Fluorimeter · Calibration procedure
Sensitivity (water Raman signal-to-noise)
Measures the Raman scatter band of pure water and compares it with the background noise. The ratio is a standard way to compare fluorimeter sensitivity.
PURPOSE
Why this is checked
Water's Raman band is always present at a fixed position and needs no fluorescent standard. A falling signal-to-noise ratio shows a weakening lamp, dirty optics or a tired detector.
Frequency
Typically every 6 or 12 months, after lamp replacement and after major repair, as defined in your calibration SOP.
Learn how the Fluorimeter worksYOU WILL NEED
Standards & tools
- Ultrapure (Type 1) water in a clean four-side-polished quartz cuvette
BEFORE YOU START
Prerequisites
- Lamp warmed up
- Settings exactly as the manufacturer's specification test (commonly λex 350 nm, slits 5 nm, a stated integration time)
PROCEDURE
Step by step
- 1Set excitation to 350 nm and scan emission from about 365 to 450 nm.
- 2Record the peak intensity of the water Raman band near 397 nm.
- 3Record the background intensity at a point with no Raman signal (e.g. 450 nm), or record the noise in a time scan as the manufacturer specifies.
- 4Calculate the signal-to-noise ratio using the manufacturer's formula.
RECORD
Example observation table
| Measurement | Wavelength (nm) | Intensity (cps) |
|---|---|---|
| Raman peak | 397 | 125,000 |
| Background | 450 | 2,500 |
CALCULATION
How to calculate the result
S/N = (Peak signal − Background) ÷ √Background (square-root method used by some manufacturers; others use RMS noise from a time scan)
- Peak = 125,000 cps; background = 2,500 cps
- √2,500 = 50
- S/N = (125,000 − 2,500) ÷ 50 = 2,450
ACCEPTANCE CRITERIA
Pass if
- S/N not less than the manufacturer's specification for the same settings and calculation method. Values vary widely between models, so compare only like with like.
IF IT FAILS
What to do
- Check cuvette cleanliness and water purity.
- Check lamp hours and replace the lamp if it is weak.
- Check slit and PMT settings; repeat.
- 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.