This Reference Material can be used to qualify the Signal to Noise Ratio of a spectrofluorometer near the limit of detection and hence indicate its ultimate sensitivity.
Description and Discussion
10mm far UV quartz fluorometer cuvette with polished faces at 90°, containing ultra-high purity water, permanently sealed by heat fusion.
Traditionally, the performance of spectrofluorometers has been tested using dilute solutions of known fluorophores such as Quinine Sulfate or Rhodamine to determine spectral response factors. Starna Certified Reference Materials are available for this purpose. To use this approach at or near the detection limit of a high-performance instrument however, is impractical because the necessary sample concentrations are so low that sample preparation becomes difficult and errorprone and there is high potential for sample contamination.
An alternative approach, employed in this Reference, is to use the phenomenon of Raman scattering. When radiation interacts with a molecule, some of the incident energy can be scattered. Most will be normal (Rayleigh) scattering, where the scattered radiation is at the same wavelength as the incident radiation. A very small proportion of the scattered photons (about 10-7) however, will exhibit Stokes Raman scattering and be emitted at a lower energy (i.e. longer wavelength) in a manner analogous to fluorescence. Because of the very small number of photons involved, the signal levels are very low and comparable to those produced by very low analyte concentrations in normal fluorescence measurements. Pure water exhibits a Raman shift of tens of nanometers when excited by UV or visible radiation, so the Raman peak can be readily identified. Uncontaminated, pure water is relatively straightforward to obtain and is therefore an ideal reference material.
The signal –to-noise ratio for a given excitation wavelength may be calculated using the instrument software.
Note that the calculated ratio will depend on the calculation protocol employed by the instrument manufacturer and therefore ‘compliance to specification’ limits will be specific to the spectrofluorometer type under test.
Typical spectra are provided with excitation at 350nm and 500nm :
Water (Raman) Fluorescence Senitivity Reference