LimsPulse
Fluorimeter · Calibration procedure

Wavelength accuracy

Checks that the excitation and emission monochromators read the correct wavelength, using sharp lines of known wavelength such as mercury emission lines.

Photo of a Fluorimeter
Photo: VGV27, CC BY-SA 4.0 (cropped), via Wikimedia Commons
PURPOSE

Why this is checked

Fluorescence methods set both an excitation and an emission wavelength. If either scale is wrong, the instrument measures off the peak, losing sensitivity and giving results that do not transfer between instruments.

Frequency

Typically every 6 or 12 months, after lamp replacement and after major repair, as defined in your calibration SOP.

Learn how the Fluorimeter works
YOU WILL NEED

Standards & tools

  • Mercury pen lamp (lines at 253.65, 365.02, 435.83 and 546.07 nm) or the instrument's built-in reference line
  • Optionally a certified fluorescence wavelength standard
BEFORE YOU START

Prerequisites

  • Xenon lamp warmed up
  • Narrow slits (e.g. 1–2.5 nm) set for sharp peaks
PROCEDURE

Step by step

  1. 1Emission: place the mercury lamp in the sample position (or use the manufacturer's accessory) and scan the emission monochromator across each line.
  2. 2Record the wavelength of each peak maximum.
  3. 3Excitation: scan the excitation monochromator using a scattering sample or the built-in reference line, as described by the manufacturer.
  4. 4Calculate the deviation of each measured peak from its reference wavelength.
RECORD

Example observation table

Hg line, reference (nm)Measured emission peak (nm)Deviation (nm)
253.65253.90+0.25
365.02364.60−0.42
435.83436.30+0.47
546.07545.70−0.37
CALCULATION

How to calculate the result

Deviation = Measured peak wavelength − Reference wavelength
  1. Hg 435.83 nm measured at 436.30 nm
  2. Deviation = 436.30 − 435.83 = +0.47 nm
  3. Within the example tolerance of ±1.0 nm: passes
ACCEPTANCE CRITERIA

Pass if

  • Each line typically within ±1.0 nm to ±2.0 nm, depending on the instrument specification and slit width. Use the limit in your SOP.
IF IT FAILS

What to do

  • Check lamp and accessory positioning and repeat with narrower slits.
  • Run the instrument's wavelength calibration routine if available.
  • Label 'Out of calibration' and call service if it still fails.
MORE

Other Fluorimeter calibrations

In LimsPulse · Calibration Management

LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.

See how