UV Vis Spectrophotometer Calibration

UV/VIS Excellence spectrophotometers are tested according to the procedures defined in the United States Pharmacopeia (USP) 43 – NF 38 and European Pharmacopeia (Ph. Eur.) Ed. 10. The tests cover the following parameters:

• Wavelength accuracy
• Photometric accuracy
• Photometric repeatability
• Photometric linearity*
• Stray light
• Resolution

Additionally, parameters not required by neither USP nor Ph. Eur. are also tested:

• Wavelength repeatability
• Photometric noise
• Baseline flatness
• Photometric drift

The comprehensiveness of the tests ensures that all aspects of the spectrophotometers' performance are assessed. Our UV7 Excellence spectrophotometers comply with the strict requirements described in more detail below.

*The photometric linearity is not longer explicitly mentioned in the 43rd edition. The photometric accuracy has to be tested over the intended operational range. Therefore, METTLER TOLEDO is still recommending the test for photometric linearity. 

Wavelength accuracy assesses the ability of the instrument to faithfully reproduce wavelengths. The measurement of the wavelength accuracy allows to determine whether shifts of misalignment of the wavelength axis are present.

Suitable reference materials for the determination of wavelength accuracy should exhibit narrow and well-defined peaks. A solution of holmium oxide is often used to this purpose.

USP and Ph. Eur. state different acceptance criteria depending on the reference material used and their wavelength range. For liquid reference materials, the following criteria apply:

USP: ±1 nm below 400 nm; ±2 nm above 400 nm
Ph. Eur.: ±1 nm below 400 nm; ±3 nm above 400 nm

According to USP and Ph. Eur., wavelength precision, or repeatability, is not required for array-based spectrophotometers. Thanks to the absence of moving part, array-based instruments are not subject to misalignment in-between measurements.

Photometric accuracy is a measure of the ability of the instrument to correctly measure absorbance and to return absorbance values as close as possible to the true value.

Photometric accuracy can be determined by measuring solid or liquid filters of known concentration and absorbance values and comparing the measured absorbance with reference absorbance values.

A 60 mg/L solution of potassium dichromate (PDC) is used for the ultraviolet range, while a 0.5 A neutral-density (ND) filter covers the visible range. The acceptance criteria are the same for both pharmacopeia and are dependent on the absorbance level of the reference material.

PDC: ±0.010 A below 1.0 A; ±1.0% (0.010*A) above 1.0 A
ND: ±0.008 A below 1.0 A; ±0.8% (0.008*A) above 1.0 A

According to USP and Ph. Eur., wavelength precision, or repeatability, is not required for array-based spectrophotometers. Thanks to the absence of moving part, array-based instrument are not subject to misalignment in-between measurements.

Photometric repeatability, also referred to as photometric precision by the USP, defines the capacity of a spectrophotometer to return absorbance values over multiple measurements in a reliable and repeatable manner.

The same reference materials used to determine photometric accuracy are used. The filters are measured 6 times and the standard deviation over all measurements is calculated to assess the repeatability.

The standard deviation, which shall not exceed 5 mA below 1 A or 0.5% above 1 A, regardless of the used reference material or the wavelength range.

Note: photometric repeatability is not required by the Ph. Eur.

Photometric linearity is a measure of the linearity of the measured values (e.g. absorbance) as a function of increasing concentration. This is especially important for quantitative applications.

Photometric linearity can be determined with the same procedures and filters used to determine photometric accuracy, however, at three concentrations/different filters covering the range between 0 A and 2 A are used.

The USP and the Ph. Eur. state different acceptance criteria for the photometric linearity:

Ph. Eur.: the correlation coefficient R2 must exceed 0.999.
USP: the measured values at all concentration/filters must fulfil the photometric accuracy criteria.

Stray light is defined as light reaching the detector, which is not part of the analysis. It causes a bias at the corresponding wavelength but does not originate from a contribution of the measured sample. Therefore, the absorbance of the sample is altered by stray light and the sample results include a systematic error.

Stray light is determined by measuring a cut-off filter (an aqueous potassium chloride solution) using both the specific wavelength method (SWM) and the solution filer ratio method (SFRM).

The acceptance criteria stated by the USP and Ph. Eur. for KCl are as follows:

SWM: Amax ≥ 2.0 A
SFRM: Amax ≥ 0.7 A

Resolution defines the resolving power of a UV Vis spectrophotometer and its capability of resolving adjacent structures in a spectrum.

Resolution is determined by measuring a solution of Toluene in n-Hexane. The absorption minimum and maximum of a sharp and well-resolved peak are used for the assessment of the resolution.

The USP states that the ratio between the minimum and maximum absorbance at respective wavelengths of 267 and 269 nm must exceed 1.3 to provide sufficient resolution, whereas the Ph. Eur. acceptance criteria are stated in the individual monograph.

Unlike scanning UV Vis spectrophotometers, array-based instruments do not have a physical slit, which mechanically defines the spectral bandwidth (SBW) of the instrument. The equivalent SBW of an array instrument can be calculated from the resolution of the instrument.
For example, the UV7 Excellence spectrophotometer's resolution is ≥ 1.9, which corresponds to a SBW ≤ 1.0 nm.

The tests defined above can be carried out either manually or automatically, however manual methods have the inherent risk of reduced accuracy. Manual workflows are time-consuming and error prone. Automated performance verification offers the following benefits:

• Faster results with traceable documentation and result validation.
• Workflow execution and data transcription errors can be avoided, tests do not need to be repeated.
• Compliance with data-integrity standards. This is a significant benefit with respect to FDA-compliant data management.
• Fast return on investment thanks to increased productivity and security.

Regulatory compliance is a key objective for all pharmaceutical manufacturers and their suppliers. According to GMP the manufacturing processes and analytical methods need to be validated, while at the same time the equipment used for production and quality control requires qualification.

In order to support customers in fulfilling this compliance requirement METTLER TOLEDO offers the EQPac for Excellence UV/VIS Spectrophotometers. The EQPac is a service solution specifically designed to address compliance concerns in today’s demanding regulatory environment. It provides an on site, fully-documented, professional installation and qualification service to ensure regulatory compliance and reliable operation.

Learn more about our equipment qualification service and our UV/VIS spectrophotometer calibration service.