Wavelength accuracy

how do you check the Wavelength accuracy in HPLC, waters and not waters ?
Do you run any injecton of susbtance (which ???) at several Wavelength and find the maximum abs ?


  • With a 2487 or 2489, that's exactly how... No autozero on wavelength changes, equilibrate columnless system w/ 10ug/mL caffeine in water, step through 200-210 and 265-280nm, local maxima should be at 206 and 273 if memory serves.
  • Waters 2998 PDA and ACQUITY PDA have a programmed calibration routine using an Erbium filter.  This accessed in the Console.
  • I think they use Holmium oxide filters...

    Also, there is NIST standard grade K2Cr2O7 that can be used to make solutions of well known molar absorbtivities at several wavelengths that can be used, if the caffeine routine lacks sufficient rigor for your taste.
  • Thank at all for the support. Do you know why anyone use caffeina or other to check accuracy instead of holmium solution 4%, like decleared in pharmacopeia for UV instruments ?

    Sorry for my english.

  • i need to check the accuracy in UV, from 200 to 365 nm.

  • Holmium Oxide does not have any useful local maximum or minimum absorbances below ~280nm, unlike caffeine, which has 2, making it a little more useful for checking in the lower UV area.
  • ok i have the solution according pharmacopea: water like eluent capillaty 2 m x 0.12 mm with solution holmium 4% 30 ul at several wavelenght from 238 to 244.
    the maximum height must be 241 +- 1 nm.