In 2008, Dr. Richard DeSa and his team started studying filled integrating spheres, known elsewhere as “integrating cavities.”1
These alternatives to cuvettes open up accurate absorbance and fluorescence work on turbid light absorbing samples.
The heightened sensitivity the oceanographers sought is a welcome second attribute. (A third advantage rarely mentioned is that the entire sample is involved in the measurement.)
Not surprisingly, volume and sensitivity are related: the higher the concentration, the less material needed; the lower the concentration, the larger the volume required.
But! Where decreasing the pathlength is one of the ‘tricks’ for handling high scattering samples, scatter is of no concern in the volume & pathlength selection in a DSPC.
For CLARiTY, the decision is entirely based on absorbance. Long pathlengths are as successful with turbidity as short pathlength. Scatter does not matter, only absorbance does.
The dynamic range of one CLARiTY spectrophotometer is determined its DSPC.
~50 uL protein in a low absorbing solvent will be measured using a 9 mL DSPC with roughly 30 cm pathlength; here, the expected dynamic range is nominally 0.001-1 Abs/cm.
2 mL test tubes are useful for samples with absorbances to 2 Abs/cm
~50 uL whole milk will be measured in a 0.2 mm pathlength FT-DSPC; here, the range is nominally 5-100 Abs/cm
Using the right DSPC – just like using the right pathlength cuvette – extends the dynamic range of the spectrophotometer. It takes moments to exchange one DSPC to another and you can always add another in future, should your interests expand.
Apparent absorbance is collected. Converting to AU/cm is achieved using an algorithm incorporated in the CLARiTY software written by Javorfi, et.al.. See the papers by the oceanographers, Javorfi, and others who prepared the way for the CLARiTY here.
Quantum Northwest produces the Peltier DSPCs for Olis instruments.
There is no magic.
Thankfully, there are integrating cavities!
Back in the 1950s, a group of oceanographers filled an integrating sphere with seawater and had success measuring exceedingly low absorbances. In their paper, they noted that ‘scatter by small particulates didn’t matter.’
Inserting solids into the DSPC can be done directly or using a test tube for ease of sample entry and removal.
A suspension-filled integrating cavity, shown external to the completed DSPC. Most are 8 mL, but larger and smaller cavities are available to support a breadth of sample types and effective pathlengths.
A standard cuvette next to the integrating cavity.
A DSPC next to the integrating cavity.
Various size integrating cavities are available to support a variety of sample types.
1 Applied Optics, 1992: “… an integrating cavity is completely filled with an absorbing sample, which generally will be considered to be an aqueous suspension or solution. Because the diffuse reflecting cavity walls have high reflectivity, the effective absorption length in the sample is many times the diameter of the integrating cavity…this integrating cavity concept is especially sensitive to small absorptions.” Another way to think of an integrating cavity is as a bright white room without windows, just two small doors. All of the measurement light, which enters through one door, and all of what would be scattered light, stays within this room, bouncing off the reflective walls, until it escapes through the second door to the detector.
This short video shows exactly what happens inside the OLIS DSPC.
DSPCs are available for a variety of uses. Each can be easily removed and replaced with a different one.