Technical

Note: 51721
Understanding Spectral Bandwidth and
Resolution in the Regulated Laboratory
Nicole Kreuziger Keppy, Michael Allen, Ph.D., Thermo Fisher Scientific, Madison, WI, USA

Introduction
Key Words Selecting the appropriate spectrophotometer to comply
• British with the complex requirements of a regulated laboratory
Pharmacopeia can be a daunting task.1 Two specifications that have
(BP) become increasingly challenging are resolution and spectral
bandwidth. Confusion over the requirements for regulatory
• European compliance has led, in particular, to two widespread myths
Pharmacopeia regarding spectral bandwidth and resolution.
(EP)
• Regulatory Myth 1: Photometric analysis requires a
Compliance spectrophotometer with a 1.0 nm spectral
bandwidth for regulatory compliance.
• Resolution
Myth 2: A smaller spectral bandwidth generates
• Spectral higher quality data.
Bandwidth
This application note will address these two myths by
• Toluene in
explaining the principles of spectral bandwidth and
Hexane Solution
resolution. Furthermore, it will address the regulatory
• U.S. requirements for these specifications in the pharmaceutical Figure 1: Gaussian intensity distribution of wavelengths emerging from the
Pharmacopeia industry as dictated by the pharmacopeias of the United monochromator. The spectral bandwidth is defined by the red boundaries and
(USP) States, Europe and Britain referred to in this document as λSB. The spectral slit width is depicted by the blue boundaries and λSSW.
the USP, EP and BP, respectively.
• UV-Visible
What is Resolution?
Spectrophotometer
What is Spectral Bandwidth? Reduced to its most basic definition, resolution is defined
When a monochromator is set to a particular wavelength, as the ability of an instrument to separate light into finite,
light with a Gaussian intensity distribution of wavelengths distinct wavelength regions and distinguish these finite
emerges from the exit slit. This intensity distribution is regions from each other. Resolution is primarily governed
depicted in Figure 1. In this figure, λ1 and the dotted vertical by the physical slit width of the instrument, in combination
line represent the wavelength set on the instrument. The with the inherent optical dispersion of light from the exit
heavy black line represents the distribution of light that of the monochromator to the detector of the instrument.
reaches the sample. The spectral bandwidth is defined as the Reducing the physical slit width decreases the spectral
width of the band of light at one-half the peak maximum bandwidth and improves the ability of the instrument to
(or full width at half maximum [FWHM]) and is represented resolve closely spaced peaks.
by the two vertical red lines and λSB on the wavelength axis. In the pharmaceutical industry, a 0.02% solution of
Spectral slit width, a related term frequently referenced in Toluene in Hexane is the currently accepted standard for
EP regulations, is defined as the total spread of wavelengths the verification of the resolution of a spectrophotometer.
represented by the blue lines and λSSW in Figure 1. The The resolving capacity of the spectrophotometer is
spectral bandwidth of the instrument will always be narrower calculated using the ratio between the absorbance maximum
than the spectral slit width. and minimum of the standard. Table 1 depicts the correlation
The spectral bandwidth of a spectrophotometer is of spectral bandwidth to resolution for a 0.02% solution
related to the physical slit-width of the monochromator of toluene in hexane as the ratio between the maximum
and, therefore, to the resolution capabilities of the and minimum absorbance of the solution.
instrument. To the best of our knowledge, there are no
formal specifications for spectral bandwidth defined in any Spectral 3.0 nm 2.0 nm 1.5 nm 1.0 nm 0.5 nm
Bandwidth ± 0.2 nm ± 0.2 nm ± 0.1 nm ± 0.1 nm ± 0.1 nm
Pharmacopeia. Some pharmacopeias do call out the
Resolution 1.0 – 1.1 1.3 – 1.4 1.6 – 1.7 1.9 – 2.0 2.3 – 2.4
advantages of a variable bandwidth instrument. Assay
optimization on a variable bandwidth instrument will be Table 1: The correlation of spectral bandwidth to resolution for a Toluene in
discussed in the following sections. Hexane Solution Standard measured at ambient temperature (25 °C)
What are the Regulatory Requirements When performing quantitative measurements it is
for Resolution? important to maximize the signal-to-noise ratio for the
• USP: No specific requirement is given in Section 851 of compound(s) of interest. This is achieved by optimizing
the USP, which provides the specifications for UV-Visible the ratio of the peak absorbance measured with respect to
spectrophotometers. the noise associated with the baseline (signal-to-noise ratio).
• EP (or Ph. Eur.): The current version of the EP does not The spectral bandwidth selected for an analysis should
contain any general instrument requirements for spectral be close to, but not less than, 1/10 of the width of the
bandwidth. It does include requirements for testing the absorbance peak at half-height. A variable bandwidth
resolution of the spectrophotometer and choosing the instrument allows the analyst to balance the need to resolve
slit-width when a variable slit-width spectrophotometer spectral features with the ability to achieve the desired limits
is used. In method 2.2.25, the EP describes these of detection and quantitation.
requirements as follows: In the following illustrations, different compounds were
– For Qualitative Analysis: “When prescribed in a analyzed at bandwidths of 0.5, 1.0, 1.5, 2.0 and 4.0 nm. The
monograph, measure the resolution of the apparatus signal-to-noise ratios of the selected peaks were calculated
as follows: record the spectrum of a 0.02 per cent V/V and the combined information was plotted. As you can see
solution of toluene R in hexane R. The minimum ratio
from Figure 2, the signal-to-noise ratio of Compound A is
of the absorbance at the maximum at 269 nm to that
maximized at 1.0 nm; however, the signal-to-noise ratio of
at the minimum at 266 nm is stated in the monograph.”
Compound B is maximized at 2.0 nm. In this case, we can
– For Quantitative Analysis: “To avoid errors due to
conclude that the analyses of these compounds can be
spectral slit-width, when using an instrument on which
optimized at different bandwidths, especially when low
the slit-width is variable at the selected wavelength,
the slit-width must be small compared with the half- limits of quantitation and detection are desired.
width of the absorption band but it must be as large
as possible to obtain a high value of I0. Therefore, a
slit-width is chosen such that further reduction does
not result in a change in absorbance reading.”
To the best of our knowledge the only monograph that
currently specifies a required resolution for the
spectrophotometer is monograph 0113 for Benzylpenicillin
potassium. This monograph specifies a resolution of at
least 1.7.
• BP: The 2008 edition of the British Pharmacopeia refers
to method 2.2.25 of the EP for instrument requirements.
Within the BP, specific monographs may require an
instrument with a bandwidth < 2.0 nm.

Advantages of a Variable Bandwidth

Spectrophotometer
For laboratories which require compliance to multiple
regulatory agencies for a variety of applications, the variable
bandwidth spectrophotometer offers a simple solution. It
is important when using a variable bandwidth instrument
to recognize that it is not ideal to use the narrowest available
bandwidth at all times. Reducing the bandwidth leads to a
reduction in energy reaching the sample. This may result
in the reduction of:
• Data Precision and Repeatability Figure 2: Graphical display of signal-to-noise ratio vs. spectral bandwidth for
Compounds A and B. The maximum of the plot for Compound A indicates the
• Accuracy of Results maximum SNR occurs at a spectral bandwidth of 1.0 nm. The maximum of
• Detection and Quantitation Limits the plot for Compound B indicates the maximum SNR occurs at a spectral
bandwidth of 2.0 nm.
• Method Robustness
Conclusion References In addition to these
1. For more information on these requirements, see Thermo Scientific offices, Thermo Fisher
Many 1.0 nm fixed bandwidth instruments are available;
Application Note AN51111 U.S. and European Pharmacopeias and Scientific maintains
however, the optimal bandwidth for an analysis is not UV-Visible Spectrophotometers.
a network of represen-
necessarily 1.0 nm. Although a narrower spectral bandwidth 2. A spectral bandwidth of 0.2 nm is only available with the Evolution™ 600
tative organizations
does improve the resolution of closely spaced peaks, it Spectrophotometer.
throughout the world.
also decreases the signal-to-noise ratio. Consequently, a
spectral bandwidth should be chosen based on both the
needs of the analysis being performed and within the
regulatory guidelines of the appropriate pharmacopeia,
when applicable.
The Thermo Scientific Evolution 300 and Evolution Africa-Other
600 variable bandwidth UV-Visible spectrophotometers +27 11 570 1840
are designed to accommodate the needs of a variety of Australia
+61 2 8844 9500
applications. These instruments have the flexibility to adjust Austria
the bandwidth of the instrument for measurements at +43 1 333 50 34 0
bandwidths of 0.2, 0.5, 1.0, 1.5, 2.0, and 4.0 nm.2 A wide Belgium
+32 2 482 30 30
range of fixed spectral bandwidth instruments are also
Canada
available for routine applications at 1.0, 1.8, 2.0 and 5.0 nm. +1 800 530 8447
An NIST-traceable Toluene in Hexane Solution Standard is China
offered for verifying the resolution of any spectrophotometer. +86 10 8419 3588
Denmark
+45 70 23 62 60
Europe-Other
+43 1 333 50 34 0
Finland / Norway /
Sweden
+46 8 556 468 00
France
+33 1 60 92 48 00
Germany
+49 6103 408 1014
India
+91 22 6742 9434
Italy
+39 02 950 591
Japan
+81 45 453 9100
Latin America
+1 608 276 5659
Middle East
+43 1 333 50 34 0
Netherlands
+31 76 579 55 55
South Africa
+27 11 570 1840
Spain
+34 914 845 965
Switzerland
+41 61 716 77 00
UK
+44 1442 233555
USA
+1 800 532 4752

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