima can be
used for high-sensitivity measurements, and a wavelength with
lower absorption on the side of the absorption band prevents
errors due to stray light.
Finally, a diode array spectrophotometer can be used in many
applications where an expensive dual-wavelength
spectrophotometer (see “Dual-wavelength design” on page 50)
was previously required.
129
Wavelength resettability Conventional mechanical scanning spectrophotometers have an

inherent wavelength resettability error (see “Wavelength
accuracy and precision” on page 55) that increases with time as
mechanical linkages wear down. They are also susceptible to
wavelength drift over long periods of time. Because in diode
array instruments no parts are moved to change wavelength or to
scan, no significant mechanical error or drift with time occurs.
Data from all parts of the spectrum, unaffected by wavelength
resettability errors, is of benefit to the analyst. It allows selection
of the optimum wavelength for improved dynamic range,
sensitivity, and selectivity in method development (see “Method
development” on page 88). Moreover, it is essential in techniques
to improve sensitivity using wavelength averaging (see
“Wavelength averaging” on page 73), to decrease sensitivity for
strongly absorbing compounds (see “Strong absorbance” on page
74), and to confirm data quality with confirmation wavelengths
(see “Confirmation wavelengths” on page 121). Other techniques
that use full spectral data include multicomponent analysis (see
“Multicomponent analysis” on page 78), method development
(see “Method development” on page 88), confirmation of data
quality (see “Confirmation wavelengths” on page 121), and
derivative spectroscopy (see “Derivative spectra” on page 14 and
“Derivative spectroscopy” on page 81).
Excellent wavelength resettability also ensures that any
differences in measurements result from the sample and not from
instrument resettability errors. Positive identification of
compounds is thus possible even when the spectra are virtually
identical but exhibit a small wavelength shift.30
Moreover, the spectra of standards can be measured, stored on
disk, and recalled days, weeks, or even months later for use in
quantitative analysis without remeasuring the standards. This
method improves productivity, especially in the case of
expensive, difficult-to-obtain, or unstable compounds that cannot
be used routinely in the laboratory.
Sensitivity The dynamic range of a diode array spectrophotometer can be

extended further at low absorption levels. Diode array
130
instruments typically are less complex and have fewer optical

surfaces than conventional ones. As a result, light throughput is
higher and noise levels are lower.
The ability to acquire full spectra quickly also can help improve
spectral sensitivity using time averaging (see “Time averaging”
on page 73) and wavelength averaging (see “Wavelength
averaging” on page 73) techniques.
Measurement statistics Diode array spectrophotometers scan so quickly that multiple

measurements normally are performed and averaged together.
Both the average and standard deviation for each data point are
calculated. The standard deviation is a measure of the reliability
of the data point and is obtained for every absorbance value in the
spectrum. It has been said that no statistical significance can be
attached to a single measurment.12 With conventional
spectrophotometers, however, obtaining multiple measurements
is too time-consuming.
The statistical data is a valuable diagnostic tool for evaluating the
quality of the analysis results (see “Statistics” on page 123) and
also should be used during method development (the software
that comes with HP spectrophotometers automatically makes use
of such data).
Ruggedness and reliability Diode array spectrophotometers are mechanically simple in that
they have almost no moving parts. Because few pieces will wear
out or break, these instruments are highly reliable. The user
benefits from minimal down-time. In addition, cost of ownership
is lower than with conventional instruments because diode array
spectrophotometers do not require regular maintenance or
recalibration. Modern diode array spectrophotometers are
estimated to have only one failure in 10 years (excluding lamp
changes).
Open sample area Owing to the reversed optics design of the instrument (see “The
diode array spectrophotometer” on page 45), the sample area of a
diode array spectrophotometer does not need to be covered
because the instrument is not susceptible to interference from
131
ambient light. Moreover, this design increases productivity by

facilitating the exchange of samples, and a wider range of sample
types can be measured because size restrictions are fewer.
Changing or adding specialized sampling accessories is also
easier.
Disadvantages of diode Because diode array spectrophotometers differ from

array spectroscopy conventional scanning spectrophotometers, many objections
have been raised concerning their performance. In this section we
address briefly these objections.
Resolution With a conventional scanning spectrophotometer, resolution can

be changed easily by varying the slit width. With a diode array
spectrophotometer, however, resolution depends on an additional
factor: the sampling interval of the diode array. The sampling
interval in turn depends on the number of diodes in the array and
on the wavelength range measured. Until recently, diode array
instruments had some resolution limitations compared with 2-nm
resolution instruments. This problem has been corrected in
modern diode arrays, such as the HP 8453 (see “Dual-beam
design” on page 48), which contain more elements. Although
diode array instruments cannot achieve resolutions of 0.5 nm or
less as can some conventional instruments, such high resolution
may not be necessary in many analyses (see “Spectral resolution”
on page 52). Moreover, with higher resolution, less light reaches
the detector and sensitivity is lower. For example, if a spectrum is
scanned with a resolution of 0.1 nm, it can take as much as an
hour or more to obtain a spectrum with good S/N.
Stray light In a sequentially scanning instrument, stray light is reduced by

moving filters into the light path in front of the monochromator
(see “Stray light” on page 56). These filters must be changed in
accordance with the wavelength of the incident light. With a
diode array spectrophotometer, using filters in this way usually
limits the range of wavelengths that can be measured
simultaneously for a spectrum. Instead, filters are placed over the
132
array itself to reduce stray light. However, because all

wavelengths of light are always in the detector area, stray light is
more problematic than it is with conventional
spectrophotometers.
Until recently, diode array spectrophotometers exhibited higher
levels of stray light than conventional instruments. However, the
HP 8453 is based on a patented process that uses the fast full
spectrum scanning capability of diode array technology to reduce
significantly stray light. First, the full spectrum is scanned. A
filter that blocks all light below 430 nm is then dropped into the
light path, and the spectrum in the region up to 430 nm is
measured. The only light detected is stray light, which is then
subtracted from the first measurement to yield a stray light
corrected spectrum. The entire process takes only 1.5 s (with two
0.5-s measurements) and reduces stray light to levels equivalent
to or better than those obtained with comparable single
monochromator conventional scanning instruments.
Sample decomposition Because a diode array spectrophotometer irradiates the sample

with all wavelengths of light, it has been argued that the sample
will be degraded. However, the intensities of light used with a
diode array spectrophotometer are no higher than with a
conventional instrument. Thus, when a full spectrum is scanned
sequentially with a conventional spectrophotometer, the total
amount of light (the sum of all light at each wavelength) to which
the sample is subjected is the same as with a diode array
spectrophotometer. If the sample is photosensitive, both
instruments will decompose it to the same extent. Some
circumstantial evidence indicates that a diode array instrument in
some cases can acquire spectra of highly photosensitive
compounds that cannot be obtained with a conventional
instrument.31
When measuring a single wavelength, a conventional instrument
should have a clear advantage over a diode array
spectrophotometer since it subjects the sample to much less
irradiation. In practice, however, problems of photodegradation
133
are extremely rare. At the time of writing, we are not aware of

any documented cases.
Complexity Some users are intimidated by diode array instruments because

they believe them to be too complicated. Yet although the
internal data processing associated with diode array
spectrophotometers is indeed far more complex than that of
conventional instruments, the user is never made aware of the
difference. And, whereas diode array spectrophotometers
generate much more data than conventional instruments, this data
is easily managed with modern software. Moreover, the
additional data creates numerous advantages, as discussed
throughout this primer.
Errors in measuring Although diode array spectrophotometers can produce incorrect

fluorescent samples results when measuring fluorescent samples, conventional
scanning instruments are subject to error as well (see
“Fluorescence” on page 85 for an explanation).
134
UVPRIMER.BK : refs.fm5 Page 135 Friday, December 20, 1996 4:12 PM
References
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15. Understanding Your Advanced ChemStation Software,

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1710–1712, 1990; General Chapter <1225>.
18. Standards in Absorption Spectrometry, Techniques in Visible
and Ultraviolet Spectrometry: Volume 1; Burgess, C.;
Knowles, A., Eds.; Chapman & Hall: London, 1981.
19. Nowicka-Jankowska, T.; Gorczynska, A.; Michalik, A.;
Wieteska, E. Comprehensive Analytical Chemistry, Volume
XIX, Analytical Visible and Ultraviolet Spectrometry;
Elsevier: Amsterdam, 1986.
20. Owen, T. Performance Verification of UV-Visible
Spectrophotometers, Hewlett-Packard Company, in
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21. Alford, J.S.; Cline, F.L. “PMA’s computer system validation
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22. EURACHEM Guidance Document No. 1/WELAC Guidance
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138
UVPRIMER.BK : uvprimer.ix Page 139 Friday, December 20, 1996 4:12 PM
Index
Chapter 8
139
Index
A chromatic aberration, 43–44 emission standards, 107

chromophore, 18–19, 68 energy
absorbance, 11, 14, 29, 89, 111 cobalt salts, 109, 114 electronic, 12
and color, 21 in nitric/perchloric acid, 112 rotational, 12
bands, 15, 19, 82, 83 collateral data, 121–124, 129 vibrational, 12
interfering, 75, 76, 80 color, 21–22 enzyme activity, 23–24, 36
measurement errors in, 59–60, 66– concave mirrors, 43, 44 enzyme kinetic assays, 36
67, 71, 85 confidence interval, 95 European Pharmacopoeia (EP), 110–
strong, 74–75, 130 confirmation wavelength, 122, 130 111, 114, 115, 118
weak, 72–74 conventional spectrophotometer, 44–45 extinction coefficient, 26–27, 28, 29,
acceptance angle, 86 correction techniques, 77–84, 98, 129 103, 104, 120
accuracy, 93–94, 106 background modeling, 79
photometric, 57–60, 104, 105, 106, derivative spectroscopy, 81–84
113, 114, 116 internal referencing, 47, 61, 70, 80,
wavelength, 55–57, 103, 107, 109, 95 F
113, 114 isoabsorbance, 78, 98
additivity, 29, 92 multicomponent analysis, 22, 29–34, filters, 41, 86, 104
amino acids, 22 78, 129, 130 neutral density glass, 112, 117
apertured cells, 66, 67 three-point correction, 80 flow-through cells, 66
ASTM (American Standard Testing correlation coefficient, 90, 91, 92, 123 fluorescence, 11, 85–86
Methods), 113–115 forward optics, 85
frequency, 11
D
B
background modeling, 79
derivative spectra, 14–18, 19–20, 28 G
derivative spectroscopy, 81–84
balance measurement, 52 detector GLP (Good Laboratory Practice), 102,
baseline flatness, 106, 119 diode array, 42–43 110, 114
Beer’s law, 24–28, 29, 89 photodiode, 41–42 GMP (Good Manufacturing Practice),
Beer–Bouguer-Lambert law, 26–27 photomultiplier tube, 41 102, 110
benzene (vapor), 109, 113, 114 deuterium arc lamp, 39, 107, 113, 114 Golay. See Savitzky-Golay polynomial
blank, 44, 47, 48, 50, 52, 70, 71 Deutsche Arzneimittelbuch (DAB), technique
Bouguer-Lambert law, 25 110
British Pharmacopoeia (BP), 110, 111 didymium oxide glass, 108
diode array detector, 42–43
diode array spectrophotometer, 45–46 H
dispersion devices, 40–41
C DNA (deoxyribonucleic acid), 22–23 holmium oxide, 115
drift, 48, 50, 61, 74, 106, 119, 130 glass, 108, 113, 114
calibration curve, 27, 89–91 dual-beam spectrophotometer, 48–49 in perchloric acid, 109, 113
cell types, 65–66 dual-wavelength spectrophotometer, 51 holmium perchlorate solution, 113,
apertured, 66, 67 114, 115, 116
flow through, 66 holographic gratings, 40–41
microcells, 66 HP 8450A spectrophotometer, 49, 50
open-topped rectangular, 65 HP 8453 spectrophotometer, 47, 48,
ultramicrocells, 66
E 115, 133
unmasked, 67 electromagnetic hydrolysis reaction
cells, 64–67 radiation, 11, 38 of sultone, 128
care of, 67 spectrum, 10
material, 64–65 electronic energy, 12
chemical derivatization, 35
140
Index
I N in sulfuric acid, 109

potassium iodide, 109, 113, 114, 117
identification, 18–19, 20, 24 NAMAS (National Measurement precision, 66, 72, 94–95, 100, 124
instrumental spectral bandwidth Accreditation Service), 102 photometric, 57–60, 104, 113
(SBW), 53–55, 115, 116, 119 natural spectral bandwidth (NBW), 54– wavelength, 55–57, 103, 107, 113
interference, 17, 28, 36 55 principle component regression (PCR),
types of, 75–84 neodymium yttrium aluminum garnet, 34
internal referencing, 47, 61, 70, 80, 95 108 prisms, 40
ISO 9000, 102 neutral density glass, 104, 108, 114 proteins, 22
isoabsorbance, 78, 98 filters, 112, 117
and multicomponent analysis, 78 nickel salts, 109, 114
in nitric/perchloric acid, 112
NIST (National Institute of Standards Q
and Technology), 47, 112, 114,
L 115, 116 qualitative analysis, 18–24
nitric acid, 112 quantitative analysis, 24–35, 64, 70,
Lambert. See Bouger-Lambert law noise, 58, 72, 94, 105, 119, 124 103, 104, 130
lamp NPL (National Physical Laboratory),
deuterium arc, 39, 107, 113, 114 47
mercury arc, 107, 114
mercury vapor discharge, 113 R
tungsten-halogen, 39
xenon, 40 O range, 97
least squares method, 17, 32–33, 34, 35, Rayleigh scattering, 76
90, 97 open-topped rectangular cells, 65 reaction
lenses, 43–44 optics, 43–44, 67 photochemical, 11, 86
light sources, 39–40 forward, 85 reference
limit of detection (LOD), 95–96 reversed, 46, 85, 86, 132 standard, 103, 104
limit of quantification (LOQ), 95–96 values, 52
linear dynamic range, 59–60, 64, 74, wavelength, 70, 78, 80, 92
129, 130 reflection, 11
linearity, 89–93, 106, 106, 113, 114 P refractive index, 71
resolution, 111, 114, 132–133
partial least squares (PLS) method, 34 spectral, 52–55
Peltier temperature controller, 69 reversed optics, 46, 85, 86, 132
M perchloric acid, 109, 112, 113, 115 rotational energy, 12
pH, 19, 23, 36, 68–69 ruggedness, 99, 131–132
matrix, 17, 70, 76, 84 pharmacopoeia
mercury arc lamp, 107, 114 British, 110, 111
mercury vapor discharge lamp, 113 European, 110–111, 114, 115, 118
metal on quartz, 108, 112, 114 United States, 100, 111–113, 114 S
method development, 88–100, 129 phosphorescence, 11
method validation, 100 photochemical reaction, 11, 86 samarium oxide
microcells, 66 photodiode detector, 41–42 in perchloric acid, 109
monochromator, 41 photometric accuracy, 57–60, 104, 105, sample decomposition, 86, 133–134
Morton-Stubbs correction. See three- 106, 113, 114, 116 sample geometry, 71
point correction photometric precision, 57–60, 104, 113 Savitzky-Golay polynomial technique,
multicomponent analysis, 22, 29–34, photomultiplier tube detector, 41 17
129, 130 polychromator, 45 scattering, 11, 17, 76–77, 90, 95, 123
and isoabsorbance, 78 potassium chloride, 109, 114, 117 Rayleigh scattering, 76
multiple least squares (MLS) method, potassium dichromate, 112, 114 Tyndall scattering, 76
34 in perchloric acid, 109 Schlieren effect, 69, 70
141
Index
Schott noise, 58 sulfuric acid, 109 X

selectivity, 35, 97–99, 130 sultone, 128
self-test, 119 system suitability, 120 xenon lamp, 40
sensitivity, 35, 66, 77, 95–97, 130, 131
signal-to-noise (S/N), 17–18, 35, 73
simple simultaneous equations method,
29–32, 33, 34, 35, 97 T
single-beam spectrophotometer, 47
single-component quantification, 28 temperature, 19, 22–23, 27, 36, 68–70
slit width, 72, 132 three-point correction, 80
sodium iodide, 109, 114, 117 time averaging, 73, 95, 131
sodium nitrite, 109, 117 toluene
solid absorption standards, 107 in hexane, 109, 114, 118
solvent transmittance, 14, 26, 57
choice of, 67–68 tungsten-halogen lamp, 39
effect of, 68–70 Tyndall scattering, 76
SOP (standard operating procedure),
121
spectral resolution, 52–55
spectrophotometer U
conventional, 44–45
diode array, 45–46 ultramicrocells, 66
dual beam, 48–49 uncertainty, 90
dual wavelength, 51 United States Pharmacopoeia (USP),
HP 8450A, 49, 50 100, 111–113, 114
HP 8453, 47, 48, 115, 133 unmasked cells, 67
single beam, 47
split beam, 50
spectrophotometric titrations, 35
split-beam spectrophotometer, 50 V
stability, 39, 49, 51, 106
standard deviation, 92–93, 94, 95, 96, vibrational energy, 12
124, 131
standard error of regression, 90
statistical methods, 34, 90
correlation coefficient, 90, 91, 92, W
123
wavelength
least squares, 17, 32–33, 34, 35, 90,
accuracy, 55–57, 103, 107, 109, 113,
97
114
multiple least squares (MLS), 34
averaging, 73, 95, 130, 131
partial least squares (PLS), 34
confirmation, 122, 130
principle component regression
precision, 55–57, 103, 107, 113
(PCR), 34
reference, 70, 78, 80, 92
simple simultaneous equations, 29–
reproducibility, 18, 46, 57, 75, 100
32, 33, 34, 35, 97
resettability, 56, 57, 130–131
standard error of regression, 90
weak absorbance, 72–74
uncertainty, 90
working curve. See calibration curve
statistics, 124, 131
stray light, 57–58, 104–105, 109, 111,
114, 117–118, 133
strong absorbance, 74–75, 130
142

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toc Page i Friday, December 20, 1996 4:12 PM

Chapter 3 Sample handling and measurement

Chapter 4 Method development and validation

Chapter 5 Routine operation

System suitability............................................................................................... 120

Appendix A Accuracy and precision

Appendix B Characteristics of diode array

 Copyright Hewlett-Packard Company,

Preface In 1988 we published a primer entitled “ The Diode-Array

Chapter 1 Principles and

Principles and applications of UV-visible spectroscopy

This chapter outlines the basic theories and principles of

UV-visible spectroscopy. These provide valuable insight into

the uses and limitations of this technique for chemical

analysis. The primary applications of UV-visible

spectroscopy are also briefly reviewed.

Ultraviolet Visible Infrared

Principles and applications of UV-visible spectroscopy

The energy associated with electromagnetic radiation is defined

where E is energy (in joules), h is Planck’s constant

Wavelength and frequency Electromagnetic radiation can be considered a combination of

where ν is frequency (in seconds), c is the speed of light

Principles and applications of UV-visible spectroscopy

Etotal = E electronic + E vibrational + E rotational

The amount of energy a molecule possesses in each form is not a

Eelectronic > E vibrational > E rotational

In some molecules and atoms, photons of UV and visible light

These transitions should result in very narrow absorbance bands

Principles and applications of UV-visible spectroscopy

However, for molecules, vibrational and rotational energy levels

electronic energy levels

Principles and applications of UV-visible spectroscopy

Absorbance is defined as follows:

Derivative spectra If a spectrum is expressed as absorbance (A) as a function of

Zero order: A = f(λ)

Figure 5 on the next page shows the effects of derivatization on a

Principles and applications of UV-visible spectroscopy

derivative has a positive and a negative band with maximum and

Principles and applications of UV-visible spectroscopy

1st derivative 2nd derivative

3rd derivative 4th derivative

is fitted by the least squares method. The coefficients a0 … al at

Principles and applications of UV-visible spectroscopy

derivative, a2 is the second derivative, and so on. Savitzky and

Applications Derivative spectra can be used to enhance differences among

Signal-to-noise An unwanted effect of the derivatization process is the decrease

Instrumental considerations The higher resolution of derivative spectra places increased

Principles and applications of UV-visible spectroscopy

larger signal errors in the derivative mode than in the absorbance

Identification—spectra UV-visible spectra generally show only a few broad absorbance

Table 1 Selected chromophores and their absorbance maxima

Chromophore Formula Example λmax (nm)

Carbonyl (ketone) RR’C=O Acetone 271

Carbonyl RHC=O Acetaldehyde 293

Amide RCONH2 Acetamide 208

Ethylene RCH=CHR Ethylene 193

Acetylene RC=CR Acetylene 173

Principles and applications of UV-visible spectroscopy

Table 1 Selected chromophores and their absorbance maxima

Nitrile RC=N Acetonitrile < 160

Nitro RNO2 Nitromethane 271

The presence of an absorbance band at a particular wavelength

Confirmation of identity Although UV-visible spectra do not enable absolute

Principles and applications of UV-visible spectroscopy

When two Gaussian bands with a 40-nm natural spectral

Color Color is an important property of a substance. The color of matter

Principles and applications of UV-visible spectroscopy

Wavelength [nm] Absorbed color Complementary color

In practice, both the generation and sensation of color are highly

Principles and applications of UV-visible spectroscopy

and instrumentation to measure color have been developed.

Other qualitative UV-visible spectroscopy can be used to determine many

Deoxyribonucleic acid (DNA) in its native state comprises two