introduction - Department of Chemistry

INTRODUCTION
Instrumental methods of analysis have become central to the study of scientific
phenomena and play a dominant role in solving problems in chemical sciences. It is
therefore important to get an understanding of the capability of these instruments and a
basic understanding of their principles of operation.
The Department of Chemistry has several sophisticated instruments and set-ups which
are accessible to you for carrying out your research program. The purpose
of this course is to expose you to an array of experimental techniques using these
instruments and to give you a feel of the capabilities of the techniques.
The write-ups in this manual are a limited introduction to the principles of the
techniques and important features of the instruments used in the experiments. Besides,
you will be asked to carry out a specific experiments using each of the techniques
indicated. You should refer to the general references given and to the specific references
for a more detail understanding of the techniques and their potential use in solving your
research problems.
References:
1) Skoog and West: Instrumental methods of analysis
2) Silverstein and Bassler: Spectroscopic identification of organic compounds
1
List of experiments
Sr. no
Title
Page no.
1
Computational Chemistry
03
2
Gas chromatography
09
3
Infra red spectroscopy
13
4
Polarimetry
19
5
Mass spectroscopy
22
6
NMR spectroscopy
28
7
Separation methods
34
8
Spectrofluorimetry
38
9
Thermal analysis
41
10
Ultra violet and visible spectroscopy
44
2
Experiment: 1
Computer Laboratory Experiment
In this experiment, you will learn the use of two softwares, namely Chem-draw and
ArgusLab (shareware). If we have time, we can Chem-Draw is mostly used to draw
molecules and visualize them. In ArgusLab, you will learn how to draw and edit
molecular structures, visualize them in three dimensions, rotate, translate and modify
atoms and molecules, and more importantly run Molecular Mechanics and semiempirical Quantum Mechanical calculations. These techniques have become handy tools
for a chemist today analogous to NMR and IR spectroscopy.
Chemdraw Ultra-10
This drawing suite includes the following applications:
- ChemDraw Ultra 10
- ChemDraw ActiveX/Plugin Viewer 10 (allows you to view and manipulate
chemical structures and molecular models online, without the capability to
print, save, copy or paste a modified drawing)
- Chem3D ActiveX Pro 10.0
- Chem3D Std 10.0
- ChemFinder Std 10.0
- E-Notebook Std 10.0
Spend the first hour of the lab trying to design and draw new molecules using the various
tools available in Chemdraw, and visualize them, and more importantly, save them in
various formats. Chemdraw comes in very handy for writing reactions using chemicals.
You should be able to make structures in various available formats.
ArgusLab
ArgusLab is a very versatile freeware available from http://www.arguslab.com. This
software was originally intended to perform only semi-empirical Quantum Mechanical
(QM) calculations on small molecules, but has now grown to be used for several types of
computation chemistry experiments as well as molecular-docking experiments useful for
biological chemists. You can use the ArgusLab installed in the PCs (Windows) of the
computer laboratory and also download the program in your home computer. Using
ArgusLab, one can easily do Molecular Mechanics (MM) (UFF/Amber force fields) and
QM semi-empirical (INDO, AMI, PM3) calculations such as evaluating single-point
energies for fixed geometry, geometry optimization in the ground state, and more
importantly obtain UV-VIS spectra of delocalized systems in gas phase and in various
dielectric environments. [Note: It is also possible to do QM ab initio calculations using
the Gaussian interface, if Gaussian set of programs are available] You should be able to
visualize molecular orbitals (MOs) such as HOMO/LUMO states, as well as electrostatic
potential (ESP) and electron density mapped surfaces.
There are two main modules: Molecule Building Option, and Calculation Option:
Molecule Building, Modifications and Visualization: Tools  Builder Tool. This
toolbox allows you to make any molecule using the mouse. If you are adding atoms or
3
molecular moieties (like a benzene ring), select that, and right click on the visualizing
panel to do so. Left click allows you to select what you want to do to the molecule that you
are building. You can select an atom or a bond (should turn yellow), and pressing the
Ctrl-key allows you to select multiple atoms/bonds. Double-Clicking on the molecule
selects the whole molecule that was built/opened. Note: These operations can be
performed only if the “selection” mode is clicked (see Fig 1 cursor). Explore the various
options such as changing atoms and their state of hybridization or the nature of the
bonds by selecting them and doing a right-click to look at the various available options.
Hydrogen atoms in the molecule can be filled by just clicking “H” button.
Many pre-made sample (molecules) already exist in Argus (look into \Program
Files\ArgusLab\Samples\), and you may choose to use them as starting points. Play with
the program and ask your TA if you have any problems. SAVE the file as a xyz coordinate
file or a protein data bank (pdb) file apart from a .AGL file (which will contain outputs).
Figure 1. Building, Editing, and Visualization of Molecules
Use the various rotations, translations, and zooming options available to you through
mouse clicks on icons on the upper left. Find out how to measure distances between
atoms, and obtain angles/dihedral angle between three/four atoms in the molecule. Find
out what the “Hand” tool does and how you can reorient two independent molecules with
respect to each other using the hand-tool.
Computational Chemistry: Do some reading to familiarize yourself with MO Theory,
Quantum Chemistry (Hartree-Fock SCF) and Molecular Mechanics calculations. It is also
important to read more on semi-empirical calculations such as INDO and AMI, and
about Configuration Interaction-Singles (CI/S) for excited state calculations.
4
As a starting example, you may make any molecule of your interest, but make sure
that the molecule is deformed, by extending bond lengths and making unrealistic bond
angles (click, select and drag) given the hybridization state of bonded atoms, and run a
quick “Clean Geometry” (click “pliars” button, beside the “hand” or Ctrl-G) which will do
a simple MM calculation and bring the molecular parameters (bond distances, bond
angles etc) close to real values. You can also perform a single point energy calculation
(CalculationEnergy or Alt-E) (see Fig 2). Alternatively, you can choose a structure after
performing “Clean Geometry” and do a “geometry optimization” (Alt-O) (see Fig 3).
Geometry optimization using QM methods is slower, but for not too large a molecule,
semi-emperical methods like INDO, AMI and PM3 are pretty fast. After geometry
optimization, you need to do UV-Vis spectra calculation (Alt-U), which would be done
using INDO-CI(S) calculations (see Fig 4). Things will get a little tricky here, and talk to
the instructor/TA about input parameters, which may change with the number of
atoms (and type of) present in the molecules. Make sure you click on “Surface
Properties” under “properties” tab, and choose the surfaces to generate and parameters
to display in the output file (see Fig2). Save your calculation (.agl) after it is done. The
outputs of the calculations can be found in the “molecule tree-view” option under “Tools”
menu: click the + folders to see the output data organized by the program (see Fig 4).
Figure 2. Single-Point Energy Calculation
Figure 3. Geometry optimization
5
Figure 4. UV-Vis Spectra Calculation
6
Each student in the group is expected to do separate calculations on different molecules
and write individual reports. Make sure you do at least one geometry optimization and
at least one UV-Vis calculation for the set of molecules you build. Ensure that you know
how to plot mapped surfaces, MOs, and HOMO/LUMO etc (Fig 5). Try to understand
what lowest-, intermediate-, highest-energy states as well as HOMO/LUMO might look
like (Number of Nodes consistent?). Finally, you have to repeat the calculations in the
presence of solvents and plot the differences in energies of transitions due to salvation
effects. If time permits, use molecules with permanent dipole moments, and then apply
external electric fields on x-, y-, and z- directions to see the effect on electric fields on the
dipole moments and transition dipole moments of the molecule. Those who are
interested in further exploring the ArgusLab software are welcome to try out Molecular
Docking experiments (The tutorials #7 / #8 are available in Help Tutorials and FAQs)
Figure 5. Plotting MOs (HOMO/LUMO) and ESP and electron density mapped surfaces,
Tabulate the energies of the UV-Vis spectrum, and display the contours of MOs of the
ground state and excited states, electron densities/electrostatic potentials. Find out the
most allowed lowest energy transitions from your calculations. Draw the correlations
between the observed MO energy levels, contours, bonding/antibonding nature of the
MOs and so on. List all the files you have created and the types of calculations performed.
References
1)
Quantum Chemistry - Levine
2)
Allen M P and Tildesley D J “ Computer stimulation of liquids “, Oxford (1987)
3)
www.arguslab.com
***********
7
Experiment: 2
Gas Chromatography
Chromatography is a technique for the separation of a mixture of compounds. Separation
is achieved by distributing the compounds between a stationary phase and a mobile
phase. In gas chromatography, the mobile phase is in the gaseous state and the stationary
phase may be either a solid or a liquid. The separation is based on the difference in
solubility or of adsorption of the various components in the mixture in the gas phase in
the stationary phase.
A mixture of compounds in the gaseous state is passed through a column containing the
stationary phase with the help of a carrier gas. This will lead to multiple adsorptiondesorption or dissolution-evaporation of the compounds leading to separation. In
principle, any compound that can be vaporized without decomposing can be separated by
this method.
Instrumentation
It consist of a cylinder containing the carrier gas, a flow control device, the injection port
for introducing the sample, the column where separation takes place, the detector and a
display/ recording device.
1)
Carrier gas: Normally, helium or hydrogen is used with thermal conductivity
detector(TCD), with some flame ionisation detector(FID), helium or nitrogen or argon is
generally used.
2)
Flow control: Flow of the carrier gas is carefully controlled with a precision needle
valve. For precise work, particularly when temperature programming is used, it is
desirable to employ a mass flow controller.
8
3)
Injection port: The injection port is located just below the column. It is heated and
microlitre quantity of the sample is injected by a syringe piercing the rubber septum. The
liquid is carried in to the column. Gaseous samples can be introduced by gas tight
syringes or by gas sampling values, several types of which are available.
4)
Column: This is the heart of the instrument where the components gets separated.
Columns packed with solid, solid coated with a liquid or open tubular capillary columns
are used.
5)
Detector: The two types are common. Thermal conductivity detector is a universal
detector that can be used for any types of samples. It makes use of the difference in
thermal conductivity of the carrier gas and the component. The sensitivity is good when
this difference is large. In the flame ionisation detector, the compounds are burnt in
hydrogen flame in the presence of air. The ionic current produced is amplified before
recording.
9
The Basic Equation
Assuming an equilibrium distribution of the components between the mobile and the
stationary phase, it can be shown that
μc = W / (Vg +Vk)
where W = volume rate of flow of the carrier gas
K = partition function of the component
Vg = volumes of the component in the mobile phase
V = volumes of the component in the stationary phase per unit length
of the column.
It can be shown that retention of a component in the column
t c = t o K V / Vg
where to = time taken by the carrier to emerge from the column.
The corrected retention time is defined as
iR = tc - to = (toEV / Vg) - to
it is seen that iR depends on K, tc and the volume ratio of the stationary phase to empty
part of the column. So it is possible to vary the parameters such as carrier gas flow rate,
column material, colmn length in order that iR of the components to be separated differ
from each other and thus achieve their separation. Another important variable is the
column temperature that determines the value of K.
Principles of column selection
10
1.
Nonpolar solutes are separated in the order of their boiling point on a
nonpolar column. The same is true for polar solutes in a polar column.
2.
Polar solutes elute more rapidly than nonpolar ones of same boiling point
on a nonpolar column.
3.
Nonpolar solutes elute more rapidly than the polar ones of same boiling
point on polar columns.
Analysis
Gas chromatography can be used for both qualitative and quantitative analysis. It is
obvious that analysis here is done by separation.
The identification of a compound is based on the determination of its retention time and
comparing it with the retention time of the possible compounds in the mixture under
identical experimental conditions.
After the instrument is stabilised, a few microlitres of the liquid is injected by a syringe.
This is repeated twice or thrice. The retention times are determined. Next pure
components are injected one at a time and their tR is also determined. The peaks due to
each component in the mixture are identified by comparision with the known compounds
Quantitative analysis is based upon the assumption that peak area is proportional to the
mole fraction of the material in the gas. Although this is not entirely correct, the relative
peak areas are a good measure of the relative amount of each component in the mixture.
The problem is then to determine the peak area. There are many methods for this. The
best solution is to use an electronic peak integrator. The cut and weigh method is useful
in the absence of this. If the guassian area can be expressed half of base multiplied by
height of the peak.
***********
11
Experiment 3
Infra Red Spectroscopy
Infrared spectroscopy is widely used in organic chemistry for the identification of
functional groups and as a ‘finger printing’ technique. In inorganic co-ordination
chemistry, where organic groups are often co-ordinated to a metal ion, infrared
spectroscopy can serve the same function. In addition, it may also be useful in
determining the bonding mode of a ligand and in favourable cases, it may indicate the
stereochemistry of a complex.
Transitions between vibrational energy levels occur in the infrared region (50-50,000
cm-1) of electromagnetic spectrum. The most useful vibrations from the point of view of
the chemist occur in the narrower range of 200-4000 cm-1 which most sophisticated
infrared spectrometers cover. The vibration of a diatomic molecule AB can be treated in
terms of a harmonic oscillator. The frequency of vibration υ, is thus related to the force
constant k (which may be related to the strength of the bond A-B), and inversely
dependent on the reduced mass of the atoms involved.
υ = (1/2πc )√k/μ
where, k = force constant
μ = reduced mass (1/μ) = (1/mA) – (1/mB)
υ = wave number υ = υ/c = frequency (s-1)/velocity of light (cms-1)
In polyatomic molecules, stretching nodes may be largely localised, accounting for the
group frequencies associated with functional groups (e.g. R2C=O, R-NH, R-OH, R-CN
etc.) Polyatomic molecules also give rise to bending vibrations (or ‘deformation’ modes)
which occur at lower energy than their stretching counterparts. In systems where
delocalisation of bonding electrons occurs, a considerable degree of ‘mixing’ of symmetry
12
co-ordinates takes place and in such cases, a given band can at best be considered to arise
primarily from one symmetry co-ordinate.
The vibrational motion of a molecule can be resolved in to ‘normal’ modes of vibration.
The number of normal modes is dependent on the number of atoms N in the molecule: a
non linear molecule has 3N-6 normal modes of vibration.
For a particular vibration to be infrared active, it must cause a change in the dipole
moment of the molecule. The stretching mode of homonuclear diatomic molecules is not
infrared active, whereas the same mode is heteronuclear diatomic molecule is infrared
active. Thus, H2, O2, N2, Cl2 do not exhibit IR spectra, whereas HCl, NO etc. do.
A further, important consequence of this selection rule is that the more highly symmetric
a molecule, the fewer IR active bands it is likely to have, since only fewer vibrations will
lead to a change in dipole moment. Usefulness of the technique in structural elucidation
can be best illustrated from the infrared spectral analysis of Ni(4CNPy)4(SCN)2 as a
typical example. One should work out the number of possile structures it may have,
assuming it to be monomeric and given that
1. 4CNPy may bond through either the CN group or the ring nitrogen
2. thiocynate may bond through either sulfur or nitrogen
3. nickel(II) forms square planar, tetrahedral, octahedral and tetragonal
complexes.
The problem should be approached by obtaining background information about the
infrared absorption bands associated with each of the groups in Ni(4CNPy)4(SCN)2 and
then using this knowledge is a basis for the investigation.
Thiocynate has 3 absorption bands of interest. CN stretching (2020 - 2170 cm-1), CS
stretching (690 - 880 cm-1) and NCS bending(400 - 490 cm-1), all of which vary in
frequency depending on whether the ion is (a) uncoordinated (ionic), (b) S-bonded, (c) N
- bonded, (d) bridging [as are found in i) KSCN, ii) K2H(SCN)4, iii) K2Co(NCS)4, iv)
CoH(SCN)4 respectively. After running the spectrum, confirmation of bonding mode can
be made from literature data 4-cyanopyridine can bond through either CN group or the
ring nitrogen atom. When pyridine co-ordinates through the ring nitrogen atom, shifts
are noted in some of the pyridine ring vibrations. This information can be obtained by
comparing the IR spectrum of pyridine and a pyridine complex, Ni(Py)4(SCN)2. but when
nitriles form complexes, the CN stretching frequency is affected.
Using the above spectral information, the bonding modes of the ligands in
Ni(4CNPy)4(SCN)2 can thus be determined. These conclusions may or not may be
sufficient to indicate the complete structure of the complex. Further confirmation of the
above findings should be sought from the far infrared spectrum < 450 cm-1. Most
bending modes involving the heavy metal atom will occur below 200 cm-1, i.e, out of the
range of the most commercial infrared spectrometers. The stretching vibrations, however
are more accessible, usually occuring in the region 200-400 cm-1 for most inorganic
complexes. The number of skeletal stretching modes for each possible structure are
indicated below:
13
Infrared active skeletal stretching modes
Complex
Point Group
(M-L)
(M-X)
[ML4]X2
Td
1
-
[ML4]X2
Tra
Ns-[ML4X2]
D4h
1
-
D4h
1
1
Cis-[ML4X2]
C2V
4
2
Examination of the actual spectrum can thus provide information about the symmetry of
the complex.
Preparation of samples and examination in an Infrared spectrometer
The spectrometer consists of a source of infrared light emitting radiation throughout the
whole range of the instrument. This light split into two beams of equal intensity and one
beam is arranged to pass through the sample to be examined. If the frequency of
vibration of the sample molecule falls within the range of the instument, the molecule
may absorb energy of this frequency from the light. The spectrum is therefore scanned by
comparing the intensity of the two beams one after one has passed through the sample to
be examined in the vapour phase, as pure liquids, in solution and in the solid state.
1.
In the vapour phase
The vapour is introduced into a special cell, usually about 10 cm long, which can then be
placed directly in the path of one of the infrared beams. The end walls of the cell are
usually made of sodium chloride, which is transparent to infrared. Most organic
compounds have two low a pressure for this phase to be useful.
2.
As a liquid
A drop of liquid is sqeezed between flat plates of sodium chloride. This is the simplest of
all procedures.
3.
In solution
The compound is dissolved, typically to give a 1 - 5% solution in CCl4 or for its better
solvent properties in chloroform free from alcohol. This solution is introduced into a
special cell 0.1 to 1 mm thick made of sodium chloride. A second cell of equal thickness,
but containing pure solvent, is placed in the path of the other beam of the spectrometer
in order that solvent absorption should be balanced. Spectra taken in such dilute
solutions in non-polar solvents are generally the most desirable, because intermolecular
forces, which are specially strong in crystalline state are minimied. On the other hand,
14
many compounds are not soluble in non-polar solvents and all solvents absorb in the
infrared. When the solvent absorption exceeds about 65% of the incident light, spectra
cannot be taken because, insufficient light is transmitted to work the detection
mechanism efficiently. CCl4 and CHCl3 fortunately, absorb over 65 % of the incident light
only in these regions ( 820 - 720 cm-1 for CCl4, 1260 - 1180 cm-1 and 900 - 625 cm-1 for
CHCl3 ) which are of little interest in diagnosis for organic compounds. In rare cases
aqueous solvents are useful: special calcium fluoride cells are used.
4.
In solid state
About 1 mg of a solid is finely ground in a small agate mortar with a drop of a liquid
hydrocarbon (nujol, kaydol), or if C-H vibrations are to be examined, with
hexachlorobutadiene. The mull is then pressed between flat plates of sodium chloride.
Alternatively, the solid is ground with 10 to 100 times its bulk of pure KBr and the
mixture pressed into a disc in a special mould and a hydraulic press. The use of KBr
eliminates the bands due to the mulling agent and tends. On the whole, to give rather
better spectra, except that a band at 3450 cm-1 from the OH group of traces of water,
always appear. Due to intermolecular interactions, band positions in the solid state
spectra are often different from those of the corresponding solution spectra. This is
particularly true of those functional groups which take part in hydrogen bonding. On the
other hand, the number of resolved lines is often greater in solid state spectrum, so that
comparison of the spectrum of, for example, synthetic and natural samples in order to
determine identity is best done in the solid state.
Instrumentation
Radiation from a source emitting in the infrared region passes alternately through the
sample and the reference before entering the monochromator. This arrangement
minimizes the effect of stray radiation. The transmitted beam is dispersed by reflection
from a grating. On passing through the slit, it gets focussed on to the detector.
Temperature and humidity in the room housing the instrument must be controlled. The
maximum permissible humidity is about 50 percent. If humidity exceeds this limit,
windows made of alkali halides gets affected. Temperature changes over a few degrees
seriously affect the accuracy of the wavelength calibration.
15
The important components of the spectrometer and their functions are discussed below:
1.
Source: The source of radiant energy is a length of ceramic tube heated by internal
metallic elements. The ceramic is heated to approximately 1100oc and produces a
continous spectum of electromagnetic energy most of which is within the infrared region.
The incandescent portion of the tubing is approximately 15 mm long and 3 mm in
diameter. The source element has a positive temperature coefficient of resistance. No
water cooling system is needed.
2.
Monochromator: The monochromator performs three basic functions:
a.
dispersion of the radiation into its wave number components
b.
restriction of the radiation arriving at the detector to a narrow wavenumber band
c.
maintenance of the energy incident at the detector at an approximately constant
level throughout the wavenumber range of the instrument when no sample is
inserted.
The monochromator optical system comprises the entrance slit, the 19o off-axis
paraboloid mirror, a plane replica grating, the exit slit and the set of optical filters. To
enable measurements to be madeover the wide range of the instrumnet, two gratings are
used mounted back to back. One has 100 and the other 25 lines per millimeter.
Sample cells are made of various kind of materials. E.g. glass, fused silica. LiF, CaF2,
NaCl. KBr, CsI, etc.
16
3.
Detectors: The detector converts radiant energy incident upon it to an electrical
potential variation, which in turn forms the input signal to the thermo-couple
transformer. In modern instruments, thermo-couple detector has a rapid response and
high sensitivity. The target is a strip (1.5mm by 0.35mm) of blackened gold leaf welded to
two pans. The incident radiation is strongly absorbed by the blackened leaf and an
increase of leaf temperature occurs. Because of the temperature difference between the
thermocouple junctions, a thermo-electric potential (the electric signal) is generated. The
sensitive element of the thermocouple is enclosed in a steel casing to eliminate pick up
and the casing is evacuated to increase the detector sensitivity.
Principle of operation
As shown in figure, the radiant energy from the source is split into the sample and
reference beams by a set of mirrors(M1, M2 and M3). The sample is placed in the sample
beam where it absorbs some of radiant energy at the characteristic vibrational
frequencies of the molecules in the sample. The sample and reference beams are reflected
to a chopper, a semicircular disc which rotates ten revolutions per second and it causes
the sample beam and the reference beam to be reflected alternately to the
monochromator grating. As the grating slowly rotates, it sends individual frequencies to
the detector thermocouple, which converts the infrared energy to electrical energy. When
the sample has absorbed light of a particular frequency, alternating current is flown from
the detector to the amplifier. The amplifier is coupled to a servo-motor which drives an
optical wedge in to the reference beam until eventually the detector receives the beam.
This movement of the wedge in and out of the reference beam shows as an absorption
band on the printed spectrum. for calibration of frequency polystyrene mark can be
checked which gives a peak at 1601 cm-1.
***********
17
Experiment No.4
Polarimetry
To measure optical rotation of given samples using polarimeter
Optical activity is the ability of a chiral molecule to rotate the plane of plane-polairsed
light. It is measured using a polarimeter, which consists of a light source, polarising
lens, sample tube and analysing lens. When light passes through a sample that can rotate
plane polarised light, the light appears to dim to the eye because it no longer passes
straight through the polarising filters. The amount of rotation is quantified as the
number of degrees that the analysing lens must be rotated by so that it appears as if no
dimming, of the light has occurred.
When rotation is quantified using a polarimeter it is known as an observed rotation,
because rotation is affected by path length (l, the time the light travels through a sample)
and concentration (c, how much of the sample is present that will rotate the light). When
these effects are eliminated a standard for comparison of all molecules is obtained, the
specific rotation, [a].
[a] = 100a / cl when concentration is expressed as g sample /100ml solution
Specific rotation is a physical property like the boiling point of a sample and can be
looked up in reference texts. Enantiomers will rotate the plane of polarisation in exactly
equal amounts (same magnitude) but in opposite directions.
Dextrorotary designated as (+), clockwise rotation (to the right) Levorotary
designated as (-), anti-clockwise rotation (to the left)
If only one enantiomer is present a sample is considered to be optically pure. When a
sample consists of a mixture of enantiomers, the effect of each enantiomer cancels out,
molecule for molecule.For example, a 50:50 mixture of two enantiomers or a racemic
mixture will not rotate plane polarised light and is optically inactive. A mixture that
contains one enantiomer excess, however, will display a net plane of polarisation in the
direction characteristic of the enantiomer that is in excess. The optical purity or the
enantiomeric excess (ee%) of a sample can be determined as follows:
Optical purity = % enantiomeric excess = % enantiomer1 - % enantiomer2 = 100 [a]mixture /
[a]pure sample
ee% = 100 ([R]-[S]) / ([R]+[S])
where [R] = concentration of the R-isomer [S] = concentration of the S-isomer
18
Procedure: Measure the optical rotation of pure D-Xylose and L-Xylose
Make samplesof L-Xylose and D-Xylose with the following compositions and
dilute it too 100ml in standard measuring flasks.
Sr.No
L-Xylose
(mg)
D-Xylose
(mg)
1
2
3
4
5
100
200
300
400
500
900
800
700
600
500
Optical
Rotation
Specific
optical
rotation
Digital Palarimeter (Jasco P-2000)
***********
19
Experiment 5:
Mass Spectrometry
Mass spectrometers are an analytical tool used for measuring the molecular weight and
structural determination of the sample.
Mass spectrometers can be divided into three fundamentals parts:
1.
Ionization source
2.
Analyser
3.
Detector.
The sample under investigation has to be introduced in to the ionization source of the
instrument. Once inside the ionization source the sample molecules are ionized, because
ions are easier to manipulate than the neutral molecules. These ions are extracted in to
the analyser region of the mass spectrometer where they are separated according to their
mass (m) to charge (z) ratios (m/z). The separated ions are detected and this signal sent
to a data system where the m/z ratios are stored with their relative abundance for
presentation in the format m/z spectrum.
The analyzer and detector of the mass spectrometer, and often the ionization source too,
are maintained under high vacuum to give ions a reasonable chance of travelling from
one end of the instrument to the other without any hindrance from air molecules. The
entire operation of the mass spectrometer, and often the sample introduction process
also, is under complete data system control on modern mass spectrometers
mass spectrometre
data system
IONISATION SOURCE
e,g.elecrospray(ESI)
MALDI
FAB
ANALYSER
mass to charge (M/Z)
e.g.quadrapole,
time of flight
DETECTOR
e.g. photomultiplier
microchannel
electron multiplier
Sample introduction
The method of sample introduction to the ionization source depends on the ionization
method being used, as well as the type and complexity of the sample.
The sample can be inserted directly in to the ionization source, or can undergo some type
of chromatography en route to the ionization source. This later method of sample
introduction usually involves the mass spectrometer being coupled directly to a high
pressure liquid chromatography (HPLC), gas chromatography (GC) or capillary
electrophoresis (CE) separation column, and hence the sample is separated in to a series
of components which then enters the mass spectrometer sequentially for individual
analysis.
Method of sample ionization
Many ionization methods are available and each has its own advantages and
disadvantages. The ionization method to be used should depend on the type of sample
under investigation and the spectrometer available.
20
Ionization methods include the followings:
1.
2.
3.
4.
5.
6.
7.
8.
Atmosphere pressure chemical ionizzation (APCI)
Chemical Ionization (CI)
Electron Impact (EI)
Electrospray Ionization (ESI)
Fast Atom Bombardment (FAB)
Fiels Desorption / Field Ionization (FD/ FI)
Matrix Assisted Laser Desorption Ionization (MALDI)
Thermospray Ionization (TSP)
With most ionization methods there is the possibility of creating both positively and
negatively charged sample ions, depending on the proton affinity of the sample. Before
embarking on an analysis the user must decide whether to detect the positively an d
negatively charged ions.
Analysis and separation of sample ions
The main function of mass analyzer is to separate, or resolve, the ions formed in the
ionization source of the mass spectrometer according to their mass to charge ratios.
There are large number of mass analysers currently available, the better known of which
include quadrapoles, time of flight (TOF) analyzers, magnetic sectors, and both fourier
transform and quadrapole ion traps.
These mass analyzers have different features including the m/z range that can be
covered, the mass accuracy and the achievable resolution. The compatibility of different
analyzers with different ionization methods varies. For example, all of the analyzers listed
above can be used in conjugation with electrospray ionization, whereas MALDI is not
usually coupled to a quadrapole analyzer.
Tandem (MS-MS) mass spectrometers
Tandem (MS-MS) mass spectrometers are instruments that have more than one analyser
and so can be used for structural and sequencing studies. Two, three and four analyzers
have all been incorporated in to commercially available tandem instruments and the
analyzers do not necessarily have to be of the same type, in which case the instrument is
hybrid one. More popular tandem mass spectrometers include those of the
quadrupole-quadrupole, magnetic sectors-quadrupole and more recently, the
quadrupole-time-of-flight geometries.
Detection and Recording of sample ions
The detector monitors the ion current, amplifies it and signal is then transmitted to the
data system where it is recorded in the form of mass spectra. The m/z values of the ions
are plotted against their intensities to show the number of components in the sample, the
molecular weight of each component, and the relative abundance of the various
components in the sample
The type of detector is supplied to suit the type of analyzer, the more common ones are
photomultiplier and the micro-channel plate detectors.
21
Instrument Description
“Q-Tof micro”: Micromass (UK)
The Q-Tof micro hybrid quadrupole and time of flight mass spectrometer is available
with electrospray ionization (ESI) and atmospheric pressure chemical ionization (ApcI)
Q-T of micro utilizes a high performance, resarch grade quadrupole mass analyzer,
incorporating a prefilter assembly to protect the main analyzer from contaminating
deposits, and an orthogonal accelaration time of flight (TOF) mass spectrometer. A
hexapole collision cell, between the two mass analyzer, can be used to induce
fragmentation to assist in structural investigations. Ions emerging from the second mass
analyzer are detected by the microchannel plate detector and ion counting system.
Ionization techniques:
Using the Micromass Z-spray atmospheric pressure ionization (API) source, two
techniques are available.
1.
Electrospray: Electrospray ionization (ESI) takes place as a result of imparting a
strong electrical charge to the eluent as it emerges from the nebulizer. An aerosol of
charged droplets emerges from the nebuliser. These undergo a reduction in size by
solvent evaporation until they have attained a sufficient charge density to allow sample
ions to be ejected from the surface of the droplet (ion evaporation)
22
The sample solution is sprayed across a high potential difference (kv) from a needle in to
an orifice in the interface. Heat and gas flow are used to desolvate the ions existing in the
sample solution. A characteristic of ESI spectra is that ions may be singly or multiply
charged. Since the mass to charge ratio compounds of high molecular weight can be
determined if multiply charged ions are formed.
Sample introduction
i.
Flow injection
ii.
LC/MS
iii.
Typical flow rates are less than 1 litre/min up to about a millilitre/min.
Benefits
i.
ii.
iii.
iv.
v.
Good for charged, polar or basic compounds
Permits the detection of high-mass compounds at mass to charge ratios
that are easily determined.
Best method for analyzing multiply charged compounds
Very low chemical background leads to excellent detection limits
Compatible with MS/MS methods
Limitations
i.
Multiply charged species require interpretation and mathematical
transformation ( can sometimes be difficult )
ii.
Complementary to ApcI. No good for uncharged, non-basic, low polarity
compounds.e.g. steroids
iii.
Very sensitive to contaminants such as alkali metals or basic compounds
iv.
Relatively low ion currents
v.
Relatively complex hardware compared to other ion sources.
Mass range
i.
low high typically less than 20,000 Da
23
2.
Atmospheric Pressure chemical Ionization
Atmospheric pressure chemical ionization (ApcI) generally produces protonated
or deprotonated molecular ions from the sample via a proton transfer (positive
ions) or proton abstraction (negative ions) mechanism. The sample is vapourized
in a heated nebuliser before emerging in to a plasma consisting of solvent ions
formed within the atmospheric source by a corona discharge. Proton transfer or
abstraction then takes place accomodated without splitting the flow. Similar
interface to that used for ESI. In ApcI, a corona discharge is used to ionize the
analyte in the atmospheric pressure region. The gas phase ionization in ApcI is
more effective than ESI for analyzing less polar species. ESI abd ApcI are
complementary methods.
Sample introduction
i.
Benefits
i.
ii.
iii.
Limitations
i.
Same as for electrospray ionization
Good for less polar compounds
Excellent LCMS interface
Compatible with MS/MS methods
Complementary to ESI
Mass range
i.
Low moderate typically less than 2000 Da
***********
Experiment 6:
Nuclear Magnetic Resonance Spectrometer
24
NMR spectroscopy is a branch of spectroscopy in which radio frequency radiations
induce transitions between the magnetic energy levels of nuclei in a molecule. These
transitions are possible when a sample is placed in a magnetic field. Under appropriate
conditions a sample can absorb electro magnetic radiations in the radio frequency region
at frequencies governed by the characteristics of the sample. A plot of frequencies of the
absorption peaks versus the peak intensities constitutes an NMR spectrum.
Theory
Only nuclei having spin quantum numbers other than zero can give rise to NMR signals.
Signal intensity depends on the concentration of nuclei and hence obviously on their
natral isotopic abundance and on the cube of gyromagnetic ratio. Thus if the signal
intensity of a proton signal is taken as unity, for carbon-13 it will be 1/6400 and hence to
detect a C-13 signal, high sensitivity of instrument is very essential.
When a nucleus with a nuclear spin quantum number I is subjected to a constant and
homogenous magnetic field HO, it can take 2I+1 orientations. For C-13, proton or
fluorine, I = 1/2, hence it can occupy one of the two energy levels1. I = + 1/2 and 2. I = 1/2. At the same time due to thermal motion it would precess around the direction of H
at a frequency called the Larmour frequency.
The difference between the energy levels is given by,
E = μ Ho / I
Where, μ= nuclear magnetic moment
The difference between the energy levels is related to the Larmour frequency by,
E=hν
And hence,
ν = Ho / h I
to detect an NMR signak, one has to use an additional frequency (rf) field H1. When the
frequency of the applied rf field H1 matches with Larmour frequency of the nucleus under
obervation, a resonance absorption occurs. The signal can thus be detected by varying the
frequency of rf field using a variable frequency radiotransmitter (frequency scan method)
or by keeping rf frequency constant and varying the magnetic field H0 (field scan)
NMR spectra are always recorded relative to a reference nucleus, say protons from
tetramethylsilane (TMS) and normalized w.r.t. the field of the instrument. The signals
are used specially for studying proton NMR spectra so that solvent proton signals would
not overlap the sample proton signals.
Ability of an instrument to detect closely spaced, signals is described as resolution. In
case of an NMR apparatus, resolution depends on
1. homogenity of applied magnetic field
25
2. on the characteristics of the sample such as crystalline character, viscosity of medium
or transverse relaxation time T2.
In case of factor 1. above, the manufacturer has to take great precautions and provide
necessary controls to get homogenous field between the pole gaps.
Applications
1. Major application of NMR spectometer lies in the area of structural
determination of compounds. This technique is highly popular and versatile with
application in synthetic organic, bio-organic chemistry, polymer chemistry and
organometallic chemistry. By judicious use of various one and two natural product
with cent percent certanity
2. NMR spectroscopy has opened new vistas in the Quality Contro Departments to
make sure the purity and to study the stability of the compounds with respect to
shelf life and temperature.
3. NMR spectroscopy has been used in Quantitative analysis to determine the molar
ratio of the components in a mixture
4. Latest development in the field of Solid State NMR spectroscopy has help in
popularizing this tecnique as a tool for elucidation of the solid state structure. The
later technique is becoming widely popular for the characterization of polymers,
rubbers, ceramics, glass and molecular sieves.
Instrumentation
Main features of the instrument
1.
Make : varian , USA
2.
Model: mercury plus spectrometer
3.
Supercon magnet:9.4 tesla
4.
Operation proton frequency: 400 MHz
5.
Two probes 5 mm for solution studies. Indirect and switcheble probes between
high frequency range (1H, 19F) and broad band frequency range (13C, 15N, 27Al, 31P,
29Se, 125Te etc)
6.
Variable temperature studies are possible in the range between -100O to +150Oc ,
provided a suitable solvent is chosen.
7.
2D NMR studies like COSY, NOESY, ROESY, HETCOR and multiple quantum
experiments are possible
8.
Typical NMR-DEPT experiments are also possible
Internal view : Varian (400MHz) mercury plus -NMR spectrometer
26
27
NMR Hardware
The figure above is schematic representation of the major systems of a nuclear magnetic
resonance spectrometer and a few of the major interconnections. This overview briefly
states the function of each component. At the top of the schematic representation, you
will find the super-conducting magnet of the NMR spectrometer. The magnet produces
the BO field necessary for the NMR experiments. Immediately within the bore of the
magnet are the shim coils for homogenizing the BO field. Within the shim coils is the
probe. The probe contains the RF coils for producing the B1 magnetic field necessary to
rotate the spins by 90 O or 180O. The sample probe is the name given to that part of the
spectrometer, which accepts the sample, sends RF energy in to the sample, and detects
the signal emanating from the sample. It contains the RF coil, sample spinner,
temperature controlling circuitry and gradient coils.
The purpose of the sample spinner is to rotate the NMR sample tube abot its axis. In
doing so, each spin in the sample located at a given position along the Z-axis and radius
from the Z-axis, will experience the average magnetic field in the circle defined by this Z
and radius. The net effect is a narrower spectral line width. The RF coil also detects the
signal from the spins within the sample. The sample is positioned within the RF coil of
the probe. Some probes also contain a set of gradient coils. These coils produce a gradient
in BO along the X, Y or Z-axis. Gradient coils are used for gradient enhanced
spectroscopy.
The heart of the spectrometer is the computer. It controls all of the components of the
spectrometer. The operator of the spectrometer gives input to the computer through a
console terminal with a mous and keyboard. Some spectrometers also have a separate
small interface for carrying out some of the more routine procedures on the
spectrometer. A pulse sequence is selected and customized from the console terminal.
The operator can see spectra on a video display located on the console and can make hard
copies of spectra using a printer.
Sample preparation
NMR sample sare prepared by dissolving an analyte in a deterium lock solvent. Several
deuterium lock solvents are available. Some of these solvents will readily absorb moisture
from the atmosphere and give water signal in your spectrum. it is therefore advisable to
keep bottles of these solvents tightly capped when not in use. Most routine high
resolution NMR samples are prepared and run in 5 mm glass NMR tubes. Always fill
your NMR tubes to the same height with lock solvent. This will minimize the amount of
magnetic field shimming required.
28
NMR Lock solvents
Acetone
CD3COCD3
Chloroform
CDCl3
Dichloromethane
CD2Cl2
Methylnitrile
CD3CN
Benzene
C6H6
Water
D2O
Diethylether (DEE)
(CD3CD2)2O
Dimethylether (DME)
(CD3)2O
N,N-Dimethylformamide (DMF)
(CD3)2NCDO
Dimethyl sulfoxide (DMSO)
CD3SOCD3
Ethanol
CD3CD2OD
Methanol
CD3OD
Tetrahydrofuran
C4D8O
Toluene
C6D5CD3
Pyridine
C5D5N
Cyclohexane
C6H12
The concentration of your sample should be great enough to give a good signal-to-noise
ratio in your spectrum, yet minimize exchange effects found at high concentrations. The
exact concentration of your sample in the lock solvent will depend on the sensitivity of
the spectrometer.
Inserting sample tube in to the probe
Before you insert the sample tube in to the specified turbine, wipe out the outer surface of
the tube with tissue paper. Check the sample volume height again with the available
height optimization tool in the NMR lab.
Recording the spectra
In order to obtain the best information out of the sample, one has to go through the
following steps one by one sitting on the computer terminal attached to the NMR
Hardware
29
Probe tunning
Variations in the polarity and dielectric constant of the lock solvent will affect the probe
tuning. For this reason the probe should be tuned whenever the lock solvent is changed.
Tuning the probe means adjusting the capacitors on the RF probe. One capacitor and the
other one is called the fine capacitor. The goal of probe tuning is to obtain the nuclei
under observation.
Sample locking
To lock the sample in the working magnetic fiels a set of parameters are being adjusted
with the help of visual inspection.
Field shimming
The purpose of shimming a magnet is to make the magnetic field more homogenous and
to obtain better spectral resolution. Shimming can be performed manually or by
computer control. Broad lines, asymmetric lines and a loss of resolution are indications
that a magnet needs to be shimmed. The shape of an NMR line is a good indication of
which shim is misadjusted. User can carry out shimming on the 400 MHz magnet by
coarse and fine way. But care must be taken while doing shimming, otherwise a lot of
instrument time will be wasted.
Data acquisition
After you end up with a good shimming acquire the final data with the necessary
parameter settings. Always save the final Fourier transform of the data with the sample
code and solvent name. it is useful for future references. Then processing can be done on
the saved data both by on-line or off-line at any time.
Printing
After the data is processed, check the printer tray for paper. Give the print command to
get all the data as hard copy.
***********
30
Experiment 7:
Separation Methods
1.
Column chromatography
Objective: Separation of o-Nitrophenol and p-Nitrophenol by column chromatography.
o-Nitrophenol and p-Nitrophenol have different polarities. Hence those can be easily
separated by column chromatography.
Procedure: A small cotton plug is inserted in a clean dry column chromatography tube.
The tube is clamped vertical and filled uniformally with silica gel 25 g. (60 to 120 mesh
size) slurry (prepared in petroleum ether). The dripping solvent is collected in dry
conical so as to maintain petroleum ether level 2 to 3 mm above the silica gel bed.
Mixture of phenol is tolune is introduced over silica gel bed and further eluted
with mixture of petroleum ether – ethyl acitate (95/5). As the elution proceeds, the two
compounds of mixture starts separating into two coloured zones, note their colours.
When the lower band has been completely eluted ; start eluting with di-ethyl ether and
collect all the fractions. Mark the every fraction collected.
2.
Thin Layer Chromatography:
Objective: To run the TLC of the components obtained from column chromatography.
TLC is most of the time used for identification and to check the purity of the compounds.
Since both the column chromatography and TLC are adsorbtion chromatography, results
from the TLC are equally application to the column chromatography.
Procedure: Place the equal size clean glass plates face toface and then immerse in a
uniformly stirred silica gel suspension in an etheyl acetate. The solvent evaporates
rapidly leaving the thin layer of silica gel. On these plates spot the difference fractions
obtained from the column chromatography. Run this plate in an appropriate solvent
system (20% ethyl acetate in pet ether) and observe it in UV light. Note the separation of
two compounds and record their Rf values.
Result:
1] Rf value of the component A =
2] Rf value of the component B =
31
3.
Fractional Distillation:
Objective: Separation of acetone and toluene from their mixture by fractional
distillation.
Separation by fractional distillation is one of the most commonly updated technique in
the organic chemistry laboratory. A mixture of two or more volatile liquids can be
purified by fractional distillation.
Apparatus: A more sophisticated Buchi glass oven 585 is used for the bulb to bulb
distillation same instrument can be used for sublimation, vacuum distillation and for
drying the samples.
Buchi glass oven 585
Procedure: Mixture of acetone and toluene (2 ml.) is taken into a small bulb which is
placed in a glass oven and connected to two other bulbs. The assembly is then placed in a
glass oven with just the first bulb is in the heating zone. The bulb is heated and assembly
is rotated with the help of small electric motor so as to avoid bumping of liquids. The
third bulb is cooled with ice bath. Required temperature is controlled by adjusting the
digital heating control. As the heating proceeds to boiling temperature of more volatile
components, it starts distilling from first bulb to second bulb. (this is only a short
32
distance hence referred as bulb to bulb distillation or ‘Kugelrohr” distillation.) The liquid
is re-distilled by moving second bulb into the heating zone and allowing the liquid to
collect in third bulb. Increase the temperature of oven so as to distill out toluene into
second bulb. Disconnect the assembly and collect two separate components.
Results:
1] Volume of component A = ___________ ml.
2] Volume of component B = ___________ ml.
4.
Melting point:
Objective: To record the melting component of three components.
Apparatus: A sophisticated Buchi melting point apparatus-545 is used.
Procedure: Three compounds were taken in separate capillary tubes and placed in the
sample chamber of the apparatus. Temperature is set with the help of heating control.
The melting temperature was recorded for all three compounds.
Buchi melting point apparatus-545
Results:
1] Melting point of compound A = ____________ 0C
2] Melting point of compound B = ____________ 0C
3] Melting point of compound C = ____________ 0C
Reference:
1] Vogel’s text book of Practical Organic Chemistry
2] L.M. Harwood, C.J.Moody and J.M.Percy’s text book of Experimental Organic
Chemistry.
***********
33
Experiment 8:
Spectrofluorimetry
Spectrofluorimeter is an useful instrument designed primarily for the study of emission
in the UV-Vis and near IR range of the electromagnetic spectrum. Emission in molecular
system in this region of the spectrum usually arises from the low lying excited electronic
states of the system and involves electronic transitions from the excited electronic state
to lower electronic states, usually the ground state of the molecular system.
Emission phenomena are widely encountered both in nature and in the laboratory and
energetic electronic states are generated in a variety of processes. Some of these
processes are listed below.
1.
Absorption of UV-Vis radiation
2.
Interaction with high energy radiation
3.
Chemical reactions ( Chemiluminescence)
4.
Biochemical reactions (bioluminiscence such as in firefuly)
5.
Electric discharge through gases.
From the chemist point of view, the study of emission provides
1. A valuable and sensitive analytical method for monitoring the concentration of the
emissive species.
2. A method to probe the energetics, dynamics, structure and properties of the
excited state molecules.
3. It allows to probe important phenomena such as energy transfer, electron
transfer, structure of fluids and in general, nature and dynamics of the
enviroment. Because of this application it has been exploited in the study of
biological phenomena.
A schematic description of the processes that occur on genaration of excited state
through absorption of electromagnetic radiation is shown in fig (1)
The energetic electronic state generated through absorption of UV-Vis radiation can
loose its energy by both radiative and radiationless modes in photophysical and
34
photochemical processes. Further it may be noted that in condensed systems, rates of
radiationless processes of decay are very high and most emissive phenomena in large
molecular systems originate from the low lying electronic states (Kashs’s Law)
Objective: The objective of this exercise is to introduce you to the principle functioning of
a spectrofluorimeter and to carry out measurements of emission spectra and determine
the concentration of anthracene in a given solution.
A schematic diagram of a spectrofluorimeter is shown in the figure.
It consists of a light source, usually a xenon or mercury xenon are lamp of high voltage
(100 W or higher). The light from this source through a collimeter and slit arrangement
goes through a monochromator, falls on the sample kept in the sample compartment.
The emission from the sample is analysed at right angles to the incident beam and the
analysing arrangement consists of a second monochromator (called emission
monochromator) for resolution of the emission and suitable PMT for measurement of
intensity of the emission. Modern instruments can provide a record of the emission
spectra v/s intensity. Solids as well as solution samples can be used for recording of the
fluorescence spectra.
The spectroflourimeter you will use is a LS-SS system. Functioning and recording of the
spectra is microprocessor controlled in this system. System also has considerable data
processing capability. Details of its operation and use will be provided during the
laboratory session. The instrument is capable of generating emission, excitation,
fluorescence and phosphorescence spectra. It also has the facility to carry out polarised
special measurements and life time and time resolved spectral measurements in
millisecond time scale.
Objective of the experiment
1. Record the emission and excitation spectra of a given solution of anthracene
2. Determine the concentration of the given solution of anthracene
3. Use of spectral information of the given solution Es1 energy of the lowest
35
excited state, stokes shift and vibrational spacing in the ground state.
Procedure
1.
Emission and excitation spectra
Prepare a dilute solution of anthracene in a suitable solvent (10-7 M in methanol) and
transfer a few millilitre to the cuvette. Record the emission spectra of the solution by
keeping the excitation monochromator “X” at a fixed wavelength (which should
correspond to a strong absorption band of the solution) and scan the emission
monochromator “M” over a range of wavelength (at least 20 nm) than the setting of the
excitation monochromator. (Never scan the excitation wavelength with emission
monochromator). You can observe a record of intensity of emission v/s wavelength of the
CRT monitor which corresponds to the emission spectrum. to obtain excitation spectrum
the emission monochromator is set at the wavelength with maximum emission intensity
and scan the excitation monochromator.
Do not scan the excitation monochromator below 250 nm.
The record of intensity v/s wavelength is the excitation spectrum. Usually excitations are
corrected and normalised for variations of lamp intensity. The corrected excitation
spectra in an ideal system is identical with the absorption spectra.
Use an intense band in the excitation spectra to obtain an emission spectra. Obtain a
hard copy of the excitation and emission spectra of your record. A typical excitation and
emission spectra of a diacetylene derivatives is shown in the figures. Use the spectra to
obtain the requisite information.
2.
Quantitative analysis
For quantitative analysis prepare a set of standard solutions with concentrations ranging
from 10-5 M - 10-8 M and using an intensity of a suitable maximum wavelength, obtain a
calibration plot, determine the intensity of the given unknown solution. Using the
calibration plot determine the concentration of anthracene.
Report your observations and result in a suitable format of your choice.
References: 1. C. A. Parkar; Fluorescence in solution, Elsevier 1968
2. R. S. Buton; Theory and interpretation of fluorescence and phospherescence, New
York, Wiley 1968
3.
G. G. Guibault; Fluorescence: Theory , instrumentation and practice,
Mercel, Dekker 1967
4.
E. J. Bowen; Luminiscence in chemistry , London 1968
5.
J.R. Lakowicz; Topics in fluorescence spectroscopy, vol I
***********
36
Experiment 9:
Thermal Analysis
Thermoanalytical methods involve techniques such as thermogravimetric analysis (TGA)
involving change in weight with respect to temperature, differential thermal analysis
(DTA) with change in heat contents with reference to temperature. The data obtained are
used to plot continuously recorded curves which may be considered as thermal spectra
i.e. a thermogram. These thermogram characterize a system in terms of temperature
dependence of its thermodynamic properties and physical chemical reaction kinetics.
TGA involves measurements of a change in weight of a system as the temperature is
increased at a predetermined rate, while DTA consists of measuring changes in the heat
content as a function of difference in temperature between the sample and reference
sample under investigation.
Diamond TG/DTA
The Diamond Thermogravimetric/ Differential Thermal Analyzer (TG/ DTA) combines
the high flexibility of the differential temperature analysis (DTA) feature with proven
capabilities of the thermogravimetry (TG) measurement technology. The combination
not only ensures that the sample is exposed to identical thermal treatment and
environment but allows one to determine whether an endothermic or exothermic
transition is associated with weight loss in contrast to a melting or crystallization process.
It has been designed to simultaneously perform thermogravimetric and differential
thermal analytic measurements on inorganic materials like ceramics and metals, as well
as on high polymer organic materials. The temperature range is from temperature to
1550OC (normally around 1300 OC). This instrument is designed for easy use, while still
maintaining the precision measurement capabilities. The Diamond TG/ DTA is
connected directly to the computer containing pyris software for Windows which controls
the analyzer.
TG/ DTA combines the thermogravimetric and differential thermal analyzers into two
modules composed of the following
1.
Measurement unit for setting and measuring the sample
2.
Base unit for processing signals sent from the measurement unit and
controlling the measurement unit temperature.
Instrument Details
1.
Make: PERKIN ELMER, USA
2.
Model: Diamond TG/ DTA
3.
Specifications:
Temp. Range : Room temperature ~ 1500OC
Weight measurement : Horizontal differential balance method
a)
b)
37
c)
d)
e)
f)
h)
i)
Sample weight : max. 200 mg of non-explosive & non-corrosive
Heating Range : 0.01OC/min. ~ 200.00OC/min
TG Measurement Range : ±200 mg
DTA Measurement Range : ±1000 µV(0.06 µV)
DTG Measurement Range : 0.5 mg/min ~ 1 g/min
Gas Flow : Max. 1000mL/min
Diagram: Diamond TG / DTA
Measurement principle
1.
TG Measurement principle
The Diamond TG/ DTA module uses a horizontal differential system balance mechanism.
Sample weight changes are measured as described below:
The sample balance beam are independently supported by a driving coil/ pivot. When a
weight change occurs at the beam end, the movement is conveyed to the opposite (rear)
end of the beam via the driving coil/ pivot, where optical position sensors detect changes
in the position of a slit. The signal from the optical position sensor is sent to the balance
circuit. The balance circuit supplies sufficient feedback current to the driving coil so that
the slit returns to the balanced position. The current running to the driving coil on the
sample side and the current running to the driving coil on the reference side is detected
and converted into weight signal
2.
DTA Measurement principle
The thermocouple (platinum-platinum rhodium 13%) for DTA measurement is
incorporated in the end of each of the balance beam ceramic tubes, and the temperature
difference between the holder on the sample side and the holder on the reference side is
38
detected. The signal is amplified and becomes the temperature difference signal used to
measure the thermal change of the sample.
Typical Applications
1.
2.
3.
4.
5.
6.
Compositional analysis
Decomposition and transition temperatures
Filler content
Heat of transition
Measurement of volatiles (e.g. water, oil)
Oxidative and thermal stabilities
Schematic diagram of TG / DTA
***********
39
Experiment 10:
Ultraviolet and Visible Spectroscopy
The absorption of ultraviolet radiation by a molecule leads to transition among the
electronic energy levels of a molecule. Although rotational and sometimes vibrational
fine structures do not appear in the liquid or the solid state, both the positions and
intensities of the rather broad absorption due to electronic transitions are characteristics
of the molecular group involved. The more mobile or loosely bound the electrons the
smaller the energy difference between the ground state and an excited electronic state
and lower the frequency of absorption i.e. longer the wavelength.
For practical purpose, the electronic spectrum is divided in to three regions:
1.
2.
3.
The visible region between 400 and 750 nm
The near ultraviolet region between 200 and 400 nm
The far (or vacuum) ultraviolet below 200 nm
The absorption by atmospheric oxygen is considerable in far UV region and spectra can
be obtained only if the spectrophotometer is carefully evacuated. Thus, commercial
instruments extend only down to about 190 nm and absorption below this range are little
used for routine chemical purpose.
The intensity of an electronic absorption is given by the equation
E = (1 / Cl) log10 (I0/I) lit mol-1 cm-1
Where
C = concentration in mol litre-1
L = path length of the sample in cm
I0 = intensity of light transmitted by the sample
E = molar extinction coefficient and the magnitude of which is governed largely by the
probability of the electronic transition and the polarity of the excited state.
E valve ranges from 5 × 105 for the strongest bands to 1 or less for very weak absorptions.
Using the above relation, absorption spectroscopy can be applied for quantitative
analysis.
The absorbing species may include transitions involving
1.
2.
3.
Sigma (σ) , pi (π) and non-bonding (n) electrons.
d and f electrons
Charge transfer electrons
1.
sigma (σ) , pi (π) and non-bonding (n) electrons
40
In chemical terms, a single bond between atoms such as C-C, C-H, O-H etc. contains only
σ electrons, a multiple bond , C=C, C=C, C=N etc contains π electrons in addition. While
atoms to the right of carbon in the periodic table viz nitrogen, oxygen and halogens
posseses n electrons. In general Χ electrons are most firmly bound to the nuclei and
hence require a great deal of energy to undergo transition. While the π and n electrons
require less energy. Thus σ - σ * transitions fall in to the vacuum ultra violet. π – σ*
appear near the border line of the near and far ultra violet and n - π* come well into the
near ultraviolet and isible regions. E values for n-π* fall in the range of 10-100 litre mol—1
cm-1. conjugation in the molecule, however , increases λmax and E values for both π - π*
and n - π*transitions . a few typical examples are NO3-(313 nm) and CO32- (217 nm) ions.
2.
d and f transitions
The colour of transition and inner transition metal compounds are usually attributed to
electronic transitions involving d or f orbitals.
Transition and inner transition elements absorb ultraviolet and visible radiation to
undergo d - d and f - f transitions. In contrast with the narrow bands observed for inner
transition elements, the bands corresponding to transition elements are broad and the
positions of the absorption bands are quite sensitive to the nature of the enviornment.
Due to Lap or te forbiddeness, octahedral complexes are less intense (E = 5-100 dm3 mol1 cm-1) than non centrosymmetric tetrahedral complexes (E = 500-5000 dm3 mol-1 cm-1)
the UV-Visible spectroscopy can thus be effectively used for predicting stereochemistry of
transition metal complexes in solution
3.
Charge transfer spectra
A charge transfer transition corresponds to the transfer of an electron from a molecular
orbital having primarily ligand character to one having primarily metal character. It thus
corresponds essentially to the ionization of the ligand in that considerable energy must
be involved to force the electron from the stable ligand orbital to the higher energy metal
antibonding orbital. It thus occurs most readily i.e. at the expense of lowest energy when
the metal is in a high oxidation state and the ligands are relatively easy to oxidise. Species
exhibiting L-M charge transfer spectra are KMnO4, K2CrO4. PbCrO4, HgS, Fe(SCN)63-,
Ce(NO3)4 etc.
The visible absorption spectra of iron(II) complexes with ligands containing the α diimine unit exhibit charge transfer band associated with the transfer ofelectron from
metal t2g orbitals to the antibonding orbitals of the α -diimine group (i.e. M-L) . Some
common examples are Fe(phen)32- etc.
In contrast to weaklly intense d - d transitions, charge transfer bands have high
extinction coefficients (E ~ 104 dm3 mol-1 cm-1) as they are fully allowed. Most
colorimetric reagents need in analysis for the detection and estimation of metal ions are
in fact ligands which form complexes having strong charge transfer bands.
Spectrophotometer:
UV Visible spectrophotometer comprises of a light source, a monochromator, a detector,
amplifier and recording devices. The source mainly incorporates a tungsten lamp
containing a small pressure of iodine vapour for wavelengths greater than 375 nm, a
deuterium lamp for values below that. The detector is usually a photomultiplier which
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utilises a thin layer of material containing alkali metal such as CS3Sb, K2CsSb, Na2KSb
with a trace of Cs. The electrons ejected from the surface by incident radiation are
focussed by an electrostatic field onto the first of a series of dyodes so as to result in
electron multiplication. The ratio of reference beam to sample beam is fed to a pen
recorder. The recorder trace of absorbance against wavelength constitutes the absorption
spectrum. The optical and electrical flow charts are shown in fig.
Principle of operation
The operation of double beam spectrophotometer can be described in the following
manner. Beam coming from the light source( deuterium lamp D2 or halogen lamp W) is
reflected by the mirrors M1 and M2 and then enters the monochromator. The slit width
of 2 nm is suitable for normal measurement. The radiation is diffracted by balzed
photographic grating of 1600 lines/mm to ensure high resolution. The beam coming out
of the monochromator encounters a rotating copper disk CH and a semicircular (half
mirror) mirror M6 also rotating that alternates the radiation between the two beams at
the frequency of 50 Hz or 60 Hz. One beam passes through a reference cuvette and the
other through an identical cuvette containing the sample. The beam which has passed the
sample cell and the reference cell alternately at 50 Hz and (or 60 Hz) is received by the
detector and passes through the pre amplifier and is finally separated in to sample side,
reference side and dark current signals. Then, the – ve voltage is controlled to make the
sum of the sample side and the refernce side signals constant irrespective of the
luminence of the light source and wavelength. The sample side and reference side signals
are subjected to A/D conversion respectively and are transmitted to the microcomputer
through the input/output port. Microcomputer calculate T% through computation of
sample reference signal and carries out base line correction. Logarithmic conversion
(Abs) thereby displating results in the form of a spectral curve.
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