Spectrofluorometry

Spectrofluorometry is commonly defined as a type of spectrophotometry deals with fluorescence, the phenomenon in which light incident on a material at a given wavelength, usually in the ultraviolet or visible spectral range, causes that material to radiate light at longer, less energetic wavelengths. To observe this effect, spectrofluorometers have dispersive optics, usually grating monochromators, both between the source and the sample for the incident light and between the sample and the detector for the exitent light. This permits the identification of the wavelengths that excite the sample and the wavelengths at which that excitation generates the fluorescence. Obviously, fluorescence can produce spurious signals in an optical system, so that fluorescent optics and their excitation and emission spectra should be recognized and accounted for. On the other hand, the fluorescence phenomenon is exploited by technologies ranging from household cleaning products to advanced biotechnology. ODA operates an Agilent Eclipse instrument to make these measurements.

Wavelength Range
Instrument Nanometer - nm Range Micron - µm Range Wavenumber - 1/cm Range
Agilent Eclipse 190 1,100 0.19 1.10 52,632 9,091

Optical Arrangement
Instrument Layout Sources Sample Compartment Detectors
Agilent Eclipse Source - monochromator - sample - monochromator - detector Pulsed xenon arc lamp Single-beam, focused Integrating PMT

Orientation

The instrument accommodates cuvettes with a standard 1 cm square cross-section. These are illuminated normally on one face; the emitted light from an adjacent face, that is, at a 90° angle from the incident beam, is detected. It can also accommodate solid samples. For the solid sample attachment for the Agilent Eclipse the excitation beam is incident at 15°; the exitent beam is therefore at 75°.


Sample Properties

Solid Samples

Flat Specular Optical Elements and Other Materials

These materials are measured with direct transmittance sample holders or with the specular reflectance accessories. Ideally, samples have diameters or square sides of 2.5 to 7.5 cm [1 to 3 inches], but samples with dimensions as small as 1 mm [0.040 inch] and as large as 0.75 m [30 inches] can be accommodated with special jigs and holders.

Curved Specular or Rough Optical Elements, Powders, and other Materials

These materials are usually measured at the transmittance or reflectance port of the appropriate integrating sphere. Ideally, samples have diameters or square sides of 2.5 to 7.5 cm [1 to 3 inches], but samples with dimensions as small as 5 mm [0.040 inch] and as large as 0.75 m [30 inches] can be accommodated with special jigs and holders. If both reflectance and transmittance must be measured at the same time ["transflectance"], the sample can be measured at the center of the sphere ["Edwards" mode], but the dimensions must not exceed 2.5 cm.

For the Cary 500E [UV-Vis-NIR], two 150-mm diameter integrating spheres, both made by Labsphere, are available.

  • The first is known as a "side-looking" sphere; the entrance and exit ports for the sample and reference beams are arrayed along the horizontally oriented "equator" of the sphere. The access port is at the "top" pole, and the detector port is at the "bottom" pole. This means that samples are mounted "vertically" on the sides of the sphere.
  • For T measurements, the sample is mounted in front of the entrance port. The specular portion of the can be included [total T] if the exit port on the opposite side is covered with a Spectralon standard identical to that of the sphere walls, or it can be excluded [diffuse T] if the exit port is covered with a black standard or light trap. Samples up to 10 cm across can be accommodated.
  • For R measurements, the specular portion of the beam can be included [total reflectance] or excluded [diffuse reflectance] , depending on whether a small portion of the sphere wall - which is on a removable plug - in the specular direction remains in place or is removed. Samples up to about two meters in maximum dimension can be accommodated.
  • For powder R measurements with this sphere, ODA employs a powder cell made by Avian Technologies with a quartz window and a Spectralon™ cavity; other arrangements can be made if necessary.
  • The second is known as a "down-looking" sphere; the entrance and exit ports for the sample beam are arrayed along the vertically oriented "equator" of the sphere. The sample beam can be switched between an entrance port at the side of the sphere or another entrance port at the "top" pole of the sphere.
  • For T measurements, the sample is placed vertically in front of the entrance port on the side of the sphere. Only total T measurements can be made, but samples up to 20 cm across can be accommodated, about double the capacity of the side-looking sphere.
  • For R measurements, the sample is placed horizontally against the port at the "bottom" pole of the sphere, and that powders and liquids can be accommodated without the use of a cell. The beam is directed downward by a mirror outside the entrance port at the top of the sphere. As with the "side-looking sphere " the specular portion of the beam can be included [total R] or excluded [diffuse R] , depending on whether a small portion of the sphere wall - which is on a removable plug - in the specular direction remains in place or is removed.

A diffuse R accessory involving specular optics and a upward-looking powder holder is also available.

For the Nicolet 560 [IR], a 102 -mm diameter sphere of gold-plated aluminum [InfraGold™] is available. The entrance port for this sphere is on the side of the sphere, so transmittance samples are mounted vertically. Because this sphere employs a specular mirror at its center to deflect the beam along a vertically oriented great circle, reflectance samples can be mounted at the bottom port, permitting the measurement of liquid or powder samples.

There is no integrating sphere capability for the Perkin-Elmer 983G.

Liquid Samples

For the UV-Vis-NIR, ODA maintains a variety of UV quartz, glass, and plastic cuvettes with path lengths ranging from 1 to 50 mm, including matched pairs.

Optical Density

In general, for transmittances down to about 0.01= 1%, measurements are made with no reference beam attenuation in a linear, transmittance mode . For transmittances below 0.01 = 1%, attenuators are placed in the rear beam and the measurements are expressed as absorptance, the negative base 10 logarithm of the transmittance. The limits for the Cary 5000 UV-Vis-NIR SPM are about A = 6.3 in the UV-Vis ranges, and about 4.5 in the NIR range. The limit of the PE 983G IR SPM is about 3 over the MIR & FIR range.

Temperature & Humidity

Most measurements are made at ambient temperature [T]. However, ODA operates a cryostat that can measure transmittance [T] at T's down to about 85K, approaching liquid nitrogen's T of 77 K, For these measurements, a diameter of 2.5 cm is standard; other sizes will incur tooling charges. ODA has measured T of samples at up to 800 °C in heater blocks for special projects. Also, T measurements have been made for fixed levels of humidity in special cells for periods of up to six weeks. ODA also performs measurement before and after exposure to environmental tests to identify failure modes. ODA can subcontract these exposures if requested.

2015, Optical Data Associates, LLC