UV-Vis · Calibration procedure
Absorbance accuracy
Checks that the absorbance reading is correct, by measuring a potassium dichromate solution of known concentration in dilute acid and comparing the specific absorbance with reference values.
PURPOSE
Why this is checked
Assays calculated from a specific absorbance A(1%, 1 cm), and any test with an absorbance limit, depend directly on the photometric scale being right. Absorbance accuracy is sometimes called photometric accuracy.
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 worksYOU WILL NEED
Standards & tools
- Potassium dichromate (CRM or primary-standard grade), dried at about 130 °C to constant weight
- 0.005 M sulfuric acid (as in Ph. Eur.) or 0.001 M perchloric acid (as in some USP procedures) as solvent and blank
- Alternatively, certified neutral-density glass filters or sealed liquid CRMs with certified absorbance values
- Matched 10 mm quartz cuvettes
BEFORE YOU START
Prerequisites
- Wavelength accuracy already passed
- Calibrated analytical balance and Class A volumetric glassware
- Lamps warmed up; cuvettes clean and matched
PROCEDURE
Step by step
- 1Accurately weigh 57.0–63.0 mg of dried potassium dichromate and dissolve it in the acid to make 1000 mL (about 60 mg/L).
- 2Zero the instrument with the acid blank at each test wavelength.
- 3Measure the absorbance of the solution at 235, 257, 313 and 350 nm (the maxima and minima of the dichromate spectrum).
- 4Convert each absorbance into specific absorbance using the exact concentration.
- 5Compare with the reference values and tolerances given in the current chapter or on the CRM certificate.
- 6If your SOP includes 430 nm, a more concentrated dichromate solution is used as the chapter describes.
RECORD
Example observation table
| Wavelength (nm) | Absorbance | A(1%, 1 cm) found | Typical reference value | Typical tolerance |
|---|---|---|---|---|
| 235 (min) | 0.748 | 124.5 | 124.5 | 122.9 – 126.2 |
| 257 (max) | 0.868 | 144.5 | 144.5 | 142.8 – 146.2 |
| 313 (min) | 0.292 | 48.6 | 48.6 | 47.0 – 50.3 |
| 350 (max) | 0.640 | 106.6 | 106.6 | 105.6 – 108.2 |
CALCULATION
How to calculate the result
A(1%, 1 cm) = A ÷ (c × b), where c is concentration in g/100 mL and b is path length in cm
- 60.06 mg of potassium dichromate in 1000 mL = 0.006006 g/100 mL
- Absorbance at 257 nm = 0.868, path length 1 cm
- A(1%, 1 cm) = 0.868 ÷ (0.006006 × 1) = 144.5
- 144.5 lies within 142.8 – 146.2, so 257 nm passes
ACCEPTANCE CRITERIA
Pass if
- Specific absorbance at each wavelength within the tolerance in the current chapter (the typical values above), or within the certified value ± its stated tolerance for a CRM.
- Where the chapter expresses the limit directly in absorbance, a typical tolerance is about ±0.010 A for readings up to 1.0 A (±1% above that); follow your SOP.
IF IT FAILS
What to do
- Check weighing, drying and dilution records, cuvette cleanliness and the blank; repeat once if an error is found and documented.
- Check stray light and lamp energy, which commonly cause low readings at low wavelengths.
- Label the instrument 'Out of calibration' and arrange service if it still fails.
- Assess the impact on assays reported since the last passing calibration.
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.