Wavelength Calibration

The UV-vis spectrum for NIST Standard Reference Material 2034 is presented in figure 1 (click here to view). The official band numbers and their wavelengths are reproduced in Table 1 (click here to view) for instrument bandwidths of 0.1 to 3nm. UV-vis spectrophotometers should be set to a suitable bandwidth for the wavelength calibration.

Since it is not necessary to measure all 14 bands of the Wavelength Calibration Solution (Holmium Oxide), six bands in figure 1 have been identified (starred) that are normally suitable with most instruments.

When using Spectrophotometers with mechanical monochrometers. Wavelength precision (resettability) is measured by moving the monochrometer 10nm away from the maximum absorbance band, then to the nominal maximum and noting the absorbance values. The percent RSD or CV is calculated for six trials. This test is not intended for use with Photo Diode Array detectors. Since no quantitation is required for wavelength determinations, we suggest using purified water as a reference standard.

Absorbance Linearity and Accuracy


The 7 Absorbance Accuracy/Linearity Solutions (potassium dichromate) supplied in the kit are chemically identical to NIST SRM 935a and is recognized as the primary reference standard for determination of absolute accuracy, and for linearity over its range.

Linearity of response is important, since demonstration of such linearity ensures that the instrument optics and electronics are operating properly and are capable of producing linear standard curves. This in turn permits the use of standards and samples over a broader range when using two-point, or even one-point calibration standards.

For spectrophotometers, NIST publishes Apparent Specific Absorbance values (extinction coefficients) for potassium dichromate (supplied in this kit) at various concentrations ranging from 0.02 – 0.20 g/kg at 5 wavelengths: 235 and 313 minima and 257, 345 and 350nm maxima.

NIST recommends that 345 nm not be used for accuracy determinations but only for linearity since it is near an isobestic point. The software supplied in this kit supports both accuracy and linearity determinations for 235, 257, 313 and 350nm.

Temperature Correction The validated software supplied with this kit allows for two different types of temperature corrections producing more reliable and accurate results. Lookup tables are provided in the software as well as other mathematical devices for making what is normally a cumbersome task into a manageable one.

Notes on Absorbance Range This kit is designed so that the highest concentration solution produces absorbance values of just over 2AU at 257nm using a 1cm cell. For most modern instruments, percent accuracy will remain at 99% or better over this range. Older instruments or instruments with wide bandwidths of 2.5nm may typically produce results that drop off in accuracy to 97-98% or more. Such trends should be evident in the linearity results of this kit.

Absorbance Precision Absorbance (photometric) Precision is determined by removing and replacing a single cuvet filled with absorbance linearity solution number 2 and calculating the precision using the Excel™ template provided with the kit.

Stray Light


SRM 2032, the official NIST standard for Stray Light determination is 1% Potassium Iodide in water. Other governing bodies utilize 1% Sodium Iodide for Stray Light. This solution is available upon request.

This kit’s Stray Light determination Step is based on the lowest official NIST wavelength (240nm). The spectrophotometer that is being tested should be referenced with purified water at 240nm using a 1.0cm cell. The absorbance of the Stray Light Solution (1% KI) is then measured at this wavelength. It is important that the temperature be kept between 21-26°C. The Stray Light Solution is prepared at 23.5°C +/- 1°C.

How is stray light determined? The absorbance of the filled cell is measured and the result is entered into the provided template in the appropriate place and the temperature corrected stray light is calculated according to the official NIST SRM 2032 values.

Typically, well designed spectrophotometers in good condition can be expected to produce values of < 0.2% at 240nm for 1% KI.

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