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Proving The Reliability Of The DH 4.0 v4 Spectrometer

by dhaffnersr | August 03, 2016 15:49 03 Aug 15:49 | #13330 | #13330

A short tutorial on Fluorescein and is Place in the Visible Spectrum

Fluorescein Sodium

Although commonly referred to as fluorescein, the dye used for fluorescein angiography is actually fluorescein sodium (C20 H10 Na2 O5). It is the water-soluble salt of fluorescein, also known as resorcinolphthalein sodium, or uranine. A member of the xanthene group of dyes, it is a highly fluorescent chemical compound synthesized from the petroleum derivatives resorcinol and phthalic anhydride. The dye was first synthesized in 1871 by Adolf von Baeyer, who later received the Nobel Prize in Chemistry (1905) for his work in organic dyes.

Fluorescein sodium absorbs blue light, with peak excitation occurring at wavelengths between 465-490 nm. The resulting fluorescence occurs at the yellow-green wavelengths of 520 to 530 nm dye concentration and pH can affect the intensity of fluorescence. Maximum fluorescence occurs at a pH of 7.4, but the pH of fluorescein sodium for angiographic use is adjusted to a range of 8 to 9.8 for stability. In powdered or concentrated solution form, fluorescein sodium appears orange-red in color.

Fluorescence is detectable in concentrations between 0.1% and 0.0000001%. In broad-spectrum illumination, diluted fluorescein sodium appears bright yellow-green in color. When illuminated with blue light, the yellow-green color intensifies dramatically.

The fluorescent properties of this dye have made it in useful in a variety of industrial, scientific, military, and medical applications. Fluorescein sodium was the first fluorescent dye used for water tracing purposes. It has been used as a visible marker for search-and-rescue operations, to track and measure flow dynamics of water sources, map subterranean water courses, track hazardous spill dispersion patterns, identify point sources of pollution, and to detect leaks or obstructions in plumbing and sewage systems.

In fact, its common use in industrial plumbing led a plumber’s union to start the tradition of using fluorescein to stain the Chicago River green for the annual St. Patrick's Day celebration.

Many of the medical and ophthalmic applications of fluorescein are analogous to its uses in plumbing or industrial flow dynamics. For example, it has been used for intraoperative assessment of blood flow in surgical resections and grafts with a Wood's Light to excite fluorescence. In ophthalmology, topical application of fluorescein sodium is routinely used for applanation tonometry, and as a vital stain in the documentation of ocular surface disorders such as corneal ulcers, abrasions, or other epithelial defects.

It is sometimes used to determine tear film breakdown time, check the fit of contact lenses, verify the patency of lacrimal passageways, and to detect leakage of aqueous humor from corneal or conjunctival wounds using the Seidel Test.

Using my Solux 4700K lamp, I followed normal procedures for capturing absorption spectra for Fluorescein Sodium, which I had previously prepared a standard stock solution of 0.1M per 200ml of distilled water with a pH of 5.5.

These are my results:



The next plot has a CFL (compact fluorescent light) calibration I acquired from Plab in July, it is interpolated on the plot to illustrate it's place in the visible spectrum as it relates to the spectrum for the fluorescein sample.


The next plot is the Absorption and Emission spectra for my fluorescein sample after dark counts removed and division by the solvent sample have been done.


An isolated view of the absorption part of the fluorescein sample indicating its absorption does indeed occur at the blue end of the spectrum.


The final graphic, is the emission plot of the fluorescein sample, with Rayleigh/Raman scattering removed and its particular place in the visible spectrum.


In conclusion, I think I have thoroughly proven that, my version of the Plab v2.5 spectrometer is a viable and also a highly reliable piece of spectroscopic equipment for a very low cost. I renamed it the DH 4.0 v4 Spectrometer, because I have put in 7 months of very hard and tedious work into it, so I feel I deserve the credit for it.

Although this is under the creative commons license agreement, I am in no way in violation of it, because I have given much credit to this detector both here and on Hackaday.



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