Demonstration of Molecular Fluorescence and Absorbance Using an Educational Spectrometer

(From Wikipedia: https://en.wikipedia.org/wiki/Fluorescence )

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

Fluorescence has many practical applications, including mineralogy, gemology, medicine, chemical sensors (fluorescence spectroscopy), fluorescent labelling, dyes, biological detectors, cosmic-ray detection, vacuum fluorescent displays, and cathode-ray tubes. Its most common everyday application is in (gas-discharge) fluorescent lamps and LED lamps, in which fluorescent coatings convert UV or blue light into longer-wavelengths resulting in white light which can even appear indistinguishable from that of the traditional but energy- inefficient incandescent lamp.

Fluorescence also occurs frequently in nature in some minerals and in many biological forms across all kingdoms of life. The latter may be referred to as biofluorescence, indicating that the fluorophore is part of or is extracted from a living organism (rather than an inorganic dye or stain). But since fluorescence is due to a specific chemical, which can also be synthesized artificially in most cases, it is sufficient to describe the substance itself as fluorescent.

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Atoms and molecules have energy levels, determined by quantum mechanics, that may interact with light. The dipole moment observed in transitions between different energy levels determines the strength of this interaction. In general, if there is a change in the wavefunction symmetry as an electron makes a transition from an initial state to a final state, there is a finite dipole moment, and therefore interaction with light. Typically, absorption results electrons to be kicked to from a lower energy level to high energy levels, and after some relaxation it decays back to the lower levels while emitting light. The wavefunctions are easy to calculate for simple atoms like hydrogen. In fact, the theory of atomic emission helped the development of quantum mechanics by supplying experimental data.

For a more simplified explanation take a look at the following:

Imagine a high schooler jumping on a trampoline (excited state) in the sunlight (absorption). They can stay up for a while, energized by the sun, but eventually gravity pulls them down (relaxation). As they descend, they might shout or laugh (emitting light), releasing some of that energy. This "shout" might be a lower-pitched squeal (lower energy, longer wavelength) than the original sunlight. That's the basic idea behind a Jablonski diagram in fluorescence!

The Jablonski diagram describes most of the relaxation mechanisms for excited state molecules. The diagram alongside shows how fluorescence occurs due to the relaxation of certain excited electrons of a molecule.(from Wikipedia)

The Trampoline Analogy:

Ground State (S0): Imagine the student standing on the ground, relaxed and at their normal energy level. This is the molecule in its non-excited state.

Absorption (A): Sunlight hits the student, giving them a boost of energy. The molecule absorbs a photon (light particle) and jumps to an excited state (S2).

Internal Conversion (IC): Like the student wiggling with excitement before jumping, the molecule might release some energy as heat before reaching the lower excited state (S1).

Fluorescence (F): The student jumps and shouts as they fall back down. The molecule relaxes from S1 to S0, emitting a photon (fluorescence) with less energy (longer wavelength) than the absorbed light.

Phosphorescence (P): If the student holds their breath and slowly releases it with a sigh, that's like phosphorescence. The molecule gets "stuck" in an excited state (T1) for a longer time before emitting lower energy light.

The Diagram Explained:

The Jablonski diagram is a fancy way to show these energy level changes visually. It has:

Horizontal lines: Each line represents an energy level (S0, S1, S2, T1).

Vertical arrows: Up arrows show absorption (energy gain), down arrows show emission (energy loss).

Wavy arrows: Internal conversion (heat loss) is shown by wavy lines.

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In general, emitted fluorescence light has a longer wavelength and lower energy than the absorbed light. This phenomenon, known as Stokes shift, is due to energy loss between the time a photon is absorbed and when a new one is emitted. The causes and magnitude of Stokes shift can be complex and are dependent on the fluorophore and its environment. However, there are some common causes. It is frequently due to non-radiative decay to the lowest vibrational energy level of the excited state. Another factor is that the emission of fluorescence frequently leaves a fluorophore in a higher vibrational level of the ground state. (from Wikipedia)

Here we explore the absorption and emission of light by a fluorescent acrylic slab. We use an educational spectrometer to quantitatively observe absorption in a transmission setup. We observe the fluorescence using a 405 nm laser pointer to excite the slab. Indeed, there is a shift between the absorption and emission, the so called Stokes shift.

An acrylic fluorescent slab (orange is used, bought from online retailers)

An educational spectrometer (e.g. Spectryx Blue, available from online retailers)

Helping hands to hold white light source and fiber

White light bulb (e.g. Maglite Xenon)

A power source (e.g. 3.3V output from an Arduino Uno) to power the white light

A 405nm near UV laser pointer

Using one crocodile of the helping hands, hold the white light bulb. Hold the fiber cable using the other crocodile. Move them close together, to about 1 cm distance. Connect the spectrometer and power source and observe light transmission on your computer. Then, get a reference spectrum, and switch to transmission measurement mode.

Place the slab in between the light source and fiber end. Measure the transmission using the computer. Save your data.

Then turn off the white light, and use the 405 nm UV laser pointer to excite the fluorescence close to the fiber input. Switch the software back to the absolute mode, and measure the fluorescent spectrum. Save your data.

Now you can use a graphing software (Excel or Octave) to plot your data. It is seen that, there is indeed a shift between the absorption peak and emission peak, that is the Stokes shift. Note that, the spectrometer measures transmission, so absorption peak shows up as a dip in the transmission.