Practical works 7

SPECTRAL ANALYSIS

A. Theoretical data about optical spectroscopy

B. Emission spectrum analysis – identification of sodium in biological
products with a three-arms spectroscope
C. Absorption spectrum analysis - the identification of hemoglobin
derivatives with the direct vision spectroscope

A. Theoretical data about optical spectroscopy

Different spectroscopic techniques have been applied in almost all technical fields of
science and technology (including devices for medical purposes) in order to explain qualitative
and quantitative facts. From all of these techniques, optical spectroscopy is used routinely to
identify the chemical composition of matter and determine its physical and chemical structure.
As you all know light is an electromagnetic radiation composed of two oscillating
vectors: electric and magnetic fields. Being emitted by a source they have the ability to transfer
energy through space as a wave.
As you know, the speed of light in a vacuum is a constant and it is equal to:
c = 3 x 108 m/s
The speed of light is slower in various transparent materials than it is in a vacuum or
outer space. The equation of its velocity is:
𝒄
𝒗 = , where n is the refraction index.
𝒏
When the light passes into a material at an incident angle (i), the light beam is bent or
refracted with an angle of refraction (r) according to Snell's law (second law of refraction) :
n1 sin i = n2 sin r,
n1 and n2 are refraction indexes (see also the practical work about refraction of light).
But also, the speed of light through a material varies slightly with the wavelength or frequency of
the light:
𝒄
𝝂 = , (λ is the wavelength and ν is the frequency of a radiation)
𝝀

With the exception of the areas of absorption, in dispersive media the refractive index
increases with the decrease in the normal dispersion wavelength. The equation
𝑛 = 𝑓(𝜆)
it is called the law of the dispersion of the medium.
Dispersion of light refers to phenomena that appear when light passes through a
dispersive medium. Thus, each wavelength is refracted at a slightly different angle when passing
through a material at the same incident angle. This spreading out of the beam of light is called
dispersion or chromatic dispersion. This can be seen when sunlight passes through a glass prism
(as shown in figure 1).
 Fig. 1 Dispersion of white light through a prism

The velocity of light in a material – and thus its index of refraction – depends on the
wavelength of the light. In general, the index of refraction is greater for shorter wavelengths.
This causes light inside materials to be refracted by different amounts according to the
wavelength or color. The wavelength of visible light is very short and often measured in
nanometers (nm), which are 1 billionth of a meter (10-9 meter) being situated between 400 nm
(violet radiation) and 800 nm (red radiation). Sunlight is often called white light, since it is a
combination of all the visible colors (is due to the superposition of an infinite number of
monochromatic light radiation. Each monochromatic radiation is characterized by a frequency ν
and by a wavelength λ. Frequency is expressed in Hertz (Hz).
The index of refraction (n) is different for each color, so, the angle of refraction will be
different for each color when the light passes from air into a glass prism. Thus, the prism can
separate different radiation component in white light.

Classification of spectra

The energy levels in atoms and ions are the key to the production and detection of
radiation. Energy levels or "shells" exist for electrons in atoms and molecules. The colors of dyes
and other compounds results from electron jumps between these shells or levels. The colors of
the surrounding world result from transfers of electrons from one shell to another. Typically the
valence electrons are the ones involved in these transfers.
Atoms have two kinds of states: a ground state and excited states. The ground state is the
state in which the electrons in the atom are in their lowest energy levels possible (atoms naturally
are in the ground state), see figure 2. This means the electrons have the lowest possible values
for "n" the principal quantum number.
 Higher energy level (excited)
E2
Absorption
E E2>E1

E1
Emission Lowest
energy level

Fig.2 Emission and absorption of radiation between two energy levels

Energy can be supplied to atoms in many different ways. It can be in the form of light
(electromagnetic radiation), an electric discharge (electric field) or heat. This received or extra
energy is emitted when the excited electrons in the atoms give off light and fall back to a lower
shell. The light emitted has wavelengths and colors that depend on the amount of energy
originally absorbed by the atoms. Usually each individually excited electron will emit only one
type of light, a monochromatic wave and its energy is equal to

ΔE = hν,

where ΔE is shown above, ν is the frequency and h is Planck’s constant (h = 6.625 x 10-34 J s).
In a sample, even in a small one, we have billions and billions of atoms so there will exist
billions of excitations and emissions. Not all atoms in a sample will absorb or be excited exactly
the same time. For example in a hydrogen atom the ground state has the electron in the n = 1
shell. The electron in some hydrogen atoms may be excited into the n = 2 level. Other hydrogen
atoms can have the electron excited into the n = 4 shell.
Emission spectra occur whenever the electrons jump from an excited state to a lower
energy level. For atoms an emission spectra looks like colored lines on a black background.
Different chemical elements emit different emission spectra when they are excited because each
type of element has a unique energy shell or energy level system. Each element has a different
set of emission colors because they have different energy levels.
In relation with their origin the emission spectra can be:
 Line spectrum - specific for atoms (isolated lines).
 Band spectrum - specific for molecules (transitions occur between very close groups of
energy levels).
 Continuous spectra – e.g. white light, the emission spectrum of all incandescent solids is
continuous.
If a light beam that has a continuous spectrum passes through an absorbent material, the
continuous spectrum will be missing lines (for atoms) or bands (molecules) that it will appear
dark on a continuous spectrum. This is an absorption spectrum.
So, we can state that spectra can also be classified as:
- emission spectrum: appears when an electron jumps back from the excited level to
the ground state. It looks like colored lines or bands (for molecules) on a dark
background;
 - absorption spectrum: results when an electron absorbs energy and jumps from the
ground level to an excited level. It looks like black lines or bands (for molecules) on
a continuous colored background.

Between emission and absorption spectra there is a correspondence given by Kirchhoff’s law:

Kirchhoff’s law of heat radiation states that emissivity of radiation bodies in

thermal equilibrium is equal to the absorptivity. Or, in other words, each
chemical substance can absorb the same wavelengths of radiations that they can
emit in the same conditions of temperature and pressure.

Due to the energy of the levels involved in a spectrum we can identify:

a. electronic spectra – if they are produced between the inner electronic levels, situated
closely to the nucleus, the spectra will be represented by X-rays (high energy spectra), or,
if they are produced between outer energetically levels it will produce visible or UV
(ultraviolet) spectra;
b. vibration spectra – characteristic to the molecules and appear due to the vibration
moments of the nuclei in the molecule near some equilibrium states, an example is IR
(infra-red) spectra;
c. rotation spectra – are also characteristic to the molecules in which there are rotation
movements of the whole molecule, for example far IR spectra.

The specificity of the optical spectra (each substance, no matter if it is solid, liquid or gas,
has its own spectrum) allows the identification of atoms and molecules involved (qualitative
analysis). If we are able to measure the intensities of the lines or of the spectral bands we can
determine the concentration of atoms or molecules in a sample (quantitative analysis).

B. Emission spectrum analysis – identification of sodium in biological products with a

three-arms spectroscope

In 1859, Gustav R. Kirchhoff, a German physicist stated that “each pure substance has its
own characteristic spectrum”. This statement represented the born of a new science: analytical
spectroscopy. Since then, spectroscopy has been used as a very helpful tool for findings of the
constituents in a material (qualitative spectroscopy) or theirs concentrations (quantitative
analysis) or to investigate their detailed structure. In a typically spectroscopic technique a
concentration of few parts per million of a chemical element in a matter can be detected by its
emission spectrum.
Usually, the device that produces and analysis a spectrum requires three elements:
 a source of light (or other electromagnetic radiation);
 a dispersive element, which separates the light into its component
wavelengths;
 a detector to sense the presence of light after dispersion.
Due to the dispersive agent we can classify the devices in spectrometers with prisms or with
diffraction gratings.
 If the detector is the eye the device is called spectroscope, if the detector is a photographic
film the device is called spectrograph and if the detector is photoelectric, thermoelectric etc. the
device is called spectrometer.
In this practical work, our goal is to determine the presence of sodium in a biological liquid
by using an emission spectrum. The spectroscope we are gone to use is a three arms spectroscope
presented in fig.3.

S2

C2

C1
F
L
S1 P

Fig. 3 Diagrammatic representation of the three arm spectroscope

The spectroscope is formed from the following constructive parts:
 C1 is a collimator consisting of a convergent lens which has its home in an
adjustable slit F, placed in front of the luminescent object to be studied. The slot
has a variable width.
 a prism P mounted on a circular support;
 a mobile telescope arm (eyepiece or bezel) that has focal adjustment and rotates
about the prism axis to permit setting on any part of the spectrum;
 an auxiliary collimator C2 projecting the image of enlightened scale
(micrometer) on the emergent side of the prism;
 light sources S1, S2

Operating principle
The collimator (C1) is used to make parallel rays from the light source S1, which fall than upon
the prism. It consists of a tube whose one end is a vertical slit with adjustable width, which is
illuminated with the source to be studied. The slot is arranged to be the principal focus of a
converging lens mounted at the other end of the tube.
The prism (P) is placed in the center of the optical system. It is an optical instrument consisting
of a highly dispersive transparent medium, that is to say, it diffuses the light given by the source.
On leaving the prism, the radiation in the spectrum is arriving on the telescope. The scale is
placed at the end of collimator (C2). It is lighted by a source of white light. The image of a
micrometer is achieved by the collimator which sends rays on the face of the prism that reflects
towards the telescope. The image of the scale is superimposed on the image of the spectrum. It is
then possible to pinpoint the location of the lines relative to the divisions of scale.
Experimental technique
Calibrating the spectroscope scale
For identifying the presence of a certain substance by spectroscopy we have to know the
precise values of the wavelength of its spectrum. The three arms spectroscope contains a scale
with arbitrary units, it is uncalibrated. In order to calibrate the scale we will project the lines of a
known spectral source onto the same plane as the unknown lines. We have to calibrate the scale
just before starting all experiments. The calibration of the spectroscope is a process by which we
establish a correspondence between the wavelengths (in nm) and the arbitrary divisions of the
scale. The calibration light sources we will use are:
 a mercury vapor tube, and
 a neon lamp.
The spectral characteristics are presented in figure 4:
12 3 4 5 6 1 2 3

492 436 408nm 640 614 585 nm

579 57754
Spectrum for mercury Spectrum for neon
1 - yellow, 1 - red,
2 -yellow, 2 – red-orange,
3 - green, 3 - yellow
4 – blue-green,
5 - indigo,
6 – violet.
Fig. 4 Lines emission spectra for mercury and neon

With the pairs of values (divisions on the scale – wavelength in nm) obtained for each
line of the spectra we will draw the calibration curve on a graph, as presented in figure 5.
 (nm)
800

750

700

650

600

550

500

450

400
30 40 50 60 70 80 90 Fig.5 Calibration curve
350
Scale divisions (arbitrary units)
 The experimental goal is the identification of sodium in a biological fluid. We will spray
the sample into a flame. Atoms will absorb the thermal energy and get excited. A part of the
excited atoms will loose the amount of energy absorbed and their electrons will fall on the
ground state, emitting the energy difference as light radiation. Looking through the telescope we
will see a characteristic emission spectrum. If the sample contains sodium, we will see a yellow
line appearing at certain division values on the scale. Using the calibration curve we have the
possibility to find out the corresponding value of the wavelength in nanometers. Usually, that
value is between 589 and 589.6 nm.

589,5 nm 589 nm

C. Absorption spectrum analysis - the identification of hemoglobin derivatives with the

direct vision spectroscope

The direct vision spectroscope is a spectroscope utilizing an Amici prism so that the
observer looks in the direction of the light source. This instrument provides direct wavelength
readings of visible spectrum. It contains:
 an Amici 3 element prism assembly;
 a variable optical slit;
 an adjustable eyepiece.

A direct vision Amici prism is a dispersive system for which the deviation is almost zero
for a given wavelength. It consists of three prisms: two "crown" (low dispersion glass of low
refraction index) and one in "flint" (glass highly dispersive of high refraction index), as shown in
figure 6.

Fig.6 Amici prisms

If the external light source is a source of white light, we will observe a series of bands of
different colors ("rainbow"), resulting from the dispersion of light by the prism. If we place the
liquid sample just before the dispersive system, we note in the visual field of the eyepiece a
series of dark lines which correspond to wavelengths absorbed by the substance in the sample.
In figure 7, it is presented the optical scheme of a direct vision spectroscope.
 SG

E
F

S2
PA
S1

Fig. 7 Diagrammatic representation of the direct vision spectroscope

S1, S2, S3 – white light sources,

E – tube with oxyhemoglobin,
F – slit
PA - Amici prisms,
SG – scale, calibrated in nanometers (nm)

Experimental technique

If a radiation that has a continuous spectrum passes through an absorbing substance, the
continuous spectrum will present dark bands. This is an absorption spectrum. We will measure
the light absorption of a solution of hemoglobin in the visible light with a direct vision
spectroscope. According to the state of the hemoglobin molecule, its light absorption
characteristics are modified, leading to different absorption spectra.
First, we have to verify the accuracy of the spectroscope with a sodium lamp. If the specific
division doesn’t match we will adjust the scale.
Second, we prepare a sample of oxyhemoglobin. If it is already prepared, we will place the tube
in front of the spectroscope. Normally, the oxyhemoglobin absorption spectrum presents two
black bands, one in green region (552-527 nm) and one in the yellow region (587-568 nm), like
presented in figure 8.

red orange yellow green blue

indigo
Fig. 8 Absorption spectra of hemoglobin