White Paper

Automated Performance Verification

Advances in UV/VIS Spectroscopy

Abstract
In UV/VIS spectroscopy, regular performance verification is essential to ensure accurate and
reliable instrument performance. Widely accepted guidelines for performance verification of
spectrophotometers are described in the US Pharmacopeia (USP). The recommended tests
include the check of photometric accuracy and repeatability, wavelength accuracy and
repeatability, instrument resolution as well as stray light measurement. The USP recently
introduced a new chapter on ultra-violet visible spectroscopy and adapted the test for stray
light. Here, we compare the methods for measuring stray light according to the current and
previous version of the USP, assess the advantages of the new test and introduce automated
optical performance verification.

Contents
1. Performance verification in UV/VIS Spectroscopy 2
™
1.1 CertiRef – Automated and Pharmacopoeia-compliant
Performance Verification 2
1.1.1 How it works in detail 2
1.1.2 What are the benefits of CertiRef? 4
1.2 Stray Light – A Fundamental Parameter 4
1.2.1 Compliance to the Latest USP Stray Light Regulations 5
1.3 Service Offering for UV/VIS Spectrophotometers 6
1.3.1 Good UV/VIS Practice 6
1.3.2 Instrument Qualification 7
1.3.3 Routine Operation 8
 1 Performance verification in UV/VIS Spectroscopy
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1.1. CertiRef™ – Automated and Pharmacopoeia-compliant

Performance Verification
Regulated environments require performance verification of UV/VIS spectrophotometers on a regular basis.
Instrument performance is the main factor directly affecting the accuracy and repeatability of measurements. It is
therefore important that it be monitored regularly and documentary evidence is provided. As the procedure for
performance verification is intricate and time consuming there are great benefits in integrating it in the analytical
workflow. For this reason METTLER TOLEDO has developed an accessory, the CertiRef, for automatic calibration
and performance tests of the UV/VIS Excellence spectrophotometer instrument line. The CertiRef module offers a
full US Pharmacopoeia compliant solution for the automated performance verification of the UV/VIS Excellence
spectrophotometers UV7, UV5 and UV5Bio.

1.1.1 How it works in detail

The quality of UV/VIS spectrophotometry measurements is quantified in its spectrum. Performance tests have
to verify:
• that the wavelength positions (x axis) are correct (wavelength accuracy) and stable
(wavelength repeatability),
• that the intensities, absorbances or transmittances (y axis) are correctly measured
(photometric accuracy) and stable (photometric repeatability), and
• that the measured shape of the spectrum is correct and not distorted
(resolution toluene, stray light).

Certified reference materials (CRMs) manufactured and certified by Starna Scientific Ltd (England), are used to
carry out the tests. The CRMs are contained in heat sealed, quartz cuvettes and accommodated in the CertiRef
module (Fig. 1). which protects the cuvettes against light and damage, allowing long-term use. The fully auto-
mated system (Fig. 2) runs the pharmacopoeia performance calibration tests in less than 10 minutes, display-
ing detailed results on the terminal screen once completed. As the results are also stored in the instrument, it is
possible to call up the control charts of up to 100 past calibrations. The results of each test can be printed or
stored as a PDF file.

When mounted, the compact design of the CertiRef module makes it an integral feature of the UV/VIS Excellence
spectrophotometer. It can remain mounted in place at all times; the CertiRef does not interfere with any manual
or automated measurements with the eight position CuvetteChanger or FillPalMini pump.

Instruments increasingly have to be accredited to internationally recognized standards of Good Laboratory

Practice, ISO/IEC 17025 or ISO 9001. This brings with it the need to provide evidence of control of the instrument
performance and is only possible if the certified reference materials used adhere to the concept of traceability.
The concept of traceability is defined in the “International Vocabulary of Basic and General Terms in Metrology
(ISO, 1993)” It states that all Certified Reference Materials must have a defined traceable path to recognized
primary materials. This ensures that compatible measurements are made across national borders, resulting in
unambiguous and reliable communication of specifications.

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Figure 1: The sealed reference material CRM containing cuvettes of the CertiRef module.
Figure 2: The CertiRef module mounted on top of a METTLER TOLEDO UV7 spectrophotometer.

Two types of CertiRef modules are available, namely a European Pharmacopeia (EP) and a United States
Pharmacopeia (USP) compliant module, respectively. All CRMs are traceable to the USA National Institute of
Standards and Technology (NIST) primary standards and are manufactured in an environment that is accredited
by UKAS under ISO Guide 34 (4001) and ISO 17025 (0659). The module can also be re-certified and the cer-
tificate is valid for a maximum period of two years from the date of issue – or sooner if specified by the user’s
own protocols. Table 1 shows the optical parameters, which can be automatically measured with the CertiRef
module. Stray light, in particular, is one of the most important factors influencing the photometric accuracy and
precision. The CertiRef USP configuration complies with the UV/VIS spectroscopy chapter <857> of the USP 40
and is described in more detail in the chapter below.

CertiRef Test Reference Material EP USP

Hexane blank • •
Resolution
Toluene in hexane • •
Wavelength accuracy
Holmium oxide • •
Wavelength repeatability
Photometric accuracy Perchloric acid blank • •
Photometric repeatability Potassium dichromate • •
Water blank • –
Stray light EP Potassium chloride, 1.2% aqueous
• –
solution
Potassium chloride 1.2% aqueous
– •
solution, 0.5 cm
Stray light USP
Potassium chloride 1.2% aqueous
– •
solution, 1 cm pathlength
Table 1: Optical parameters that can be automatically measured with the CertiRef module either in the European or
United States Pharmacopeia (EP, USP) compliant version.

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 1.1.2 What are the benefits of CertiRef?
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In addition to traceable regulatory compliance, the CertiRef module offers the benefit of fully automating the
tedious and error-prone manual workflow of repetitive measurements of the certified reference materials (CRM)
in single sealed cuvettes, which include the following steps:
• Preparatory work: Instrument warm-up and preparing CRM cuvettes
• Running of tests: Changing CRM and blank cuvettes
• After run handling: Cleaning and storing of CRM cuvettes
• Calculation: Calculation and validation of results
• Reporting: Compiling data and creating formal report

Important optical parameters such as resolution, wavelength accuracy/repeatability photometric accuracy/re-

peatability and straylight can be measured within 10 minutes, fully unattended. With CertiRef the time to result,
including traceable documentation and secure validation, is approximately 3 times faster than manually executed
performance measurements. Workflow execution and performance data transcription errors can be excluded
avoiding test repetition. In total, precious time is saved for other productive analytical tasks. Comparing the regu-
lar automated, mainly monthly, performance check of the instrument with manual checks using single CRMs in
sealed cuvettes, the CertiRef investment pays back within a reasonable time frame of 1.5–2 years. This is depen-
dent on the number of performance tests per individual run. As the unit can reside on the UV/VIS Spectrophotom-
eter without interfering with sample measurements, performance checks prior to each sample series measure-
ments can be considered. The execution is controlled simply with a One Click shortcut on the instrument's
terminal. A further level of automation is possible when connecting the instrument to a PC with LabX software.
The automated performance check with CertiRef can be scheduled as an automatic task by the LabX task sched-
uler before the actual measurements takes place. This ensures that the instrument is always in the condition
required for accurate spectroscopic measurements.

1.2. Stray Light – A Fundamental Parameter

Stray light, or stray radiant energy, is a common confounding factor in spectrophotometric measurements. It is
defined as light from a source other than the instrument’s light source, which does not follow the optical path.
Although the measurement of absorbance or transmittance is, in theory, independent of monochromatic source
intensity (it is a ratio measurement of intensities); in practice it affects linearity. When stray light reaches the
detector it causes the negative deviation of the linear relationship between concentration and absorbance
(Beer-Lambert law), especially at high concentrations (figure 3). The resulting light intensity is measured as
higher than it should be. Conversely, the absorbance of the sample appears lower. The results of sample
measurements affected by stray light include a systematic error that can easily go unnoticed. Furthermore, the
adverse effects of stray light tend to increase over time with aging of optical components and lamps in the spec-
trophotometer. The stringent requirements of the US and EU Pharmacopoeias require that stray light be smaller
than 1% transmittance. Due to its significant effect on practical measurements, stray light is detected at a given
wavelength with a suitable liquid filters (CRMs), as part of regular instrument performance verification.

4.0 0.00% Stray-light

3.5 0.01% Stray-light
Measured Absorbance [AU]

3.0 0.10% Stray-light

1.00% Stray-light
2.5
2.0
1.5
1.0
0.5

0.0 Figure 3: Effects of stray light on

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 the linearity of optical
True Absorbance [AU] spectrophotometer.

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1.2.1. Compliance to the Latest USP Stray Light Regulations
In accordance with the pharmacopoeias, the CertiRef EUP and CertiRef USP apply ASTM E387-04 standard
methods to test for stray light. The limits specified in both the European Pharmacopoeia (EP 8, chapter 2.2.25)
and US Pharmacopeias (USP version 38 and 39, [3, 4]) are tested with the two methods described below:

1. Specific Wavelength Method (SWM) The specific wavelength method is well established and complies with
the current EP 8, chapter 2.2.25 and the older USP 38. It was regarded as the preferred method for stray light
testing by the US Pharmacopeia for many years. The method is based on measuring a material with a sharp
cut-off wavelength against water as compensation liquid (blank). In theory, measurement of a cut-off filter
would lead to infinite absorbance below the cut-off wavelength (all light is absorbed). In practice, the light that
is detected results from stray light (figure 3). The METTLER TOLEDO CertiRef EUP applies the specific wavelength
method.

2. Solution Filter Ratio Method (SFRM) This method has been introduced in the USP 39 and is implemented
in the METTLER TOLEDO CertiRef USP. It uses the same cut-off filter materials as the SWM, but the filter material
is measured with a path length of 1 cm against the same material with a pathlength of 0.5 cm as blank. With
smaller wavelengths towards the cut-off wavelength, the absorbance of the measurement increases and shows
a peak whose height serves as the criterion for the amount of stray light present (figure 4).

The two pharmacopoeia specific CertiRef modules are summarized below:

Definition “…Stray light may be detected at a given wavelength with suitable filters or solutions: for example the
absorbance of a 12 g/L solution of potassium chloride in a 1 cm cell should be greater than two at
198 nm when compared with water as the compensation liquid.”
Limits >2.0 absorbance at 198 nm (KCl)
Test SWM using KCl in a 1 cm cuvette against water as a blank in a 1 cm cuvette.
Table 2: CertiRef EUP stray light test as described in the European Pharmacopoeia. (EP 8, chapter 2.2.25)

Definition “…Stray light can be detected at a given wavelength with a suitable liquid filter. These solutions are
available as CRMs…This procedure simply requires the 10 mm cell measurement to be referenced
against the 5-mm cell (filled with the same filter)…”
with the same filter)…”
Spectral Range of Selected Material for Monitoring Stray Light
Spectral Range (nm) Liquid or Solution
190 – 205 Aqueous potassium chloride (12 g/L)

Limits ≥0.7 absorbance at the peak maximum (KCl)

Test SFRM using KCl in a 1 cm cuvette against KCl blank in a 0.5 cm cuvette
Table 3: CertiRef USP stray light test as described in the US Pharmacopoeia. (USP 39 chapter 857)

The SFRM, as required by the USP39, is a more sensitive method than the specific wavelength method. The
peak absorbance is measured in contrast to a selected wavelength close to the cut-off wavelength. This results
in more accurate absorbance measurements than the specific wavelength method. A further advantage is that
measurements are taken at lower absorbance values.

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1 cm path length
2.5 0.5 cm path length
Difference
2.0
Absorbance [A]

1.5

1.0

0.5

0.0

195 200 205 210 215 220 225

Wavelength [nm]
Figure 4: Comparison of the specific wavelength method (SWM) and the solution filter ratio method (SFRM). The blue line shows
the measurement of KCl in a 1 cm cuvette against water as blank (SWM), the orange line shows the measurement of KCl in a
1 cm cuvette against a 0.5 cm cuvette as blank (SFRM).

1.3. Service Offering for UV/VIS Spectrophotometers

With UV/VIS spectrophotometry, the ability to demonstrate consistent and comparable results is imperative
to achieving compliance with international standards such as GLP; ISO; FDA; HACCP and Pharmacopeia.
Our services ensure your instrument is professionally installed in a suitable environment and is kept in good
working order to provide accurate and dependable results you can trust.

1.3.1. Good UV/VIS Practice

Good UV/VIS Practice (GUVP™) improves measurement quality by minimizing risks through a 5-step lifecycle
program. It provides professional evaluation & selection tools, comprehensive installation & qualification ser-
vices and training and maintenance programs. Each step is thoroughly documented to support traceability and
regulatory compliance.

Step 1 & 2 – Evaluation and Selection

The GUVP design qualification tool DQmate helps you to define
your current and future requirements. Based on a thorough analy-
sis of your workflows, our experienced consultants will help you
evaluate and select the best suitable UV/VIS system including in-
strument, accessories and PC software.

Step 3 – Installation and Qualification

In order to fulfill regulatory requirements, qualification of the in-
strument is required at installation with easy to understand and
traceable documentation. Our professional equipment qualifica-
tion service complies with strictest regulatory requirements also
includes basic training to support correct operation, cleaning and
maintenance of the instrument. Figure 5: Good UV/VIS Practice (GUVP™).

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Step 4 – Training
Correctly trained users make fewer errors. Guided by a specialist, the EduPac training package explains efficient
and safe operation with practical exercises. In addition, our application literature, technical publications and cus-
tomer seminars will all help to ensure expertise in the lab.

Step 5 – Routine Operation

Keeping your instruments in good working order is an important component of routine operation. With our onsite
Preventive Maintenance and Calibration services, your instruments are properly cared for, ensuring reliable and
accurate results. For routine testing by the user, the CertiRef™ module provides automated USP- or EP-compliant
performance verification based on NIST-traceable certified reference materials.

1.3.2 Instrument Qualification

EQPac
The METTLER TOLEDO EQPac provides a comprehensive installation and fully documented qualification solution
for UV/VIS spectrophotometers. It complies with the strictest regulatory requirements regarding installation,
operation, and performance qualification. The UV/VIS EQPac is the tool of choice for regulated environments:

• IQ: Installation Qualification – Verification of complete delivery and correct installation in suitable
environmental conditions.

• OQ: Operational Qualification – Functional, operational and performance verification tests to confirm
the accurate performance of your instrument in the installed location.

• Initial Calibration – Traceable calibration certificate performed using the Pharmacopoeia-compliant

CertiRef module to meet quality, industry & regulatory requirements.

• User familiarization – Educational overview to operators and maintenance personnel to ensure

correct operation of the instrument and accessories.

• Lifecycle Support – Comprehensive testing and documentation supporting PQ & MQ for regulated
environments.

• Comprehensive documentation – Detailed documentary evidence of equipment qualification,

suitable for highly-regulated industries.

• Compliance with: GMDP/GLP; ISO; FDA; HACCP; USP; EP

StarterPac
If comprehensive fully documented qualification is not required, the StarterPac provides an easy and
quickly executed solution to document complete shipment and correct installation and functioning of the
UV/VIS instrument. Users receive a basic introduction to the operation and care of the instrument.
The UV/VIS StarterPac is the ideal choice for your internal quality management system.

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 1.3.3. Routine Operation
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Preventive maintenance
Preventive maintenance supports the requirement of accurate and reliable results. When performed by factory
trained and certified technicians, it reduces the risk of breakdown by regularly monitoring equipment deteriora-
tion and restoring equipment to proper condition. Regular preventive maintenance will help to extend the lifetime
of your equipment.

Preventive maintenance includes:

• Inspection and cleaning
• Functional and operational checks
• Documentary evidence of service, including equipment condition

Calibration and certification

If an instrument adjustment or external calibration certificate is required for your audit, or for customers who do
not have their own CertiRef module, our skilled service technicians can provide certification using an indepen-
dent USP or EP-compliant CertiRef module. With regular instrument calibration and adjustment using certified
reference materials, you can trust your measurements, be compliant and avoid the costs of repeat analysis due
to inaccurate results.

www.mt.com/uv-vis
Mettler-Toledo GmbH, Analytical For more information
Sonnenbergstrasse 74
CH-8603 Schwerzenbach, Switzerland
Tel. +41-44 806 77 11
Fax +41-44 806 73 50

Subject to technical changes.

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