Abstract: in this post we describe the application of the Thunder Optics SMA Spectrometer and the Spectragryph software in absorbance measurements. We will apply the absorption spectroscopy technique to the study of some liquid solutions of colored compounds.
In the 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 absorption spectroscopy. In particular we will use this technique to analyze the optical characteristics of a set of liquid solutions of colored compounds of particular interest.
The Measurement of Absorbance
Absorbance (formerly optical density, indicated with D) is a measure of the electromagnetic radiation that is absorbed by a substance, in spectroscopy it is defined as the opposite of the logarithm of the transmittance.
A = -log10T = -log10(Φt / Φ0) = log10(Φ0) – log10(Φt)
Where Φ0 and Φt are respectively the light incident and light emerging from the sample under examination. The absorbance, for solutions having a sufficiently low concentration, have a linear relationship with the concentration of the sample, according to the Lambert-Beer law. With this relationship, the absorbance measurement is the basis of the quantitative chemical analysis carried out with spectrophotometric technique.
Spectragryph software automates the calculation of absorbance. The selected measurement mode defines the type of y-axis of the measured live spectrum: intensity, transmittance, reflectance, absorbance (Fig. 1). We will choose the absorbance mode.
Fig. 1 – 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. 2 – Dark spectrum assignment
Fig. 3 – Reference spectrum assignment
Fig. 4 – 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, mode: intensity
- 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.
The reference light source should have a spectrum as flat as possible over the entire range of wavelengths of interest. Lamps of this type are for example halogen or xenon lamps. If such a lamp is not available, a different light source can also be used, as long as it has a constant intensity over time. We used Thunder Optics Mini Light Source.
After the definition of the reference spectrum, the measurement mode of the spectrometer can be switched to absorbance, where the absorbance calculated from the raw measurement and the reference spectrum is shown directly on the graph. 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 absorbance is the following:
Absorbance: Live = – log10 ( (Raw – Dark) / (Reference – Dark) ) – Blank
Materials and Methods
To measure the absorbance of liquid solutions you can use the TO spectrometer coupled directly with the cuvette holder and with the reference light source (Fig. 5 a) or use the optical fiber with SMA connector (Fig. 5 b). Using the fiber it is recommended to use fibers with cores of at least 200 μm in order to collect enough light. Using a halogen or xenon source, intrinsically more intense, would allow the easy use of even “thinner” fibers, for example 100 μm.
Fig. 5 a – Setup with direct coupling (without fiber) for absorbance measurements
Fig. 5 b – Setup with fiber for absorbance measurements
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 (Fig. 6).
Fig. 6 – (a) Cuvette with chlorophyll (b) Cuvette with phthalocyanine
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. Two main types of chlorophyll can be identified:
- Chlorophyll a, it mainly absorbs blue-violet and red light (absorption peaks at 430 nm and 663 nm)
- Chlorophyll b, it mainly absorbs blue and orange light (absorption peaks at 480 nm and 650 nm)
For our absorbance measurement (Fig. 7) we used spinach which was chopped and left to macerate in ethanol. Our sample is therefore a solution of chlorophyll in ethanol.
Fig. 7 – Chlorophyll absorbance spectrum
Phthalocyanine is a heterocyclic compound whose chemical structure is similar to that of natural porphyrins. Phthalocyanine, characterized by an intense blue-green color, is widely used as a dye. Phthalocyanine forms coordinated complexes with many elements of the periodic table. These compounds are also intensely colored and find various applications as dyes and pigments. The absorption spectrum, reported in Fig. 8, shows strong absorbance between 550 nm and 750 nm.
Fig. 8 – Phthalocyanine absorbance spectrum
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 solution shows a strong absorption in the band ranging from 400 nm to 550 nm (Fig. 9).
Fig. 9 – Ru(bpy)3 absorbance spectrum
Olive Oil Spectroscopy
The absorption spectrum of extra virgin olive oil, acquired by the spectrometer and shown in Fig. 10, shows remarkable similarity with the spectrum of chlorophyll, a sign that this substance is present within the olive oil. The absorption band from 400 nm to 500 nm is also a typical characteristic of the carotenoid category.
Fig. 10 – Olive Oil absorbance spectrum
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 (yellow-green, very characteristic) when excited by ultraviolet at 254 nm and in the blue range 465-490 nm. The absorption spectrum shown (Fig. 11) highlights the strong absorbance between 450 and 500 nm.
Fig. 11 – Fluorescein absorbance spectrum
Bromothymol Blue Spectroscopy and pH Measure
Bromothymol blue is an organic compound used as a pH indicator. In concentrated ethanol solution it is a a blue-green liquid. In its normal (acidic) form it is yellow in color, while its conjugate base is blue; due to this color difference between the two forms, bromothymol blue is used as a pH indicator. It has a color change range between pH 6.0 and pH 7.6.
The protonated form, i.e. with acid pH, of bromothymol blue has the absorption peak around 450 nm, therefore transmits yellow light in acidic solutions, while the deprotonated form, i.e. with basic pH, has the absorption peak around 600 nm , thus transmitting blue light in basic solutions. The color change is also evident in the absorption spectrum acquired by the spectrometer and shown in Fig. 12.
Fig. 11 – Bromothymol Blue absorbance spectrum depending on pH solution
Our apparatus consisting of Thunder Optics SMA Spectrometer and mini light source proved to be more than adequate for the qualitative and quantitative analysis of the absorbance of the solutions we examined. Analyzes of this type generally make use of halogen or xenon light sources, which guarantee an intense emission with a “flat” spectrum compared to an incandescent lamp. The spectrometer and the spectragryph software have however adequately compensated the non-constant spectrum of the light source used, allowing to obtain excellent results.
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