ATOMIC AND

MOLECULAR PHYSICS
Advanced Physics lab – I (MSPH 201): Department of Applied Physics

Submitted to
Prof. MS Mehta & Dr Yogendra Meena

Submitted by
Gurmeet Singh | 2K21/MSCPHY/15
MSc Physics – Semester 3

DELHI TECHNOLOGICAL UNIVERSITY

 INDEX

[Link]. AIM OF THE EXPERIMENT PAGE REMARKS

1. To perform single-point energy and
vibrational frequency calculations using
variational principles, on the simple
Molecule (7HQ).
2. To record absorption spectra of a given
molecule in wavelength scale and estimate
the possible parameters.
3. To verify Beer – Lambert’s Law.
4. To introduce the principles of fluorescence
in a molecule and demonstrate how the
fluorescence spectrum of a fluorophore is
affected by the fluorophore concentration
(Inner filter effects).
- References
 EXPERIMENT – 1

AIM: To perform single-point energy and vibrational frequency calculations using variational principles, on the
simple molecule (7HQ).

APPARATUS/SOFTWARE USED: Gaussian Software version 09W, Gauss-View 6.

THEORY: 7-hydroxyquinoline or quinoline-7-ol is a mono-hydroxyl quinoline, i.e., quinolone substituted by a

hydroxy group at position 7. It derives from a hydride of quinoline. 7- hydroxyquinoline (7-HQ) exists as enol
and keto forms and is interconverted through isomerization. It possesses antifungal, antibacterial, antimalarial,
cardiotonic, anthelmintic andanti-inflammatory activities.

Fig. 1. Structure of 7HQ molecule

Energy Optimization: A molecule is a small chemical element that is made up of two or moreatoms held together
by chemical bonds. These bonds are formed as a result of the exchange or sharing of electrons among atoms of
the molecule. Atoms tend to attain more electrons thereby increasing stability. Bond length is the distance between
the nucleiof two bonded atoms whereas bond angle is the angle formed between two adjacent atoms in amolecule.
Bond angle and bond length are the two important parameters which determine the shape and size of a molecule.
Thus, the geometry of a molecule can be characterized by analyzingthe bond length and bond angle. A dihedral
angle or torsion angle defines the conformations around rotatable bonds. Dihedral angles of amino acids are
biologically important since they define the backbone for protein in protein structure prediction. The main objective
of molecular mechanics is to find the lowest energy conformation of a molecule and this process is termed energy
minimization.
The lowest energy conformation can be calculated from the bond lengths and angles with the smallest steric
energy. The system makes several changes in the atom’s position through rotationand calculates energy in every
position. This process is repeated many times to find the position with the lowest energy until an overall minimum
energy is attained. In every move the energy is kept lower, otherwise, the atom will return to its original position.
The one full round of an atom rotation is called the minimization step or iteration. By applying a force field, the
minimum energy of a molecule in its stable conformation can be calculated, which is always taken as a negative
derivative of the energy function with respect to the coordinates of the atom. When the molecule is optimized with
its geometry, it normally attains the most stable conformation.

Z – Matrix: The Z-matrix is a way to represent a system built of atoms. A Z-matrix is also known as an internal
coordinate representation. It describes each atom in a molecule in terms of its atomic number, bond length, bond
angle, and dihedral angle, the so-called internal coordinates. However, it is convenient to write a Z-matrix in
terms of bond lengths, angles and dihedrals since this will preserve the actual bonding characteristics. The name
arises because the Z-matrix assigns the second atom along the Z-axis from the first atom, which is at the origin.
Each line of a Z-matrix gives the internal coordinates for one of the atoms within the molecule. The most-used
Z-matrix format uses the following syntax: Element-label, atom 1, bond length, atom 2, bond angle, atom 3, and
dihedral angle.
Element-label is a character string consisting of either the chemical symbol for the atom or its atomic number. If
the elemental symbol is used, it may be optionally followed by other alphanumeric characters to create an
identifying label for that atom.

Atom1, Atom2, and Atom3 are the labels for previously-specified atoms and are used to define the current atoms’
position. As an initial example, consider hydrogen peroxide. A Z-matrix for this structure would be:
H
O 1 0.9
O 2 1.4 1 105.0
H 3 0.9 2 105.0 1 120.0

The first line of the Z-matrix simply specifies hydrogen. The next line lists an oxygen atom and specifies the
internuclear distance between it and the hydrogen as 0.9 Angstroms. The third line defines another oxygen with
an O-O distance of 1.4 Angstroms (i.e., from atom 2, the other oxygen) and having an O-O-H angle (With atoms
2 and 1) of 105 degrees. The fourth and final line is the only one for which all three internal coordinates need to
be given. It defines the other hydrogen as bonded to the second oxygen with an H-O distance of 0.9 Angstroms, H-
O-O angle of 105 degrees and an H-O-O-H dihedral angle of 120 degrees.

METHODS OF ITERATION:

I. DFT (Density Functional Theory): Density-functional theory (DFT) is computational quantum

mechanical modelling method used in physics, chemistry and materials science to investigate the
electronic structure (or nuclear structure) (principally the ground state) of many-body systems, in
particular atoms, molecules, and the condensed phases. Using this theory, the properties of a many-
electron system can be determined by using functionals, i.e. functions of another function. In the
case of DFT, these are functionals of the spatially dependent electron density. DFT is among the
most popular and versatile methods available in condensed-matter physics, computational physics, and
computational chemistry.

DFT has reduced the problem of calculating the ground state characteristics of a many- electron system
in a local external field to the solution of the Hartree-like one-electron equations. At first DFT was
limited to consideration of only the ground states of many- electron systems, leaving aside their
dynamic properties, which are closely related to the systems behavior in the time-dependent external
fields. This limitation of DFT was surmounted by Runge and Gross by transforming DFT into the
so-called Time-Dependent Density Functional Theory (TDDFT), which in fact is a quite natural
generalization of DFT. Both DFT and TDDFT are based on the one-to-one correspondence between
the particle densities of the considered systems and external potentials acting upon these particles.
In the context of computational materials science, initially (from first principles) DFT calculations
allow the prediction and calculation of material behavior on the basis of quantum mechanical
considerations, without requiring higher-order parameters such as fundamental material properties. In
contemporary DFT techniques the electronic structure is evaluated using a potential acting on the
system's electrons. This DFT potential is constructed as the sum of external potentials Vext, which is
determined solely by the structure and the elemental composition of the system, and an effective
potential Veff, which represents interelectronic interactions. Thus, a problem for a representative
supercell of a material with n electrons can be studied as a set of n one-electron Schrödinger-like
 equations, which are also known as Kohn–Sham equations.
II. Hartree Fock Method: In computational physics and chemistry, the Hartree–Fock (HF) method is a
method of approximation for the determination of the wave function and the energy of a quantum many-
body system in a stationary state. The Hartree–Fock method often assumes that the exact N-body wave
function of the system can be approximated by a single Slater determinant (in the case where the
particles are fermions) or by a single permanent (in the case of bosons) of N spin-orbitals. By invoking
the variational method, one can derive a set of N-coupled equations for the N spin orbitals. A solution
of these equations yields the Hartree–Fock wave function and energy of the system.
The Hartree–Fock method is typically used to solve the time-independent Schrödinger equation for a
multi-electron atom or molecule as described in the Born–Oppenheimer approximation. Since there are
no known analytic solutions for many-electron systems (there are solutions for one-electron systems
such as hydrogenic atoms and the diatomic hydrogen cation), the problem is solved numerically. Due
to the nonlinearities introduced by the Hartree–Fock approximation, the equations are solved using a
nonlinear method such as iteration, which gives rise to the name "self-consistent field method".
Unfortunately, HF method has a number of difficulties in its application. For instance, one needs a whole
set of single-particle wave functions to calculate the single-electron nonlocal potential. It is also necessary
to follow a rather intricate procedure to include the correlation corrections that are beyond the HF
framework. As a result, in the case of complex many- electron systems, starting with molecules, that
include at least several atoms, the calculations become too complicated.

Basis Sets: A basis set is a set of wave functions that describes the shape of atomic orbitals (AOs). The molecular
orbitals (MOs) are computed using the selected theoretical model by linearly combining the AOs (LCAO).
Not all theoretical models require the user to choose a basis set to work with. For example, PMn (n=3,...,6)
models use an internal basis set, while HF or density functional theory require a basis set specification. The level
of approximation of your calculation is directly related to the basis set used. The choice to make is a trade-off
between accuracy of results and CPU time. A basis set in theoretical and computational chemistry is a set of
functions (called basis functions) that is used to represent the electronic wave function in the Hartree–Fock
method or density-functional theory in order to turn the partial differential equations of the model into algebraic
equations suitable for efficient implementation on a computer.
Various types of Basis sets are:
➢ STO – 3G: Both Slater Type Orbitals (STOs) and Gaussian Type Orbitals (GTOs) are used to describe
AOs. STOs describe the shape of AOs more closely than GTOs, but GTOs have an unbeatable
advantage: they are much easier to compute. In fact, it is faster to compute several GTOs and combine
them to describe an orbital than to compute one STO. This is why combinations of GTOs are
commonly used to describe STOs, which in turn, describe AOs. STO-nG (n=2,...,6) means that n
GTOs are used to decribe one STO, and only one STO is used to describe an AO (single Zeta). Usually,
n < 3 gives too poor results, so STO-3G is called the minimal basis set. Minimal basis sets are used
for either qualitative results, very large molecules or quantitative results for very small molecules
(atoms)[7]. Commonly used minimal basis sets of this type are:
a. STO-3G
b. STO-4G

➢ 3 – 21G: These basis sets are also called Pople basis sets and allow to specify the number of GTO’s
to use for core and valence electrons separately (size adjustable). These are double Zeta (2 functions
per AO) or triple Zeta. The notation is as follows:
 K-LMG
Where, K = number of sp-type inner shell GTOs
L = number of inner valence s- and p-type GTOs
M = number of outer valence s- and p-type GTOs
G = indicates that GTOs are used
The notation for the split-valence basis sets arising from the group of John Pople is typically X-YZG.
Commonly used split-valence basis sets of this type are:
a. 3-21G
b. 4-21G
c. 6-31G

PROCEDURE:

1. First, we have to install Gaussian Software (any version) and GuassView Software for simple
approach towards molecule creation.
2. Then, design the given molecule of 7HQ in GuassView Software by Builder → Element fragment
option and Modify bond options with precise bond lengths and bond angle.
3. After creating the molecule, go to Calculate → Gaussian Calculation Setup. There a window of
different setups opened.
4. In this experiment, you have to go Job Type → Optimization, Method → Ground State, DFT/Hartree
Fock, Basis Set → STO-3G, 3-21G etc.
5. After setting up all the variables, have to click submit button in bottom side.
6. After clicking submit, A processing page appears which take 3-4 minutes to compute and optimize
the given molecule (Timing depends on the molecule structure)
7. After computation/processing, two files are received (one of .chk file and other is .log file). From
.log file, we can navigate the summary of results which gives optimization energy, dipole moment
& basis set etc. We can get the value of optimized energy from here.
8. After extracting multiple energies, create a chart of optimization energies in excel sheet.
9. Repeat the steps from iv to viii to get the values for optimized frequency also.
INPUT OBSERVATIONS:
• INPUT FILE:

FIG. 2. INPUT STRUCTURE OF OPTIMIZED 7HQ

TABLE 1. Z-MATRIX OF INPUT STRUCTURE

Center Atomic Atomic Coordinates (Angstroms)

Number Number Type X Y Z
C1 6 0 -2.112133 -0.210115 0.000003
C2 6 0 -1.035592 -1.105583 0.000000
C3 6 0 0.314947 -0.617349 -0.000002
C4 6 0 0.553920 0.809476 0.000000
C5 6 0 -0.578063 1.703069 0.000004
C6 6 0 -1.871512 1.216774 0.000005
H7 1 0 -1.200506 -2.191190 -0.000001
C8 6 0 1.915852 1.265583 -0.000001
H9 1 0 -0.394544 2.786618 0.000005
H10 1 0 -2.737518 1.891814 0.000008
C11 6 0 2.939890 0.332118 -0.000005
C12 6 0 2.612201 -1.065843 -0.000006
H13 1 0 2.124559 2.344852 0.000000
H14 1 0 3.993305 0.640379 -0.000006
H15 1 0 3.434019 -1.803133 -0.000009
N16 7 0 1.355971 -1.597993 -0.000005
17 8 0 -3.466874 -0.603071 0.000005
18 1 0 -3.413181 -1.627608 0.000004
OUTPUT & CALCULATIONS:

ENERGY OPTIMIZATION

METHOD: HARTREE FOCK, BASIS SET: STO – 3G

FIG. 3 OPTIMIZED STRUCTURE

Symbolic Z – matrix (Charge = 0 Multiplicity = 1) Gaussian Calculation Summary

C 2.89974 0.34643 -0.00008
File Type .chk
C 1.89568 1.24749 -0.00002
C 0.54198 0.7831 0.00003 Calculation Type SP
C 0.31549 -0.60351 0.00011
Calculation RHF
C 2.57855 -1.04922 0.00000 Method
H -0.4073 2.73011 -0.00002 Basis Set STO – 3G
H 3.9382 0.64774 -0.00019
Charge 0
H 2.08909 2.31376 -0.0001
C -0.58227 1.66097 0.00003 Spin Singlet

C -1.01942 -1.10072 0.00004 Solvation None

H 3.39384 -1.7702 -0.00008
Electronic Energy -468.278295 Hartree
C -2.07571 -0.23769 0.00002
C -1.84527 1.17449 0.00007 RMS Gradient 0.000000 Hartree /
H -1.18195 -2.16943 0.00002 Norm Bohr
Imaginary Freq
H -2.69685 1.84332 0.00000
O -3.36695 -0.76606 -0.00011 Dipole Moment 1.208610 Debye
H -3.97176 001607 -0.00017
Point Group
N 1.35996 -1.54441 0.00003

TABLE 2: Z- MATRIX OF OPTIMIZED STRUCTURE TABLE 3: CALCULATION SUMMARY

METHOD: DFT, BASIS SET: STO – 3G

FIG. 5 OPTIMIZED STRUCTURE

TABLE 4: Z – MATRIX OF OPTIMIZED STRUCTURE TABLE 5: CALCULATION SUMMARY

Symbolic Z – matrix (Charge = 0 Multiplicity = 1) Gaussian Calculation Summary
C -2.11213 -0.21012 0.00000
File Type .chk
C -1.03559 -1.10558 0.00000
C 0.31495 -0.61735 0.00000 Calculation Type SP
C 0.55392 0.80948 0.00000
Calculation Method RB3LYP
C -0.57806 1.70307 0.00000
C -1.87151 1.21677 0.00001 Basis Set STO – 3G

H -1.20051 -2.19119 0.00000 Charge 0

C 1.91585 1.26558 0.00000
Spin Singlet
H -0.39454 2.78662 0.00001
H -2.73752 1.89181 0.00001 Solvation None
C 2.93989 0.33212 0.00000
Electronic Energy -471.120054 Hartree
C 2.6122 -1.06584 -0.00001
H 2.12456 2.34485 0.00000 RMS Gradient Norm 0.000000 Hartree
H 3.99331 0.64038 -0.00001 / Bohr
Imaginary Freq
H 3.43402 -1.80313 -0.00001
N 1.35597 -1.59799 -0.00001 Dipole Moment 0.874811 Debye
O -3.46687 -0.60307 0.00001
Point Group
H -3.41318 -1.62761 0.00000
METHOD: DFT, BASIS SET: 3 – 21G

FIG. 6 OPTIMIZED STRUCTURE

TABLE 6: Z – MATRIX OF OPTIMIZED STRUCTURE TABLE 7: CALCULATION SUMMARY

Symbolic Z – matrix (Charge = 0 Multiplicity = 1) Gaussian Calculation Summary

C -2.08564 -0.21064 0.00000 File Type .chk
C -1.02514 -1.02514 0.00000
C 0.30812 -0.6144 0.00000 Calculation Type SP
C 0.54609 0.79715 0.00000 Calculation RB3LYP
C -0.56836 1.68094 0.00000 Method
C -1.84854 1.19106 -0.00001 Basis Set 3-21G
H -1.16477 -2.16319 0.00000 Charge 0
C -1.89285 1.23953 0.00000
H -0.39272 2.75123 0.00001 Spin Singlet
H -2.71076 1.84435 0.00001 Solvation None
C 2.91264 0.31357 0.00000
C 2.58189 -1.06411 -0.00001 Electronic Energy -474.513306 Hartree
H 2.10211 2.3041 0.00000 RMS Gradient 0.000000 Hartree
H 3.95145 0.61799 -0.00001 Norm / Bohr
H 3.37681 -1.8037 -0.00001 Imaginary Freq
N 1.33658 -1.52709 0.00000 Dipole Moment 0.814970 Debye
O -3.41462 -0.59293 0.00001
H -3.48466 -1.58307 0.00000 Point Group
FREQUENCY OPTIMIZATION

METHOD: HARTREE FOCK, BASIS SET: STO – 3G

Mode Frequency
(cm-1)
C1-C2, C12-H15, C8-C11 138.35
C1-O17-H18 319.16
C6-H10, C12-H15 998.29
C2-H7 1050.07
C12-H15 1125.72
C6-H10 1186.55
C12-H15 3691.76
C12-H15 3691.76
C6-H10, C5-H9 3743.49
O17-H18 4263.24

TABLE 8: VIBRATIONAL MODES OF OPTIMIZED STRUCTURE FIG. 7 OPTIMIZED STRUCTURE

Symbolic Z – matrix (Charge = 0 Multiplicity = 1)

C 1.03457 -0.21092 0.00000
C 1.03457 -1.08282 0.00000
C -0.30961 -0.5981 0.00000
C -0.54738 0.78539 0.00000
C 0.57296 1.6767 0.00000
C 1.83764 1.20366 -0.00001
H 1.19555 -2.15209 0.00000
C -1.90244 1.23582 0.00000
H 0.38469 2.74346 -0.00001
H 2.69245 1.86707 -0.00001
C -2.89987 0.32377 0.00000
C -2.56648 -1.06511 0.00001
H -2.10755 2.29988 0.00000
H -3.94059 0.61689 0.00000
H -3.37305 -1.79556 0.00001
N -1.34029 -1.54814 0.00001
O 3.41712 -0.59604 -0.00001
H 3.40462 -1.58467 -0.00001

TABLE 9: Z – MATRIX OF OPTIMIZED STRUCTURE

METHOD: HARTREE FOCK, BASIS SET: 3 – 21G

Mode Frequency
(cm-1)
O17-H18 382.91
C5-C6 478.71
C2-H7, C8-H13, C5-H9 565.90
C5-H9, C2-H7 938.46
C11-H14 1006.06
C2-H7 1083.49
C2-H7 3385.36
C11-H14, C8-H13 3396.49
C6-H10 3405.61
017-H18 3912.38

TABLE 10: VIBRATIONAL MODES OF OPTIMIZED STRUCTURE FIG.8 OPTIMIZED STRUCTURE

Symbolic Z – matrix (Charge = 0 Multiplicity = 1)

C 2.0638 -0.21154 -0.00001
C 1.02349 -1.07991 0.00001
C -0.30466 -0.60183 -0.00002
C -0.5415 0.78226 -0.00001
C 0.56758 1.66573 0.00000
C 1.8317 1.1848 -0.00001
H 1.16477 -2.14119 0.00002
C -1.88435 1.22231 0.00001
H 0.39117 2.72368 0.00001
H 2.68438 1.83016 -0.00002
C -2.89033 0.30972 0.00002
C -2.55688 -1.06213 -0.00004
H -2.0917 2.27494 0.00002
H -3.91808 0.60785 0.00004
H -3.336 -1.7979 0.00002
N -1.32936 -1.49788 0.00001
C 2.0638 -0.21154 -0.00001
C 1.02349 -1.07991 0.00001

TABLE 11: Z – MATRIX OF OPTIMIZED STRUCTURE

METHOD: DFT, BASIS SET: STO – 3G

Mode Frequency
(cm-1)
C8-C11 293.68
C12-H15 546.27
C12-H15, C5-H10 815.42
C6-H10 874.44
C2-H10 900.71
C12-H15 984.47
C5-H10 1027.79
C12-H15 3415.00
O17-H18 3725.04

TABLE 12: VIBRATIONAL MODES OF OPTIMIZED STRUCTURE FIG.9. OPTIMIZED STRUCTURE

Symbolic Z – matrix (Charge = 0 Multiplicity = 1)

C -2.11213 -0.21012 0.00000
C -1.03559 -1.10558 0.00000
C 0.31495 -0.61735 0.00000
C 0.55392 0.80948 0.00000
C -0.57806 1.70307 0.00000
C -1.87151 1.21677 0.00001
H -1.20051 -2.19119 0.00000
C 1.91585 1.26558 0.00000
H -0.39454 2.78662 0.00001
H -2.73752 1.89181 0.00001
C 2.93989 0.33212 -0.00001
C 2.6122 -1.06584 -0.00001
H 2.12456 2.34485 0.00000
H 3.99331 0.64038 -0.00001
H 3.43402 -1.80313 -0.00001
N 1.35597 -1.59799 -0.00001
O -3.46687 -0.60307 0.00001
H -3.41318 -1.62761 0.00000

TABLE 13: Z – MATRIX OF OPTIMIZED STRUCTURE

METHOD: DFT, BASIS SET: 3 – 21G

Mode Frequency
(cm-1)
C12-H15 436.86
C6-H10 706.20
C2-H7 837.03
C12-H15 3171.26
C8-H13, C5-H9 3189.63
C2-H7 3211.72
C11-H14 3224.85
C6-H10, C5-H9 3235.24
O17-H18 3522.29

TABLE 14: VIBRATIONAL MODES OF OPTIMIZED STRUCTURE FIG.10. OPTIMIZED STRUCTURE

Symbolic Z – matrix (Charge = 0 Multiplicity = 1)

C -2.08564 -0.21064 0.00000
C -1.02514 -1.08889 0.00000
C 0.30812 -0.6144 0.00000
C 0.54609 0.79715 0.00000
C -0.56836 1.68094 0.00000
C -1.84854 1.19106 0.00001
H -1.16477 -2.16319 0.00000
C 1.89285 1.23953 0.00000
H -0.39272 2.75123 0.00001
H -2.71076 1.84435 0.00001
C 2.91264 0.31357 -0.00001
C 2.58189 -1.06411 -0.00001
H 2.10211 2.3041 0.00000
H 3.95145 0.61799 -0.00001
H 3.37681 -1.8037 -0.00001
N 1.33658 -1.52709 -0.00001
O -3.41462 -0.59293 0.00001
H -3.48466 -1.58307 0.00000

TABLE 15: Z – MATRIX OF OPTIMIZED STRUCTURE

RESULTS:
Optimized Energy (in Hartree) for various methods/basis sets are as follows:

Method / Basis Sets STO – 3G 3 – 21G

Hartree Fock -468.27 -471.55
DFT -471.12 -474.51

Optimized Frequency(cm-1) of O17-H18 for various methods/basis sets are as follows:

Method / Basis Sets STO – 3G 3 – 21G

Hartree Fock 4263.24 3912.38
DFT 3725.04 3522.29

SOURCES OF ERROR AND PRECAUTIONS:

1. Carefully create the structure by taking care of bond lengths and bond angles.
2. Always save the .log file for better understanding of whole iterations takes place during
processing.

3. Number of bonds at particular atom should be maintained according to their valence electron
configurations.
 EXPERIMENT – 2

AIM: To record absorption spectra of a given molecule(7HQ) in wavelength scale and estimate the possible
parameters.

APPARATUS/SOFTWARE USED: Gaussian Software version 09W, Gauss-View 6.

THEORY:

Gaussian Software: Gaussian is a general-purpose computational chemistry software package initially

released in 1970 by John Pople and his research group at Carnegie Mellon Universityas Gaussian 70. It has
been continuously updated since then. The name originates from Pople'suse of Gaussian orbitals to speed
up molecular electronic structure calculations as opposed to using Slater-type orbitals, a choice made to
improve performance on the limited computing capacities of then-current computer hardware for Hartree–
Fock calculations. Gaussian process(GP) modelling is commonly used for fitting metamodels in simulation
experiments since it provides a flexible model and model-based estimate of prediction error even if the
simulation itself is deterministic.

UV – Visible Spectroscopy: UV-Vis spectroscopy is an analytical technique that measures theamount of

discrete wavelengths of UV or visible light that are absorbed by or transmitted througha sample in comparison
to a reference or blank sample. This property is influenced by the sample composition, potentially providing
information on what is in the sample and at what concentration. Since this spectroscopy technique relies
on the use of light, let’s first consider the properties of light.

Light has a certain amount of energy which is inversely proportional to its wavelength. Thus, shorter
wavelengths of light carry more energy and longer wavelengths carry less energy. A specific amount of
energy is needed to promote electrons in a substance to a higher energy state which we can detect as
absorption. Electrons in different bonding environments in a substancerequire a different specific amount
of energy to promote the electrons to a higher energy [Link] is why the absorption of light occurs for
different wavelengths in different substances. Humans are able to see a spectrum of visible light, from
approximately 380 nm, which we seeas violet, to 780 nm, which we see as red.1 UV light has wavelengths
shorter than that of visible light to approximately 100 nm. Therefore, light can be described by its
wavelength, which canbe useful in UV-Vis spectroscopy to analyze or identify different substances by
locating the specific wavelengths corresponding to maximum absorbance.

UV – Vis Spectra: UV-Vis spectroscopy information may be presented as a graph ofabsorbance, optical
density or transmittance as a function of wavelength. However, the information is more often presented as
a graph of absorbance on the vertical y axis and wavelength on the horizontal x axis. This graph is typically
referred to as an absorption spectrum.

The absorbance (A) is equal to the logarithm of a fraction involving the intensity of light beforepassing
through the sample (Io) divided by the intensity of light after passing through the sample

(I). The fraction I divided by Io is also called transmittance (T), which expresses how much light has passed
through a sample. However, Beer–Lambert's law is often applied to obtain the concentration of the sample
(c) after measuring the absorbance (A) when the molar absorptivity (ε) and the path length (L) are known.
Typically, ε is expressed with units of L mol-1 cm-1, L has units of cm, and c is expressed with units of mol
L-1. As a consequence, A has no units. Beer– Lambert's law is especially useful for obtaining the
concentration of a substance if a linear relationship exists using a measured set of standard solutions
containing the same substance.
 Equation 1: A set of equations showing the relationships between absorbance A, Beer–Lambert's law, the
light intensities measured in the instrument, and transmittance.

UV/Vis spectra originate from the excitation of electrons, and therefore UV/Vis spectroscopy is often also
referred to as “electron spectroscopy”. The term “electron spectroscopy” encompasses the excitation of electrons
by ultraviolet or visible radiation. Hence UV/VIS spectra belong to the molecular spectra. The total energy of a
molecule is represented as the sum of its translational, rotational, vibrational and electronic energies.
Etotal = Etranslational + Erotational + Evibrational + Eelectronic

The various forms of energy differ with regard to the amount of energy they contain; within a molecule the order
is:
Eelectronic > Evibrational > Erotational > Etranslational

In molecules or atoms whose electrons are excited by UV/Vis radiation, transitions take place between the
various energy levels of the electrons, the individual excitation wavelengths being determined by the distribution
of electrons in the molecule or atom. In the ground-state molecule, the electrons will normally be in a bonding
singlet state. However, when an electron is excited, it changes to an anti - bonding singlet state. Such a change
is associated with a transition, as a rule at room temperature, from the vibrational ground state of the electronic
ground state to an excited vibrational state of the excited electronic state. When the energy of the incident light
corresponds to the energy of transition between two electronic states this results in excitation and, hence, transition
of the electron from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital
(LUMO). The energy that this requires is withdrawn from the incident light, and this process is referred to as
absorption. In diatomic molecules such as the hydrogen molecule H2, this transition is fairly easily described.
In polyatomic molecules, however, description of the transitions is limited to the electrons in transition.
Saturated organic molecules, for example the alkanes, require considerable excitation energy so that transition
takes place only in the far UV (approx. 160 nm). As a consequence, the compounds are colourless.

PROCEDURE:

1. First, we have to install Gaussian Software (any version) and GaussView Software for simple approach towards
molecule creation.

2. Then, design the given molecule in GaussView Software by Builder Element fragment option and Modify
bond options with precise bond lengths and bond angle.
3. After creating the molecule, go to Calculate Gaussian Calculation Setup. There a window of different setups
opened.

4. In this experiment, you have to go Job Type → Optimization, Method → TD-SCF, DFT/Hartree
Fock, Basis Set → STO-3G, 3-21G etc.
5. After setting up all the variables, have to click submit button in bottom side.
6. After clicking submit, A processing page appears which take 3-4 minutes to compute and optimize the given
molecule (Timing depends on the molecule structure)
7. After computation/processing, two files are received (one of .chk file and other is .log file). From .log file, we
can navigate the summary of results which gives optimization energy, dipole moment & basis set etc.
8. We can also navigate the UV-Vis Spectra of particular molecule.

INPUT OBSERVATIONS

INPUT FILE:

FIG.1. INPUT STRUCTURE OF OPTIMIZED 7HQ

Center Atomic Atomic Coordinates (Angstroms)

Number Number Type X Y Z
1 6 0 -2.112133 -0.210115 0.000003
2 6 0 -1.035592 -1.105583 0.000000
3 6 0 0.314947 -0.617349 -0.000002
4 6 0 0.553920 0.809476 0.000000
5 6 0 -0.578063 1.703069 0.000004
6 6 0 -1.871512 1.216774 0.000005
7 1 0 -1.200506 -2.191190 -0.000001
8 6 0 1.915852 1.265583 -0.000001
9 1 0 -0.394544 2.786618 0.000005
10 1 0 -2.737518 1.891814 0.000008
11 6 0 2.939890 0.332118 -0.000005
12 6 0 2.612201 -1.065843 -0.000006
13 1 0 2.124559 2.344852 0.000000
14 1 0 3.993305 0.640379 -0.000006
15 1 0 3.434019 -1.803133 -0.000009
16 7 0 1.355971 -1.597993 -0.000005
 17 8 0 -3.466874 -0.603071 0.000005
18 1 0 -3.413181 -1.627608 0.000004

TABLE 1. Z – MATRIX OF INPUT STRUCTURE

CALCULATIONS

METHOD: HARTREE FOCK, BASIS SET: STO-3G

FIG.2. OPTIMIZED STRUCTURE

TABLE 2: Z – MATRIX OF OPTIMIZED STRUCTURE TABLE 3: CALCULATION SUMMARY

Symbolic Z – matrix (Charge = 0 Multiplicity = 1)
Gaussian Calculation Summary
C 2.07877 -0.21092 0.00000
File Type .chk
C 1.03457 -1.08282 0.00000
C -0.30961 -0.5981 0.00000 Calculation Type FOPT
C -0.54738 0.78539 0.00000
Calculation RTD – FC
C 0.57296 1.6767 0.00000 Method
C 1.83764 1.20366 -0.00001 Basis Set STO – 3G
H 1.19555 -2.15209 0.00000
Charge 0
C -1.90244 1.23582 0.00000
H 0.38469 2.74346 -0.00001 Spin Singlet

H 2.69245 1.86707 -0.00001 Solvation None

C -2.89987 0.32377 0.00000
Electronic Energy -468.094842 Hartree
C -2.56648 -1.06511 0.00001
H -2.10755 2.29988 0.00000 RMS Gradient 0.000080 Hartree /
H -3.94059 0.61689 0.00000 Norm Bohr
Imaginary Freq
H -3.37305 -1.79556 0.00001
N -1.34029 -1.54814 0.00001 Dipole Moment 1.219520 Debye
O 3.41712 -0.59604 -0.00001
Point Group
H 3.40462 -1.58467 -0.00001
METHOD: HARTREE FOCK, BASIS SET: 3 - 21G

FIG.3. OPTIMIZED STRUCTURE

TABLE 3: Z – MATRIX OF OPTIMIZED STRUCTURE TABLE 4: CALCULATION SUMMARY

Symbolic Z – matrix (Charge = 0 Multiplicity = 1) Gaussian Calculation Summary

C 2.0638 -0.21154 0.00000
File Type .chk
C 1.02349 -1.07991 0.00001
Calculation FOPT
C -0.30466 -0.60183 -0.00002
Type
C -0.5415 0.78226 -0.00001 Calculation RTD – FC
C 0.56757 1.66573 0.00000 Method
Basis Set 3-21G
C 1.8317 1.1848 -0.00001
H 1.16477 -2.14119 0.00002 Charge 0
C -1.88435 1.22231 0.00001
Spin Singlet
H 0.39117 2.72368 0.00001
H 2.68438 1.83016 -0.00002 Solvation None

C -2.89033 0.30972 0.00002 Electronic -471.369421 Hartree

C -2.55688 -1.06213 -0.00004 Energy
RMS 0.000078 Hartree /
H -2.0917 2.27494 0.00002
Gradient Bohr
H -3.91808 0.60785 0.00003 Norm
H -3.336 -1.7979 0.00002 Imaginary
Freq
N -1.32936 -1.49788 0.00001 Dipole 0.723491 Debye
O 3.38217 -0.59017 0.00001 Moment
Point Group
H 3.50059 -1.54746 0.00002
METHOD: DFT, BASIS SET: STO – 3G

FIG.4. OPTIMIZED STRUCTURE

TABLE 5: Z – MATRIX OF OPTIMIZED STRUCTURE TABLE 6: CALCULATION SUMMARY

Symbolic Z – matrix (Charge = 0 Multiplicity = 1) Gaussian Calculation Summary

C -2.11213 -0.21012 0.00000 File Type .chk
C -1.03559 -1.10558 0.00000
C 0.31495 -0.61735 0.00000 Calculation FOPT
Type
C 0.55392 0.80948 0.00000 Calculation RB3LYP
C -0.57806 1.70307 0.00000 Method
Basis Set TD-FC
C -1.87151 1.21677 0.00001
H -1.20051 -2.19119 0.00000 Charge 0
C 1.91585 1.26558 0.00000
Spin Singlet
H -0.39454 2.78662 0.00001
H -2.73752 1.89181 0.00001 Solvation None
C 2.93989 0.33212 -0.00001
Electronic -470.988901 Hartree
C 2.6122 -1.06584 -0.00001 Energy
H 2.12456 2.34485 0.00000 RMS Gradient 0.000107 Hartree /
Norm Bohr
H 3.99331 0.64038 -0.00001
Imaginary Freq
H 3.43402 -1.80313 -0.00001
N 1.35597 -1.59799 -0.00001 Dipole Moment 2.384187 Debye
O -3.46687 -0.60307 0.00001 Point Group
H -3.41318 -1.62761 0.00000
METHOD: DFT, BASIS SET: 3 – 21G

FIG.5. OPTIMIZED STRUCTURE

TABLE 7: Z – MATRIX OF OPTIMIZED STRUCTURE TABLE 8: CALCULATION SUMMARY

Symbolic Z – matrix (Charge = 0 Multiplicity = 1)

Gaussian Calculation Summary
C -2.08564 -0.21064 0.00000
File Type .chk
C -1.02514 -1.08888 0.00000
C 0.30812 -0.6144 0.00000 Calculation Type FOPT
C 0.54609 0.79715 0.00000
RB3LYP
C -0.56836 1.68094 0.00000
C -1.84854 1.19106 0.00001 Basis Set TD-FC
H -1.16477 -2.16319 0.00000
Charge 0
C 1.89285 1.23953 0.00000
H -0.39272 2.75123 0.00001 Spin Singlet

H -2.71076 1.84435 0.00001 Solvation None

C 2.91264 0.31357 0.00000
C 2.58189 -1.06411 -0.00001 Electronic Energy -474.382491 Hartree

H 2.10211 2.3041 0.00000 RMS Gradient 0.000065 Hartree /

H 3.95145 0.61799 -0.00001 Norm Bohr
H 3.37681 -1.8037 -0.00001 Imaginary Freq

N 1.33658 -1.52709 0.00000

Dipole Moment 2.755494 Debye
O -3.41462 -0.59293 0.00001
H -3.48466 -1.58307 0.00000 Point Group
OBSERVATIONS:

FIG.6. Z – COMBINED UV – VIS PLOT FOR VARIOUS METHODS AND BASIS SETS

Method / Basis Sets STO – 3G 3 – 21G

Hartree Fock 202.8 nm 266.2 nm
DFT 291.2 nm 306.6 nm
TABLE 9: WAVELENGTHS CORRESPONDING TO HIGHEST PEAKS

RESULTS:
The wavelength of absorption at highest peak is calculated at normal termination ofoptimization as 306.6
nm (maximum value)

SOURCES OF ERROR AND PRECAUTIONS:

1. Carefully create the structure by taking care of bond lengths and bond angles.
2. Always save the .log file for better understanding of whole iterations takes placeduring
processing.
 EXPERIMENT – 3

AIM: To verify Beer – Lambert’s Law.

APPARATUS/ SOFTWARE USED: Virtual simulator, ImageJ software.
THEORY: Wavelength and the intensity of electromagnetic radiation in the visible region of the spectrum. It is
used extensively for identification and determination of concentrations of substances that absorb light. Two
fundamental laws are applied: that of a French scientist, Pierre Bouguer, which is also known as Lambert’s law,
which relates the amount of light absorbed and the distance it travels through an absorbing medium; and Beer’s
law relates light absorption and the concentration of the absorbing substance. The two laws may be combined and
expressed bythe equation log I0/I = kcd, where Io = intensity of the incident beam of light, I = transmitted
intensity,c = the concentration of absorbing substance, d = the distance through the absorbing solution, andk = a
constant, dependent upon the absorbing substance, the wavelength of light used, and the units used to specify c
and d.
Beer’s Law: Beer Law states that the light-absorbing capacity of a dissolved substance is directlyproportional to
its concentration in the solution. Mathematically this law is given by
𝐴 = −𝑙𝑜𝑔10𝑇 = 𝜀𝜆𝑙𝑐

where, A = absorbance,
T = transmittance = I / Io (where Io = intensity of incident beam and I = intensity of theemergent beam)
𝜀𝜆 = molar absorptivity or extinction coefficient of the dissolved coloured substance (which is aproperty of the
substance and the solvent at a particular wavelength λ)
𝑙 = sample path length
c = concentration of the substance in the sample
Therefore, A = constant × c (for a given path length of a sample)
One can verify validity of Beer Law by using a spectrophotometer and preparing a few different concentrations
of solutions of a light absorbing substance in a suitable solvent. If one plots A vs.c for a sample of a given path
length, then a straight line passing through origin and slope should be obtained. It should be noted here that often
deviations from the linearity occur at higher concentrations of the substance in a solvent. Nevertheless, Beer Law
is accurate for a rangeof low concentrations of chromophores in many solvents and is therefore widely used in
quantitative spectroscopy. If the molar absorptivity or extinction coefficient is known for particular substance,
the unknown concentration of that substance in a solution can be calculated from the above equation. In the case
of non - availability of molar absorptivity or extinction coefficient, the unknown substance concentration can be
determined using a "calibration" straight line (or the regression equation for the straight line) derived by plotting
the absorbance vs. concentration from a few standard solutions.
Lambert’s Law: We know that many organic molecules do not absorb electromagnetic radiationin the ultraviolet-
visible (UV-VIS) regions (~wavelengths range 190 nm - 800 nm). However, there are some compounds which
absorb a portion of the radiation when a radiation passes through them. There is a relationship between the amount
of light absorbed and the distance the light travels through the absorbing medium. This relationship is known as
the Lambert law (Bouguer-Lambert law).
According to the Lambert law, each layer of equal thickness of the medium absorbs an equal fraction of the light
traversing it. In other words, the fraction of light absorption is proportional to the sample path length. Say, a beam
of monochromatic radiation of intensity Io, falls on a sample. For a traversal of a small path length dℓ (with unit
cross-section, ℓ is the path length) thedecrease in the intensity is dI due to the absorption of radiation.
Then according to the Lambert law,
ⅆ𝐼
= −𝑘𝜆 ⅆ𝑙
𝐼
Where, 𝐼𝜆 = intensity in the wavelength interval λ and λ+dλ

𝐾𝜆 = absorption coefficient (practically independent of path length but depends on thewavelength)

Rearrangement and integration yield the following equation:

𝐼
= exp⁡(−𝑘𝜆 𝑙)⁡
𝐼𝑜
Where 𝐼 = intensity of beam of radiation leaving the sample after absorption.
𝐼
− 𝑙𝑜𝑔 ( ) = 𝑘𝑙
𝐼0
The fraction of the amount of radiation absorbed is known as transmittance,
𝐼
𝑇=
𝐼0
Another parameter, absorbance, A, is defined as,
𝐼
𝑙𝑜𝑔 (𝐼 ) ⋅ 𝑘𝑙
0
𝐴 = − 𝑙𝑜𝑔10 𝑇 = − =
2.303 2.303
Therefore, A = (constant)× ℓ. The measurement of A at a particular wavelength gives a measure
of the absorption capacity and in turn transparency of the medium with respect to the [Link] using a
spectrophotometer and a few cuvettes of different path lengths, one can verify this law that the fraction of the
light absorbed is proportional to the path length. In other words, one can find that the absorbance (A) varies
linearly with the path length, ℓ.
Beer-Lambert’s Law: The Beer–Lambert law, also known as Beer's law, the Lambert–Beer law,or the Beer–
Lambert–Bouguer law relates the attenuation of light to the properties of the materialthrough which the light is
travelling. The law is commonly applied to chemical analysis measurements and used in understanding
attenuation in physical optics, for photons, neutrons, orrarefied gases.
A common and practical expression of the Beer–Lambert law relates the optical attenuation of aphysical material
containing a single attenuating species of uniform concentration to the optical path length through the sample
and absorptivity of the species.
This expression is
𝐴 = 𝜀𝑙𝑐
Where, A = absorbance,
𝜀 = molar attenuation coefficient
𝑙 = optical path length
c = concentration of attenuating species

PRODECURE
A. To get the absorption vs. wavelength curve
 a. Prepare a standard K2Cr2O7 solution of strength nearly 3.16x10 -3 M. This is used as stocksolution.
From the stock solution, prepare six different concentrations of K2Cr2O7. Click on concentration bar
to select the concentration of K2Cr2O7.
b. Turn on the instrument clicking on the power button and wait for 30 min for initialization of the
instrument.
c. Click and drag on the concentration bar to choose the appropriate concentration of the solution whose
absorbance has to be measured. One should start with the lowest concentration solution first.
d. Click on the beaker to take a clean, dry beaker.
e. Click on the volumetric flask to pour the solution into the clean, dry beaker.
f. Click on the micropipette to collect appropriate quantity of solution from the beaker.
g. Take a cuvette (quartz cuvette with a given path length, say, 1 cm) by clicking on it.
h. Pour the solution from the micropipette into the cuvette by clicking on the micropipette. (In real
measurements, the cuvette is filled to two-third of its volume.)
i. Click on the spectrophotometer lid to open it.
j. Click on the cuvette to place it in the sample holder. One has to use water as the sample blank or
reference in an identical cuvette for this measurement. Here a double beam spectrophotometer
is shown. In this case, one can place the sample in the sample holder (often the front one) and the
sample bank or reference in the reference holder (often the back one) simultaneously.
k. Run the wavelength scan by clicking on the computer monitor first and then on the Scan button there
to observe the wavelength scan. In the real spectrophotometer, an appropriatewavelength range of
incident light for the sample can be chosen and the wavelength scan are run via the accompanied
computer software. One can run the scan in absorbance or transmittance mode. The scan data is
stored in the computer. If the spectrophotometer is asingle beam instrument, then first the sample
blank or reference is taken in a cuvette and the wavelength scan is run followed by the sample. One
has to subtract the reference data from the sample data for respective wavelengths.
l. Click on Reset button to start new measurement.
m. Select next higher concentration and repeat the measurement. Every time one should rinsethe cuvette
taking a small portion of the solution from the solution that will be measured next. Similarly, one
can repeat it for all the concentrations one after another.
n. Collect all data by clicking on the Data tab.
o. Plot the absorbance of the sample at various wavelengths for different concentrations anddetermine
the wavelength of maximum absorptions i.e., spectral peak-positions.
p. Find out the wavelengths of maximum absorbance (λmax), the absorbance at λmax and at another
wavelength (say, 335 nm) for all the concentrations of K2Cr2O7 and make a table containing these
data.
q. Plot absorbance vs. concentration. Connect the points first with line segments. Then attempt linear
fits and calculate linear regressions.

B. For the calculation of 𝝀𝒎𝒂𝒙 and corresponding absorbance (Using ImageJ)

a. Drag the first output curve picture of the first concentration in the ImageJ software.
b. Click on the line icon on the ImageJ tab.
c. Click on 400 (on the x – axis) and drag the line upto 450 such that the angle of the line is, 0 degrees.
d. Click on analyze and select the set scale option.
e. Enter the known distance according to the length selected and enter the unit as nm forwavelength.
f. Click ok.
g. Click on the peak of the curve and drag it to the x – axis parallel to the y- axis such that the angle is 90
degrees.
 h. Click Ctrl + D.
i. Zoom in the x – axis.
j. Pointing at the origin, drag the line to the point of intersection of the tangent at the x-axis keeping the
angle 0 degree.
k. Click Ctrl + M.
l. A dialog box showing the length from the origin to the intersection point appears.
m. Add this length to 325 (as it is the starting point of the x – axis).
n. This gives us the value of wavelength for maximum absorbance.
o. Similarly, set the scale for y-axis.
p. Click Ctrl + M.
q. Again, following the same steps we can get value of length from the origin to the point ofmaximum
absorbance.
r. This length gives us the value of maximum absorbance at the calculated wavelength.

OBSERVATIONS

FIG. 1. EXPERIMENT SETUP FOR THE SIMULATION

FIG. 2. ABSORPTION SPECTRUM OF K2Cr2O7 WITH WATER (C=0.158mM)

FIG. 3. ABSORPTION SPECTRUM OF K2Cr2O7 WITH WATER (C=0.316mM)

FIG. 4. ABSORPTION SPECTRUM OF K2Cr2O7 WITH WATER (C=0.474mM)

FIG. 5. ABSORPTION SPECTRUM OF K2Cr2O7 WITH WATER (C=0.632mM)

FIG. 6. ABSORPTION SPECTRUM OF K2Cr2O7 WITH WATER(C=0.790mM)

FIG. 7. ABSORPTION SPECTRUM OF K2Cr2O7 WITH WATER(C=0.948Mm)

 TABLE 1: OBSERVED VALUES OF 𝜆𝑚𝑎𝑥 AND CORRESPONDING ABSORBANCE

[Link]. Concentration Wavelength (𝝀𝒎𝒂𝒙) Absorbance (A)

(c) (mM) (nm)
1. 0.158 348.743 0.5
2. 0.316 349.074 0.818
3. 0.474 349.294 0.99
4. 0.632 349.719 1.186
5. 0.790 349.581 1.34
6. 0.948 349.444 2.964

FIG. 8. VARIATION OF ABSORBANCE WITH CONCENTRATION

CONC. V/S ABSORBANCE

1.6
= 1.2962x + 0.3524
1.4
R² = 0.9802
1.2
ABSORBANCE (A)

0.8

0.6

0.4

0.2

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

CONCENTRATION (mM)

RESULTS:
1. The wavelength for which maximum absorbance is observed
(𝜆𝑚𝑎𝑥)independent of concentration of the sample.
2. The absorbance increases with the increase in concentration of
the sample linearly up to 0..948Mm
 3. The regression coefficient r2 is calculated to be 0.9802.

SOURCES OF ERROR AND PRECAUTIONS:

1. Beer-lambert’s law is successful in describing absorbance of dilute solutions
onlybecause for higher concentrations, intermolecular interactions can occur
betweenmolecules which can alter 𝜀.
2. Absorptivity also depends on refractive index of the medium, that can be
altered byvarying concentration.
 EXPERIMENT – 4

AIM: To introduce the principles of fluorescence in a molecule and demonstrate how the fluorescence
spectrum of a fluorophore is affected by the fluorophore concentration (Inner filtereffects).

MATERIALS REQUIRED: Virtual software, ImageJ software

THEORY:
Fluorescence spectroscopy analyzes fluorescence from a molecule based on its fluorescent properties.
Fluorescence is a type of luminescence caused by photons exciting a molecule, raising it to an electronic excited
state. Fluorescence spectroscopy uses a beam of light that excites the electrons in molecules of certain
compounds, and causes them to emit light. That light is directed towards a filter and onto a detector for
measurement and identific ation of the molecule or changes in the molecule. Fluorescence spectroscopy
(also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy thatanalyzes
fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons
in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A
complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence
spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs
of fluorophores. Devices that measure fluorescence are called fluorometers.
Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with
electronic and vibrational states. Generally, the species being examined hasa ground electronic state (a low
energy state) of interest, and an excited electronic state of higherenergy. Within each of these electronic states there
are various vibrational states. In fluorescence,the species is first excited, by absorbing a photon, from its ground
electronic state to one of the various vibrational states in the excited electronic state. Collisions with other
molecules cause theexcited molecule to lose vibrational energy until it reaches the lowest vibrational state from
the excited electronic state. This process is often visualized with a Jablonski diagram. The moleculethen drops
down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the
process.[1] As molecules may drop down into any of several vibrational levels in the ground state, the emitted
photons will have different energies, and thus [Link], by analysing the different frequencies of
light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different
vibrational levels can be determined.
For atomic species, the process is similar; however, since atomic species do not have vibrationalenergy levels, the
emitted photons are often at the same wavelength as the incident radiation. Thisprocess of re-emitting the absorbed
photon is "resonance fluorescence" and while it is characteristic of atomic fluorescence, is seen in molecular
fluorescence as well. In a typical fluorescence (emission) measurement, the excitation wavelength is fixed and
the detection wavelength varies, while in a fluorescence excitation measurement the detection wavelength is fixed
and the excitation wavelength is varied across a region of interest. An emission map is measured by recording the
emission spectra resulting from a range of excitation wavelengths and combining them all together. This is a three
dimensional surface data set: emission intensity as a function of excitation and emission wavelengths, and is
typically depicted as a contour map.
At low concentrations the fluorescence intensity will generally be proportional to the concentration
of the fluorophore. Unlike in UV/visible spectroscopy, ‘standard’, device independent spectra are not easily
attained. Several factors influence and distort the spectra, and corrections are necessary to attain ‘true’, i.e.
machine-independent, spectra. The different types of distortions will here be classified as being either instrument-
or sample-related. Firstly, the distortion arising from the instrument is discussed. As a start, the light source
intensity and wavelength characteristics varies over time during each experiment and between each experiment.
Furthermore, no lamp has a constant intensity at all wavelengths. To correct this, a beam splitter can be applied
after the excitation monochromator or filter to direct a portion of thelight to a reference detector.
Additionally, the transmission efficiency of monochromators and filters must be taken into account. These may
also change over time. The transmission efficiency of the monochromator also varies depending on wavelength.
This is the reason that an optional reference detector shouldbe placed after the excitation monochromator or filter.
The percentage of the fluorescence pickedup by the detector is also dependent upon the system.
Furthermore, the detector quantum efficiency, that is, the percentage of photons detected, varies between different
detectors, with wavelength and with time, as the detector inevitably deteriorates. Fluorescence spectroscopy is used
in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. There has
also been a report of its use in differentiating malignant skin tumors from benign. Atomic Fluorescence
Spectroscopy (AFS) techniques are useful in other kinds of analysis/measurement of a compound present in air
or water, or other media, such as CVAFS which is used for heavy metals detection, such as mercury. Fluorescence
can also be used to redirect photons, see fluorescent solar collector. Additionally, Fluorescence spectroscopy can
be adapted to the microscopic level using microfluorimetry. In analytical chemistry, fluorescence detectors are used
with HPLC. In the field of water research, fluorescence spectroscopy can be used to monitor water quality by
detecting organic pollutants.[14] Recent advances in computer science and machine learning have even enabled
detection of bacterial contamination of water.

PROCEDURE:
1. Prepare six standard quinine sulfate solutions of following concentrations: 0.0001, 0.001, 0.01, 0.05,
0.1 and 1 ppm (parts per million) quinine in 0.05 M H2SO4. These solutions areprepared via dilution
from a 10 ppm quinine stock solution in 0.05 M H2SO4. To prepare the stock solution, first exactly
6 mg of quinine sulfate dihydrate is quantitatively transferred to a 500 mL volumetric flask, dissolved
by adding 25 mL of 1 M H2SO4 and finally made up to the mark with deionized water. Quinine sulfate
decomposes under [Link], the stock solution should be made fresh for measurements and
stored in brownbottles in a cool place. Here all the solutions, including an unknown concentration
solution, are shown on a concentration bar. Note that the supplied unknown sample was made as
follows. A 1 mL of tonic water sample (containing an unknown concentration of quinine sulfate) was
diluted to the mark with aq. 0.05 M H2SO4 in a 100 mL volumetric flask.
2. Carry out the excitation and emission measurements of the solutions as follows.
3. To choose an appropriate concentration of the solution whose fluorescence has to be measured, click
on the pointer below the concentration bar and drag it to the desired value. In real experimental
measurements, one should start with the lowest concentration solutionfirst and proceed to next higher
concentration and so on. (Why?)
4. Click on the volumetric flask containing the experimental solution to take it to the instrument table.
5. Take an all-side-transparent quartz cuvette (path length, 1 cm x 1 cm) by clicking on it.
6. Click on the 5 mL-capacity pipette to collect 3 mL of the experimental solution which will be
transferred into the quartz cuvette. In real operation, one has to set the volume to 3 mLin the pipette
and an appropriate tip should be attached prior to dipping it in the solution.
7. Click on the pipette to draw the solution into it.
8. Click on the pipette to take it out of the volumetric flask.
9. Click on the pipette again to transfer the solution into the cuvette.
10. Turn on the spectrofluorimeter by clicking on the power button. In real operation, it takesapprox. 30
min for initialization of the instrument.
11. Click on the lid of the sample chamber of the spectrofluorimeter to open it for placing thesample in
the instrument.
12. To place the cuvette in the sample holder in the chamber, click on the cuvette.
13. Click on the lid of the sample chamber to close it.
14. To run the Excitation Spectral Scan of the sample, open the instrument set-up screen by clicking on
the fluorescence measurement icon on the computer monitor.
15. Select the Excitation Scan Mode on the screen.
16. On the screen, enter the Emission wavelength: 450 nm, Excitation Start Wavelength: 270nm and
Excitation End wavelength: 500 nm. One chooses the Excitation Slit(nm) and Emission Slit(nm)
values (here 5 nm/5 nm) and the scan speed value (here "medium") also.
17. To run the wavelength scan for excitation spectrum, click on 'OK' button on the set-up screen.
18. Click on 'Close' button when spectral scan is complete. In real operation, the scan data arestored in
the computer. The instrument stores data and therefore asks for the Sample File name. One enters a
file name to save the data.
19. To run the Emission Spectral Scan of the sample, open the instrument set-up screen by clicking on
the fluorescence measurement icon on the computer monitor.
20. Select the Emission Scan Mode on the screen.
21. On the screen, enter the Excitation wavelength: 350 nm, Emission Start Wavelength: 360nm and
Emission End wavelength: 600 nm. One chooses the Excitation Slit(nm) and Emission Slit(nm) values
(here 5 nm/5 nm) and the scan speed value (here "medium") also.
22. To run the wavelength scan for emission spectrum, click on 'OK' button on the set-up screen. One
has to be sure that the solvent blank does not fluoresce in the wavelength range of interest.
23. Click on 'Close' button when spectral scan is complete. In real operation, the scan data arestored in
the computer. The instrument stores data and therefore asks for the Sample File name. One enters a
file name to save the data.
24. To take the cuvette out of the sample chamber, first click on the sample chamber lid to open it and
then on the cuvette.
25. Click on the lid of the sample chamber to close it.
26. Click on 'Reset' button to start over the measurements.
27. Select the next higher concentration solution for measurement by clicking on the concentration
selection bar and carry out the Emission scan. In real measurements, if one uses the same cuvette for
all the measurements, every time one should rinse the cuvette bytaking a small portion of the solution
to be analysed prior to filling up the cuvette with thesolution.
28. Repeat the Emission scan measurements for the rest of the solutions including the unknown concentration
solution.
29. Collect all data by clicking on the Data tab.
30. Tabulate the maximum emission wavelengths for all the concentrations. If the maximum emission
wavelengths vary by a few nanometres from sample to sample, then one should note down the
intensities at only one fixed wavelength which should be ideally very near to most of the maximum
emission wavelengths.
31. Construct a calibration plot by plotting the fluorescence intensity values at a given wavelength
against the corresponding standard concentrations. Check whether linearity relationship is valid or
not. Compare the linear fits first taking only the lowest five concentrations and then taking all six
concentrations of quinine sulfate. Choose the appropriate straight line as the calibration line.
 32. By using the calibration line, determine (both graphically and mathematically) the concentration of
the unknown sample analysed and then the concentration of the original tonic water sample.

OBSERVATIONS

FIG. 1. EXPERIMENT SETUP FOR THE SIMULATION

FIG. 2. EXCITATION SPECTRUM OF QUININE SULFATE(0.0001ppm)

FIG. 3. EMISSION SPECTRUM OF QUININE SULFATE(0.0001ppm)

FIG. 4. EMISSION SPECTRUM OF QUININE SULFATE(0.001ppm)

FIG. 5. EMISSION SPECTRUM OF QUININE SULFATE(0.01ppm)

FIG. 6. EMISSION SPECTRUM OF QUININE SULFATE(0.1ppm)

FIG. 7. EMISSION SPECTRUM OF QUININE SULFATE(0.05ppm)

FIG. 8. EMISSION SPECTRUM OF QUININE SULFATE(1ppm)

 FIG. 9. EMISSION SPECTRUM OF QUININE SULFATE (UNKNOWN CONC.)

* TABLE 1: OBSERVED VALUES OF 𝜆𝑚𝑎𝑥 AND CORRESPONDING INTENSITY IN EMISSIONSPECTRUM

[Link]. Concentration Wavelength Intensity(a.u.)

(c)(ppm) (𝝀𝒎𝒂𝒙)(nm)

1. 0.0001 415.308 7.940

2. 0.001 414.474 9.209

3. 0.01 415.351 26.197

4. 0.1 415.308 199.500

5. 0.05 415.708 104.722

6. 1 415.749 1769.118

FIG. 10. VARIATION OF INTENSITY WITH CONCENTRATION

CONC. V/S INTENSITY

2000
1800 y = 1757.8x + 12.623
1600 R² = 0.9999
1400
INTENSITY (a.u.)

1200
1000
800
600
400
200

0 0.2 0.4 0.6 0.8 1 1.2

CONCENTRATION (ppm)
RESULTS
1. The wavelength for which maximum intensity is observed (𝜆𝑚𝑎𝑥) independent of
concentration of the sample.
2. The intensity increases with the increase in concentration of the sample linearly upto
1ppm.
3. The regression coefficient r2 is calculated to be 0.9999.
SOURCES OF ERROR AND PRECAUTIONS:
1. Fluorescence is linearly proportional to dye concentration in dilute samples. However, if the
concentration is too great, quenching occurs and the relationship between fluorescence and
concentration becomes curvilinear.
2. Contamination from the sample collection is one of the biggest sources of error: if the sample is
not properly collected or hands/gloves are not clean, this can tamper with the concentration. Also,
making sure the glassware and equipment is clean from any sourcesof contamination.
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