The Chemical Solutions UV-vis Kit™ is a series of NIST traceable solutions and an Excel™ template designed for easy data recording and reporting of the actual data with acceptance criteria. This will allow you to qualify or calibrate a UV-vis spectrophotometer easily and fully in laboratories. In-house performance qualification (PQ) of these instruments is easy and saves money and time. Fully DIY.

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What does it test?

The UV-vis Kit™ tests any UV or visible wavelength detectors/spectrophotometers for the following performance characteristics: 

  • Absorbance Accuracy
  • Absorbance Linearity
  • Photometric Precision
  • Wavelength Accuracy
  • Wavelength Precision
  • Stray Light (based on NIST SRM 2032)

What are the methods suggested in this Kit based on?

  • ASTM 1657-94: “Standard Practice for Testing Variable-Wavelength Photometric Detectors Used in Liquid Chromatography”
  • ASTM 925-83: “Standard Practice for the Periodic Calibration of Narrow Bandpass Spectrophotometers”
  • NIST Standard Reference Material 935a: “Crystalline Potassium Dichromate for Use as an Ultraviolet Absorbance Standard”
  • NIST Standard Reference Material 2034: “Holmium Oxide Solution Wavelength Standard from 240nm to 650nm”
  • NIST Standard Reference Material 2032: “Crystalline Potassium Iodide Heterochromatic Stray Radiant Energy Standard for Ultraviolet Absorption Spectrophotometry”

What are the concentrations of the solutions provided in this Kit?

  • Absorbance Solution No. 1. nominal 0.02 mg/mL Potassium Dichromate 30ml bottle
  • Absorbance Solution No. 2. nominal 0.04 mg/mL Potassium Dichromate 30ml bottle
  • Absorbance Solution No. 3. nominal 0.06 mg/mL Potassium Dichromate 30ml bottle
  • Absorbance Solution No. 4. nominal 0.08 mg/mL Potassium Dichromate 30ml bottle
  • Absorbance Solution No. 5. nominal 0.10 mg/mL Potassium Dichromate 30ml bottle
  • Absorbance Solution No. 6. nominal 0.12 mg/mL Potassium Dichromate 30ml bottle
  • Absorbance Solution No. 7. nominal 0.14 mg/mL Potassium Dichromate 30ml bottle
  • Reference Solution Diluent 0.001N Perchloric Acid used to prepare linearity standards 60ml bottle
  • Wavelength Calibration Solution*, 4% Holmium Oxide 4ml bottle
  • Stray Light Solution, 1% Potassium Iodide in water 30ml bottle

* The Holmium Oxide solution is chemically identical to NIST SRM 2034 and is directly qualified against NIST standards during manufacture.

Principles of Operation; Wavelength Calibration, Absorbance Linearity and Accuracy & Stray Light

The UV-vis spectrum for NIST Standard Reference Material 2034 is presented is shown below. The official band numbers and their wavelengths are reproduced in Table 1 (shown below) 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 (*) that are normally suitable with most instruments.

When using spectrophotometers with mechanical monochrometers. Wavelength precision (reset ability) 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. Since no quantitation is required for wavelength determinations, we suggest using purified water as a reference standard. This test is not intended for use with photo diode array detectors.

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 is determined by removing and replacing a single cuvette 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 appropriate place in the provided template. The temperature corrected stray light is then 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.

Official Absorbance Values for Holmuim Oxide


Spectrum of NIST SRM 2034 (lot95) using an HP 8453 spectrophotometer (bandwidth 1nm). Official values are rounded to nearest nanometer.
The bands with a * are recommended for use as calibration bands for HPLC and or CE detectors. 
Download Holmium Oxide Spectrum

Certified Wavelengths for Holmium Oxide (14 bands)


Table 1

Certified wavelengths (nm) of minimal transmittance for 14 absorbance bands for holmium oxide (Cat. No. PQAHOX01) 4% in 10% perchloric acid at six spectral bandwidths.
Source NIST SRM 2034

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