LimsPulse
UV-Vis · Calibration procedure

Resolution

Checks the instrument's ability to separate closely spaced spectral features, using the ratio of the toluene absorbance maximum near 269 nm to the minimum near 266 nm.

Photo of a UV-Vis
Photo: Wiki210397, CC BY-SA 4.0 (cropped), via Wikimedia Commons
PURPOSE

Why this is checked

If the spectral bandwidth is too wide, fine structure in a spectrum is smoothed out and maxima appear lower. Identification by spectrum shape and assays measured at sharp maxima both rely on adequate resolution.

Frequency

Typically every 6 months (some labs quarterly), and after lamp replacement, major repair or relocation, as defined in your calibration SOP.

Learn how the UV-Vis works
YOU WILL NEED

Standards & tools

  • Toluene, 0.02% v/v in hexane (UV grade)
  • Hexane (UV grade) as the blank
  • Or a sealed certified toluene-in-hexane resolution cell
BEFORE YOU START

Prerequisites

  • Wavelength accuracy already passed
  • Spectral bandwidth set to the value used by your methods or stated in the monograph
  • Prepare the solution in a fume hood; keep the cuvette capped to avoid evaporation
PROCEDURE

Step by step

  1. 1Record a baseline with hexane in both cell positions.
  2. 2Scan the toluene solution from about 250 to 280 nm.
  3. 3Read the absorbance at the maximum near 269 nm and at the minimum near 266 nm.
  4. 4Divide the maximum by the minimum to obtain the ratio.
RECORD

Example observation table

FeatureWavelength (nm)Absorbance
Maximum2690.512
Minimum2660.298
Ratio (max ÷ min)—1.72
CALCULATION

How to calculate the result

Resolution ratio = A(max, about 269 nm) ÷ A(min, about 266 nm)
  1. A at 269 nm = 0.512; A at 266 nm = 0.298
  2. Ratio = 0.512 ÷ 0.298 = 1.72
  3. Typical requirement is not less than 1.5, so the instrument passes
ACCEPTANCE CRITERIA

Pass if

  • Ratio typically not less than 1.5, unless a monograph or your SOP states a different value for the bandwidth used.
IF IT FAILS

What to do

  • Confirm the bandwidth setting and repeat with freshly prepared solution.
  • If it still fails, use a narrower bandwidth (if available and allowed) or have the monochromator serviced.
  • Do not use the instrument for tests that depend on fine spectral features until it passes.
MORE

Other UV-Vis 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