[ 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 exploited by technologies ranging from household cleaning products to advanced biotechnology.

ODA owns one spectrofluorometer, the Varian Eclipse, and operates another, the Spex Fluoro-Max 2, under an agreement with the University of Arizona. The attributes of these two instruments are compared in the following tables:

Wavelength Range

Optical Arrangement

Orientation

Both instruments can accommodate 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.

Both instruments can also accommodate solid samples. For the solid sample attachment for the Varian Eclipse the excitation beam is incident at 15°; the exitent beam is therefore at 75°. For the Spex Fluoro-Max 2, the excitation beam is incident at 20°; the exitent beam is therefore at 70°.

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 ["transflec-tance"], 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 now available.

• The first is known as a "side-looking" sphere; the entrance and exit ports for the sample and reeference 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 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 emplys a powder cell made by Avian Technologies 2with a quartz window and a Spectralon™ cavity; other arrangements can eb 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, such 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 specualr 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.

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

For the Nicolet 560 [IR], a 102 -mm diameter sphere 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.

Gas Samples

ODA maintains a single gas cell with a 10 cm path length.

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 500E 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.