Application Note
01142
Wavelength Accuracy –
Measurement and Effect on Performance
in UV-Visible Spectrophotometry
Introduction
Key Words
• Wavelength
Accuracy
• Mercury Light
Source
• Deuterium Light
Source
• Emission Lines
• Holmium Filter
• Didymium Filter
• Rare Earth Oxides
Any quality control procedure will require that the
performance of the instrument be checked on specified
occasions. Verification of the wavelength accuracy will
form part of any such performance check.
This application note details how the wavelength
accuracy of a Thermo Electron Corporation UV-Visible
spectrophotometer may be checked and recalibrated
if necessary.
Several methods are listed below, in order of preference.
Methods 1 and 2 are primary methods employing fundamental constants, and should be used when available.
1. Emission lines from a Mercury source.
2. Emission lines from a Deuterium source.
3. Calibrated traceable Holmium & Didymium glasses.
4. Rare Earth Oxides in acid solution.
• Calibration
• NTRM
Factors Affecting Wavelength Accuracy
• NIST
Physical Shock
Accuracy may be affected if the spectrophotometer is
jolted violently, dropped, or subjected to severe or
prolonged vibration.
• CRM
• NPL
• ISO/IEC Standard
17025
• Neutral Density
Filters
• Wavelength
Calibration
• Holmium Oxide in
Perchloric Acid
• Calibrating
Solutions
Temperature
Problems may be found if the operating temperature is
significantly different from the temperature at which the
spectrophotometer was last calibrated. All instruments are
calibrated at room temperature and wavelength differences
of between 0.1 and 0.3 nm per 10°C are not uncommon.
It is therefore essential that instruments are warmed up
to normal operating temperature before use and before
performing any instrument verification test.
Replacement of Key Optical Components
The instrument will need recalibrating if any of the key
optical components are replaced.
Mechanical Wear
Thermo UV-Visible spectrophotometers have been
designed to minimize the effects of wear. Therefore this
should not be a problem.
Requirements for Wavelength Standards
A standard method should be:
• Readily available.
• Easily assembled or prepared.
• Easy and safe to handle.
• Suited to the optical properties of the instrument e.g.
spectral bandwidth, etc.
• Unaffected by environmental conditions, e.g. good
chemical stability, small temperature coefficients, etc.1
Instrumentation
Application 1
Use of Emission Lines from a Mercury Source
Mercury sources are available as optional accessories for
the following Thermo spectrophotometers:
INSTRUMENT
Nicolet® Evolution™ 300
Nicolet Evolution 500
UV Series 300 and 500
The mercury lamp fits into the third lamp position
within the optical system of the instrument, and is powered
from the instrument power supply. Once installed it can
be left in place and used when required.
Method: The emission spectrum of a low-pressure mercury
lamp has a number of intense lines that cover a large part
of the UV and visible range. The frequencies of these lines
are fundamental physical properties of the source element,
and therefore the wavelengths are invariant. The mercury
emission spectrum is a primary wavelength standard.
nm
253.65
296.73
404.66
435.84
546.07
760.95
871.68
To obtain an emission spectrum, in addition to selecting
the required wavelength range, it is important to select
appropriate parameters for the scan method. Suitable
values are as follows:
LOCAL CONTROL SOFTWARE
Scan Type
Mode
Bandwidth
Speed
Data Interval
Lamp Change
Standard Scan
I (Intensity Mode)
0.5 nm (if available)
30 nm/min
0.1 nm
Hg
VISION SOFTWARE
Type
Data Mode
Bandwidth
Scan Speed
Data Interval
Lamp Change
Standard
Intensity
0.5 nm (if available)
30 nm/min
0.1 nm
Mercury
When using an instrument with a photomultiplier
(PMT) detector, the FLAT EHT profile must be used for
this procedure.
Sample beam intensity (l units)
1.0
253.65
Sample beam intensity (l)
0.9
0.8
0.7
0.6
435.84
0.5
365.02
0.4
656.1
200000
486.0
150000
100000
50000
0
470
404.66
0.3
250000
490
510
530
546.07
0.2
550
570
590
610
630
650
670
Wavelength (nm)
0.1
576.96
Figure 2: Deuterium lamp emission spectrum
0.0
200
250
300
350
400
450
500
550
600
Wavelength (nm)
Figure 1: A mercury emission spectrum (rescaled)
Most of the energy in the mercury spectrum is emitted
in the 253.65 nm line, the intensity of which is much greater
than the other lines. When the spectrum is first scanned
only the 253.65 nm line will be apparent, because the
vertical scale will have been expanded to accommodate it.
When the scan is completed the spectrum can be
rescaled as follows, in order to see all the peaks. Select
MANIPULATE then rescale. Set GRAPH HIGH to 1 and
GRAPH LOW to 0. The top of the line at 253.65 nm is
off the scale but the lower energy lines are now clearly
visible. Values for the wavelengths can be obtained from
the Peak Table.
The permitted tolerances for wavelength accuracy
are quoted in the specifications of Thermo UV-Visible
spectrophotometers. The mercury lamp accessory enables
the user to demonstrate that the wavelength accuracy of
the instrument is within specification quickly and conveniently, whenever necessary.
If the required tolerances are not met, a wavelength
calibration is necessary. Under normal circumstances this
would only be expected following replacement of a major
optical component. The mercury lamp calibration remeasures all seven relevant emission lines, then recalculates
and replaces the software compensation equation.
The permitted tolerances for wavelength accuracy
are quoted in the specifications of Thermo UV-Visible
spectrophotometers. If the required tolerances are not
met, a wavelength calibration is necessary. Under normal
circumstances this would only be expected following
replacement of a major optical component.
The Deuterium lamp recalibration uses the Deuterium
emission line at 656.1 nm to adjust the software compensation equation and is intended to remove the effects of
small movements of optical components and shifts due
to temperature, shock or vibration. If the errors found
are linear and no more than 0.5 nm then a Deuterium
calibration can be used. The process will take about five
minutes and is fully described in the appropriate software
manual. This option only applies where an existing
calibration is present.
Application 3
Use of Rare Earth Glasses
A calibrated/traceable set of holmium & didymium glasses
is available as follows:
9423 185 03111 Set of 2 calibrated/traceable wavelength filters
• Certified Reference Materials (CRM’s).
• Holmium filter traceable to National Physical
Laboratory (NPL) and National Institute of Standards
and Technology (NIST) via ISO/IEC Standard 17025
calibration processes.
Application 2
• Didymium filter traceable to NPL only via ISO/IEC
Standard 17025 calibration process.
Use of Emission Lines from a Deuterium Source
• Supplied with calibration certificate.
Wavelength verification and recalibration can be performed
using the deuterium lamp in UV-Visible instruments having
a deuterium source (e.g. Nicolet Evolution 500, UV Series,
Spectronic® UV Helios range).
• UKAS accreditation number 0521.
Method: The emission lines produced from the deuterium
lamp are again characteristic of the source element; and as
a fundamental physical standard, these wavelengths are
invariant. However, unlike the mercury source, only two
characteristic lines are usable. These are at 486.0 and
656.1 nm.
The emission spectrum (over the desired range) can be
easily obtained by selecting scan method parameters as
above but with lamp change set to Deuterium (D2).
When using an instrument with a photomultiplier
(PMT) detector, the FLAT EHT profile must be used for
this procedure.
• Suitable for use with any appropriate Visible or
UV-Visible spectrophotometer.
• Includes wooden storage box.
Further details are also available in our Standards for
UV-Visible Spectrometry publication.2
NIST Traceable Reference Materials
9423 CRM 9300E NIST Traceable Reference Material
(NTRM) filter set 1
• Comprises 3 neutral density filters, a clear filter holder
and a wavelength check filter.
• Suitable for checking the photometric calibration of
UV-Visible spectrophotometers in absorbance or
transmittance.
• Supplied with calibration certificate.
• Includes filters of nominal values 0.5, 0.7 and 1A at
546.1 nm.
• Calibration values are given at 5 wavelengths.
• Holmium glass wavelength filter with certified
wavelengths.
• Includes transport case.
9423 CRM 1930E NTRM filter set 2
• Comprises 3 neutral density filters, a clear filter holder
and a wavelength check filter.
Wavelength accuracy checking using a traceable
holmium oxide glass filter can be conveniently carried out
using one of the following performance qualification
accessories:
PART NUMBER
DESCRIPTION
9423 UV6 1400E
Calibration Validation Carousel (NPL) for Spectronic
Helios Alpha/Beta
Intelligent Calibration Validation Carousel (NPL) for
Nicolet Evolution 300
Calibration Validation Carousel (NIST) for Spectronic
Helios Alpha/Beta
Intelligent Calibration Validation Carousel (NIST) for
Nicolet Evolution 300
Calibration Validation Unit (NPL) for Nicolet
Evolution 500/UV Series
Calibration Validation Unit (NIST) for Nicolet
Evolution 500/UV Series
10 01 0601
9423 UV6 1460E
• Suitable for checking the photometric calibration of
UV-Visible spectrophotometers in Absorbance or
transmittance.
10 01 0701
• Supplied with calibration certificate.
9423 UV6 1250E
• Includes filters of nominal values 0.3, 1.5 and 2A at
546.1 nm.
9423 UV6 1260E
• Calibration values are given at 5 wavelengths.
• Holmium glass wavelength filter with certified
wavelengths.
Application 4
• Includes transport case.
Use of Rare Earth Oxide Solutions
9423 CRM 9400E NTRM filter sets 1 and 2
Method: This technique uses solutions of rare earth oxide,
prepared by dissolution in acid media. The most frequently
used is holmium oxide in perchloric acid. The reference
wavelengths have been well-defined3, and a suitable
solution is available from various sources.
Once the solution has been obtained, the spectrum
(over the desired range) can be quickly obtained by selecting the normal scan mode. This procedure does not allow
for recalibration of the instrument, but its speed of implementation compared to Applications 1 & 2 makes it ideal
for routine checking of the wavelength calibration.
NB: The peaks produced when the holmium compound
is in solution are much sharper than those produced by
the glasses. They are therefore much more sensitive to
spectral bandwidth. On a variable bandwidth instrument,
0.5 nm should be used. Compensation tables are available
for use at other known bandwidths.3
• Suitable for checking the photometric calibration of
UV-Visible spectrophotometers in absorbance or
transmittance.
• Supplied with calibration certificate.
• Includes filters of nominal value 0.3, 0.5, 0.7, 1.0, 1.5,
and 2.0A at 546.1 nm.
• Calibration values are given at 5 wavelengths.
• Holmium glass wavelength filter with certified
wavelengths.
• Includes transport case.
Method: This technique uses glasses manufactured by
fusing the appropriate rare earth oxide in silica. The two
most frequently used are holmium and didymium (a mixture
of neodymium and praseodymium). Manufacturing can
cause batch-to-batch variation in these standards. This is
overcome by calibrating each standard glass against
certified reference glasses from the NPL and NIST
(Holmium) or NPL only (Didymium) via an ISO/IEC
standard 17025 procedure.
With the appropriate filter available the spectrum
(over the desired range) can be quickly obtained by
selecting the normal scan mode.
This procedure does not allow for recalibration of the
instrument, but its speed of implementation compared to
Applications 1 & 2 makes it ideal for routine checking of
the wavelength calibration.
NB: On a variable bandwidth instrument, 0.5 nm
should be used as the spectral bandwidth.
Results:
536.4
1.4
1.2
640.3
1.0
Absorbance
• Comprises 6 neutral density filters, clear filter holders
and a wavelength check filter.
0.8
278.1
361.2
485.1
0.6
0.4
249.7
385.3
0.2
0
245
295
345
395
445
495
Wavelength (nm)
Figure 3: Holmium oxide solution in perchloric acid
545
595
645
Far UV Wavelength Standard
This standard consists of a solution of a rare earth oxide
dissolved in dilute sulfuric acid, supplied in a permanently
heat-sealed Far UV quartz cell. The absorbance spectrum
of this rare earth contains a series of characteristic peaks
extending into the Far UV region.
Specification
Material
Solution of rare earth oxide in dilute
sulfuric acid. Consists of one sealed
cell with certified peaks at spectral
bandwidth values of 0.1, 0.2, 0.5,
1.0, 1.5, 2.0 and 3.0 nm.
Usable Range
200 – 300 nm, instruments with
spectral bandpass less than 5 nm
9423 UV9 5540E
UV Wavelength filter
Thermo Electron Corporation
has direct subsidiary offices in
North America, Europe and
Japan. To complement these
direct subsidiaries, we maintain
a network of representative
organizations throughout the
world. Use this reference list or
visit our Web site to locate the
representative nearest you.
References
Australia
Tel: +61 (0)2 9898 1244
1. C. Burgess & A. Knowles, UV Spectrometry Group, Standards in
Absorption Spectrometry, Chapman & Hall, ISBN 0-412-22470-4.
Austria
Tel: +43 (0)1 333 50 34 0
2. Data Sheet 18, “Standards for UV-Visible Spectrometry” (Thermo Electron
Corporation).
Belgium
Tel: +32 3890 4770
3. Holmium Oxide Solution Wavelength Standard From 240 to 640 nm,
SRM 2034 NBS Special Publication, 260-102.
Canada
Tel: +1 905 890 1034
China
Thermo ARL:
Tel: +86 10 6833 6715
Thermo Elemental:
Tel: +86 10 6592 0232
Thermo Nicolet:
Tel: +86 10 6597 3388 ext 2912
France
Tel: +33 (0)1 39 30 53 00
Germany
Tel: +49 (0)6102 3671 0
Italy
Tel: +39 02 6601 6351
Japan
Thermo Elemental:
Tel: +81 774 201245
Thermo Nicolet:
Tel: +81 45 450 1112
Netherlands
Tel: +31 76 5724840
Nordic
Thermo Nicolet:
Tel: +358 9 3291 00
Thermo ARL:
Tel: +46 (0)8 556 468 31
South Africa
Tel: +27 (0) 11 570 1840
Spain
Tel: +34 (91) 657 49 30
www.thermo.com/spectronic
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Madison, WI 53711-4495
Tel: +1 800 201 8132, +1 608 276 6100
Fax: +1 608 273 5046
Email: [email protected]
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Cambridge, CB5 8HY, UK
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Email: [email protected]
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case postale, CH-1024, Ecublens
Tel: +41 (0)21 694 71 11
Fax: +41 (0)21 694 71 12
Email: [email protected]
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