TRACEABILITY OF SPECTROPHOTOMETRIC MEASUREMENTS
TO THE HIGHEST STANDARD OF CALIBRATION
R.D. Pepenene, E.M. Coetzee
National Metrology Institute of South Africa (NIMSA)
Private Bag X34, Lynnwood Ridge, Pretoria, 0040, South Africa
rpepenene@nmisa.org
Phone: 012 841 4260 Fax: 012 841 3131
ABSTRACT
UV-Visible spectrophotometers are versatile instruments that measure a variety of different
samples in diverse laboratory settings. The technique is mainly used quantitatively (although
some qualitative analysis can also be performed). For any type of critical determination,
whether it be clinical, pharmaceutical or industrial quality control, environmental analysis or
research, it is essential that the instrument is verified to ensure that its measurements are
traceable to appropriate measurement standards maintained at the National Metrology
Institute. Traceability ensures equivalent measurements are made regardless of the instrument
or the region of the world where the measurement is made.
In this paper, the principal calibration of secondary reference materials (Holmium Oxide
filters and Neutral density filters) used in the verification of laboratory spectrophotometers
for accurate absorption spectroscopy will be discussed. Other tests (e.g., the linearity of the
absorption scale, baseline measurement and absorbance stability) will be used to test the
performance of the Transfer Reference Spectrophotometer used to maintain and disseminate
the national transmittance/absorbance scale.
Keywords: UV-Visible Spectrophotometer; Traceability of Measurement Results; Certified
Reference Materials (Holmium Oxide Filter and Neutral Density Filter).
1. INTRODUCTION
Through the years, ultraviolet and visible spectrophotometry has been the method of choice
in most laboratories concerned with the identification and measurement of organic and
inorganic compounds in a wide range of products and processes e.g. in foodstuffs,
pharmaceuticals, mineral oils and in paint. For any type of critical determination, it is
essential that the wavelength accuracy and the photometric accuracy (absorbance accuracy)
of an instrument are verified for the accurate peak wavelength determination and correct
absorbance measurement [1]. Secondary reference materials used for this purpose need to be
certified by either a national metrology institute (NMI) or an accredited calibration laboratory
to ensure traceability of the measurement results to the highest standard. It is within the
National Metrology Institute of South Africa (NMISA’s) mission to maintain the national
transmittance/absorbance scale, and disseminate it for the benefit of laboratories, industries,
and others who need the highest accuracy measurements.
2. ABSORPTION SPECTROSCOPY
Ultraviolet-visible spectrophotometry (UV/Vis) refers to absorption spectroscopy in the
ultraviolet-visible spectral region. This method of analysis is based upon the fact that the
absorbance of the compounds is measured precisely at the specific wavelength of the
absorption peak (λmax). The Beer-Lambert law states that the amount of light radiation
absorbed by a sample is directly related to the concentration of the coloured compound in the
sample [2]. Thus, for a fixed path length, UV/V is spectroscopy can be used to determine the
concentration of the absorber in a solution (equation 1). This makes it important that the
wavelength accuracy and the photometric accuracy (absorbance accuracy) of an instrument
be checked for the accurate peak wavelength determination and absorbance measurement.
𝐼
𝐴 = log10 (𝐼0 ) = 𝜀. 𝑐. 𝑙…………………………………………………………………....... (1)
1
Where 𝐴 is the measured absorbance, in Abs,𝐼0 is the intensity of the incident light at a given
wavelength, 𝐼1 is the transmitted intensity, 𝑙 the path length through the sample, and c the
concentration of the absorbing species. For each species and wavelength, ε is a constant
known as the molar absorptivity which is a measurement of how strongly a chemical species
absorbs light at a given wavelength).
3. UV-VISIBLE SPECTROPHOTOMETER
The Reference Transmittance Spectrophotometer (RTS) housed in the NMISA is the national
measurement standard which establishes the national scale for regular spectral transmittance
measurements at controlled conditions of humidity and temperature. This instrument is linked
to the primary transmittance scale by means of optical filter reference materials manufactured
and calibrated at the Nation Physical Laboratory (NPL) in the United Kingdom. It consists of
a source of radiant energy, a double dispersing system to provide monochromatic radiation,
and a detector system to measure the amount of radiation through the instrument [3] (see
figure 1).The readout consists of adjusting the response to a value of 100% for the reference
or standard. With the sample in place, the response of the detector represents the percentage
of the sample’s transmittance to that of the reference.
The path of radiation is split into two parts within the instrument to provide a sample beam
and a reference beam. When a sample is placed in the sample beam, the equality of the two
beams is broken and the detector senses the difference and relates that to the transmittance of
the sample at that wavelength.
Figure 1.Optical diagram of the Hitachi U-3400 recording spectrophotometer
Since the illumination in the instrument is monochromatic, the spectral-power distribution of
the source is not a factor as long as the intensity is sufficient to allow a satisfactory signal-tonoise ratio in the response [4]. Any attenuation of radiation other than that of the sample,
such as lenses, mirrors, prisms, gratings and so on; are cancelled out because of the relativity
of the measurement. The readout is displayed graphically with the percentage of
transmittance plotted as a function of wavelength, and the absorbance (Abs) can be obtained
by the log conversion of the transmittance.
100
𝐴 = log10 (
𝑇
) ………………………………………………………………………….….(2)
Where A is the absorbance, in (Abs) and T is the transmittance in percentage (%).
4. REFERENCE MATERIAL
Laboratory spectrophotometers are available in a multitude of optical designs, specifications,
spectral measurement ranges, capabilities, and operational configurations. Through
calibration, equivalent measurements traceable to the national transmittance scale are
possible regardless of the instrument design or region of the world where measurements are
made. The practical way of calibrating the spectrophotometer will be to send it to the
calibration laboratory. This process has proven to be costly and sometimes cumbersome. The
availability of secondary reference standards is the ideal and most hassle-free approach to
calibrating the instrument.
The wavelength and photometric reference standard of preference is the one which exhibits
the most certified absorption peaks and transmittance/absorbance values in the same spectral
region of the analytical method. For any wavelength standard, if no certified absorption peaks
fall within the spectral region of interest, the use of a wavelength reference material that
exhibits a certified absorption peak closest to the analytical wavelength of interest is
acceptable [5]. Optical neutrality is a desirable spectral characteristic of a photometric
reference material because it relaxes the restrictions on finite spectral bandwidth specification
and wavelength accuracy to validate transmittance/absorbance measurements. A photometric
reference material is considered optically neutral if its transmittance/absorbance spectrum
varies little with wavelength.
Holmium oxide solution is superior to the holmium oxide glass filter as a UV/VIS
wavelength reference material because of its higher certification accuracy and better UV
spectral range coverage [5]. Having several certified absorption peaks in the 650 nm to 900
nm spectral range, the didymium oxide glass filter is preferred for validation of the
wavelength scale in the near infrared spectral region. Solid photometric reference materials
exhibit a higher degree of optical neutrality than photometric reference solutions [5]. The
optical neutrality attribute is one of several reasons why solid photometric standards are
preferred over photometric reference solutions for most photometric validations. Because of
the lack of optical neutrality in their respective absorption spectra, the
transmittance/absorbance certification for the photometric reference solutions is restricted to
their fixed standard wavelengths.
5. VERIFICATION OF A REFERENCE TRANSFER SPECTROPHOTOMETER
WITH PRIMARY STANDARD FILTERS
Verification of the wavelength accuracy and photometric scale on the Reference
Transmittance Spectrophotometer is performed by using the standard reference material
traceable to the National Physical Laboratory (NPL).
5.1. Baseline correction
It is important to perform a baseline correction before the start of every measurement, as this
will affect the accuracy of the photometric scale of the spectrophotometer (figure 2) and it is
always good practice to verify the wavelength scale of the instrument before the photometric
scale and the linearity of the absorption scale can be performed.
120
Transmittance (%)
100
80
60
Before Correction
40
After Correction
20
0
380
480
580
Wavelength (nm)
680
780
Figure 2.Effect of baseline correction
5.2. Primary Wavelength standard filters
To cover the wide spectral measurement range used in the different laboratory set-ups, the
operational performance and data quality of the reference transmittance spectrophotometer is
optimised to include a wide range of spectral regions. The results in figure 3 show the
nominal wavelength of absorption maximum of the primary wavelength standard filters
covering the UV-Visible range. The measurements were performed at a bandwidth setting of
2 nm and the wavelength peak values obtained at each calibration interval were compared
with the certified values obtained from the NPL calibration certificate. The red error bars
indicate the expanded (k=2) claimed by the NPL, which was ± 0,14 nm for wavelengths
230,59 nm and 292 nm. The black error bar indicate the expanded uncertainty (k=2) claimed
by the NMISA which was 0.4 nm for all the indicated wavelengths.
Wavelength (nm)
230.9
(a) Wavelength of absorption maxima (230,59 nm)
230.7
230.5
230.3
NMISA
230.1
NPL
229.9
229.7
229.5
2001
2004
2007
2010
Calibration Interval
2013
Wavelength (nm)
293.3
293.1
292.9
292.7
292.5
292.3
292.1
291.9
291.7
291.5
2001
(b) Wavelength of absorption maxima (292,83 nm)
NMISA
NPL
2004
2007
2010
2013
Calibration Interval
Wavelength (nm)
355
(c) Wavelength of absorption maxima (354,22 nm)
354.8
354.6
354.4
NMISA
354.2
NPL
354
353.8
353.6
2001
2004
2007
2010
2013
Wavelength (nm)
Calibration Interval
482.5
482.3
482.1
481.9
481.7
481.5
481.3
481.1
480.9
480.7
480.5
2001
(d) Wavelength of absorption maxima (481,32 nm)
NMISA
NPL
2004
2007
2010
2013
Wavelength (nm)
Calibration Interval
589.6
589.4
589.2
589
588.8
588.6
588.4
588.2
588
2001
(e) Wavelength of absorption maxima (588,57 nm)
NMISA
NPL
2004
2007
2010
Calibration Interval
2013
Wavelength (nm)
749.3
749.1
748.9
748.7
748.5
748.3
748.1
747.9
747.7
747.5
2001
(f) Wavelength of absorption maxima (748, 37 nm)
NMISA
NPL
2004
2007
2010
2013
Calibration Interval
Figure 3.The nominal wavelength of selected absorption maxima of a holmium oxide filter
(a-f) measured at the scheduled calibration interval.
The results shows that the Reference Transmittance Spectrophotometer can measure the
absorption peaks within the NPL claimed uncertainty values. The wavelength accuracy over
scheduled intervals indicates that the performance of the moving mechanical components
(which is responsible for positioning the monochromator) is stable.
5.3. Primary Neutral density filters
5.3.1. Precision and accuracy check
Like the wavelength standard filters, the photometric standard filters are available in different
transmittance levels to cover a wide range of transmittance percentages. Figure 4 shows the
selected transmittance filters having different nominal transmittance levels (low, medium and
high) used to verify the photometric scale on the reference spectrophotometer. The
transmittance values presented are the average of the values measured over a period of nine
years. The measurements were made at a spectral bandwidth setting of 2 nm and the values
were compared with the one obtained from the calibration certificate. The uncertainty of the
measurements claimed was ± 0.4 % transmittance.
100.00
NMISA AA03
90.00
NMISA AC03
Tranmittance (%)
80.00
NMISA AF03
70.00
NMISA AK03
60.00
NMISA AL03
50.00
NPL AA03
40.00
NPL AC03
30.00
NPL AF03
20.00
NPL AK03
10.00
NPL AL03
0.00
380
480
580
Wavelegnth (nm)
680
780
Figure 4.Average transmittance values of the selected filters measured at the scheduled
calibration interval
As indicated in figure 4, the stability of the photometric scale over the scheduled calibration
interval is adequate, thus the variations that might occur during the calibration of filters will
not limit the precision and accuracy of the measurement data.
5.3.2. Linearity of the photo-detectors
Comprehensive validation of the spectrophotometer at periodic intervals includes a
verification of photo-detector linearity using the full transmittance/ absorbance range of the
photometric reference material standards. Figure 5 show the neutral density filters with
different transmittance/ absorbance levels used in the range of the calibration (near ultraviolet
to visible). The measured transmittance values are plotted against the known NPL calibration
values. The transmittance values vary from 0.08 %T to 80 %T and this corresponds to
absorbance values ranges from 0.09 to 3 absorbance units. The results show that the photodetector linearity is appropriate over the transmittance/absorbance range of the calibration.
AA03
AB03
90
AC03
NMISA Transmittance (%)
80
AD03
70
AE03
60
AF03
50
AG03
AH03
40
AJ03
30
AK03
20
AL03
10
AM03
0
0
20
40
60
NPL Transmittance (%)
80
100
Figure 5.Linearity of the photo-detectors covering the near ultraviolet-visible region
6. DISSEMINATION OF NATIONAL TRANSMITTANCE SCALE
The periodic calibration of laboratories’ spectrophotometers requires measurement of
wavelength accuracy and absorbance. As mentioned in section 2, traceable measurements of
the instrument are achieved by sending the secondary reference material standards for
calibration. The calibrated filters are then used by the laboratory to verify the instrument used
to perform the daily analysis.
6.1 Calibration of secondary neutral density filter
The absorbance of the neutral density filter was measured at wavelengths of 420 nm, 480 nm,
546 nm, 600 nm and 700 nm using a bandwidth of 2 nm. The result in figure 6 shows the
calibration of the filter over a period of nine years. The expanded uncertainties of the
absorbance values measured are given in Table 1.
Table 1.Uncertainty of the measurement
Absorbance
Expanded Uncertainty
(k=2) [Abs]
[Abs]
A < 1,0
± 0,005
1,0 < A < 2,0
± 0,01
2,0 < A < 2,5
± 0,02
(a) Absorbance at 420 nm
Absorbance (Abs)
2.6
2004
2005
2006
2007
2008
2009
2010
2012
2013
2.58
2.56
2.54
2.52
2.5
419
421
(b) Absorbance at 480 nm
2.38
Absorbance (Abs)
420
Wavelength (nm)
2004
2005
2006
2007
2008
2009
2010
2012
2013
2.36
2.34
2.32
2.3
2.28
478
482
(c) Absorbance at 546 nm
2.4
Absorbance (Abs)
480
Wavelength (nm)
2004
2005
2006
2007
2008
2009
2010
2012
2013
2.38
2.36
2.34
2.32
2.3
544
546
Wavelength (nm)
548
(d) Absorbance at 600 nm
2.4
2004
Absorbance (Abs)
2.39
2005
2.38
2006
2.37
2007
2.36
2008
2009
2.35
2010
2.34
2012
2.33
2013
2.32
598
600
Wavelength (nm)
602
(e) Absorbance at 700 nm
1.85
2004
2005
2006
2007
2008
2009
2010
2012
2013
Absorbance (Abs)
1.84
1.83
1.82
1.81
1.80
1.79
1.78
698
700
Wavelength (nm)
702
Figure 6.Absorbance values of the neutral density filter measured at the wavelength a) 420
nm, b) 480 nm, c) 546 nm, d) 600 nm, and e) 700 nm.
The result shows that the measured absorbance values done at the scheduled calibration
intervals are within the claimed uncertainty shown in Table.1. Thus the spectrophotometer is
capable of reproducing the measured values through the years.
6.2 Calibration of secondary Holmium Oxide filter
The spectral absorbance of the Holmium Oxide filter was measured from 200 nm to 800 nm
using a bandwidth of 2 nm. The result in figure 7 shows nominal wavelength of selected
absorption maxima. The expanded uncertainty (k=2) of the wavelength values was ±0,4 nm.
Wavelength (nm)
(a) Wavelength of absorption maxima (278,8 nm)
279.6
279.4
279.2
279
278.8
278.6
278.4
278.2
2002
2004
2006
2008
2010
Calibration Year
2012
2014
(b) Wavelength of absortion maxima (287,5 nm)
Wavelength (nm)
288.5
288
287.5
287
286.5
2002
2004
2006
2008
2010
Calibration Year
2012
2014
Wavelength (nm)
(c) Wavelength of absorption maxima (361,0 nm)
361.6
361.4
361.2
361
360.8
360.6
360.4
360.2
2002
2004
2006
2008
2010
Calibration Year
2012
2014
Wavelength (nm)
(d) Wavelength of absorption maxima (419,0 nm)
419.4
419.2
419
418.8
418.6
418.4
418.2
418
2002
2004
2006
2008
2010
Calibration Year
2012
2014
(e) Wavelength absorption maxima (446,2 nm)
Wavelength (nm)
446.8
446.6
446.4
446.2
446
445.8
445.6
2002
2004
2006
2008
2010
Calibration Year
2012
2014
(f) Wavelength absorption maxima (460,3 nm)
Wavelength (nm)
461
460.5
460
459.5
459
2002
2004
2006
2008
2010
Calibration Year
2012
2014
Wavelength (nm)
(g) Wavelength absorption maxima (536,7 nm)
537.4
537.2
537
536.8
536.6
536.4
536.2
536
2002
2004
2006
2008
2010
Calibration Year
2012
2014
Wavelength (nm)
(h) Wavelength absorption maxima (637,4 nm)
638.6
638.4
638.2
638
637.8
637.6
637.4
637.2
2002
2004
2006
2008
2010
Calibration Year
2012
2014
Figure 7.The nominal wavelength of selected absorption maxima of a holmium oxide filter
(a-h) measured at the scheduled calibration interval.
The result shows that the measured peak wavelength values are within the claimed
uncertainty. Thus the spectrophotometer is capable of reproducing the measured values
through the years.
7. CONCLUSION
The verification tests in Section 5.2 and 5.3 facilitate the acceptance and use of the reference
transmittance spectrophotometer to disseminate the transmittance scale with great confidence.
In a laboratory setting, the performance verification (PV) tests on the field spectrophotometer
will be performed using secondary reference materials (RMs) and the results in section 6.1
and 6.2 indicates that they are traceable to primary reference standards.
It is important that reference materials used are accurate and certified in the spectral
wavelength region of analytical interest. This is important because the operational
performance and data quality of any given spectrophotometer may be optimal in certain
spectral regions, but compromised in other spectral regions. The use of traceable secondary
reference materials has also been considered an important tool to investigate potential
instrument-related systematic errors that may impact on the quality and reliability of the data.
Partnering with a reputable UV/VIS measurement science and standards organisation will
reinforce or enhance the laboratory’s quality consciousness and reputation, which will further
support its commitment to analytical excellence in the competitive global marketplace.
REFERENCES
1. Technical Guide, “UV/VIS spectrophotometer calibration procedures, ASTG4, May
2005, International Accreditation New Zealand, ISBN: 0908611595
2. http://www.biochrom.co.uk/ basic UV/Visible Spectrophotometry, 15 July 2013
3. Hitachi Ltd, Instruction manual for model U-3400 recording spectrophotometer, 1985,
page 2-2
4. Franc Grum and C. James Bartleson, Optical radiation measurements volume 2, 1980,
Academic Press, Inc. ISBN 0-12-304902-4 (v.2), page 339 - 355
5. http://www.siphotonics.com/pages/products/product_Literature/Reference%20Materials.pdf, 15 July 2013