Pigments and Colors: Extraction, Purification and Characterization using different modern analytical tehcniques

1. DOS&R IN ORGANIC CHEMISTRY
TUMKUR UNIVERSITY
By
PRUTHVIRAJ K
Faculty
DOS&R in Organic Chemistry
KPR. DOS&R in ORGANIC CHEMISTRY TUT
Pigments and colours-
Purification and Characterization

11. UV, FTIR, FTNMR, LC-MS,
GC-MS, CHNS analysis

19. PURIFICATION TECHNIQUES

21. The main principle involved in column chromatography is adsorption of the solutes of a
solution through a stationary phase and separates the mixture into individual components.
This is based on the affinity towards the mobile phase and stationary phase. The molecules
which are more affine towards the stationary phase elute later and which are less affine
towards the stationary phase elutes first

28. HPLC works following the basic principle of thin layer chromatography or column
chromatography, where it has a stationary phase ( solid like silica gel) and a mobile
phase (liquid or gas). The mobile phase flows through the stationary phase and carries the
components of the mixture with it. Different components travel at different rates. Thus the
components separated and found in different region in chromatography to separate,
identify and quantify
HPLC is a improved form of column chromatography. The difference is, here instead of
dripping solvent under gravity a pressure of up to 400 atmosphere is applied on the
chromatography to have a quick separation. And a very smaller particle size of column
packing material is used. Thus the separation is much better in HPLC.

31. ELECTROMAGNETIC SPECTRUM
SPECTROSCOPY
Branch of Science that deals with the study of interaction of
electromagnetic radiation with matter
The Electromagnetic Spectrum is a continuum of all the electromagetic waves
arranged according to their frequency and wavelength

32. A. UV Visible Spectroscopy

33. When ultraviolet radiations are absorbed, this results in the excitation of the
electrons from the ground state towards a higher energy state. The theory
revolving around this concept states that the energy from the absorbed
ultraviolet radiation is actually equal to the energy difference between the
higher energy state and the ground state
UV spectrophotometer principle follows the Beer-Lambert Law. This law states
that whenever a beam of monochromatic light is passed through a solution with
an absorbing substance, the decreasing rate of the radiation intensity along with
the thickness of the absorbing solution is actually proportional to the
concentration of the solution and the incident radiation
The concept and principle of UV spectrophotometer have several applications. For
instance, this is used to detect a functional group. It can be used to detect the absence or
the presence of chromophore in a complex compound.
This can also be used to detect the extent of conjugation in polyenes. When there is an
increase in double bonds, the absorption shots to the longer wavelength. In addition, UV
spectroscopy may be used to identify unknown compounds. The spectrum of an
unknown compound is going to be compared with the spectrum of a reference
compound. If both spectrums coincide, this unknown compound will be successfully
identified

34. Setup & Diagram
UV-Spectra of Vitamin A

35. Energy Transition and Order

37. FT-IR stands for Fourier Transform InfraRed, the preferred method of infrared
spectroscopy. In infrared spectroscopy, IR radiation is passed through a sample.
Some of the infrared radiation is absorbed by the sample and some of it is passed
through (transmitted). The resulting spectrum represents the molecular
absorption and transmission, creating a molecular fingerprint of the sample.
Like a fingerprint no two unique molecular structures produce the same infrared
spectrum. This makes infrared spectroscopy useful for several types of analysis
Fourier transform infrared spectroscopy is preferred over dispersive or filter
methods of infrared spectral analysis for several reasons: • It is a non-destructive
technique • It provides a precise measurement method which requires no external
calibration • It can increase speed, collecting a scan every second • It can increase
sensitivity – one second scans can be co-added together to ratio out random noise
• It has greater optical throughput • It is mechanically simple with only one
moving part

38. Mode of vibration

41. FTIR Spectrum of some Compounds

42. Nuclear magnetic resonance, NMR, is a physical phenomenon of resonance transition
between magnetic energy levels, happening when atomic nuclei are immersed in an
external magnetic field and applied an electromagnetic radiation with specific frequency.
By detecting the absorption signals, one can acquire NMR spectrum. According to the
positions, intensities and fine structure of resonance peaks, people can study the
structures of molecules quantitatively. The size of molecules of interest varies from small
organic molecules, to biological molecules of middle size, and even to some
macromolecules such as nucleic acids and proteins. Apart from these commonly utilized
applications in organic compound, NMR also play an important role in analyzing
inorganic molecules, which makes NMR spectroscopy a powerful technique

43. Nuclear spin and the splitting of energy levels in a magnetic field
Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axes.
In many atoms (such as 12C) these spins are paired against each other, such that the nucleus of
the atom has no overall spin. However, in some atoms (such as 1H and 13C) the nucleus does
possess an overall spin. The rules for determining the net spin of a nucleus are as follows;
 If the number of neutrons and the number of protons are both even, then the nucleus
has NO spin.
 If the number of neutrons plus the number of protons is odd, then the nucleus has a half-
integer spin (i.e. 1/2, 3/2, 5/2)
 If the number of neutrons and the number of protons are both odd, then the nucleus has an
integer spin (i.e. 1, 2, 3)
The overall spin, I, is important. Quantum mechanics tells us that a nucleus of spin I will have
2I + 1 possible orientations. A nucleus with spin 1/2 will have 2 possible orientations. In the
absence of an external magnetic field, these orientations are of equal energy. If a magnetic field
is applied, then the energy levels split. Each level is given a magnetic quantum number, m
When the nucleus is in a magnetic field, the initial populations of the energy levels are
determined by thermodynamics, as described by the Boltzmann distribution. This is very
important, and it means that the lower energy level will contain slightly more nuclei than
the higher level. It is possible to excite these nuclei into the higher level with
electromagnetic radiation. The frequency of radiation needed is determined by the
difference in energy between the energy levels

44. Nuclear Energy Levels in a Magnetic Field
Energy Level Spin Distribution

45. Relaxation processes
How do nuclei in the higher energy state return to the lower state? Emission of radiation is
insignificant because the probability of re-emission of photons varies with the cube of the
frequency. At radio frequencies, re-emission is negligible. We must focus on non-radiative
relaxation processes (thermodynamics!).
Ideally, the NMR spectroscopist would like relaxation rates to be fast - but not too fast. If
the relaxation rate is fast, then saturation is reduced. If the relaxation rate is too fast, line-
broadening in the resultant NMR spectrum is observed.
There are two major relaxation processes;
Spin - lattice (longitudinal) relaxation
Spin - spin (transverse) relaxation
Spin-Lattice Relaxation
Nuclei in an NMR experiment are in a sample. The sample in which the nuclei are held is
called the lattice. Nuclei in the lattice are in vibrational and rotational motion, which
creates a complex magnetic field. The magnetic field caused by motion of nuclei within the
lattice is called the lattice field. This lattice field has many components. Some of these
components will be equal in frequency and phase to the Larmor frequency of the nuclei of
interest. These components of the lattice field can interact with nuclei in the higher energy
state, and cause them to lose energy (returning to the lower state). The energy that a
nucleus loses increases the amount of vibration and rotation within the lattice (resulting in
a tiny rise in the temperature of the sample)

46. The relaxation time, T1 (the average lifetime of nuclei in the higher energy state) is
dependent on the magnetogyric ratio of the nucleus and the mobility of the lattice. As
mobility increases, the vibrational and rotational frequencies increase, making it more likely
for a component of the lattice field to be able to interact with excited nuclei. However, at
extremely high motilities, the probability of a component of the lattice field being able to
interact with excited nuclei decreases
Spin - spin relaxation
Spin - spin relaxation describes the interaction between neighbouring nuclei with identical
precessional frequencies but differing magnetic quantum states. In this situation, the nuclei
can exchange quantum states; a nucleus in the lower energy level will be excited, while the
excited nucleus relaxes to the lower energy state. There is no net change in the populations
of the energy states, but the average lifetime of a nucleus in the excited state will decrease.
This can result in line-broadening
Chemical shift
The magnetic field at the nucleus is not equal to the applied magnetic field; electrons
around the nucleus shield it from the applied field. The difference between the applied
magnetic field and the field at the nucleus is termed the nuclear shielding

55. D. Mass Spectrometry

56. Mass spectrometry is a powerful analytical technique used to quantify known materials, to
identify unknown compounds within a sample, and to elucidate the structure and chemical
properties of different molecules. The complete process involves the conversion of the
sample into gaseous ions, with or without fragmentation, which are then characterized by
their mass to charge ratios (m/z) and relative abundances.
This technique basically studies the effect of ionizing energy on molecules. It depends upon
chemical reactions in the gas phase in which sample molecules are consumed during the
formation of ionic and neutral species
he first step in the mass spectrometric analysis of compounds is the production of gas phase
ions of the compound, basically by electron ionization. This molecular ion undergoes
fragmentation. Each primary product ion derived from the molecular ion, in turn,
undergoes fragmentation, and so on. The ions are separated in the mass spectrometer
according to their mass-to-charge ratio, and are detected in proportion to their abundance.
A mass spectrum of the molecule is thus produced. It displays the result in the form of a
plot of ion abundance versus mass-to-charge ratio. Ions provide information concerning the
nature and the structure of their precursor molecule. In the spectrum of a pure compound,
the molecular ion, if present, appears at the highest value of m/z (followed by ions
containing heavier isotopes) and gives the molecular mass of the compound.