Spectrometer Complements ENG

Diffraction
Grating
Spectrometer
Design and Collected
Spectra
Theremino System
Theremino System Spettrometro_ENG Page 1
Table of contents
Design and Components.................................................................................................................................... 6

Theory of Diffraction Grating ........................................................................................................................ 6
Diffraction Grating Specifications .................................................................................................................. 8
Portable Spectrometer Construction ................................................................................................................ 9
Benchtop Spectrometer Construction............................................................................................................. 10
Spectra of Lamps ............................................................................................................................................. 11
Spectra of Flames and Plasma ......................................................................................................................... 14
Emission and Atomic Absorption spectra........................................................................................................ 15
LED Spectra ...................................................................................................................................................... 16
Laser Spectra ................................................................................................................................................... 18
Sun Spectrum .................................................................................................................................................. 20
Wien Law / Black body radiation ................................................................................................................. 22
Comparison with Reference Spectra ............................................................................................................... 23
CFL Lamp...................................................................................................................................................... 23
Neon Lamp .................................................................................................................................................. 24
Sodium-vapor Lamp..................................................................................................................................... 25
Absorption Spectroscopy ................................................................................................................................ 26
Absorption Spectrometer Design ................................................................................................................ 26
Water ........................................................................................................................................................... 27
Red Wine ..................................................................................................................................................... 27
Blue Food Dye.............................................................................................................................................. 28
Yellow Food Dye .......................................................................................................................................... 28
Blue Ink ........................................................................................................................................................ 28
Fluorescence Spectroscopy ............................................................................................................................. 29
Fluorescence Spectrometer Design ............................................................................................................. 29
Fluorescence Excitation Sources ................................................................................................................. 30
Fluorescence Theory ................................................................................................................................... 31
Spectroscopy of Organic Pigments .................................................................................................................. 33
Chlorophyll .................................................................................................................................................. 33
Phycoerythrin .............................................................................................................................................. 35
Anthocyanins as pH Indicator ...................................................................................................................... 36
Carotenoids ................................................................................................................................................. 38
Betalaine ...................................................................................................................................................... 39
Spectroscopy of Fluorescent Dyes................................................................................................................... 41

Stilbenes Triazine (Optical Brightener) ..................................................................................................... 41
Fluorescein Sodium (Uranine) ..................................................................................................................... 42
Eosin ............................................................................................................................................................ 43
Acridine Orange ........................................................................................................................................... 44
Rhodamine B ............................................................................................................................................... 45
Rhodamine 6G ............................................................................................................................................. 46
Green Fluorescent Dye ................................................................................................................................ 47
Methylene Blue ........................................................................................................................................... 48
Crystal Violet................................................................................................................................................ 49
Methylene Blue Malachite Green Crystal Violet Acriflavine (Preparation for Fungal infections) ...... 50
Phthalocyanine ............................................................................................................................................ 51
Erythrosine .................................................................................................................................................. 52
Coumarin ..................................................................................................................................................... 53
Spectroscopy of Edible Oils ............................................................................................................................. 54
Extra Vergine Olive Oil ............................................................................................................................. 54
Peanut Oil .................................................................................................................................................... 55
Sunflower Oil ............................................................................................................................................... 56
Corn Oil ........................................................................................................................................................ 56
Soybean Oil .................................................................................................................................................. 56
Almond Oil ................................................................................................................................................... 57
Sesame Oil ................................................................................................................................................... 57
Castor Oil ..................................................................................................................................................... 57
Spectroscopy of Vitamins ................................................................................................................................ 58
Cyanocobalamin (Vitamin B12) ................................................................................................................... 58
Riboflavin (Vitamin B2) and Pyridoxine (Vitamin B6) .................................................................................. 59
Retinol (Vitamin A) ...................................................................................................................................... 59
Spectroscopy of Food Substances ................................................................................................................... 60
White Wine .................................................................................................................................................. 60
Wine Vinegar ............................................................................................................................................... 60
Isoflavones (Soy Sauce) ............................................................................................................................... 61
Green Walnut Extract (Liqueur Nocino) ...................................................................................................... 61
ProtoPorphyrin (Egg Shell) .......................................................................................................................... 62
Honey........................................................................................................................................................... 63
Rose Siroop .................................................................................................................................................. 64
Rhubarb Extract ........................................................................................................................................... 64

Ale Beer ....................................................................................................................................................... 65
Dark Beer ..................................................................................................................................................... 65
Stout Beer .................................................................................................................................................... 65
Pistachio Extract .......................................................................................................................................... 66
Green Tea .................................................................................................................................................... 66
Spectroscopy of Drugs ..................................................................................................................................... 67
Hematoporphyrin ........................................................................................................................................ 67
Sodium Salicylate ......................................................................................................................................... 68
Piroxicam ..................................................................................................................................................... 69
Spectroscopy of Various Compounds.............................................................................................................. 70
Pyranine ....................................................................................................................................................... 70
Perfume ....................................................................................................................................................... 70
Bleaching Detergent .................................................................................................................................... 70
Methyl Salicylate ......................................................................................................................................... 71
Quinine ........................................................................................................................................................ 71
Urine ............................................................................................................................................................ 72
Hemoglobin ................................................................................................................................................. 73
Gasoline ....................................................................................................................................................... 74
Anthracene .................................................................................................................................................. 74
Tetracene (Naphthacene)............................................................................................................................ 75
Motor Oil ..................................................................................................................................................... 75
Bergamot Essential Oil................................................................................................................................. 76
Chamomile Essential Oil .............................................................................................................................. 77
Orange Essential Oil..................................................................................................................................... 78
Orange Blossoms Water .............................................................................................................................. 78
Curcumin Essential Oil ................................................................................................................................. 79
Spectroscopy of Inorganic Compounds ........................................................................................................... 80
Strontium Aluminate ................................................................................................................................... 80
Uranium glass .............................................................................................................................................. 81
Halogen Inner Bulb ...................................................................................................................................... 81
Raman Spectroscopy ....................................................................................................................................... 82
Chemiluminescence Spectroscopy .................................................................................................................. 83
Blue LightStick ............................................................................................................................................. 84
Green LightStick........................................................................................................................................... 84
Yellow LightStick .......................................................................................................................................... 84

Purple LightStick .......................................................................................................................................... 85
Orange LightStick ......................................................................................................................................... 85
Red LightStick .............................................................................................................................................. 85
Design and Components
Webcam : NEW TRUST MEGAPIXEL WEBCAM PRO 1.3MP 1024x1280

Diffraction gratings : holographic 1000 linee/mm 600 linee/mm
Collimating lens
slit 100micron / slits 0.10.5-1mm built with razor blades / micrometric adjustable slit
Portable Spectrometer
Theory of Diffraction Grating

A monochromatic light beam that is incident on a grating gives rise to a transmitted beam and various
diffracted beams , at angles that depend on the ratio between the distance between the lines of the grating
and the wavelength of the light.
So, if the light beam is composed of multiple wavelengths, the decomposition of the beam into its
components is obtained.
The light with a longer wavelength is deflected to a larger angle with respect to the incident direction
(angle of diffraction ) . For each wavelength more rows can be observed. The number of rows that are
counted from the middle line, which is not skewed with respect to the incident beam and is taken as a
reference , it is said "order" and is often denoted by the letter m.
The diffraction gratings can act both for transmission and for reflection of the incident light depending on
the light scattering takes place on the same side or the opposite side of the light source . The transmission
gratings are composed of a transparent plate on which are created many small strips that do not allow the
passage of the radiation. In this way you get many small slits whose figure generated on a screen is solved
by a method analogous to that used for the interference .
The reflection gratings are constituted by a reflective layer (mirror) on which are created many small strips
or grooves that do not allow the reflection of the radiation. They are used in monochromators and
spectrometers .
The distance between the slits, known as "grating pitch" , in the gratings used in spectroscopy is of the
same order of magnitude as the wavelength of the light to be analyzed . In practice, the patterns are
usually characterized by the number of engravings per unit length , often expressed in lines per millimeter
(l/mm).
The fundamental property of the gratings is that the angle of deviation of all the refracted beams depends
on the wavelength of the incident light. Thus, a grating separates a beam of polychromatic light in its
wavelengths components, so the grating is a dispersive tool.
When a light beam is incident on a grating is diffracted in different beams. The beam corresponding to the
direct transmission is called zero-order diffraction . The convention in use denote the not deflected beam
with m = 0 . Respect to the direction identified by the reference beam is possible to measure the diffraction
angle that characterizes each deflected beam . m can assume positive or negative values depending on that
the deflected beam is to the right or left of the zero order beam (this depends on the convention used for
the sign of the angles).
Denoting by d the grating pitch and the wavelength of the incident radiation can be written :
When the beam hits at angle i the grating. The sign in the formula depends on the choice of the
Convention on the sign of the angles.
From the previous relation it can be seen that a beam of polychromatic light is divided into its components
from violet ( which is the color characterized by shorter wavelength ) till to red; instead in a glass prism the
angle of deviation is greater for violet, so the sequence of colors is reversed.
Comparison between the spectra obtained from a diffraction grating (1), and
from a refraction prism (2). Longer wavelengths (red) are deflected more,
while the shorter ones (violet) are deflected less.
The light from a lamp seen through a transmission grating which shows
three diffraction orders. The order m = 0 corresponds to the direct
transmission through the grating.
In the first positive order (m = +1), colors with longer wavelength (from violet to red) are deflected at
higher angles.
The diffracted beams of different colors and corresponding to consecutive orders can overlap, this
phenomenon becomes more likely to grow in the order of diffraction. Moreover, in an experiment the
observed diffraction lines are never infinitely narrow ( as expected from the theory ) , this phenomenon is a
consequence of the not ideal experimental conditions and because of the Doppler effect.
The equation of the grating shows that the diffraction angle depends only on the pitch of the grating and
not by the shape of the slits. The efficiency of the grating can also depend on the polarization of the
incident light .
Diffraction Grating Specifications

Type of
Grating
Lines/mm
Paton
grating
Holographic
grating
Diffraction for different wavelengths
600
Space
between
lines
(nm)
1600
1000
1000
400nm
500nm
600nm
700nm
800nm
900nm
14
18
22
26
30
34
24
30
37
44
53
64
Gratings in use
Holographic grating with 1000 lines/mm and holographic Paton grating with 600 lines/mm
Portable Spectrometer Design :
Grating
40o
Slit
Webcam
Benchtop Spectrometer Design :

Collimator lens
Webcam
Grating
Slit
Webcam Lens
Portable Spectrometer Construction
Grating
Webcam
Details of webcam and diffraction grating
0.1mm slit, 0.5mm slit and 1mm slit
Scattering screen
Benchtop Spectrometer Construction
Lens
Webcam
Grating
Inside view with collimating lens, grating and webcam
Slit
Detail of the micrometric slit and the spectrometer assembled

Calibration CFL lamp
Spectra of Lamps
Infrared Lines
CFL lamp just lit presence of lines broadened by phosphorus infrared emission ( > 750nm)
436nm
546nm
576nm
579nm
CFL lamp after warm-up (detail 400nm 800nm) no infrared emission
Incandescent lamp 25W continuous spectrum with maximum at ~ 800nm
Halogen lamp 70W continuous spectrum more emission at shorter (temperature is increased)
Low pressure neon lamp discrete spectrum with many narrow lines
Wood lamp UV emission at 370nm with tiny

visible tail beyond 380nm
Sodium-vapor lamp (street lamp) evidence of the sodium double absorption line at a 589nm further
lines at 568nm, 616nm, 514nm, 498nm all corresponding to emission lines of the sodium spectrum
Xenon lamp
Due to the broad extension of the emission, from 350nm to 950nm, this lamp is used in absorption and
fluorescence spectroscopy
Spectra of Flames and Plasma
Candle emission mainly at longer wavelengths
Plasma from high voltage arc great emission of UV radiation

Atmospheric nitorgen emission spectrum (from
reference spectra). Corespondance of the emission
peaks at 400nm and 430nm. Thus we can infer that
the plasma emission is mainly due to the nitrogen
emission.
Emission and Atomic Absorption spectra
Sodium doublet
In the first spectrum of HV plasma with sodium chloride is evident the sodium line at 589nm. In the
second spectrum the detail shows the sodium doublet at 589,0nm and 589,6nm. The third and fourth
spectra show the detail of the absorption line in a sodium-vapor lamp
Potassium doublet
Spectrum of potassium chlorate match evidence of potassium doublet at 770nm
Sodium emission
590nm
Potassium emission
770 and 780nm
HV plasma spectrum with sodium and potassium emission lines overlapping thermal background
LED Spectra
Infrared LED
Red LED
Yellow RED
Green LED
Blue LED
Violet LED
White LED cold light
Laser Spectra
Violet laser diode ~ 410nm
Red laser diode emission 650nm
Green laser diode detail of the emission line with two peaks at 532 and 530nm
Green laser diode the pumping emission lines are shown
532nm green laser diode functioning scheme. There is IR pumping emission at 800nm. Nd:YVO4 crystal
converts this wavelength into 1064nm emission, the latter is frequency-doubled, at 532nm, from a KTP
crystal
Sun Spectrum
O2 Atm
H2O Atm
O2 Atm
H
Mg
Na
Fe
Sun spectra at different times
Maximum at ~ 530nm => T = 5500Ko (from black body radiation / Wien law)
Evidence of UV (<400nm) and IR (>750nm)
Evidence of the following absorption bands / lines :
Atmospheric oxygen absorption band O2 760nm Fraunhofer A
Atmospheric water vapor absorption band 720nm

Atmospheric oxygen absorption band O2 684nm Fraunhofer B
Absorption H 657nm (Balmer series) Fraunhofer C

Absorption H 480nm (Balmer series) Fraunhofer F
Absorption H 430nm (Balmer series) Fraunhofer G
Sodium absorption line at 589nm Fraunhofer D
Iron absorption line at 530nm Fraunhofer E
Magnesium absorption line at 520nm Fraunhofer b
Fraunhofer main lines
Sun spectrum 650nm - 800nm with absorption bands of atmospheric gases
Wien Law / Black body radiation

Wien's displacement law states that the black body radiation curve for different temperatures peaks at
a wavelength inversely proportional to the temperature. The shift of that peak is a direct consequence of
the Planck radiation law which describes the spectral brightness of black body radiation as a function of
wavelength at any given temperature. However it had been discovered by Wilhelm Wien several years
before Max Planck developed that more general equation, and describes the entire shift of the spectrum of
black body radiation toward shorter wavelengths as temperature increases.
,
Sun spectrum with absorption bands of

atmospheric gases
Comparison with Reference Spectra

CFL Lamp
Peak number
Wavelength of peak
(nm)
Species producing peak
Actual line location (nm)
405.4
mercury
402 - 404
2
3
4
5
436.6
487.7
542.4
546.5
437
488
542
546
577.7
580.2
584.0
587.6
Mercury
terbium from Tb3+
terbium from Tb3+
Mercury
likely terbium from Tb3+ or
mercury
mercury or terbium from
Tb3+
possibly terbium from
Tb3+ or europium in
Eu+3:Y2O3
likely europium in Eu+3:Y2O3
576
579
584
587
10
11
12
13
14
15
16
17
18
19
20
21
22
593.4
599.7
611.6
625.7
631.1
650.8
662.6
687.7
693.7
707 and 709
712.3
760.0
811.0

europium in Eu+3:Y2O3
likely terbium from Tb3+
likely argon
likely argon
592
600
612
625
630
649
661
689
693
707 709
712
Neon Lamp
Neon lamp reference spectrum

Correspondence of all emission lines. More than 25 different lines are shown
Sodium-vapor Lamp
Sodium-vapor lamp evidence of the double sodium absorption line at 589nm lines at 568, 616, 514,
498 e 467 nm all correspondent to sodium emission lines
Absorption Spectroscopy
The absorption spectra are obtained by means of an halogen lamp and a xenon lamp with constant
emission over a wide wavelength range. The variations in the emission intensity are compensated with a
software algorithm. The useful bandwith starts at 370nm till to 1000nm.
Absorption Spectrometer Design
Xenon / Halogen
lamp
Sample
Spectrometer
Sample holder
Halogen lamp and sample cuvette
holder
Halogen
lamp
Xenon lamp
Halogen Lamp Linear Reference
Linear reference from 400nm to 1000 nm

Xenon Lamp Linear Reference
Linear reference from 370nm to 700 nm
Water
IR absorption
Water absorption (transmission) spectrum. Water absorbs from near IR
Red Wine
Anthocyanins
absorption
Red wine absorption (transmission) spectrum

Main absorption band with shorter than 550nm due to the content of anthocyanins
Blue Food Dye
Absorption
band
Blue food dye E131 (blue patent V) E514 (sodium solfate) absorption (transmission) spectrum
between 500nm and 700nm are absorbed
Yellow Food Dye
Absorption
band
Yellow food dye E102 (tartrazine) E110 (orange yellow S)

(transmission) spectrum
shorter than 550nm are absorbed
E514 (sodium solfate) absorption
Blue Ink
Blue ink absorption (transmission) spectrum

between 470nm and 700nm are absorbed
Fluorescence Spectroscopy
Fluorescence Spectrometer Design
Sample
405nm Laser
532nm Laser
650nm Laser
Wood lamp 370nm
Laser
holder
Spectrometer
Cuvette
holder
Construction scheme with excitation

lasers and cuvette holder
Example of fluorescence of a sample (olive oil) excited with a

violet laser at 405nm inside a cuvette
Fluorescence Excitation Sources
Violet Laser
Power: 20mW
Wavelength: 405nm
Current: <280mA @ 2.9 ~ 3V input
Color: blue violet
Green Laser
Power: 30mW
Wavelength: 532nm
Voltage: 2.9 ~ 3V input
Color: green
Red Laser
Power: 5mW
Wavelength: 650nm
Voltage: 2.9 ~ 3V input
Color: red
Wood Lamp
Wavelength: 370nm
Fluorescence Theory
At room temperature most molecules occupy the lowest vibrational level of the ground electronic state,
and on absorption of light they are elevated to produce excited states. The simplified diagram below shows
absorption by molecules to produce the first S1 excited state.
Excitation can result in the molecule reaching any of the
vibrational sub-levels associated with each electronic state.
Since the energy is absorbed as discrete quanta, this should
result in a series of distinct absorption bands. However, the
simple diagram above neglects the rotational levels associated
with each vibrational level and which normally increase the
number of possible absorption bands to such an extent that it
becomes impossible to resolve individual transitions. Therefore,
most compounds have broad absorption spectra.
Having absorbed energy and reached one of the higher
vibrational levels of an excited state, the molecule rapidly loses
its excess of vibrational energy by collision and falls to the
lowest vibrational level of the excited state. In addition, almost all molecules occupying an electronic state
higher than the second undergo internal conversion and pass from the lowest vibrational level of the upper
state to a higher vibrational level of a lower excited state which has the same energy. From there the
molecules again lose energy until the lowest vibrational level of the first excited state is reached.
From this level, the molecule can return to any of the vibrational levels of the ground state, emitting its
energy in the form of fluorescence. If this process takes place for all the molecules that absorbed light, then
the quantum efficiency of the solution will be a maximum, unity. If, however, any other route is followed,
the quantum efficiency will be less than one and may even be almost zero.
One transition, that from the lowest vibrational level in the ground electronic state to the lowest
vibrational level in the first excited state, the 0 - 0 transition, is common to both the absorption and
emission phenomena, whereas all other absorption transitions require more energy than any transition in
the fluorescence emission. We can therefore expect the emission spectrum to overlap the absorption
spectrum at the wavelength corresponding to the 0 0 transition and the rest of the emission spectrum to
be of lower energy, or longer wavelength.
In practice, the 0-0 transitions in the absorption and emission spectra rarely coincide exactly, the difference
representing a small loss of energy by interaction of the absorbing molecule with surrounding solvent
molecules. This difference in wavelength is called stokes shift.
em > a
a and em are absorption and emission spectra peaks
Max Em. 537nm

Max Abs. 525nm
Example of Stokes Shift

Absorption and emissione spectra of acridine orange. From the pictures we can see that the difference
between the maxima is rather small : stokes shift = 537 525 = 12nm
The absorption of energy to produce the first excited state does not perturb the shape of the molecule
greatly and this means that the distribution of vibrational levels is very similar in both the ground and first
excited states. The energy differences between the bands in the emission spectrum will be similar to those
in the absorption spectrum and frequently the emission spectrum will be approximate to a mirror image of
the absorption spectrum.
Mirror Rule
Max Em. 620nm

Max Abs. 620nm
Example of Mirror Spectra

Absorption and emission spectra of hematoporphyrin. There is coincidence of the first peaks of emission
and absorption spectra. The shape of the two spectra are in a good agreement to the mirror-image rule
Since the emission of fluorescence always takes place from the lowest vibrational level of the first excited
state, the shape of the emission spectrum is always the same, despite changing the wavelength of exciting
light. This is also known as the Kasha rule
UV exc.
532nm exc.
Example of Emission Spectra Excited by different wavelength

Emission spectra of fluorescein. There is coincidence of the maximum and shape of emission spectrum
despite different exciting wavelength. In the second spectrum are shown both the anti-stokes (negative
shift) emission and the stokes emission (positive shift)
The Kasha rule does not always apply and is violated in many simple molecules, an example is the compost
azulene which emits fluorescence from transition S2 - S0 instead of the usual transition S1 - S0. For details
see the paragraph on the chamomile essential oil.
Fluorescence is also influenced by the structure of the molecule. For example the rigid molecules that
present systems of conjugated double bonds , are well suited to the fluorescence : in particular molecules
where there are aromatic structures, in which the resonance phenomenon of the double bonds are
scattered throughout the structure, if excited give rise to * transitions, and thus facilitate the
fluorescence.
Spectroscopy of Organic Pigments

Chlorophyll
Chlorophyll is a green pigment found in chloroplasts of algae and plants.
Chlorophyll is an extremely important biomolecule, critical
in photosynthesis, which allows plants to absorb energy from light.
Chlorophyll absorbs light most strongly in the blue portion of
the electromagnetic spectrum, followed by the red portion. Conversely,
it is a poor absorber of green and near-green portions of the spectrum,
hence the green color of chlorophyll-containing tissues.
Measurement of the absorption of light is complicated by the solvent
used to extract it from plant material, which affects the values obtained.
In diethyl ether, chlorophyll A has approximate absorbance maximum of
430 nm and 662 nm, while chlorophyll B has approximate maximum of 453 nm and 642nm.
Chlorophyll A fluoresces at 673 nm (maximum) and 726 nm. The different absorption spectra of Chlorophill
A and B achieve a better absorption of the sun radiation in order to enhance the efficiency of the
photosynthesis.
The alcoholic chlorophyll solution has been obtained with spinach leaves grinded and macerated in ethanol
95%
Reference
Chlorophyll B
Chlorophyll A
Chlorophyll B
Chlorophyll A
Detail
Absorption (transmission) spectrum of chlorophyll solution in 95% ethanol
The green band from 500nm till to 600nm passes without absorption. There are two main absorption
bands, the first one on the red the second one on the blue. Two maxima between 600nm and 700nm
correspond to absorption peaks of chlorophyll A and chlorophyll B. The absorption on the blue band can be
explained with the sum of the absorptions of the two chlorophyll types
Chlorophyll A
Fluorescence
Excitation
II
III
Excitation
Raman
Scattering
Excitation
Chlorophyll A
Fluorescence
Chlorophyll A
Fluorescence
I : Chlorophyll fluorescence spectrum excited with laser emission at 405nm

Emission peak at 670nm due to Chlorophyll A with a second peak around 710nm and tail till to 740nm
II : Fluorescence spectrum excited with laser emission at 532nm
Emission peak at 670nm due to Chlorophyll A
III : Fluorescence spectrum excited with laser emission at 650nm
Emission peak at 670nm due to Chlorophyll A
Phycoerythrin
Phycoerythrin (PE) is a red protein-pigment complex from the light-harvesting phycobiliprotein family,
present in red algae rhodophytes, accessory to the main chlorophyll pigments responsible
for photosynthesis.
Red algae, like palmaria palmata, are red because of the presence of the pigment phycoerythrin; this
pigment reflects red light and absorbs blue light. Because blue light penetrates water to a greater depth
than light of longer wavelengths, these pigments allow red algae to photosynthesize and live at somewhat
greater depths than most other "algae". Some rhodophytes have very little phycoerythrin, and may appear
green or bluish from the chlorophyll and other pigments present in them.
Absorption peaks in the visible light spectrum are measured at 495 and 545/566 nm, depending on the
chromophores bound and the considered organism. A strong emission peak exists at 575 10 nm.
(i.e., phycoerythrin absorbs slightly blue-green/yellowish light and emits slightly orange-yellow light.)
The aqueous phycoerythrin solution has been obtained with palmaria palmata leaves grinded and
macerated in water
Phycoerythrobilin is the typical chromophore in
phycoerythrin. It is similar to porphyrin of chlorophyll for
example, but tetrapyrrole is linear, not closed into ring
with metal ion in the middle.
Riboflavin
Fluorescence
Phycoerythrin
Fluorescence
Chlorophyll A
Fluorescence
Polyphenols
Fluorescence
I
Phycoerythrin
Fluorescence
II
I : Fluorescence spectrum excited with laser emission at 405nm
Different fluorescence bands are shown : around 670nm, due to chlorophyll, around 570nm due to
phycoerythrin. The two bands at 475nm and 535nm are likey due to polyphenols and riboflavin
II : Fluorescence spectrum excited with laser emission at 532nm
The 532nm excitation shows the maximum at 570nm due to the phycoerythrin chromophore
Anthocyanins as pH Indicator
Anthocyanins are water-soluble vacuolar pigments that may
appear red, purple, or blue depending on the pH. They belong to a
parent class of molecules called flavonoids synthesized via
the phenylpropanoid pathway; they are odorless and nearly
flavorless,
contributing
to
taste
as
a
moderately astringent sensation.
Anthocyanins
occur
in
all tissues of higher plants, including leaves, stems, roots, flowers,
and fruits. Anthoxanthins are clear, white to yellow counterparts of
anthocyanins occurring in plants.
Anthocyanins are derived from anthocyanidins by adding pendant
sugars. Anthocyanins are glucosides of anthocyanidins, the basic chemical structure of which is shown in
the picture.
The absorbance pattern responsible for the red color of anthocyanins may be complementary to that of
green chlorophyll in photosynthetically active tissues. Together with carotenoids the red color become
evident in autumn when the chlorophyll degradates and the remaining anthocyanins and carotenoids
contribute to the color of the leaves.
In addition to their role as light-attenuators, anthocyanins also act as powerful antioxidants.
The alcoholic anthocyanins solution has been obtained with red cabbage leaves grinded and macerated in
ethanol 95% and with violet carrots also grinded and macerated in ethanol
Anthocyanins (violet carrot extract) fluorescence spectrum excited with laser emission at 405nm
It is evident a red fluorescence that can be attribuited to anthocyanins because the carrot extract does not
contain chlorophyll
Ethanol 95% anthocyanins solution in
neutral solution, centre of the picture, in
acidic solution on the left and in alkaline
solution, on the right
Acidic pH
Neutral pH
Alkaline pH
Reference Spectrum
Reference
Anthocyanins (red cabbage juice) in neutral pH absorption (transmission) spectrum
Neutral pH
min 470nm
Anthocyanins (red cabbage juice) in acidic pH absorption (transmission) spectrum
Acidic pH
min 500nm
Anthocyanins (red cabbage juice) in alkaline pH absorption (transmission) spectrum
Alkaline pH
min 620nm
Carotenoids
Carotenoids are organic pigments that are found in
the chloroplasts and chromoplasts of plants and some
other photosynthetic
organisms,
including
some bacteria and some fungi.
There are over 600 known carotenoids; they are split
into two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and
contain no oxygen)
In general, carotenoids absorb wavelengths ranging from 400-550 nanometers (violet to green light). They
serve two key roles in plants and algae: they absorb light energy for use in photosynthesis, and they protect
chlorophyll from photodamage. In humans, three carotenoids (beta-carotene, alpha-carotene, and betacryptoxanthin) have vitamin A activity (meaning that they can be converted to retinal), and these and other
carotenoids can also act as antioxidants.
Carotenoids belong to the category of tetraterpenoids (i.e., they contain 40 carbon atoms, being built from
four terpene units each containing 10 carbon atoms). Structurally, carotenoids take the form of
a polyene hydrocarbon chain which is sometimes terminated by rings, and may or may not have additional
oxygen atoms attached.
Carotenoids with molecules containing oxygen, such as lutein and zeaxanthin, are known
as xanthophylls.
The unoxygenated (oxygen free) carotenoids such as -carotene, -carotene, and lycopene, are
known as carotenes.
Their colour, ranging from pale yellow through bright orange to deep red, is directly linked to their
structure. Xanthophylls are often yellow, hence their class name. The double carbon-carbon bonds interact
with each other in a process called conjugation, which allows electrons in the molecule to move freely
across these areas of the molecule. As the number of conjugated double bonds increases, electrons
associated with conjugated systems have more room to move, and require less energy to change states.
This causes the range of energies of light absorbed by the molecule to decrease. As more frequencies of
light are absorbed from the short end of the visible spectrum, the compounds acquire an increasingly red
appearance.
The carotene solution has been obtained by grinded carrots heated in oil.
carotene absorption spectrum : two absorption peaks at 480nm and 450nm
carotene
absorption
Reference carotene absorption spectrum
Betalaine
Betalains are a class of red and yellow indole-derived pigments found in
plants of the Caryophyllales, where they replace anthocyanin pigments.
Betalains also occur in some higher order fungi. They are most often
noticeable in the petals of flowers, but may color the fruits, leaves, stems,
and roots of plants that contain them. They include pigments such as
those found in beets.
The name "betalain" comes from the Latin name of the common beet
Betanin
(Beta vulgaris), from which betalains were first extracted. The deep red
color of beets, bougainvillea, amaranth, and many cactuses results from
the presence of betalain pigments. The particular shades of red to purple
are distinctive and unlike that of anthocyanin pigments found in most
plants. There are two categories of betalains:
Betacyanins include the reddish to violet betalain pigments. Among the betacyanins present in
plants include betanin, isobetanin,probetanin, and neobetanin.
Betaxanthins are those betalain pigments which appear yellow to orange. Among the betaxanthins
present in plants include vulgaxanthin, miraxanthin, portulaxanthin, and indicaxanthin.
Plant physiologists are uncertain of the function that betalains serve in those plants which possess them,
but there is some preliminary evidence that they may have fungicidal properties. Furthermore, betalains
have been found in fluorescent flowers.
The aqueous betalain solution has been obtained with red beetroot grinded and macerated in water
Absorption spectrum
Betaxantine
absorption
480nm
Betacyanin
absorption
540nm
Absorption into visible band centered at 500nm and for wavelengths shorter than 400nm
Fluorescence spectrum of fresh excited with UV emission
Excitation
Emission
Fluorescence
emission detail
Fluorescent bands at 476nm 523nm 603nm. The green-yellow fluorescence, caused by betaxantine is
partially absorbed by betacyanin, which absorbs a band around 540nm
Absorption spectrum of the betalain solution after ageing (betacyanin has been degradated)
Betaxantine
absorption
480nm
Betaxantine absorption band with peak at 480nm
Fluorescence spectrum of the betalain solution after ageing (betacyanin has been degradated)
Betaxantine
emission
410nm Exc.
532nm Exc.
The fluorescence maximum at 540nm is due to the betaxantine. This fluorescence band is visible in the
aged solution because the concentration of betacyanine has been reduced. In the fresh solution this peak
is not visible because the emission is absorbed by betacyanine, whose absorption peak corresponds to
540nm. Absence of betacyanine is also evident in the absorption spectrum which shows the betaxantine
absorption peak only. The 532nm excitation produces only a weak fluorescence at longer wavelength.
Spectroscopy of Fluorescent Dyes

Stilbenes Triazine (Optical Brightener)
Optical brighteners, optical brightening agents (OBAs), fluorescent
brightening agents (FBAs) or fluorescent whitening agents (FWAs) are
chemical compounds that absorb light in the ultraviolet and violet region
(usually 340-370 nm) of the electromagnetic spectrum, and re-emit light in
the blue region (typically 420-470 nm). Fluorescent activity is a short term or
Stilbene
rapid emission response, unlike phosphorescence, which is a delayed
emission. These additives are often used to enhance the
appearance of color of fabric and paper, causing a "whitening"
effect, making materials look less yellow by increasing the overall
amount of blue light reflected.
Triaz.
The most common classes of chemicals with this property are
the stilbenes-triazine and older, fluorescent chemical such as
umbelliferone, which absorb energy in the UV portion of the spectrum and re-emit it in the blue portion of
the visible spectrum. A white surface treated with an optical brightener can emit more visible light than
that which shines on it, making it appear brighter. The blue light emitted by the brightener compensates for
the diminishing blue of the treated material and changes the hue away from yellow or brown and toward
white.
Stilbenes triazine aqueous solution absorption spectrum
Reference
Absorption
peak 380nm
Stilbenes triazine aqueous solution fluorescence spectrum excited with UV emission

Stilbene
Fluorescein Sodium (Uranine)

Fluorescein is a synthetic organic compound available as a dark orange/red
powder slightly soluble in water and alcohol. It is widely used as
a fluorescent tracer for many applications.
Fluorescein is a fluorophore commonly used in microscopy, in a type of dye
laser as the gain medium, in forensics and serology to detect latent blood
stains, and in dye tracing. Fluorescein has an absorption maximum at 494nm
and emission maximum of 521nm (in water). The major derivatives are
fluorescein isothiocyanate (FITC). The disodium salt form of fluorescein is
known as uranine or D&C Yellow no. 8. The fluorescence of this molecule is very intense; peak excitation
occurs at 494nm and peak emission at 521nm.
Fluorescein aqueous solution absorption spectrum. Second spectrum with detail in the UV band
Fluorescein aqueous solution fluorescence spectrum, excited from UV radiation and from 532nm laser
Excitation
Excitation
Anti-Stokes
Emission
Stokes
Emission
e
Eosin
Eosin is a fluorescent acidic / negative compound that binds to and forms
salts with basic, or eosinophilic, compounds containing positive charges (such
as proteins that are basic / positive due to the presence of amino acid
residues such as Arginine and Lysine) and stains them dark red or pink as a
result of the actions of bromine on fluorescein. In addition to staining
proteins in the cytoplasm, it can be used to stain collagen and muscle
fibers for examination under the microscope. Structures that stain readily
with eosin are termed eosinophilic. There are actually two very closely
related compounds commonly referred to as eosin. Most often used is Eosin Y The other eosin compound
is Eosin B it has a very faint bluish cast. The two dyes are interchangeable, and the use of one or the other
is a matter of preference and tradition. Eosin Y is a tetrabromo derivative of fluorescein. Eosin B is a
dibromo dinitro derivative of fluorescein.
Eosin absorption spectrum
Reference
Eosin fluorescence spectrum excited from UV radiation and 532nm laser
Excitation
Excitation
Acridine Orange
Acridine orange is an organic compound. It is used as a nucleic acidselective fluorescent cationic dye useful for cell cycle determination.
It
interacts
with DNA and RNA by intercalation or electrostatic
attractions respectively. When bound to DNA, it is very similar
spectrally to fluorescein, with an excitation maximum at 502 nm and
an emission maximum at 525 nm (green). When it associates with RNA, the excitation maximum shifts to
460 nm (blue) and the emission maximum shifts to 650 nm (red). Acridine orange has been widely accepted
and used in many different areas, such as epifluorescence microscopy.
Acridine orange aqueous solution absorption spectrum
Reference
Acridine orange aqueous solution fluorescence spectrum excited from UV radiation and 532nm laser
Excitation
Excitation
Rhodamine B
Rhodamine B is a chemical compound and a dye. It is often used as
a tracer dye within water to determine the rate and direction of flow
and transport. Rhodamine dyes fluoresce and can thus be detected
easily and inexpensively with instruments called fluorometers.
Rhodamine dyes are used extensively in biotechnology applications
such as fluorescence microscopy. Rhodamine B is used in biology as
a staining fluorescent dye.
Rhodamine B is tunable around 610 nm when used as a laser dye. Its
luminescence quantum yield is 0.65 in basic ethanol, 0.49 in ethanol, 1.0, and 0.68 in 94% ethanol. The
fluorescence yield is temperature dependent.
Rhodamine B absorption spectrum
Reference
Rhodamine B fluorescence spectrum excited by UV emission and by 532nm laser

Excitation
532nm Exc.
Rhodamine 6G
Rhodamine 6G is a highly fluorescent rhodamine family dye. It is often
used as a tracer dye within water to determine the rate and direction
of flow and transport. Rhodamine dyes fluoresce and can thus be
detected
easily
and
inexpensively
with
instruments
called fluorometers. Rhodamine dyes are used extensively in
biotechnology applications such as fluorescence microscopy, flow
cytometry, fluorescence correlation spectroscopy and ELISA.
Rhodamine 6G is also used as a laser dye, or gain medium, in dye
lasers, and is pumped by the 2nd (532 nm) harmonic from an Nd:YAG laser or nitrogen laser. The dye has a
remarkably high photostability, high fluorescence quantum yield (0.95), low cost, and its lasing range has
close proximity to its absorption maximum (approximately 530 nm). The lasing range of the dye is 555 to
585 nm with a maximum at 566 nm.
Rhodamine 6G absorption spectrum
Reference
Rhodamine 6G fluorescence spectrum excited by UV emission and by 532nm laser
Excitation
Excitation
Green Fluorescent Dye

Green fluorescent dye is a blend of fluorescein and cresyl brilliant blue.
Fluorescein is a synthetic organic compound.
Fluorescein has an absorption maximum at 494nm and emission maximum of
521nm (in water). See the paragraph on the fluorescein for the details.
Brilliant cresyl blue (shown in the picture) is a dye used in biology for
counting reticulocyte
Absorption spectrum. Second spectrum with UV detail
Fluorescein
absorption
Cresyl blue
absorption
Fluorescence spectrum excited from UV emission and 532nm laser

Excitation
Excitation
Methylene Blue
Methylene blue (CI 52015) is a heterocyclic aromatic chemical
compound with the molecular formula C16H18N3SCl. It has many uses
in a range of different fields, such as biology and chemistry. At room
temperature it appears as a solid, odorless, dark green powder, that
yields a blue solution when dissolved in water. The hydrated form has
3 molecules of water per molecule of methylene blue. Methylene blue should not be confused with methyl
blue, another histology stain, new methylene blue, nor with the methyl violets often used as pH indicators.
Methylene blue has a wide range of biological applications.
Methylene blue absorption spectrum (transmission)
Absorption band between 550nm and 700nm with peaks at 665nm and 610nm
Absorption
band
Methylene blue fluorescence spectrum excited from UV emission, 532nm laser and 650nm laser.
Fluorescence band at 690nm
Fluorescence
700nm
UV
Excitation
Excitation
532nm
Raman
scattering
Excitation
650nm
Fluorescence
700nm
Fluorescence
700nm
Crystal Violet
Crystal
violet or gentian
violet (also
known
as methyl
violet
10B, hexamethyl pararosaniline chloride) is a triarylmethane dye. The dye is
used as a histological stain and in Gram's method of classifying bacteria.
Crystal violet has antibacterial, antifungal, and anthelmintic properties and
was formerly important as a topical antiseptic. The medical use of the dye
has been largely superseded by more modern drugs, although it is still listed
by the World Health Organization. The name "gentian violet" was originally
used for a mixture of methyl pararosaniline dyes (methyl violet) but is now
often considered a synonym for crystal violet. The name refers to its colour,
being like that of the petals of a gentian flower; it is not made from gentians or from violets.
When dissolved in water the dye has a blue-violet colour with an absorbance maximum at 590nm. The
colour of the dye depends on the acidity of the solution. At a pH of 1.0 the dye is green with absorption
maxima at 420 nm and 620 nm while in a strongly acidic solution (pH of 1), the dye is yellow with an
absorption maximum at 420 nm.
Crystal violet absorption spectrum (transmission)
Absorption
band
Absorption band between 450nm and 650nm

Crystal violet fluorescence spectrum excited from 532nm laser
Raman
scattering
Fluorescence
610nm
Weak fluorescence at 610nm
Methylene Blue Malachite Green Crystal Violet Acriflavine

(Preparation for Fungal infections)
Methylene blue (CI 52015) is a heterocyclic aromatic chemical compound.
Malachite green is an organic compound that is used as a dyestuff and has emerged as a controversial
agent in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather,
and paper.
Crystal violet or gentian violet (also known as methyl violet 10B, hexamethyl pararosaniline chloride) is a
triarylmethane dye.
Acriflavine (shown in the picture) is a topical antiseptic. It has the form
of an orange or brown powder with green fluorescence. It may be
harmful in the eyes or if inhaled. It is a dye and it stains the skin and
may irritate. Commercial preparations are often mixtures
with proflavine. It is known by a variety of commercial names.
Acriflavine is also used as treatment for external fungal infections of aquarium fish.
Absorption spectrum of the mixture
Reference
Acriflavine
absorption
Malachite green
crystal violet
absorption
Methylene
blue absorption
Fluorescence spectrum of the mixture
Excitation
Acriflavine
emission
Phthalocyanine
Phthalocyanine is an intensely blue-green-coloured aromatic macrocyclic
compound that is widely used in dyeing. Phthalocyanines form coordination
complexes with most elements of the periodic table. These complexes are also
intensely colored and also are used as dyes or pigments.
Phthalocyanines
are
organic
compounds
closely
related
to
other tetrapyrrole macrocyles including porphyrins and porphyrazines.
Phthalocyanines strongly absorb light between 600 and 700 nm, thus these
materials are blue or green. Approximately 25% of all artificial organic
pigments are phthalocyanine derivatives.
Absorption spectrum
Reference
Phtalocyanine
absorption
Three absorption bands are shwon : <450nm, 620nm, 720nm
Erythrosine
Erythrosine, also known as Red No. 3, is an organoiodine compound,
specifically a derivative of fluorone. It is cherry-pink synthetic, primarily
used for food coloring. Its maximum absorbance is at 530 nm in an
aqueous solution, and it is subject to photodegradation.
It is used as a food coloring, printing ink, biological stain and dental
plaque disclosing agent, just to mention a few.
Erythrosine aqueous solution absorption spectrum (transmission)
Reference
Erythrosine
absorption
Absorption band with a peak at 530-540nm and a shoulder at 500nm

Fluorescence spectrum excited by UV emission and 532nm laser
Excitation
Excitation
Coumarin
coumarin is an aromatic compound. At room temperature is in the form of
colorless crystals, with characteristic odor.
Isolated for the first time from Dipteryx odorata, whose name was indeed
coumarin, coumarin is present in more than 27 families of plants, and is
responsible for sweet smell of freshly cut grass.
It is the first of a class of compounds - called coumarins - that share the coumarin chemical structure.
Even idrossicumarine are present in many plants : umbelliferone , esculetin and scopoletin are the most
common in nature. More complex coumarins as furanocoumarins are limited to a few families (Rutaceae
and Apiaceae); typical example are the phototoxic psoralens which are present in the essential oil of
Bergamot (bergaptene).
Coumarin is also used as a gain medium in some dye laser and as a sensitizer in photovoltaic technologies.
Coumarin absorbs wavelengths less than 400nm and gives strong fluorescence at about 460nm.
Fluorescence spectrum of coumarin alcoholic excited from UV emission at 405nm
Fluorescenza
ca 460nm
Spectroscopy of Edible Oils

Food oils (olive oil, seed oil) have interesting optical properties (absorption, fluorescence) due to the
content of optically active compounds, such as chlorophyll, beta-carotene and others. These properties can
be used to recognize and characterize the various types of oil.
In the spectrum of Extra Vergine olive oil, obtained by cold pressing you notice the absence of the
products of peroxidation of fatty acids, which give fluorescence at about 470nm. This happens both
because the oil is cold worked and because the high content of natural anti-oxidants (carotenes and
polyphenols ) prevents oil from oxidative degradation.
Seeds oils show all instead, to varying degrees, a clear fluorescence at about 470nm, a sign of the content
in peroxides resulting from oxidative degradation of fatty acids, both because they are supposedly hotworked and because the lower content of molecules anti-oxidants.
These features make the seeds oils less suitable for use at high temperature.
Extra Vergine Olive Oil
Chlorophills
Carotenoids
Chlorophills
Olive oil absorption (transmission) spectrum

Absorption maxima in the red band at around 660-670nm and in the blue band, less than 500nm. Olive oil
absorption is due to carotenoids and chlorophills
Chlorophill A
Chlorophill A
Excitation
Olive oil fluorescence spectrum excited by UV emission at 405nm (first spectrum) and by 532nm laser
(second spectrum)
The maximum at 680nm is due to chlorophill A
Peroxides
emission
Chlorophill A
Olive oil after

heating
Fluorescence spectrum of olive oil subjected to heating (thermal degradation) excited by UV emission
The chlorophill fluorescence has been reduced whilst it appeared the signal of the presence of peroxides
due to thermal degradation of fatty acids
Peanut Oil
Chlorophill A
Excitation
Peanut oil fluorescence spectrum excited by UV emission at 405nm

The fluorescence band at 664nm is due to the content in chlorophill. The fluorescence band with peaks at
463nm and 475nm presumably is due to the products of peroxidation (degradation) of polyunsaturated
fatty acids in the oil (oleic and linoleic)
Peroxides
emission
Peanut oil after

heating
Fluorescence spectrum of peanut oil subjected to heating (thermal degradation) excited by UV emission
We note the increase in the intensity of fluorescence at 470nm - 530nm due to thermal degradation of
fatty acids and the consequent production of peroxides. The chlorophyll fluorescence has virtually
disappeared because the chlorophyll itself has been degraded by heating
Sunflower Oil
Excitation
Sunflower oil fluorescence spectrum excited by UV emission at 405nm

The fluorescence band with peaks at 464nm and 476nm ca presumably is due to the products of
peroxidation (degradation ) of polyunsaturated fatty acids in the oil (oleic and linoleic)
Corn Oil
Excitation
Corn oil fluorescence spectrum excited by UV emission at 405nm

The fluorescence band with the peak at 464nm is presumably due to the products of peroxidation
(degradation) of polyunsaturated fatty acids present in the oil (oleic and linoleic acid). The attribution of
the fluorescence band with the peak at 520nm is uncertain and may be also linked to the process of
thermal degradation of fatty acids. Alternatively (or in addition) may be due to the compounds of vitamin
B2 (flavin)
Soybean Oil
Excitation
Chlorophill A
Soybean oil fluorescence spectrum excited by UV emission at 405nm

(degradation ) of polyunsaturated fatty acids present in the oil (oleic and linoleic) . The same applies to the
band with a peak at 533nm, to assess the contribution of isoflavones on the emissions of the green
fluorescence. See section on soybeans
Almond Oil
Excitation
Almond oil fluorescence spectrum excited by UV emission at 405nm

(degradation ) of polyunsaturated fatty acids present in the oil (oleic and linoleic acid). The fluorescence
band with peak at 533nm and 596nm is presumably due to the compounds of vitamin B2 (flavins) - See also
section on the fluorescence of riboflavin
Sesame Oil
Excitation
Chlorophill A
Sesame oil fluorescence spectrum excited by UV emission at 405nm

The fluorescence band with the peak at 475nm is presumably due to the products of peroxidation. The
attribution of the fluorescence band with the peak at 533nm is uncertain and may be also linked to the
process of thermal degradation of fatty acids, or due to the compounds of vitamin B2 (flavin)
Castor Oil
Castor oil fluorescence spectrum excited by UV emission at 405nm

Spectroscopy of Vitamins
Cyanocobalamin (Vitamin B12)
Vitamin B12, vitamin B12 or vitamin B-12, also called cobalamin, is a
water-soluble vitamin with a key role in the normal functioning of
the brain and nervous system, and for the formation of blood. It is
one of the eight B vitamins. It is normally involved in
the metabolism of every cell of the human body, especially
affecting DNA synthesis
and
regulation,
but
also fatty
acid metabolism and amino acid metabolism.[1] Neither fungi, plants,
nor animals are capable of producing vitamin B12.
Only bacteria and archaea have the enzymes required for its
synthesis, although many foods are a natural source of B12 because
of bacterial symbiosis. The vitamin is the largest and most structurally
complicated vitamin and can be produced industrially only through
bacterial fermentation-synthesis.
Cyanocobalamin is the most widely manufactured vitamin in the
vitamin B12 family (the family of chemicals that function as B12 when
put into the body), because cyanocobalamin is the most air-stable of
the B12 forms. It is the easiest to crystallize and, therefore, easiest to
purify after it is produced by bacterial fermentation, or synthesized in
vitro. It can be obtained as dark red crystals or as an amorphous red powder. Cyanocobalamin is very
hygroscopic in the anhydrous form, and sparingly soluble in water (1:80). It is stable to autoclaving for short
periods at 121 C.
Absorption
Band
Cyanocobalamin absorption (transmission) spectrum.
Riboflavin (Vitamin B2) and Pyridoxine (Vitamin B6)

Riboflavin (vitamin B2) is part of the vitamin B group. It is the central
component of the cofactors FAD and FMN and as such required for a variety
of flavoprotein enzyme reactions including activation of other vitamins.
Riboflavin is a yellow-orange solid substance with poor solubility in water. It is
best known visually as it imparts the color to vitamin supplements and the
yellow color to the urine of persons taking it. It shows a strong green
fluorescence The name "riboflavin" comes from "ribose" (the sugar
whose reduced form, ribitol, forms part of its structure) and "flavin", the ring-moiety which imparts the
yellow color to the oxidized molecule (from Latin flavus, "yellow").
Pyridoxine is one form of vitamin B6. Its hydrochloride salt pyridoxine hydrochloride is used as vitamin
B6 dietary supplement.
Excitation
Riboflavin
Pyridoxine
Fluorescence spectrum of a multivitamin compound with group B vitamines excited by UV emission
Retinol (Vitamin A)
Vitamin A is a group of unsaturated nutritional organic
compounds, that includes retinol, retinal, retinoic acid, and
several provitamin A carotenoids, among which beta-carotene is
the most important. Vitamin A has multiple functions: it is
important for growth and development, for the maintenance of
the immune system and good vision. Vitamin A is needed by the retina of the eye in the form of retinal,
which combines with protein opsin to form rhodopsin, the light-absorbing molecule necessary for both lowlight (scotopic vision) and color vision.
Retinol
Fluorescence
Excipients
fluorescence
Retinol fluorescence spectrum (with excipients) excited by UV emission with 405nm laser
Spectroscopy of Food Substances

White Wine
White wine fluorescence spectrum excited by UV emission
Excitation
Stilbenes
Fluorescence
emission detail
Flavonoids
Fluorescence band around 478nm

Fluorescence band around 520-540nm
The fluorescence bands are difficult assignment. The set of organic compounds contained in white wine, for
example, stilbenes, flavonoids and polyphenols all contribute to determine the final output. The complexity
of the spectrum can also be deduced from jagged edges of the bands, indicating that there are more
compounds that emit fluorescence at different wavelengths slightly different from each other.
Wine Vinegar
Wine Vinegar fluorescence spectrum excited by UV emission

The Wine Vinegar fluorescence bands are very similar to the wine bands
Isoflavones (Soy Sauce)

Isoflavones comprise a class of organic compounds, often naturally
occurring, related to the isoflavonoids class compounds. Many act
as phytoestrogens in mammals. Some are termed antioxidants because of
their ability to trap singlet oxygen. Some isoflavones, in particular soy
isoflavones, when studied in populations eating soy protein, have
indicated that there is a lower incidence of breast cancer and other
common cancers because of its role in influencing sex hormone
metabolism and biological activity through intracellular enzymes, protein
synthesis, growth factor actions, malignant cell proliferations, differentation and angiogenesis. However,
the risk reduction in breast cancer from soy isoflavones was only shown in Asian populations (Shanghai
Breast Cancer Survival Study). Isoflavones are produced almost exclusively by the members of
the Fabaceae (i.e.,Leguminosae, or bean) family.
Soy sauce absorption spectrum
Soy sauce fluorescence spectrum excited by UV emission
Green Walnut Extract (Liqueur Nocino)

Fluorescence spectrum excited from UV emission
Excitation
Fluorescence bands at 480nm, at 540nm and at 600nm

The fluorescence bands are difficult assignment. The set of organic compounds contained in the extract, for
example flavonoids and polyphenols all contribute to determine the final output
ProtoPorphyrin (Egg Shell)

Protoporphyrins are tetrapyrroles containing the following side chains:
methyl (4)
propionic acid (2)
vinyl (2)
Protoporphyrin IX is a biochemically widely used carrier molecule for divalent
cations. Together with iron (Fe 2+) the body of the heme- group
of hemoglobin, myoglobin and many other heme-containing enzymes like
cytochrome c and catalase are formed. Complexed with magnesium-ions (Mg 2+)
the main part of the Chlorophylls are formed. Complexed with zinc-ions (Zn 2+) it forms Zinc
protoporphyrin. Protoporphyrin IX as a direct precursor of heme is accumulated by patients
of erythropoietic protoporphyria, which is one of the genetic disorders of the biosynthesis of the heme pathway. It causes a severe photosensivity against visible light.
The sensitivity of protoporphyrin IX against light is also used as a therapy against different forms of cancer
(photodynamic therapy, PDT).
Protoporphyrins are deposited in the shells of the eggs of some birds as a brown or red pigment, either as a
ground colour or as spotting. Protoporphyrins strengthen the egg shell, and are deposited where the shell
is too thin as a result of calcium shortage. Spotting therefore tend to be heavier where the local soil is
calcium-deficient, and in the eggs laid last in a clutch.
The solution of protoporphyrin analyzed was derived from the dissolution of egg shell fragments in
hydrochloric acid.
Protoporphyrin absorption spectrum
Protoporphyrin fluorescence spectrum excited by UV emission
Excitation

Honey
Honey absorption spectrum: absorption bands from 430nm till to 490nm and beyond 550nm
Reference
Acacia honey
Tarassaco honey
Honey fluorescence spectrum

Excitation
Acacia honey
Excitation
Tarassaco honey
We can identify two emission peaks : around 475nm and 520-540nm
From the literature the band of green fluorescence at around 520nm could be attributed to the content of
flavins (see paragraph on the fluorescence of vitamin B2) , while the band around 470nm may be attributed
to the enzyme substance NADH
Rose Siroop
Excitation
Fluorescence spectrum excited by UV emission

We can identify two emission peaks : around 480nm and 540nm
The band of green fluorescence from 520nm to 540nm could be attributed to the content of flavins , while
the band at around 480nm could be attributed to the substance NADH
NADH
Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. The compound is a
dinucleotide, because it consists of two nucleotides joined through their phosphate groups. One nucleotide
contains an adenine base and the other nicotinamide. Nicotinamide adenine dinucleotide exists in two
forms, an oxidized and reduced form abbreviated as NAD+ and NADH respectively.
NADH, also known as coenzyme 1, is a naturally occurring biological substance. The "H" after NAD stands
for hydride, or "high-energy hydrogen". Both NAD+ and NADH strongly absorb ultraviolet light because of
the adenine, NAD+ and NADH differ in their fluorescence. NADH in solution has an emission peak at 460 nm
and a fluorescence lifetime of 0.4 nanoseconds, while the oxidized form of the coenzyme does not
fluoresce.
Flavine
Flavin (from Latin flavus, "yellow") is the common name for a group of organic compounds based
on pteridine, formed by the tricyclic heteronuclear organic ring isoalloxazine. The biochemical source is the
vitamin riboflavin. The flavin moiety is often attached with anadenosine diphosphate to form flavin adenine
dinucleotide (FAD), and, in other circumstances, is found as flavin mononucleotide(or FMN),
a phosphorylated form of riboflavin. It is in one or the other of these forms that flavin is present as
a prosthetic group inflavoproteins. In aqueous solution, flavins are yellow-coloured when oxidized, taking a
red colour in the semi-reduced anionic state or blue in the neutral (semiquinone) state, and colourless
when totally reduced.
Rhubarb Extract
Fluorescence spectrum excited by UV emission at 405nm

Emission peak at around 600nm due to the compound anthraquinone
Ale Beer
Excitation

We can identify two emission peaks at about 480nm and 530nm
The fluorescence bands are difficult assignment. The set of organic compounds contained in beer, such as
polyphenols and flavonoids all contribute to determine the final output. However, we can speculate that
the blue band is due to polyphenols while the band on the green is due to riboflavin.
Dark Beer

We can identify three emission peaks at about 480nm, 530nm and 600nm
Stout Beer
Excitation

We can identify three emission peaks at about 480nm, 530nm and 600nm
Pistachio Extract
Chloropyll
Fluorescence Spectrum excited from UV emission at 405nm

Emission peak at about 670nm
The red fluorescence band at about 670nm is due to chlorophyll which give pistachio the light green color
Green Tea
Fluorescence spectrum of green tea infusion excited by UV emission
Flavonoids
Chlorophyll

The band at 680nm is attributed to the content of chlorophyll and the band on the blue-green at 540nm is
presumably due to the complex organic substances contained in the leaf of the tea plant, among which
polyphenols and flavonoids.
Spectroscopy of Drugs
Hematoporphyrin
Hematoporphyrin is a porphyrin compound. Differs from protoporphyrin
hemoglobin for the presence of two hydroxyethyl groups instead of two vinyl
groups . Its importance is especially historic because it was the first prepared for
porphyrin synthesis (starting at emine) . Its biological role seems minor and his
presence in the wild is not currently certain. The hematoporphyrin is used as
photosensitizer in photodynamic therapy (PDT ).
Hematoporphyrin absorption spectrum and UV band detail in the second picture
Hematoporphyrin fluorescence spectrum excited by UV emission and 532nm laser
410nm Exc.
532nm Exc.
Fluorescence bands at 620nm and 682nm
Sodium Salicylate
Sodium salicylate is the sodium salt of salicylic acid .
At room temperature is presented as a white crystalline solid odorless .
Is obtained by reacting acetylsalicylic acid (the common aspirin) in an aqueous
suspension with a basic solution of sodium hydroxide, at the end of the reaction
yields a solution of sodium salicylate of pale blue color. It shows blue fluorescence
when exposed to UV radiation .
Absorption spectrum
Reference
Absorption
< 400nm

Excitation
Fluorescence
emission detail
Fluorescence band at around 470nm
Piroxicam
Piroxicam is a non-steroidal anti-inflammatory drug , which belongs to the
series of oxicams , which has anti-inflammatory , analgesic and antipyretic .
It is an amphoteric compound with properties of both the weak acid is of
weak base.
Fluorescence spectrum
Excitation
Spectroscopy of Various Compounds

Pyranine
Pyranine fluorescence spectrum excite from UV emission

Pyranine is a hydrophilic, pH-sensitive fluorescent dye from the group of
chemicals known as arylsulfonates. Pyranine is soluble in water and has
applications as a coloring agent, biological stain, optical detecting reagent,
and a pH indicator. One example would be the measurement of intracellular
pH. Pyranine is also found in yellow highlighters, giving them their
characteristic fluorescence and bright yellow-green colour. It is also found in
some types of soap.
Perfume
Excitation
Fluorescence spectrum of a perfume excited from UV emission

The fluorescence is due to coumarin
Bleaching Detergent
Excitation
Fluorescence spectrum of detergent excited by UV emission at 370nm.

The blue fluorescence is due to the content of stilebene
Methyl Salicylate
Methyl salicylate (oil of wintergreen or wintergreen oil) is an organic ester
naturally produced by many species of plants, particularly wintergreens. It is also
synthetically produced, used as a fragrance, in foods and beverage.
Numerous plants produce methyl salicylate in very small amounts. Some plants
such as gaultheria, produce more. It is used in low concentrations (0.04% and
under) as a flavoring agent in chewing gum and mints. When mixed with sugar and
dried it is a potentially entertaining source of triboluminescence, gaining the
tendency to build up electrical charge when crushed or rubbed. This effect can be observed by crushing
wintergreen Life Savers in a dark room. Compare with the section on sodium salicylate.
Absorption spectrum
Absorption
< 400nm
Faint fluorescence band around 476nm
Quinine
Quinine fluorescence spectrum (tonic water) excited from UV emission at 370nm

Quinine is a natural white crystalline alkaloid having antipyretic (fever-reducing),
antimalarial, analgesic (painkilling), and anti-inflammatory properties and a bitter
taste. It is a stereoisomer of quinidine, which, unlike quinine, is
an antiarrhythmic. Quinine contains two major fused-ring systems:
the aromatic quinoline and
the bicyclic quinuclidine.
Quinine
is
highly fluorescent (quantum yield ~0.58) in 0.1 M sulfuric acid solution and it is
widely used as a standard for fluorescence quantum yield measurement.
Urine
Reference
Bilirubin
Urine absorption (transmission) spectrum
Evident the absorption peak at around 460nm due to bilirubin
Bilirubin is a pigment of yellow-reddish color , contained in bile and is a
product of the catabolism of hemoglobin.
The word comes from the Latin bilis, bile, and ruber, red.
Excitation
Excitation
Urine fluorescence spectrum

Band at around 480nm, due to NAD(P)H. Band at around 520nm, due to flavine
Hemoglobin
Reference
Abs. Max 579nm

Abs. Max 540nm
Hemoglobin
Abs. Max 413nm
Diluted Hemoglobin
Hemoglobin absorption (transmission) spectrum
Evident the absorption peaks at 579nm, at 540nm and at 413nm and for shorter than 390nm
Hemoglobin is a globular protein whose
quaternary structure consists of four subunits . It is soluble, red (is a chromoprotein),
and is present in the red blood cells of
vertebrates, excluding some Antarctic fish. It
is responsible for the transport of molecular
oxygen from one compartment with high
concentration of O2 to the tissues that need
it. Each of its four protein cells, called
globin, contains inside a molecule of
protoporphyrin coordinating an iron ion Fe
(II), located slightly outside the plane of the
molecule, collectively called Eme Group.
When it binds to oxygen hemoglobin is
called oxyhemoglobin, and it is called
deoxyhemoglobin in the unbound form .
Hemoglobin Absorption
Gasoline
Excitation
Gasoline fluorescence spectrum excited by UV emission

Emission peak at 445nm
The fluorescence of gasoline is due to its content of polyaromatic hydrocarbons. The spectrum that is
obtained is a mixture of distinct peaks of varying intensity :
Monocyclic compounds ( benzene, toluene , ethylbenzene , xylene ) : 250-290nm (known as BTEX)
- Polycyclic compounds (known as IPA)
o Two ring compounds (naftalene) : 310-330nm
o Phenanthrene (three rings) : 345-355nm
o Anthracene : 447nm
o Tetracene (Naftacene) : 473nm, 498nm
The spectrum is obtained by excitation at 405nm and then are shown only the emissions at longer
wavelengths, among which recognize the peaks of anthracene and tetracene
Anthracene
Anthracene is a polycyclic aromatic hydrocarbon solid compound (IPA)
consisting of three benzene rings condensed and it has a linear structure . The
double bonds between the carbon atoms give rise to fluorescence.
The anthracene is used in the dye industry for the synthesis of the dye of
Alizarin Red. It was also used as a preservative in the timber, thanks to its
insecticidal properties. Anthracene is colorless , but has blue fluorescence if hit by a source of ultraviolet
light. Anthracene has found lately space as the organic semiconductor , it is also used as a scintillator for
the detection of electrons and alpha particles.
Excitation
Fluorescence spectrum of diphenylanthracene (from glowstick) excited by UV emission at 405nm
Tetracene (Naphthacene)
Tetracene, also called naphthacene, is a polycyclic aromatic
hydrocarbon. It has the appearance of a pale orange powder.
Tetracene is the four-ringed member of the series of acenes, the
previous one being anthracene (tricene) and the next one
beingpentacene.
Tetracene is a molecular organic semiconductor, used in organic field-effect transistors (OFETs) and organic
light-emitting diodes (OLEDs). In May 2007, researchers from two Japanese universities, Tohoku
University in Sendai and Osaka University, reported an ambipolar light-emitting transistor made of a single
tetracene crystal. Ambipolar means that the electric charge is transported by both positively
charged holes and negatively charged electrons. Tetracene can be also used as a gain medium in dye
lasers as a sensitiser in chemiluminescence.
Fluorescence spectrum of tetraphenyl naphthacene (from glowstick) excited by UV emission at 405nm
Motor Oil
Excitation
Fluorescence spectrum excited by 532nm laser

Lubricating oils for engines are added with fluroescent dyes in order to facilitate the search for leaks. From
the spectra can be seen the band fluorescence due to these additives . The band on the blue-green color is
probably due to a mixture of stilbene / triazine while the orange band could be due to a dye similar to
acridine orange.
Bergamot Essential Oil

The essence is a clear liquid (sometimes there is a deposit formed by waxes) of color ranging from green to
greenish yellow. From a chemical point of view it consists for the most part (average 95 %) of a volatile
fraction and a fraction (or residual) non-volatile (the remaining 5%). It is a highly complex mixture of many
classes of organic substances, particularly terpenes, esters, alcohols and aldehydes, for the volatile fraction.
Oxygenated heterocyclic compounds, coumarins and furanocoumarins, for the non-volatile fraction.
The main components of the non-volatile fraction consist of coumarins (citropten, 5-Geranyloxy-7methoxycoumarin) and furanocoumarins (bergaptene, Bergamottin). Furanocoumarins (also known as
psoralen) are phototoxic.
Chlorophyll
Coumarins
Furanocoumarins
II
Chlorophyll
Excitation
III
Chlorophyll
Excitation
I : Fluorescence spectrum excited from laser emission at 405nm.

Emission peak at about 680nm due to chlorophylls.
Blue-green-yellow fluorescence bands likely due to coumarins and furanocoumarins.
II : Fluorescence spectrum excited from laser emission at 532nm.
III : Fluorescence spectrum excited from laser emission at 650nm.
Absorption spectrum of bergamot essential oil

Absorption maxima at about 480 and 660nm due to chlorophyll
Chamomile Essential Oil

Chamomile essential oil contains interesting substances among which we mention
azulene. Azulene is an aromatic hydrocarbon, naphthalene isomer. It is also a
monoterpene. At room temperature is a crystalline solid of intermediate color between
blue-violet and black, insoluble in water.
It is mainly used for coloring and in the cosmetics industry.
Its molecule is planar and is composed of two condensed rings, one of 5 carbon atoms, the other of 7. A
peculiar feature of azulene is its anomalous fluorescence. Indeed the molecule emits fluorescence from
transition S2 - S0 instead of the usual transition S1 - S0.
Chamomile essential oil absorption spectrum
Azulene
Flavonoids
Chamomile essential oil fluorescence spectrum excited from UV emission
Orange Essential Oil

The essential oil of orange (Citrus aurantium) is extracted from the rind of the fruit namesake tree , through
cold pressing or distillation. Using fruits naturally ripened. Depending on the used fruit type, we have sweet
orange essential oil or essential oil of bitter orange.
Chlorophyll
Excitation
Flavonoids
Fluorescence Spectrum excited from UV emission
Orange Blossoms Water

From the distillation of orange blossoms it is obtained a liquid which is composed of two phases : an
aqueous phase, which stratifies in the bottom as it is heavier, and an oily phase, which brings high because
lighter. Separated from each other, the aqueous phase is precisely the water of orange blossoms, while the
oily, most valuable, is the essential oil of orange blossom. The essential oil is mainly obtained from the
flowers of the bitter orange (neroli oil). The violet - blue fluorescence is due to the content of methyl
anthranilate. Methyl anthranilate is the methyl ester of anthranilic acid. It is used as a repellent for birds. It
is considered one of the first artificial flavors, but is present in nature in many essential oils.
Excitation
Methyl
Anthranilate
Fluorescence Spectrum excited from UV emission
Curcumin Essential Oil

Curcumin is the principal curcuminoid of turmeric, which is a
member of the ginger family (Zingiberaceae). Turmeric's
other two curcuminoids are desmethoxycurcumin and bisdesmethoxycurcumin. The curcuminoids are natural
phenols that are responsible for the yellow color of turmeric.
Curcumin can exist in several tautomeric forms, including a 1,3-diketo form and two equivalent enol forms.
The enol form is more energetically stable in the solid phase and in solution.
Curcumin can be used for boron quantification in the curcumin method. It reacts with boric acid to form a
red-color compound,rosocyanine. Curcumin is a bright-yellow color and may be used as a food coloring. As
a food additive, its E number is E100.
Reference
Curcumin
Absorption
Absorption spectrum of curcumin ethanol solution

Absorption maxima at about 420nm and at UV wavelength
Excitation
Curcumin
Fluorescence
Fluorescence spectrum of curcumin ethanol solution excited from UV emission

Maxima at about 535nm
Spectroscopy of Inorganic Compounds

Strontium Aluminate
Strontium aluminate (SRA, SrAl, SrAl2O4) is a solid odorless, nonflammable, pale yellow powder, heavier
than water. It is chemically and biologically inert. When activated with a suitable dopant (e.g. europium,
then it is labeled SrAl2O4:Eu), it acts as a photoluminescent phosphor with long persistence
of phosphorescence.
Strontium aluminate is a vastly superior phosphor to its predecessor, copper-activated zinc sulfide; it is
about 10 times brighter and 10 times longer glowing, however about 10 times more expensive than ZnS:Cu.
It is frequently used in glow in the dark toys, where it displaces the cheaper but less efficient ZnS:Cu.
However, the material has high hardness, causing abrasion to the machinery handling it; coating the
particles with a suitable lubricant is usually used when strontium aluminate is added to plastics.
Strontium aluminate phosphors produce green and aqua hues, where green gives the highest brightness
and aqua the longest glow time. The excitation wavelengths for strontium aluminate range from 200 to
450nm. The wavelength for its green formulation is 520nm, its blue-green version emits at 500nm, and the
blue one emits at 490nm. Colors with longer wavelengths can be obtained from the strontium aluminate as
well, though for the price of some loss of brightness.
The wavelengths produced depend on the internal crystal structure of the material. Slight modifications in
the manufacturing process (the type of reducing atmosphere, small variations of stoichiometry of the
reagents, addition of carbon or rare-earth halides) can significantly influence the emission wavelengths.
Strontium aluminate phosphor is fired at about 1250 C. Subjecting it to temperatures above 1090 C is
likely to cause loss of its phosphorescent properties.
The glow intensity depends on the particle size; generally, the bigger the particles, the better the glow.
Excitation
Emission peak detail at around 520nm
Uranium glass
Excitation
Fluorescence spectrum of uranium glass excited by UV emission
Halogen Inner Bulb
Excitation
Fluorescence spectrum of halogen inner bulb excited by UVemission

The fluorescence is supposedly due to the quartz of the inner ampoule which encloses the tungsten
filament
Raman Spectroscopy
Raman spectroscopy is a spectroscopic technique used to observe vibrational, rotational, and other lowfrequency modes in a system. It relies on inelastic scattering, or Raman scattering, of monochromatic light,
usually from a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with
molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser
photons being shifted up or down. The shift in energy gives information about the vibrational modes in the
system. Infrared spectroscopy yields similar, but complementary, information. Raman spectroscopy is
commonly used in chemistry to provide a fingerprint by which molecules can be identified.
Typically, a sample is illuminated with a laser beam. Electromagnetic radiation from the illuminated spot is
collected with a lens and sent through a monochromator. Elastic scattered radiation at the wavelength
corresponding to the laser line (Rayleigh scattering) is filtered out, while the rest of the collected light is
dispersed onto a detector by either a notch filter or a band pass filter.
Spontaneous Raman scattering is typically very weak, and as a result the main difficulty of Raman
spectroscopy is separating the weak inelastically scattered light from the intense Rayleigh scattered laser
light.
The Raman effect occurs when electromagnetic radiation impinges on a molecule and interacts with the
polarizable electron density and the bonds of the molecule in the phase (solid, liquid or gaseous) and
environment in which the molecule finds itself. For the spontaneous Raman effect, which is a form of
inelastic light scattering, a photon(electromagnetic radiation of a specific wavelength) excites (interacts
with) the molecule in either the ground rovibronic state or an excited rovibronic state.
When photons are scattered from an atom or molecule, most photons are elastically scattered (Rayleigh
scattering), such that the scattered photons have the same energy (frequency and wavelength) as the
incident photons. A small fraction of the scattered photons (approximately 1 in 10 million) are scattered by
an excitation, with the scattered photons having a frequency different from, and usually lower than, that of
the incident photons.
Raman scattering spectrum of ethanol 95% - Excitation 532nm laser
Raman
Scattering
Rayleigh
Scattering
Raman scattering spectrum of chlorophill alcoholic solution - Excitation 532nm laser
Rayleigh
Scattering
Raman
Scattering
Chemiluminescence Spectroscopy
Chemiluminescence (sometimes "chemoluminescence") is the emission of light (luminescence), as the
result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an
excited intermediate P
A + B P* P + h
In practice, the reaction leads to the product P in an excited state and the decay to the ground state does
not lead to the formation of heat but of a photon (hv) . It is therefore necessary that the radiative decay
mechanisms are competitive compared to non-radiative.
An example of a reaction that leads to chemiluminescence is that of luminol with hydrogen peroxide and a
metal catalyst.
When this phenomenon occurs in biological systems, for example in fireflies, it is called bioluminescence. In
these cases the reactions are catalyzed by enzymes .
Chemiluminescence is the base of the so-called Glowstick.
The lightstick, also known as starlight, cyalume, glowstick or more simply chemical light, is a cylinder of
silicone, or other soft plastic material, self-luminous variable in size from a few cm to over 30 cm.
The main chemical components of a glow sticks are hydrogen peroxide , diphenyl oxalate (an ester of oxalic
acid) and a fluorescent pigment which determine the color : yellow, blue , green , purple, red , orange or
white.
To turn the light is sufficient bending the wand thus breaking the vial contained within it . This makes
mixing the chemicals and initiates the luminescent reaction. The mechanism consists in the reaction
between the hydrogen peroxide and the ester which provide the energy required to excite electrons of the
fluorescent dye . The electrons flow from the fundamental energy level in the excited and returning to the
fundamental level emit light.
Glowsticks
Blue LightStick
Blue chemiluminescence spectrum

The blue chemiluminescence is obtained from the substance (fluorescent dye) diphenyl anthracene
Green LightStick
Green chemiluminescence spectrum

The green chemiluminescence is obtained from the substance (fluorescent dye) bis phenylethynyl
anthracene. This aromatic compound has a strong fluorescence and is also used in OLED components
Yellow LightStick
Yellow chemiluminescence spectrum

The yellow chemiluminescence is obtained from the substances (fluorescent dyes) chloro
bis(phenylethynyl) anthracene and tetraphenyl naphthacene.
Purple LightStick
Purple chemiluminescence spectrum

The purple chemiluminescence is presumably obtained by mixing the fluorescent dyes diphenyl anthracene
(blue) and violantrone (orange)
Orange LightStick
Orange chemiluminescence spectrum

The orange chemiluminescence is presumably obtained from the fluorescent dye violantrone (orange)
Red LightStick
Red chemiluminescence spectrum

The red chemiluminescence is probably obtained from a derivative of the substance fluorescent dye
violantrone

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Diffraction

Theremino System Spettrometro_ENG Page 1

Design and Components.................................................................................................................................... 6

Spectroscopy of Fluorescent Dyes................................................................................................................... 41

Rhubarb Extract ........................................................................................................................................... 64

Yellow LightStick .......................................................................................................................................... 84

Theremino System Spettrometro_ENG Page 5

Design and Components

Webcam : NEW TRUST MEGAPIXEL WEBCAM PRO 1.3MP 1024x1280

Theory of Diffraction Grating

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Diffraction Grating Specifications

Diffraction for different wavelengths

Benchtop Spectrometer Design :

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Portable Spectrometer Construction

Details of webcam and diffraction grating

0.1mm slit, 0.5mm slit and 1mm slit

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Benchtop Spectrometer Construction

Inside view with collimating lens, grating and webcam

Detail of the micrometric slit and the spectrometer assembled

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CFL lamp after warm-up (detail 400nm 800nm) no infrared emission

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Incandescent lamp 25W continuous spectrum with maximum at ~ 800nm

Wood lamp UV emission at 370nm with tiny

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Spectra of Flames and Plasma

Candle emission mainly at longer wavelengths

Plasma from high voltage arc great emission of UV radiation

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Emission and Atomic Absorption spectra

Spectrum of potassium chlorate match evidence of potassium doublet at 770nm

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White LED cold light

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Violet laser diode ~ 410nm

Red laser diode emission 650nm

Green laser diode the pumping emission lines are shown

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Sun spectra at different times

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Atmospheric water vapor absorption band 720nm

Absorption H 657nm (Balmer series) Fraunhofer C

Fraunhofer main lines

Sun spectrum 650nm - 800nm with absorption bands of atmospheric gases

Theremino System Spettrometro_ENG Page 21

Wien Law / Black body radiation

Sun spectrum with absorption bands of

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Comparison with Reference Spectra

Species producing peak

Actual line location (nm)

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