COLORIMETRY

The light having wavelength between 400-800 nm is called visible light because human eyes
can perceive this light. The perception is called as color. The color of the light depends on
wavelengths of the incoming light. White light contains the whole visible region. Objects can
absorb some wavelengths of the white light and remaining lights constitutes the color of the
objects. For example, an object that absorbs light around 420-440 nm appears yellow.
Objects which do not absorb light in the visible region appear white while objects which
absorb the whole visible region appear black.

There are three things that can happen when a light come through an object: it can be
absorbed (subsequently the object can fluoresce) or it can be scattered by the object or it
can pass through the object without any interaction. The light that are not absorbed by the
substances (emissions are also included in case of fluorescence) constitutes the color of the
substances. Therefore, there is a relationship between light absorption and the color. In the
following table this relationship is tabulated. One can guess the region of light absorption of
a substance by looking the color of it.

Table 1. The relationship between absorbed light and color

Absorbed light (nm) Absorbed color Color
380-420 Violet Yellow-green
420-440 Violet-blue Yellow
440-470 Blue Orange
470-500 Blue-green Red
500-520 Green Purple
520-550 Yellow-green Violet
550-580 Yellow Violet-blue
580-620 Orange Blue
620-680 Red Blue-green
680-780 Purple Green

We make some daily-life analyses using the color of substances. For example, the color can
give an idea about whether a food is fresh or even delicious or not. The color of a tea tells us
how much strong it is and how much aromatic compounds it contains.

It is possible to conduct quantitative analysis by using color intensity as well as qualitative

analysis of materials by looking at the color. Because the color intensity is proportional to
the concentration of the substances. The analysis in which the concentration of compounds
is determined by the color of them is called colorimetry.

In laboratories, colorimetric analyses are generally conducted indirectly using

spectrophotometers. However, in some areas where precise color measurements are very
important such as clothing industry, special colorimetry instruments are used. Moreover,
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there are softwares that can measure the color intensity from photographs and using these
softwares, colorimetric analysis can also be conducted by using computers or even mobile
phones.

Colorimetric Fe3+ Analysis

In this experiment, the concentration of a Fe3+ sample will be roughly determined by naked
eye detection. In this method, the concentration of the sample is determined by comparing
the color intensity of it with the color intensities of standards. This method can be used
where very precise results are not necessary and simple analyzes are preferable.

Standard Fe3+ solution:

• In a beaker weigh 0.006 g FeCl3.6H2O and add 3 mL of concentrated HCl.

• Transfer the solution to a 50 mL volumetric flask and dilute it to 50 mL with distilled
water.
• Transfer 1, 2, 3, 4 and 5 mL standard solution to five different test tubes and dilute
them to 10 mL using distilled water.
• Calculate the Fe3+ concentration in each test tube.

Fe3+ sample:

• Transfer 10 mL sample to a test tube.

6.5 mM K4[Fe(CN)6].3H2O

• Prepare 3.0 mM 10 mL K4[Fe(CN)6].3H2O solution

The experiment:

• Add 1 mL of 3.0 mM K4[Fe(CN)6].3H2O into the test tubes and shake the tubes well.
• Comparing the color intensities of the sample and the standards, determine the
concentration range of the sample. (Molecular weights: K4[Fe(CN)6].3H2O = 422.39
g/mol, FeCl3.6H2O=270.30 g/mol, Fe = 55.85 g/mol)

4 FeCl3 + 3 K4[Fe(CN)6] → Fe4[Fe(CN)6]3 + 12 KCl

Prussian blue

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 FLUORESCENCE SPECTROSCOPY
Electrons occupy the lowest energy regions of atoms and molecules when they are not
excited. This state is called “ground state”. When species are exposed to an external energy,
their electrons can absorb this energy and move to higher energy orbitals. This state is an
unstable state and it is called “excited state”. Since excited state is unstable, species have
tendency to lose this excess energy and return to the ground state. The process of losing this
excess energy in the form of light is called luminescence and the technique investigating the
luminescence is called luminescence spectroscopy1. Luminescence has two different
mechanisms depending on the spin of the excited electron; fluorescence and
phosphorescence. If in excited electron has opposite spin of ground state electron, this is
called singlet state and electrons are paired in this state (Figure 1). On the other hand, if the
excited electron has the same spin with the ground state electron, then this state is called
triplet state and electrons are not paired in this state (Figure 1)

Ground Excited Excited

singlet state singlet state triplet state

Figure 3. Schematic diagram of possible states of electrons in ground and excited states.

When atoms or molecules are exposed to energy, they mostly transformed from ground
singlet state into excited singlet state. The excited singlet state species lose its energy by
emitting light (called fluorescence) in a very short timescale (~10 nanosecond). This
phenomena (fluorescence) is used in highlighter pens, fluorescence and neon lamps as well
as it can occur in nature such as in some kind of scorpions, corals and mushrooms.

The relaxation from excited triplet state to ground state by emitting light takes place over a
relatively long time (from 1 millisecond to 1 second) and it is called phosphorescence.
Phosphorescence is more rarely observed than fluorescence. Some species have very long
phosphorescence emission time. For example, glow in the dark toys and light switches show
long-term phosphorescence. Moreover, some types of jellyfishes, crabs and squids naturally
have phosphorescences.

Luminescence can be classified according to the types of external energy. For example,

• when light is used for excitation of molecules, it is called photoluminescence,

• when the electrical energy is used for excitation of molecules, it is called
electroluminescence,

1
Spectroscopy is a science which investigate relation between light and substance.

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 • when the energy from a chemical reaction is used for excitation of molecules, it is
called chemiluminescence,
• when the energy of biomolecules is used for excitation of molecules, it is called
bioluminescence.
As well as excited species can lose their energies in form of light (fluorescence and
phosphorescence) for returning to the ground states, there are also some other non-
radiative relaxation mechanisms to lose their energy. Generally, the loss of energy from
excited state is the mixture of radiative and non-radiative process. If radiative relaxation is
higher than non-radiative relaxation, then more intense fluorescence / phosphorescence will
be observed. On the contrary, when the non-radiative relaxations are dominant, then
fluorescence or phosphorescence may not be observed. In non-radiative relaxations, the
excited electron generally loses its energy in the form of heat, using transitions between
vibrational levels.

In fluorescence emission, molecules emit lower energy of light than that they absorbed due
to the non-radiative relaxations. Thus, in fluorescence spectroscopy, fluorescence emission
is observed at longer wavelengths than the excitation2. This phenomenon is called Stokes
shift. Molecular structure determines the fluorescence of a molecule and it might be
possible to get an idea of what the material is using the wavelength of emitted light.
Therefore, fluorescence spectroscopy can provide qualitative information (but generally it is
solely not enough to give a decision).

Although the main factor that determines the fluorescence is molecular structure, some
external factors such as solvent type, temperature, pH, dissolved oxygen highly affect the
fluorescence intensity. In diluted solutions, the concentration of the molecule is proportional
to the fluorescence intensity:

2
Planck equation about the energy of a photon: E = hc/λ
E is the energy of the photon, h is the Planck constant, c is the speed of the light and λ is the wavelength of the
light. The equation shows that the wavelength of the light is inversely proportional to energy of light. It means
the higher the wavelength the lower the energy.
Human eye can see the “visible light” between 400-800 nm.
Ultraviolet, X-rays and gamma rays regions, which have shorter wavelengths than this region, have increasingly
energetic zones. On the other hand, infrared, microwave and radio waves have longer wavelengths than the
visible region, and they have decreasing energies.

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 F=kC

here F is the fluorescence intensity, k is the fluorescence constant for the substance and C is
the concentration of the substance. This equation shows that quantitative analysis can be
performed using fluorescence intensities in diluted solutions.

The instruments used in fluorescence spectroscopy are similar to those of UV-Visible

absorption spectroscopy but there are some differences. Firstly, since in absorption
spectrophotometers the light which is not absorbed by the sample is measured, light source,
sample and detector placed in the same direction. On the other hand, in fluorescence
spectrophotometers, since only emitted light is measured, detector is placed 90 o angle to
the sample compartment. Thus, the light which is not absorbed by the sample cannot reach
the detector, just the emitted light from the sample is recorded. Secondly and finally, two
wavelength selectors (generally monochromators) before and after the sample
compartment are used while in absorption spectrophotometers there is only one
wavelength selector which is placed before the sample compartment.

Following is the schematic representation of a fluorescence spectrophotometer:

Wavelength selector

Light source Sample

compartment

90o

Wavelength selector

Detector
Recorder Signal
(Computer) amplificator

Figure 4. Schematic representation of fluorescence spectrophotometer

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SPECTROFLUORIMETRIC RIBOFLAVIN (VITAMIN B2) DETERMINATION

Weigh 2.5 mg pure riboflavin and dissolve it in 50 mL acetic acid solution (0.02 M) (If
dissolving takes too much time, it can be accelerated by heating or using ultrasonication. Use
suitable glassware if it is heated!) The concentration of this solution is 50 µg/mL. Then,
prepare 10 mL solution of 0.2, 0.5, 1.0, 1.5 and 2.0 µg/mL riboflavin solutions by diluting the
above solution (calculation for dilutions will be done by students). In fluorescence
spectrophotometer, adjust the excitation wavelength to 450 nm and emission range to 460-
800 nm. Choose both slits as 5 nm and voltage for photomultiplier tubes as 700 V. Measure
the fluorescence spectra of the standard solutions and record the fluorescence intensities at
526 nm which is the peak of the emission band. Use these data in linear regression. The
linear regression will be used for quantification of unknown samples.

The riboflavin containing unknown samples (multivitamin tablets, energy drinks etc.) is
measured using same parameters in the instrument. The concentration of the sample is
calculated using linear regression equation.