METTLER TOLEDO GmbH, AUGUST 2017
Regulatory and Practical Aspects of Wavelength
Accuracy in UV/VIS Spectrophotometers
Mario Crevatin and Heiner Saßmannshausen
Mettler-Toledo GmbH
Abstract—Wavelength aspects of UV/VIS spectropho-
tometers are discussed in this paper. The wavelength re-
quirements of different pharmacopeias, ASTM standards
and applications are outlined.
Wavelength calibration and adjustment1 with mercury
argon, deuterium, and xenon lamps as well as different
certified reference materials (CRMs), which are liquid
solutions or glass containing holmium-, didymium- or
other rare earth oxides, are analyzed with the focus
on their wavelength-range coverage, accuracy limits and
usability.
Three different pharmacopeia-compliant scanning and
array spectrophotometers are tested with respect to wave-
length accuracy and repeatability. The role of resolution,
the number of data points on the wavelength axis, software
features and calibration procedures are investigated and
recommendations based on practical experiences are given.
I. I NTRODUCTION
W AVELENGTH accuracy and repeatability are key
performance aspects of UV/VIS instruments. An
accurate calibration of the wavelength axis is crucial for
every application relying on a spectroscopic technique
and is the basis for its use as a reliable analytical tool.
For example, the typical UV/VIS measurement methods
of fixed-wavelength or scanning deliver two types of
results both sensitive to the wavelength accuracy of the
instrument: Absorbance or transmittance values at pro-
grammed wavelengths and wavelengths values of spec-
tral peaks or valleys. If there is an error in the wavelength
x-axis, both results will deviate from the “true values”. In
the first case, the measurement actually takes place at a
wavelength different from the programmed wavelength.
In the second case, the position of the spectral peak is
wrongly translated into a wavelength value.
1
Calibration and Adjustment [1]: In this document we use cali-
bration for the evaluation of the deviation, in particular wavelength
deviations, of values compared to target values from a reference. An
adjustment is the process to reduce these deviations to the minimum
possible and store the correction for future calibrations.
1
The same sensitivity towards wavelength accuracy
remains in the higher-level time-dependent kinetic or
concentration-dependent quantification methods.
This article is organized as follows: In section II,
the wavelength-accuracy requirements for UV/VIS spec-
trophotometers stated in pharmacopeias and for high-
demanding applications are presented. In these docu-
ments, calibration standards are recommended. In section
III, these standards, ranging from gas-discharge lamps
to rare earth solutions and rare earth ion doped glass,
are discussed and compared. Based on measurements
on three different instruments, section IV is dedicated
to an analysis of theoretical and practical aspects re-
lated to wavelength accuracy and calibration procedures.
The section contains valuable information for users of
UV/VIS spectroscopy to better understand the instru-
ments’ behavior and performance. The article ends with
a short conclusion.
II. R EGULATORY AND APPLICATION REQUIREMENTS
To understand the requirements on wavelength aspects
we first need to understand the needs of regulatory
standards and applications.
A. Pharmaceutical and ASTM requirements
Pharmaceutical regulations, so called pharmacopeias
[2]–[5], list quantitative requirements of UV/VIS spec-
trophotometers. These requirements are separated into
the four independent categories of wavelength accuracy
and repeatability; photometric accuracy, repeatability and
linearity; resolution; and straylight. The first require-
ments concern wavelength accuracy, and the perfor-
mance tests for the latter requirements are carefully
designed to eliminate influences from it. These phar-
macopeia wavelength accuracy requirements are sum-
marized in Table I. Most countries and laboratories are
referring to and testing in line with the European or
US Pharmacopeia (USP). Among the pharmacopeias, the
US version has the most detailed outline on the test
parameters to be used.
2 METTLER TOLEDO GmbH, AUGUST 2017

TABLE I TABLE II
WAVELENGTH REQUIREMENTS OF DIFFERENT P HARMACOPEIA P HARMACOPOEIA & ASTM APPLICATIONS WITH THIGH
AND ONE ASTM STANDARD TOLERANCES ON WAVELENGTH ACCURACY & REPRODUCIBILITY

Pharmacopeia Range Accuracy Repeatability Application Range Accuracy Rep.

EU & UK, UV range 200 − 400 nma ±1 nm - Cyanocobalamin peaks 278 & 361 mm ±1 nm -
EU & UK, VIS range 400a− 800 nm ±3 nm - USP peak 550 nm ±2 nm -
EU & UK single wavelengthb ±2 nm - Aspartame peaks 247 & 252 nm ±1 nm -
US, UV range 200 − 400 nm ±1 nm 0.5 nm stdc USP peaks 258 & 264 nm ±1 nm -
US, VIS range 400 − 700 nm ±2 nm 0.5 nm stdc Thiophene in Benzene 589 nm - ≤1.0 nm
CN, UV range - ±1 nm - ASTM D1685-00
CN, VIS range 500 nm region ±2 nm - Monoethylene Glycol 220.6 nm ±0.5 nm ≤0.3 nm
JP, optical filters 200 − 800 nm ±0.5 nm ±0.2 nmd ASTM E2193-02
JP, Hg or De peaks 200 − 800 nm ±0.3 nm ±0.2 nmd Petroleum Products 220 − 400 nm ±0.2 nm ≤0.2 nm
ASTM E925-09 [6] 200 − 700 nm ±1.0 nm ±0.1 nm ASTM D2008-12
a No exact transition wavelength between UV and VIS defined in the EU
pharmacopeia.
b Accuracy criteria if only a single wavelength is required to be measured. TABLE III
c Standard deviation (calculated from six measurements) not to be exceeded. N UMBER OF PEAKS RECOMMENDED FOR
No requirement for array instruments. WAVELENGTH - CALIBRATION STANDARDS IN PHARMACOPEIAS
d When the measurement is repeated three times, each value obtained needs
to be within this tolerance around the mean. Pharmacopeia EU US CN JP
calibration source & UK
Mercury argon lamp 10 8 11 4 optional
B. Application requirements
Deuterium lamp 1 2 2 2 optional
The requirements on the wavelength accuracy coming Holmium oxide solution 4 14 11 not quantified
from applications are illustrated by six examples pre- Holmium oxide glass - 11 optional 9 -
sented in Table II. These examples comprise two mea- Rare earth & Didymium - optional - -
surements which are part of the USP, and three examples
selected from 12 available ASTM applications2 . They are
selected among many because they impose high demands line sources. Next, we will describe the most relevant
on wavelength accuracy for UV/VIS spectrophotometers. certified reference materials (CRMs). These CRM stan-
It should be noted that many applications have require- dards are typically delivered in the form of 1 cm optical
ments on the photometric accuracy, which, particularly path length cuvettes from different vendors.
for applications with narrow peaks and valleys, translates
into a tolerance for wavelength accuracy. Nevertheless, A. Mercury argon reference lamps - HgAr
most applications include broad spectra and are therefore In gas discharge lamps, atoms in the gas phase are
not particularly sensitive to wavelength deviations (e.g. electronically excited using an electric discharge and
ASTM D4046, where the absorbance is to be measured then decay upon emission of light at characteristic
at 452 nm ± 5 nm) or include a quantification method wavelengths. Consequently, the intensity spectra of such
with multiple standards. For these reasons, many appli- lamps consist of narrow lines, the wavelength positions
cations are insensitive to wavelength deviations. of which trace back to the internal energy structure
of the corresponding gas-phase atoms. In contrast to
III. WAVELENGTH - ADJUSTMENT STANDARDS AND broad absorption bands of solutions, those positions are
WAVELENGTH - ACCURACY TEST METHODS stable, precise and traceable back to the metric SI unit.
Those are the main reasons why these lamps are used as
In this section, the technical limits and uncertain-
a primary wavelength-adjustment standard for UV/VIS
ties as well as practical aspects of the most common
spectrophotometers.
wavelength-accuracy test and calibration methods are in-
HgAr lamps in particular, containing a mixture of
vestigated. The focus lies on the standards recommended
mercury vapor and argon gas, are used because of several
in the different pharmacopeias, as presented in Table III.
distinct advantages. They are widely available, easy to
The numbers in this table denote the stated number of
use and durable. Their line spectrum is well understood
test wavelengths for each method. First, we will discuss
and features many narrow lines spanning the full UV/VIS
the use of mercury argon, deuterium and xenon atomic
part of the electromagnetic spectrum. 19 positions are
2
ASTM C25, D1685, D2008, D2269, D3231, D4046, D4590, known to very high precision, with uncertainties being
E533, E819, E1615, E1784, E2193 on the order of 10−4 nm [7]. Their importance is revealed
METTLER TOLEDO GmbH, AUGUST 2017
when considering that the spectrophotometers used to
determine certified reference substances are themselves
calibrated with HgAr lamps (see, e.g., [11]).
Conceptually simple, wavelength adjustment with
HgAr lamps exhibits some practical complications: i)
The light from the external lamp needs to be inserted into
the beam path of the spectrophotometer. In the design
of some (mostly scanning) spectrophotometers this is
solved by special slots to insert pen-shaped HgAr lamps
(e.g. LOT Oriel, Pen-Ray line sources). A convenient so-
lution for array-based instruments are HgAr lamps which
send the light through an optical fiber to an adapter that
directly fits into the standard 1-cm cuvette holder (e.g.
Ocean Optics, HG-1 Mercury Calibration Source). ii)
The intensity differences among the lines of HgAr lamps,
particularly between the very intense lines of mercury
and the much weaker lines of argon, are typically larger
than the dynamic range of the spectrophotometers. This
requires separate measurements with adopted illumina-
tion times for the differently-intense spectral regions.
iii) Instrument-specific behavior that has an influence
on the wavelength-adjustment, i.e. wavelength-dependent
detection sensitivity, resolution-dependent shapes of the
recorded emission lines and geometrical aspects. These
complications all dictate the use of special algorithms
outside of the instrument’s application toolbox to fully
exploit the HgAr lamp’s advantages for wavelength
adjustment.
Typically, the wavelength adjustment with an HgAr
lamp requires the following successive steps: i) Selection
of reference lines and definition of their positions. ii)
Recording the lamp spectrum, usually involving different
illumination times for the differently-intense spectral re-
gions. iii) Spectral smoothing and interpolation. iv) Peak
finding of the chosen reference peaks, thereby connecting
the wavelength λ with an instrument parameter x, such
as pixel number (in case of array instruments) or a
generalized position of a dispersive element (for scan-
ning instruments). v) Fitting of a parametrized formula
λ(x), which can be a simple polynomial of the form
λ(x) = a0 + a1 x + a2 x2 + a3 x3 , in order to obtain this
relationship not only on the positions of the reference
lines but over the whole spectrum. The determination of
parameters with physical meaning have been suggested
instead of polynomial coefficients [8].
Such HgAr-lamp based adjustments can achieve a
wavelength accuracy of 0.1 nm over the whole spectrum
on instruments with 0.2 nm data intervals, using sophis-
ticated calibration algorithms (but are limited by the in-
strument’s actual technical capabilities, see section IV-C)
[8], [9].
3
B. Instrument-internal tungsten-deuterium and xenon
lamps - TgDe / Xe
The majority of spectrophotometers relies on tungsten-
deuterium or xenon lamps as a light source. The emission
spectra of these lamps also feature sharp lines that can be
used for wavelength calibration. Using the internal lamp
for calibration requires no additional equipment, no test
substances, and no time-consuming calibration setup and
installation by the user. Because of these advantages, it
has become an established procedure.
However, the disadvantage lies in the fact that only
a few lines have accurately known spectral positions
and can actually be used as reference lines. In case
of the deuterium lamp there are two peaks at 486.0 nm
and 656.1 nm which are recommended as references in
the pharmacopeias, and in case of Xe lamps various
single peaks are used, but none is clearly established
in regulations. Effectively, this results in a two- or one-
point calibration or adjustment, which cannot compete in
performance with the previously mentioned HgAr lamp.
C. Holmium oxide solution
Holmium oxide (4%) in perchloric acid (10%), sealed
in 1 cm optical path length cuvettes, is typically mea-
sured against air as blank (no cuvette at all) for cal-
ibration. Intensive work has been done on this stan-
dard by Travis et al. [10]. They report on tests that
were performed on 17 instruments in 14 countries with
spectral bandwidths of 0.1 nm, 1.0 nm and 3.0 nm and a
wavelength sampling interval of 0.1 nm. Peak positions
were determined to the nearest 0.01 nm by use of peak-
location algorithms. All instruments were adjusted with
respect to atomic spectral lines prior to the measure-
ments. Figure 1 shows the spectrum of an holmium oxide
solution in the top panel.
In the following, the paper of Travis et al. [10] is
summarized:
a) Temperature sensitivity: The positions of ab-
sorption maxima of holmium oxide solutions have shown
to be very temperature insensitive. Peak shifts < 0.05 nm
have been reported in the range between 25◦ C − 45◦ C.
Nevertheless it is recommended to use holmium oxide
solution between 20◦ C and 30◦ C.
b) Concentration sensitivity: Changes in the con-
centration between 10 − 60 g/l result in peak shifts of
less than 0.05 nm.
Proper concentration adherence is in the responsibility
of the certified reference material supplier. It is recom-
mended that concentration variations shall be within 20%
compared to the nominal value of 40 g/l.
4 METTLER TOLEDO GmbH, AUGUST 2017
0.8 Holmium oxide solution
0.6
0.4
0.2
0 spectrum is shown in the middle panel of Figure 1. These
Absorbance
Holmium oxide glass
1.5
1
0.5
0 sorption cut-off effect of the glass itself for wavelengths
Didymium oxide glass
2
1 with such a filter on a Mettler-Toledo UV7 spectropho-
0
200 300 400 500 600 700 800
Wavelength [nm]
Fig. 1. Spectra of different certified reference materials measured
on a Metter-Toledo UV7 spectrophotometer. Upper panel: Holmium
oxide solution; middle panel: holmium oxide glass; lower panel:
didymium-oxide glass. Sharp peaks are highlighted with stars ∗ and
optional peaks with larger uncertainties in their peak position are
denoted with ◦.
c) Contamination sensitivity: Baseline absorbance
values at 230 nm greater than 0.3 A (<50%T) indicate
problems with contamination of the solution or of the
cuvette surface.
It is therefore crucial to prevent finger prints or other
contaminations on the cuvette surfaces.
d) Resolution sensitivity: Based on a mathematical
model, the positions of 14 absorption peaks are provided
for 0.1 nm steps of the spectral bandwidths (SBW)
between 0.1 nm up to 3.0 nm SBW (and 1 nm SBW steps
up to 10 nm SBW). The calculated values have 95%
confidence interval of 0.05 nm. Because significant shifts
of the peak positions can occur, it is crucial to know the
instruments spectral bandwidth (resolution, slit-width) to
apply the correct reference values from the holmium
certificate. This aspect is discussed in more detail in
section IV-B.
e) Accuracy limits: The 95% confidence interval
for final peaks for 13 instruments is smaller than ± 0.05
nm. The prediction intervals P 95 for measuring holmium
peaks on a single instrument on the basis of a single
spectrum are between ±(0.16 − 0.26) nm for a SBW of
1 nm.
Starna Scientific Ltd. and Hellma GmbH & Co. KG state
on their holmium oxide solution certificates uncertainties
(k = 2, 95%) of ±0.11 nm [11] and ±0.20 nm [12],
respectively.
D. Holmium oxide glass
Solid holmium oxide glass filters (fitting in stan-
dard 1-cm-cuvette holders) have been used since 1961
as wavelength-calibration references [13]. A measured
filters provide an alternative to the holmium oxide solu-
tion, but only provide 11 wavelengths in the same spec-
tral range. In a recent publication [14], several problems
of these filters are outlined, among which are the ab-
below 300 nm and the necessity of regular recertification
due to their hygroscopic nature. Practical experiments
tometer have shown that the lowest wavelength peak at
900 241.7 nm can still be just resolved on a huge absorption
background from the glass. Towards longer wavelengths,
the peaks at 536.5 nm and 638.0 nm are very broad
and not recommended as reliable calibration standards.
Consequently, 8 reference peaks remain in the range
from 279.4 nm to 460 nm. The peak positions are found
in the vicinity but not at the same wavelengths as those
of a holmium oxide solution [15]. Regarding this type of
holmium oxide glass filter, the possibility of differences
between glass batches is mentioned in the USP. The
uncertainty of the certified wavelength positions lies in
the range of ±0.11 nm [16] to ±0.20 nm [15].
E. Rare earth & didymium glass filters
Rare earth and didymium glass filters expand
the wavelength range towards the lower and upper
wavelength range [17]. USP refers to the didymium
filters (solution and glass) as an option to expand
the wavelength range up to 865 nm if the application
requires it. The lower panel of Figure 1 shows the
spectrum. Measurements with a didymium glass filter
on a Mettler-Toledo UV7 spectrophotometer have
shown that some peaks (at 573 nm, 741 nm, 807 nm,
and 879 nm) only have a partially resolved substructure,
or lie at a shoulder (431 nm). Practically, that restricts
the applicability at longer wavelength up to 749 nm.
Rare earth or didymium glass filters may be useful for
calibration for applications in the low-UV range or the
VIS to NIR range, respectively.
F. Holmium-didymium oxide solutions
For a dense and broad wavelength-range coverage,
mixtures of holmium oxide and didymium oxide in solu-
tion are available as CRMs [18], [19]. The recommended
reference peaks range from 241.12 nm to 794.16 nm and
include 7 peaks of holmium and 7 peaks of didymium.
METTLER TOLEDO GmbH, AUGUST 2017 5

TABLE IV A. Optical setup

DATA FROM CERTIFICATES OF WAVELENGTH - CALIBRATION
STANDARDS AND PRACTICAL LIMITS
There are two main categories of UV/VIS spectropho-
tometers that can be differentiated by their basic optical
Calibration source Rangea #WLb Uncertainty design:
Mercury argon lamp 253 − 922 nm 19 10−4 nmc a) Scanning: The original and most common type
Deuterium lamp ∗486.0, 656.1 nm 2 - are scanning instruments. Such systems scan the wave-
Holmium solution ∗241 − 641 nm ∗14 0.11 / 0.20 nmd length over the desired range by moving an optical
Holmium oxide glass ∗280 − 460 nm 11, ∗8 0.11 / 0.20 nme element inside the instrument. More specifically, a light
Rare earth solution 201 − 253 nm 5 -
beam of the selected wavelength passes through the
Didymium solution 731 − 864 nm 5 0.20 / 0.40 nmf
Didymium glass ∗473 − 749 nm 11, ∗6 0.11 / 0.20 nmg
sample and is measured by a detector. Sequentially,
Ho-didymium solution 241 − 794 nm 14h/ 7i 0.20 / 0.40 nmi and in tunable increments, the wavelength positions are
a Wavelength range according data sheets, or, if marked with an ∗, practical scanned in a pre-defined interval, typically taking a
recommended limits.
b Number of wavelengths total and practical (indicated by ∗).
scan time of seconds up to minutes. More time-efficient
c Value taken from [7]. measurements are available in “fixed wavelength” modes
d Certificates of Starna Scientific Ltd. [11] and Hellma Analytics [12].
e Certificate of Starna Scientific Ltd. [16].
where the absorbance is determined only at a single
f Certificates of Starna Scientific Ltd. [11] and Hellma Analytics [20]. wavelength. Many pharmaceutical test procedures are
g Certificates of Hellma Analytics [21] and Starna Scientific Ltd. [22].
written according to the specific behavior of scanning
h Recommended by Starna Scientific Ltd., private communication.
i Certificate of Hellma Analytics [23]. instruments.
Due to the mechanical moving parts of scanning
instruments they are prone to misalignments and, as a
Because the spectrum of the mixture is spectrally dense, result, a loss of wavelength accuracy or repeatability
it is recommended to use Ho-didymium mixed solutions over time. To counteract these drifts, the tested TE-
only for instruments with a high resolution better than 260 instrument and, as reported in [24], a Perkin Elmer
1 nm. instrument perform a wavelength calibration with the
peaks of the internal light source as reference at start-up
In Table IV, the findings of this section are sum-
(TE-260: xenon lamp peaks at 484.45 nm and 541.90 nm
marized. The table contains information from different
or Perkin Elmer deuterium lamp peak at 656.1 nm).
sources combined with recommendations based on our
This process takes around half a minute for two Xe
own practical experiences (indicated with a star).
wavelengths on the TE-260.
b) Array: Array based instruments are capable of
recording the full spectral range in a single measure-
IV. P RACTICAL I NSTRUMENT A SPECTS ment. These instruments do not have mechanical moving
components but comprise a spectrograph with an array
In this section, wavelength aspects related to the spec- of sensors dedicated to the different wavelengths. To
troscopic instrument used, the optical setup and software obtain the full spectrum, white light of all wavelengths is
features are outlined. We have performed measurements passed through the sample and later spectrally analysed.
on a Thermo Fisher Scientific Evolution 260 Bio (TE- The measurement time only depends on the required
260) scanning instrument and two array based systems, averages (i.e. the signal-to-noise ratio), and typically
Agilent 8453 (A-8453) and Mettler Toledo UV7 (MT- takes between sub seconds and a few seconds. Because
UV7) to demonstrate practical wavelength related issues array systems have no moving parts they are generally
of UV/VIS spectrophotometers. This chapter is not in- less prone to the wavelength stability problems outlined
tended to compare the performance or features of these for scanning instruments. This has been recognized by
particular instruments, but to study instrument-dependent the USP, which requires only a test of wavelength
aspects related to wavelength accuracy and their impact accuracy at a single wavelength and no repeatability
on applications. The Agilent and Thermo Fisher Scien- test for array instruments [4]. Regardless, both tested
tific instruments were not updated to the latest software array instruments provide means for calibration with
version, nor were they adjusted by a service technician the holmium oxide solution and with the internal light
prior to our measurements. For this reason, no judgment sources.
related to a specific brand is intended and future software However, wavelength-accuracy changes can also occur
releases may outdate any brand-specific statements. Our with array spectrographs, primarily because of mechani-
intent is to provide general, but practical considerations cal stress released by temperature changes. To minimize
and information when using these or similar instruments. this effect, spectrographs often undergo an extensive
6 METTLER TOLEDO GmbH, AUGUST 2017
tempering process over a wide temperature range in pro-
duction to compensate the internal stress due to mount-
ing. After tempering, tests on a MT-UV7 instrument
show no measurable temperature dependence of the 14
peak positions of an holmium oxide solution in the range
of 10◦ C − 40◦ C [25]. These results confirm the tempera-
ture insensitivity of the holmium oxide solution standard
and the stability of the array technology. However, small
shifts due to large thermal differences were observed in
measurements taken with instruments immediately after
significant temperature changes (e.g., unloading from
cold/hot car and transfer into the laboratory), without
giving the equipment the time to equilibrate.
For best practices, we recommend using the internal
start-up calibration for scanning systems and to always
ensure temperature equilibrium with the environment
before wavelength calibration.
B. Resolution dependency
The wavelength positions and photometric values of
absorption peaks are important for many different appli-
cations in UV/VIS spectroscopy. Using analyte-specific
peak positions is beneficial for several reasons: The
relevance of spectral peaks is easily understood; they are
easily determined numerically; and, the first derivative of
the signal being zero, peak absorbance values are stable
with respect to small wavelength fluctuations between
the measurements.
However, in the case of asymmetric lines and band-
shapes, which often occur in solution spectroscopy, the
peak positions depend on the instrument’s spectral res-
olution, which can lead to unintended peak shifts after
adjustment procedures.
As indicated in section III-C, Travis et al. [10] have
determined 14 peak positions of the holmium oxide
solution as a function of the spectral bandwidth (SBW)
of the measurement. A part of their results is summarized
in Fig. 2, where the range of peak-position differences
compared to a SBW of 1 nm, measured with SBWs
between 0.1 and 3 nm, are presented. The positions of
SBWs of 1 nm, 1.5 nm and 2 nm are commonly available
on certificates and are highlighted by black bars, green
dots and blue crosses, respectively, as shown in Fig. 2.
Shifts up to 0.3 nm occur between measurements at
SBW of 1 nm and 2 nm. Systematic wavelength shifts
on the order of a third of the SBW change are clearly
measurable. Consequently, the reference peak values
need to be adopted to the instruments specific SBW in
the wavelength adjustment and calibration processes.
For the Mettler-Toledo UV/VIS spectrophotometers
UV7, UV5Nano and UV5/UV5Bio, with spectral res-
olutions around 1 nm, 1.5 nm and 1.7 nm, respectively,
the TE-260 with 1 nm or 2 nm and the A-8453 with
1.5 nm, the holmium oxide solution reference values
given in Table V are used for calibration. Using the
correct reference values, taking into account even sub-
tle physical effects, minimizes systematic, instrument-
independent deviations.
The described resolution dependency of peak positions
occurs generally with asymmetric lines, and care has to
be taken when comparing application results obtained on
instruments with different SBWs.
0.8
SBW = 0.1 - 3 nm (1 nm center)
SBW = 1.5 nm
0.6 SBW = 2 nm
[nm]
0.4
peak, SBW = 1 nm
0.2
0
-0.2
-0.4
200 300 400 500 600 700
peak
at SBW = 1 nm [nm]
Fig. 2. Sensitivity of 14 commonly used Ho reference peak positions
towards the spectral resolution (in the form of a spectral bandwidth)
of the measurement. The horizontal axis shows the wavelength
positon of the peaks and the vertical axis shows the wavelength shift
of the peak when measured with SBW between 0.1 and 3 nm. The
shift is given with respect to the measurement at a SBW of 1 nm.
The green and blue marks highlight the shifts at SBW of 1.5 nm and
2.0 nm, respectively. Data taken from Ref. [10].
C. Technical limitations to peak determination
This section is dedicated to the fundamental accuracy
and repeatability limits that are a direct result of a
discrete measured spectrum. In a discrete spectrum,
the absorbance values are measured in a given interval
along the wavelength axis. In scanning instruments, the
wavelength interval λI can be tuned in certain limits,
whereas in array instruments it is given by the number of
single sensors per nanometer. In both cases, interpolation
can be used to decrease the wavelength interval.
To simulate an application, and to demonstrate
wavelength-interval-related accuracy issues, a holmium
oxide solution was measured around the absorption
peak at 333.48 nm on the three tested instruments. In
Fig. 3, the spectra obtained with the three instruments
are depicted. The solid vertical line shows the reference
wavelength at 333.48 nm. The wavelength interval λI
METTLER TOLEDO GmbH, AUGUST 2017 7

TABLE V
H OLMIUM OXIDE SOLUTION ABSORPTION PEAKS , MEASURED AT DIFFERENT SPECTRAL BANDWIDTHS (SBW S ) AND USED AS
REFERENCES WAVELENGTHS FOR CALIBRATION , e.g. 1.0 NM FOR THE MT-UV7 & TE-260, 1.5 NM FOR MT-UV5NANO & A-8453 AND
2 NM FOR THE MT-UV5/UV5B IO SPECTROPHOTOMETERS . DATA TAKEN FROM R EF. [10], [11].

SBW
1.0 nm 241.12 249.89 278.13 287.22 333.48 345.38 361.25 385.61 416.25 451.45 467.82 485.23 536.56 640.50
1.5 nm 241.13 249.98 278.11 287.40 333.49 345.38 361.18 385.70 416.42 451.33 467.86 485.26 536.71 640.62
2.0 nm 241.12 250.03 278.10 287.52 333.47 345.42 361.12 385.80 416.57 451.32 467.90 485.25 536.86 640.79

0.2
Reference 333.48 nm
b) Wavelength repeatability: Whereas for the abso-
1 nm, Agilent A-8453
0.2 nm, Mettler Toledo UV7 lute wavelength accuracy a possible error in the order of
0.1 nm, ThermoFisher TE-260
0.15
λI /2 can never be ruled out, the situation is different for
the corresponding repeatability measurement. Repeata-
Absorbance

bility is the standard deviation of several measurements

0.1
0.05
0 cific measurement from the average of all measurements,
330 331 332 333 334
Wavelength [nm]
Fig. 3. Holmium peak at 333.48nm measured on three instruments
with different data intervals of 1 nm, 0.2 nm and 0.1 nm. The mea-
surements were taken against air as blank and only the wavelength
positions and not the photometric absorbance values can be compared
between the measurements.
for the TE-260 scanning instrument was set to 0.1 nm,
whereas the A-8453 and MT-UV7 array instruments
have a fixed wavelength interval of 1 nm and 0.2 nm,
respectively.
a) Wavelength accuracy: The measurement was
repeated ten times on each instrument. For each mea-
surement, the peak positions λi determined by the in-
struments’ own algorithms were extracted. The average
values λ̄ = n1 · i λi are shown in Table VI. The
P
wavelength accuracy is calculated as the difference be-
tween the average measured peak position λ̄ and a given
reference value λref according to
λaccuracy = λ̄ − λref .
For the two array instruments A-8453 and MT-UV7, the
reference wavelength is located almost in the middle
between two data points in our example (see Fig. 3). This
is the reason why the observed deviations λaccuracy of
the measured peak positions from the reference value
correspond closely to λacc. limit = λI /2, which is the
maximal wavelength-accuracy error resulting from the
finite wavelength interval λI . The TE-260 reports the
peak position at 333.60 nm independent of the data
interval.
of the peak position. In the calculation of the standard
deviation, the average λ̄ of the measurements is used
instead of the known reference value. With n being the
number of measurements at a specific wavelength and
∆λi = λ̄ − λi being the wavelength deviation of a spe-
335 336
the repeatability is calculated as
v
u n
u 1 X
λrepeatability = t (∆λi )2 .
n − 1 i=1
Independent of the data interval λI , the value of
λrepeatability can be small, even zero, if the instru-
ments measure with high photometric accuracy the same
absorption values repeatedly at the same wavelength.
This can be seen also in Table VI, where both array
instruments have obtained the same peak value in ten
successive measurements, leading to a standard deviation
of zero.
Photometric noise is always present to some degree,
and it is visible in the TE-260 spectrum, but to a lesser
extent also in the MT-UV7 and the A-8453 spectra.
Due to the photometric fluctuations from measurement
to measurement, the peak position can toggle between
two, or even more, wavelength points. This effect is
enhanced in case of broad peaks, with many wavelength
points having similar photometric values. In case that
measured peak positions toggle evenly between two
neighboring wavelength points, the resulting effect of the
wavelength interval λI on the wavelength repeatability
can be calculated as
n λI
r
λrep. toggling = · ,
n−1 2
where ∆λi = λI /2 was inserted in the general
equation of λrepeatability .
For the three instruments discussed here, we can
calculate this repeatability λrep. toggling on the basis of
8 METTLER TOLEDO GmbH, AUGUST 2017
TABLE VI
R ESULTS OF MEASUREMENTS OF THE HOLMIUM OXIDE
ABSORPTION PEAK AT 333.48 NM ON THREE DIFFERENT
SPECTROPHOTOMETERS : WAVELENGTH ACCURACY AND
REPEATABILITY
A-8453 TE-260 MT-UV7
Data interval λI 1 nma 0.1 nm 0.2 nm
Accuracy (n = 10)
λ̄ 333.00 nma 333.60 nmb 333.40 nm
λaccuracy −0.49 nmc +0.12 nm −0.09 nm
λacc. limit ±0.50 nm ±0.05 nm ±0.10 nm
Repeatability (n = 10)
λrepeatability 0 nmd 0.02 nmb 0 nmd
λrep. toggling 0.53 nmf 0.05 nmf 0.11 nmf
λrep. s.o. 0.32 nme 0.03 nme 0.06 nme
a Peak/valley finding without further mathematical functions.
b Use of interpolation.
c Reference value of 333.49 nm for 1.5 nm resolution [26].
d All obtained wavelength values are equal.
e One single data point deviates from the others
by λI .
fThe peak positions of multiple measurements are evenly distributed
over two neighboring data points.
the instrument-specific λI values (see results listed in
Table VI). The number of measurements n does not have
a significant impact on this value, that asymptotically
approaches λI /2 with increasing measurements.
In another scenario, where the peak is found only once
out of n measurements on a neighboring wavelength
data point, λrep. s.o. is smaller than λrep. toggling but still
surprisingly large. It can be expressed as λrep. s.o. = √λnI
(where “s.o.” abbreviates “single outlier”). Both repeata-
bility limits, λrep. toggling and λrep. s.o. , are given in
Table VI for the three tested instruments.
c) Interpolation: In the peak-position determina-
tion, some instruments additionally interpolate the ab-
sorbance in between the wavelength points. Thereby, the
above described artifacts can be reduced. The A-8453
and TE-260 instruments use interpolation algorithms in
the calibration procedure3 for best performance results.
In normal applications, the A-8453 does not use the same
interpolation function by default, but it can be activated
by the user. The TE-260 provides the interpolation-based
peak-finding procedure automatically in all measurement
modes. The MT-UV7 does not provide an interpolation
beyond the wavelength data interval of 0.2 nm.
d) Peakfinding sensitivity: During the measurement
of the holmium oxide solution on the tested instruments
it was revealed that the instruments did not find the
same number of peaks for each scan. Generally the user
is interested in an application-specific, clearly defined
number of peaks. Therefore, it is beneficial that the peak
finding algorithms of the instruments can be improved
3
For the A-8453 instrument [27]: ”99-point spline function is used”
by parametrisation. Even with these improvements, some
desired peaks were still omitted or picked wrongly by the
peak-finding algorithm. The complication for automated
peak finding originates from the difficult distinction
between peaks which result from photometric noise or
those that result solely from the absorption of the sample.
A pragmatic, but powerful solution to this problem, is
the definition of specific search areas in the application
method. This can be particularly helpful for scanning
instruments to reduce the overall measurement time.
To conclude, the analysis of the tests has shown that
there are differences regarding wavelength accuracy and
repeatability between different UV/VIS spectrophotome-
ters, but also between different measuring modes, peak-
finding and data-interval parametrisation on the same
instrument. These differences were traced back primarily
to the instrument-specific wavelength data intervals and
if additional interpolation was performed on the data.
In order to cover the vast majority of applications, it
is sufficient to obtain a wavelength data interval of
≤ 0.2 nm, which translates into a maximal technically-
limited wavelength error of λacc. limit = ±0.10 nm. That
accuracy value exceeds the pharmacopeia requirements
and is of a similar magnitude as the uncertainty of
reference materials.
D. Application software features
In practice during above outlined measurements, it
was found that for all applications, particularly for
typical applications with broad spectral peaks, further
measurement aspects are as important as the performance
specifications. The authors’ suggestions for more time-
efficient working procedures are listed next:
• The possibility of application method programming,
e.g., to allow for a pre-defined number of wave-
length positions to be measured, to obtain only the
desired peak positions.
• In order to prevent error-prone manual data anal-
ysis, modern UV/VIS instruments should provide
mathematical functions capable of calculating de-
rived application-specific values on the basis of the
measured data (e.g., the wavelength deviation from
a target wavelength, differences or ratios between
absorbances measured at pre-defined wavelength
positions or peaks, or statistics over multiple mea-
surements).
• A simple way of exporting the spectral data (in
case the mathematical functions do not deliver the
required results) as well as derived quantities.
• Acceptance limits to validate the calculated results
according to boundaries set by company or regula-
tory standards.
METTLER TOLEDO GmbH, AUGUST 2017 9

E. Wavelength adjustment & calibration TABLE VII

WAVELENGTH CALIBRATION AND ADJUSTMENT FEATURES
Among other tests such as stray light, resolution, and
photometric accuracy, all instruments provide special Wavelength test A-8453 TE-260 MT-UV7
software features for the user to execute wavelength Accuracy
calibration and repeatability tests. In this short section, Holmium #wavelengths 14 6 14
oxide solution time [min] 1:45a 8:20 0:10
the possibilities offered by the three tested instruments
Internal lamp lamp Tg Xe Xe
are presented (see Table VII). On the Agilent instru-
wavelength(s) 486.0 nm 542 nm 260.6 nm
ment, these tests are part of a defined performance-test 656.1 nm 528.6 nm
sequence. For the other instruments, the tests are freely time [min] 0:15 0:40 0:05
configurable for the user. As outlined in Table VII, the External lamp HgAr − optionalc −
instruments provide the possibility to execute perfor- Repeatabilityb
mance tests with holmium oxide solution or solely with Holmium #wavelengths 14 1 14
the internal light source. For both array instruments the oxide solution 536.6 nm
time [min] 1:45a 3:15 0:25
holmium oxide solution tests are executed for all 14
Internal lamp lamp Xe Xe
wavelength as stated by USP in Table III. All instruments
wavelength(s) − 542 nm 260.6 nm
do provide a performance test report for traceability. As time [min] 6:10 0:25
expected, the array instruments perform these perfor- Adjustment
mance tests faster than the scanning instruments. External lamp HgAr − optionalc serviced
As stated in section III-A on mercury argon lamps, time [min] 0:10
special software features are required to perform the Internal lamp time [min] 0:20 5:10 −
a
wavelength adjustment and calibration procedures, which Time for the holmium oxide wavelength accuracy and repeatability part of
an extended performance test.
are always tailored to a specific reference lamp or b All instrument perform 10 measurements and calculate the standard devi-

material. On the A-8453 and TE-260 instruments, the ation.

wavelength adjustment is accessible for the customer.
There, selected peaks in the spectrum of the internal
lamp are used as reference. For the UV7 instrument,
wavelength adjustment is part of the instrument’s service.
It is performed on the basis of 13 reference lines of an
external HgAr lamp.
V. C ONCLUSION
Typical applications in UV/VIS spectroscopy for
liquids and solids have broad peaks. Pharmacopoeia
standards therefore require a wavelength accuracy of
±1 nm in the 200 − 400 nm UV range and ±2 nm
in the 400 − 800 nm VIS range. For the wavelength
repeatability a standard deviation value of 0.5 nm of
at least six replicates is needed. Specific countries like
Japan, or a few ASTM standards, as outline in this
paper, have higher requirements.
To reach these specifications, wavelength adjustment
using mercury argon lamps is recommended. These
lamps provide more than a dozen sharp peaks with uncer-
tainties in the order of 10−4 nm. Elaborated adjustment
algorithms allow for wavelength-accuracy values down
to 0.1 nm. The instrument internal tungsten deuterium
or xenon lamp peaks provide means for a simple one to
two point calibration or adjustment. In case of scanning
instruments, the internal lamps provide good means for
a start-up performance check.
c Optionally available as Thermo Scientific EvolutionTM Mercury Lamp
Accessories.
d Executed by a service technician and HgAr lamp tool.
In line with many regulatory standards, the holmium
oxide solution, certified and sealed in 1 cm optical
path length cuvettes, is the first choice for instrument
calibration. It has advantages in the user friendliness,
robustness, reproducibility and temperature insensitivity.
14 peaks in the 241 − 641 nm range can be certified
with uncertainties of 0.11 nm or 0.20 nm, depending
on the manufacturer. The cuvettes shall be clean and
used between 20◦ C and 30◦ C. Other substances like
didymium or rare earth oxides can be used to expand
the wavelength range from 201 nm to 864 nm with larger
uncertainties up to 0.40 nm. A mixture of didymium with
holmium oxide solution is available as well.
The differences in the wavelength-range coverage
and uncertainties among the wavelength adjustment
and calibration procedures are large. It is therefore
important that the instrument suppliers clearly state
which procedures are offered and over which wavelength
range the specifications can be guaranteed with these
procedures.
The resolution of an instrument can have an impact on
measured peak positions. Therefore, the resolution needs
to be considered in wavelength calibration procedures
and when comparing application results of different
10 METTLER TOLEDO GmbH, AUGUST 2017

instruments. For the calibration, the correct resolution ACKNOWLEDGMENT

specific reference values have to be taken from the The authors would like to thank John P. Hammond
reference material certificates. If values of the wrong - Technical & Marketing Manager, Starna Scientific
spectral bandwidth are applied, systematic errors of Ltd, Hainault, UK and Birgit Kehl - Compliance Man-
0.3 nm and more are possible. All tested instruments ager Calibration Laboratory, Hellma GmbH & Co. KG,
have these values incorporated in their automated test Müllheim, Germany for their valuable inputs. Further-
sequences for 14 or 6 reference values of the holmium more, we would like to thank Rujuta Bhandarkar, In-
oxide solution. Preferably, wavelength repeatability is ternational Market Support Group, Mettler-Toledo India,
tested at all reference wavelengths. As a compromise for her contribution on the ASTM applications.
for a faster measuring process on scanning instruments,
a subset of the wavelengths might be applied, which
should preferably cover a broad wavelength range.
To reduce the effects of wavelength sampling to the
same level as the uncertainty of certified reference ma-
terials, an instrument shall provide a data point on the
wavelength x-axis every 0.2 nm. This will prevent instru-
ment specific wavelength errors greater than ±0.1 nm
and reproducibility errors greater than 0.11 nm. Inter-
polation can reduce these technical errors further, but
it needs to be available in both the calibration and
application modes.
For fast and application specific wavelength mea-
surements, the instrument should provide comprehensive
software support. The software should enable the user
to scan at application specific peak locations, to use
mathematical result calculations, and to check accep-
tance criteria.
The three tested instruments, among many others,
fulfill the wavelength-accuracy requirements imposed
by the different pharmacopeias. In the measurements
presented here, those instruments performed even better
than specified with wavelength accuracies below 0.5 nm.
With further technical improvements it can be envi-
sioned, that the instrumental accuracy will soon approach
the uncertainties of commonly used and accepted refer-
ence substances, such as the holmium oxide solution. In
the future, it would be desirable that a broader wave-
length range could be addressed with a single certified
reference material containing a combination of rare earth
oxides in solution. Such a combination would need to be
intensely tested and should be incorporated in different
regulatory standards. It would also be desirable to define
common reference peaks of xenon, which can then be
used in efficient calibration procedures of modern xenon
lamp based spectrophotometers.
METTLER TOLEDO GmbH, AUGUST 2017 11

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