Fluorescence Spectroscopy

Abstract: in this post we describe the application of the Thunder Optics SMA spectrometer and the Spectragryph software in fluorescence measurements. We will apply the fluorescence spectroscopy technique to the study of some chemical compounds of particular importance and interest.

Introduction on Fluorescence

Fluorescence is the property of some substances to re-emit (at a longer wavelength and therefore with a lower energy) the electromagnetic radiation received, in particular to absorb radiation in the ultraviolet and re-emit it in the visible. The mechanism of fluorescence is the following: an incident radiation excites the atoms of the fluorescent substance, promoting an electron to an energetic level (orbital) that is less bound, more energetic and therefore more “external”. Within a few tens of nanoseconds, the excited electron returns to the previous level in two or more phases, that is, passing through one or more excited states at intermediate energy. All decays except one are usually non-radiative, while the last emits light with a longer wavelength than the incident radiation: this emission is called fluorescence (Fig. 1).

Fig. 1 – Absorption of excitation light and emission of fluorescence radiation

We can therefore expect that the emission spectrum partially overlaps the absorption spectrum at the wavelength corresponding to the fluorescence transition, while the rest of the emission spectrum will be at lower energy or, in other words, at greater wavelength. In practice, the transitions involved in the absorption and emission spectra never exactly coincide, the difference represents a small loss of energy that occurs due to the interaction of the absorbent molecule with the surrounding solvent molecules. This difference is called the stokes shift.

In a previous post: Thunder Optics Spectrometer & Spectragryph, we described the Thunder Optics SMA spectrometer (hereafter referred to as the TO spectrometer) and used it to acquire the spectra of some light sources. We now continue the experimentation activity by “exploring” with this equipment, with its accessories and with the spectragryph software, the technique of fluorescence spectroscopy. In particular we will use this technique to analyze the fluorescence of a set of compounds of particular interest.

The Measurement of Fluorescence

Spectragryph software facilitates the measure of fluorescence. The selected measurement mode defines the type of y-axis of the measured live spectrum: intensitytransmittancereflectanceabsorbance (Fig. 2). We will choose the absorbance mode.

Fig. 2 – Measurement Mode selection

Depending on the chosen modality, one or more auxiliary spectra may be required. The auxiliary spectra are the Dark spectrum, the Reference spectrum and the Blank spectrum. Each of these can be set, removed and viewed at any time. As soon as they are registered, they are stored and kept ready for later use. To update them, simply set them up again with a newly measured live spectrum. The Dark spectrum (Fig. 2) and Blank spectrum (Fig. 4) are optional, so their use must be activated by clicking on the respective button. The Reference spectrum (Fig. 3) is always mandatory except for the intensity mode, when necessary it is used automatically by the system.

Fig. 3 – Dark spectrum assignment

Fig. 4 – Reference spectrum assignment

Fig. 5 – Blank spectrum assignment

Measurement mode of auxiliary spectra:

  • Dark spectrum: light source off, shutter closed, no light reaches the detector, mode: intensity
  • Reference spectrum: light source on, full light (100% level) reaching the detector, modeintensity
  • Blank spectrum: with “blank” sample (eg pure solvent or buffer in the sample container), with the final measurement mode selected

Each spectrum should be re-measured after changing the exposure time, furthermore the reference spectrum should be updated after any change in intensity of the excitation light.

To improve the accuracy of the measurement it is always recommended to acquire the dark spectrum and activate its use, the same applies to the blank spectrum. Generally for each measurement context it is necessary to evaluate which auxiliary spectra has to be acquired and used. In general, the formula used by the software for calculating the intensity is the following:

Intensity: Live = Raw – Dark – Blank

Materials and Methods

In general, in the detection of fluorescence, the excitation light source and the detector for measuring the emission are placed at right angle to each other (Fig. 6). For specific quantitative measurements, optical filters are often used, the filters have the purpose of discriminating between the excitation source and the fluorescent emission. The light detector could be, for example a photodiode or, when the light intensity levels are very low, a photomultiplier.

Fig. 6 – Basic scheme for the measurement of fluorescence compared with the method of measurement of absorbance

Fluorescence radiation has its own characteristic spectrum whose intensity depends on the intensity and wavelength of the excitation radiation. In our measurements we used a fixed wavelength excitation source and we used the TO spectrometer to record the emission spectrum. As source of excitation we adopted a DPSS (diode pumped solid state) laser with emission at about 406 nm and one with emission at 445 nm.
The solutions to be examined were inserted in the classic spectrophotometry cuvettes (12x12x45 mm). For the alcoholic solutions we used a quartz cuvette, for the aqueous solutions we used disposable plastic cuvettes. During the measurement the tubes are placed in the cuvette holder. Fig. 7 shows the experimental setup.

Fig. 7 – Experimental setup

As can be seen in the detailed image of Fig. 8, the excitation laser and the fiber for collecting the emission light are placed at right angle to each other.

Fig. 8 – Experimental setup – cuvette holder and DPSS Laser

The intensity of the fluorescence can vary greatly depending on the excitation wavelength, its intensity, the concentration of the solution (higher concentrations give greater fluorescence but also greater self-absorption) and obviously depends on the substance under examination. In Fig. 9 for example, we see the strong bright red fluorescence produced by chlorophyll.

Fig. 9 – Fluorescence in cuvette holder

Chlorophyll Fluorescence Spectroscopy

Chlorophyll is a green pigment present in the chloroplast grains of plant cells or in prokaryotic organisms that carry out chlorophyll photosynthesis. The structure of the molecule is characterized by the presence of a porphyrin heterocycle, at the center of which a Mg ion is coordinated. For our fluorescence measurement (Fig. 10) we used spinach which was chopped and left to macerate in ethanol. Our sample is therefore constituted by an ethanol solution. The emission peak occurs at 674 nm (red) and extends into the infrared region till about 800 nm. The measurement was made with both the 406 nm laser and the 445 nm laser: as you can see from the diagram, the emission spectrum is practically the same except for some small variations in intensity.

Fig. 10 – Chlorophyll fluorescence spectrum

Ru(bpy)3 Fluorescence Spectroscopy

The ruthenium salt known as Ru(bpy)3 is a red crystalline solid, soluble in water and in polar organic solvents. Its cation [Ru(bpy)3]2+ is one of the most studied chemical complexes in photochemical laboratories. The reason for this interest lies in a unique combination of chemical stability, redox properties, luminescence and reactivity in the excited state. Processes involving this compound are often referred to as an example of artificial photosynthesis. In solution, the compound takes on a yellow-orange-red color depending on the molar concentration The fluorescence spectrum is shown in the graph of Fig. 11. As excitation source we used the blue laser with emission at 445 nm, corresponding to the absorption peak. The fluorescence is intense in the red-orange band with the maximum around 610 nm.

Fig. 11 – Ru(bpy)3 fluorescence spectrum

Olive Oil Fluorescence Spectroscopy

Even extra virgin olive oil, thanks to the high content of chlorophyll, shows an intense red fluorescence when excited by a light source in the UV band or in the blue band. The graph in Fig. 12 shows the spectrum obtained by irradiating the oil with the 445 nm laser.

Fig. 12 – Olive oil fluorescence spectrum

Fluorescein Fluorescence Spectroscopy

Fluorescein is the “prototype” of fluorescent dyes. At room temperature it appears as an odorless red-brown solid, very soluble in water, which emits an intense fluorescence in the 520-530 nm range (Fig. 13) when excited by ultraviolet at 254 nm and in the blue range 465-490 nm.

Fig. 13 – Fluorescein fluorescence spectrum

Coumarin Fluorescence Spectroscopy

Coumarin is an aromatic compound. At room temperature it comes in the form of colorless crystals, with a characteristic odor. Isolated for the first time from Dipteryx odorata, whose popular name was coumarin, coumarin is present in more than 27 plant families, and is responsible for the sweet smell of freshly cut grass. Coumarin is also used as a gain medium in some dye lasers and as a sensitizer in photovoltaic technologies. It absorbs at wavelengths below 400 nm and has strong fluorescence at 460 nm (Fig. 14).

Fig. 14 – Coumarin fluorescence spectrum

Ruby Fluorescence Spectroscopy

the ruby ​​crystal exhibits spectacular red fluorescence when excited by violet or blue-green light, with a double emission at 692.80 and 694.30 nm, which appears as a single line. In the cover image and in the image of Fig. 15 we show our ruby ​​crystal illuminated by the violet laser at 406 nm. The crystal emits a strong red fluorescence (Fig. 16). This emission is interesting for its intensity, its very narrow emission line (responsible for the brightness and color of the crystal) and for the long duration of the fluorescence.

Fig. 15 – Ruby Fluorescence

Fig. 16 – Ruby fluorescence spectrum

Quantum Dots Fluorescence Spectroscopy

A quantum dot is a nanostructure formed by an inclusion of a semiconductor material, with a certain band gap and with typical dimensions comparable to the De Broglie wavelength, inside another semiconductor with largest forbidden band. This structure generates a three-dimensional potential well that confines the charge carriers (electrons and holes) to a small region of space where energy levels become discrete. This latter property has led to the association between quantum dots and atoms generating the pseudonym of “artificial atoms”.

In our test we want to measure the fluorescence produced by a colloidal solution of quantum dots, excited by a 406 nm DPSS laser. In particular, we intend to show that the wavelength of the emission is linked to the size of the Quantum Dot: the greater the size, the longer the wavelength of the fluorescent radiation. For this experiment, colloidal solutions of CdTe type QuantumDots (Cadmium – Tellurium) produced by PlasmaChem (Fig. 17) with the following characteristics were used:

CdTe hydrophilic quantum dots Declared Emission max (nm) CdTe radius (nm) Average molar weight (Da)
PL-QDN-520 520 2,04 16’000
PL-QDN-570 570 3,12 59’000
PL-QDN-600 600 3,39 76’000
PL-QDN-640 640 3,66 96’000
PL-QDN-680 680 4,22 146’000

Fig. 17 – Colloidal solutions of quantum dots

The fluorescence spectra obtained with the TO spectrometer and the Spectragryph software are shown in Fig. 18. It can be seen that as the radius of the QD increases, the fluorescence emission shifts towards longer wavelengths, from 540 nm and till 680 nm.

Fig. 18 – QD fluorescence spectra

Europium Fosforescence Spectroscopy

Phosphorescence is the phenomenon of radiative emission from some materials following the absorption of energy through ultraviolet rays (very energetic) and the subsequent re-emission in the form of visible light (with lower energy). The phosphorescent materials continue to emit light even up to many hours after the end of external illumination. When all the accumulated energy runs out, the material no longer emits light. The radiative emission derives from the decay of electrons to quantum levels of lower energy. It is distinguished from fluorescence because in the latter the effect is immediate and stops as soon as the energy source is interrupted, while in phosphorescence the effect continues even after. The principle, simplified, is the same: an energy source, generally composed of visible light or ultraviolet radiation, excites the atoms, causing some electrons to jump to an outermost orbit. When these return to internal orbit they emit light.

Europium activated Strontium aluminate is a new material that exhibits high luminosity and long persistence phosphorescence. The excitation wavelengths for strontium aluminate vary between 200 and 450 nm. The green emission wavelength is 520 nm, for the green-blue version it is 505 nm, while for the blue version it is 490 nm. The following diagram (Fig. 19) shows the spectrum of the phosphorescence excited by the DPSS laser at 406 nm.

Fig. 19 – Europium based compound phosphorescence spectrum

The phosphorescence decays slowly and this allows us, by recording the spectra at regular intervals, to evaluate the temporal trend of the intensity of the emission. The diagram of Fig. 20 shows the spectra recorded, at intervals of 5 seconds from each other, after the excitation source has been switched off. We see that the intensity decreases, first quickly and then more slowly. In greater detail, in the graph of Fig. 21, we have reported the intensity of the emission at the wavelength of 520 nm with respect to time. The decay curve is usually approximated with a power law.

Fig. 20 – Phosphorescence spectra recorded at intervals of 5 sec.

Fig. 21 – Trend decay of phosphorescence intensity


Our apparatus consisting of Thunder Optics SMA Spectrometer and cuvette holder proved to be more than adequate for the qualitative and quantitative analysis of the fluorescence of the solutions we examined. If the fluorescence intensity is high, the measurement can be made with the optical fiber, if the intensity is low and a fiber of large core diameter is not available, it can also be operated by directly coupling the spectrometer and cuvette holder. The results obtained are however excellent, demonstrating the goodness of the experimental setup.

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