A holmium-oxide glass filter is used to validate the wavelength accuracy. The filter can be moved into the light path in front of the entrance slit for the wavelength validation. The detector determines the wavelengths of several absorption peaks in the resulting spectrum and compares them to the nominal holmium oxide values that are stored in the detector firmware.

A validation can be performed for all lamp configurations (both lamps turned on, only UV lamp turned on, and, but least favorable, only VIS lamp turned on (if available)).

The accuracy is verified for the wavelengths that are stated in the declaration of conformity for the holmium-oxide glass filter. For information about the declaration, see NIST Compliance.

A wavelength validation can also be performed with an external standard, such as a caffeine solution. In this case, an accuracy of ± 1 nm can be achieved.

When

A wavelength validation is recommended in the following situations:

  • After moving the detector

  • After the flow cell has been exchanged

  • After a lamp has been replaced

Preparations

Before you start the wavelength validation or wavelength calibration, observe the following notes:

  • Allow the flow cell to warm up for 5 minutes.

  • Ensure that the baseline is sufficiently stable. The baseline may become unstable, for example, if the solvent composition has been modified, or if air bubbles are present in the light path.

  • Verify that the solvent flowing through the flow cell is not strongly absorbing in the wavelength range of the holmium-oxide glass filter that is to be verified.

    This problem occurs, for example, if the flow cell is filled with a mixture of 96% hexane and 4% ethyl acetate. Thermo Fisher Scientific recommends using degassed LC/MS-grade water.

  • Allow the lamps to warm up and reach operating temperature. The lamps should be running for at least 1 hour before you start the wavelength validation or wavelength calibration. A lamp spectrum changes significantly during the first few minutes after a lamp has been turned on.

To perform a wavelength validation

  1. Turn on the flow at the flow rate that will be used for the analysis.

  2. In the chromatography data system, in the Commands window, execute the WavelengthValidation command.

  3. If the wavelength validation fails, perform a wavelength calibration.

To perform a wavelength calibration

  1. In the chromatography data system, in the Commands window, execute the WavelengthCalibration command.