Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Analytical
Microbiology

Book list
1
...
Spectroscopic techniques
Spectroscopy: The study of spectra or the interaction of matter with the electromagnetic spectrum is called
spectroscopy
...

Spectrophotometry: Spectrophotometry is the branch of spectroscopy that deals with measurement of the radiant
energy transmitted or reflected by a body as a function of the wavelength
...
It is the device for measuring the brightness of various
portions of spectra
...

Electromagnet: An electromagnet is a type of magnet in which the magnetic field is produced by an electric current
...
The main advantage of an electromagnet over a permanent
magnet is that the magnetic field can be quickly changed by controlling the amount of electric current in the winding
...

Electromagnets are widely used as components of other electrical devices, such as motors, generators, relays,
loudspeakers, hard disks, MRI machines, scientific instruments and magnetic separation equipment
...

Radiation: Radiation is the emission or transmission of energy in the form of waves or particles through space or
through a material medium
...

The nature of light: Electromagnetic radiation (light) exhibits both wave-like properties and particle-like properties
...
When viewed as a wave,
electromagnetic radiation consists of perpendicular oscillating, electric and magnetic fields (figure 1)

Fig: The perpendicular, oscillating, electric and magnetic fields associated with electromagnetic radiation
The wavelength describes the distance between adjacent peaks of an oscillating field, while the frequency describes the
number of wavelengths that pass a particular point in space per unit time
...
This inverse relationship is
summarized in the following equation where frequency (ν) wavelength (λ) are inversely proportional
...
The energy of each
photon is directly proportional to its frequency:

2

E = hν
Where h is Planck’s constant (h = 6
...
The range of all possible frequencies is known as the
electromagnetic spectrum, which is arbitrarily divided into several regions by wavelength
...


The nature of matter: Matter, like electromagnetic radiation, also exhibits both wave-like properties and particle-like
properties
...
According to the principles of quantum
mechanics, the energy of a molecule is quantized
...
When the motor is turned on, the tire
begins to rotate and a measuring device is used to determine the rate of rotation
...
It would
be inconceivable to suggest that a measurable rotation rate would be unattainable by the laws of physics
...
06251 revolutions per second, there is nothing preventing us from doing so
...
The rotation of a molecule appears to be restricted to specific energy levels
...
Other rotation rates are
simply not allowed by the laws of physics
...

Molecules can store energy in a variety of ways
...
Each of these forms of energy is quantized
...
The horizontal lines in the diagram represent allowed
vibrational energy levels for a particular bond
...
The difference in energy (ΔE) between allowed energy levels is determined
by the nature of the bond
...
If a photon of light possesses exactly this amount of energy, the bond can absorb the
photon to promote a vibrational excitation
...
Each form of spectroscopy uses a different region
of the electromagnetic spectrum and involves a different kind of excitation
...
EMR is classified by wavelength into radio, microwave, infrared, the visible region that we perceive as light,
ultraviolet, X-rays and gamma waves
...
Their frequency is related
to the wavelength by the expression:

ν=

𝑐
𝜆

or c

= νλ

Where,
ν = frequency in Hz (hertz, cycles per second)
C = velocity of light, 3 × 108 ms⸺1
λ = wavelength in meter

3

The behavior of EMR depends on its wavelength
...
When EMR interacts with single atoms and molecules, its behavior depends on the amount
of energy per quantum it carries
...

What happened when electromagnetic radiation is passed through a substance
When a beam of electromagnetic radiation is passed through a substance, the radiation can be either absorbed or
transmitted, depending upon its frequency and the structure of the molecules
...
Just how much energy it gains depends upon the frequency
of the radiation; the higher the frequency (the shorter the wavelength) the greater the gain in energy
...
626 × 10⸺34 Js
ν = frequency, in hertz (cycles per second)
The energy gained by the molecule in this way may bring about increased vibration or rotation of the atoms, or may
raise electrons to higher energy levels
...
The
spectrum of a compound is a plot that shows how much electromagnetic radiation is absorbed or transmitted at each
frequency
...

Absorbance vs transmittance
Absorbance and transmittance are two related but different quantities used in spectrometry
...

As light passes through a material, it is absorbed by molecules in the material
...
Transmittance through a sample solution
is easily measured by measuring the intensities of incident and transmitted light
...

Transmittance (T): Transmittance is a measurement of how much light passes through a substance
...
Transmittance is defined as the ratio of the intensity
of incident light to the intensity of transmitted light i
...
if the intensity of incident light is I0 and the intensity of
transmitted light is I, then
Transmittance T =

𝐼
𝐼0

At times, this fraction may be represented as a percentage (%) where it is called the percentage transmittance (%T)
...
So we can write,
A = ecI

4

Where, e is a constant called the molar absorptivity
...
This is an extremely
useful relationship which allows concentrations in unknown solutions to be found by measuring the absorbance of light
through a sample
...
It uses light in the visible and adjacent near ultraviolet and near infrared
ranges
...
The method is used in a quantitative
way to determine concentrations of an absorbing species in solution, using the Beer Lambert law:
A = log

𝐼
𝐼0

= ecL

Where A is the measured absorbance
I0 is the intensity of the incident light at a give wavelength
I is the transmitted intensity
L is the length of the cell
c is the constant known as the extinction coefficient
Lambert’s law: There are two laws which govern the absorption of light by the molecules
...

2
...


Lambert’s law: When a beam of monochromatic radiation passes through a homogenous absorbing medium,
the rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to the intensity
of the incident radiation
...
Its value depends upon the nature of the absorbing
medium
Let, I0 is the intensity of the radiation before entering the absorbing medium (x = 0)
Then, I, the intensity of radiation after passing through any thickness x of the medium can be calculated as:

5

The intensity of the radiation absorbed, Iabs is given by:
Iabs = I0 ⸺ I
= I0 (1 ⸺ e⸺kx)
Equation of Lambert’s law can also be written by changing the nature of logarithm to the base 10
...
303

Beer’s law: When a beam of monochromatic radiation passes through a homogenous absorbing medium, the rate of
decrease of intensity of radiation with thickness of absorbing medium is proportional to the intensity of the incident
radiation as well as the concentration of the solution
...
303

= molar extinction coefficient of the absorbing solution

6

Beer’s law can also be written as: When a monochromatic light is passed through a solution of an absorbing substance,
its absorption remains constant when the concentration (c) and the thickness of the absorption layer (x) are changed in
the inverse ratio
...
If I is less than
I0, it means that the sample has absorbed some of the light
...

The relationship between the absorbance A, and the two intensities is given by empirical expression known as BeerLambert law
...

An absorbance of 0 at some wavelength means that no light of that particular wavelength has been absorbed
...
Multiple samples with
known properties are measured and graphed, which then allows the same properties to be determined for unknown
samples by interpolation on the graph
...

Standard curves are most commonly used to determine the concentration of a substance such as glucose, protein, DNA
etc
...

Example: The O (ortho)-toluidine method utilizes a specific color reaction with glucose
...
Concentration of stock glucose standard solution is 300
mg/100 mL
...
5 mL centrifuge tubes are set up
↓
5 μL diluted standards are transferred to appropriately labeled tubes
↓
500 μL reagents are transferred to each tube
↓
The tubes are closed tightly and mixed

7

↓
The tubes are placed in a tube holder and heated in a boiling water bath or heat block for 8 minutes
...
Bubble formation is avoided carefully
...
The energy change associated with this transition provides information on the
structure of a molecule and determines many molecular properties such as color
...
When σ electron is promoted to antibonding σ* orbital, it is represented as σ to σ* transition
...
Likewise, promotion of an electron of a π bonding orbital to
an antibonding π orbital* is denoted a π → π* transition
...
This process is denoted as n
→ π* transition
...


Chromophore: A chromophore is a part of a molecule responsible for its color
...
It is defined as any isolated
covalently bonded group that shows a characteristic absorption in the UV or visible region
...

Auxochrome: An auxochrome is a functional group of atoms to the chromophore which modifies the ability of the
chromophore to absorb light, altering the wavelength or intensity of the absorption
...
Example: ⸺OH, ⸺OR, ⸺NH2
...

Hypsochromic shift: Shift of the absorption spectrum towards smaller wavelength
...

Hypochromic shift: A decrease in the absorption intensity
...
Such type of bands arise in
compounds like dienes, polyenes, enonones etc
...
The intensity of K band is usually more than 104
...
K band is obtained
normally from π → π* transition
...

R band: Such types of band is obtained from the transition n → π* transition of a single chromophore group
and having at least one lone pair of electrons on the hetero atom
...

B band: This type of band arise due to π → π* transition in aromatic or heteroaromatic molecules
...
When a chromophoric group is attached to the benzene ring,
the B band is observed at longer wavelength than the more intense k band
...
These are further characterized as E1 and E2 bands
...
E1 band, which appears at lower wavelength is usually
more intense than the E2 band for the same compound which appears at longer wavelength
...
The standard unit of molar absorptivity
is liters per mole per centimeter (L mole⸺1 cm⸺1)
Using the Beer-Lambert law
A = log

I
I0

= εcL
Where A is the measured absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted
intensity, L is the length of the cell and c is the concentration of the absorbing species
...

A = εcL
Or ε =

𝐴
𝑐𝐿

It allows us to make comparisons between compounds
...
05 mol/L
...
5
...
5

0
...
A dilute
solution of sample is always prepared for the spectral analysis
...
Non-polar
solvent has very less effect on intensity shift
...

Woodward-Fieser rules: In 1945, Robert Burns Woodward gave certain rules for correlating λmax for a given structure
by relating the position and degree of substitution of chromophore
...


•

Heteroannular diene: Cyclic diene having conjugated double bonds in different rings
...


•

Exocyclic double bond: Double bond in which one of the double bonded atoms is a part of a ring system
...
Ring B has only one endocyclic double bond
...

2
...

4
...

6
...

8
...

10
...

Quantitative analysis for an analyte
...

Rate of chemical reaction
...

Metal analysis
...

Detection of a functional group
...

Solvent’s purity etc
...
The absorption
pattern is then compared to the infrared spectra of known substances for identification
...
The movement of atoms
and the chemical bonds like spring and balls (vibration)
...
When energy, in the form of infrared radiation is applied, then it causes the vibration between the atoms of
the molecules and when, applied infrared frequency = natural frequency of vibration, then absorption of IR radiation
takes place and a peak is observed
...
Therefore, IR spectrum of a chemical substance is a fingerprint of a molecule for its
identification
...

•
•
•

The higher energy near-infrared, approximately 14000 – 4000 cm⸺1 (0
...
5 μm wavelength) can excite
overtone or harmonic vibrations
...
5 – 25 μm wavelength) may be used to study the
fundamental vibrations and associated rotational-vibrational structure
...


Types or modes of vibration: The simplest type or modes of vibrational motion in a molecule that are infrared-active
are the stretching and bending modes
...
For molecules with N number of
atoms in it, linear molecules have 3N⸺5 degrees of vibrational modes, whereas nonlinear molecules have 3N⸺6
degrees of vibrational modes (also called vibrational degrees of freedom)
...

Identification of functional groups with IR spectroscopy: For each and every bond in a molecule, the energy gap
between vibrational states is very much dependent on the nature of the bond
...


Both bonds will absorb IR radiation but the C⸺H bond will absorb a higher energy photon
...
We
simply irradiate the compound with all frequencies of IR radiation and then detect with frequencies were absorbed
...

In this way IR spectroscopy can be used to identify the presence of functional groups in a compound
...
A plot is then constructed showing which
frequencies were absorbed by the sample
...

Several techniques are used for preparing a sample for IR spectroscopy
...
These expensive plates are made of NaCl and are used because they are transparent to IR radiation
...
If the compound is a solid at room temperature, it can be dissolved in a suitable
solvent and placed in between two salt plates
...
All of these sampling techniques are commonly used
for IR spectroscopy
...
This plot is called an absorption spectrum (figure below)
...
The location of each signal on the spectrum
can be specified either by the corresponding wavelength or by the corresponding frequency of radiation that was
absorbed
...

currently, the location of each signal is more often reported in terms of a frequency-related unit, called wavenumber
(𝜈̃)
...
Wavenumber
is proportional to frequency, and therefore, a large wavenumber represents higher energy
...
Every signal in an IR spectrum has three characteristics: wavenumber, intensity and shape
...
The impact of these two factors
can be rationalized when we treat a bond if it were a vibrating spring connecting two weights:

12

Using this analogy, we can construct the following equation, derived from Hooke’s law, which enables us to
approximate the frequency of vibration for a bond between two atoms of mass m1 and m2:

In this equation, f is the force constant of the spring, which represents the bond strength of the bond, and m red
is the reduced mass of the system
...
Notice that mred appears in the denominator
...
For example, compare
the following bonds
...


While mred appears in the denominator of the equation, the force constant (f) appears in the numerator
...
For example, compare the following bonds
...


Single bonds (except for X⸺H bonds) appear on the right side of the spectrum (below 1500 cm ⸺1) because
single bonds are generally the weakest bonds
...
And finally, the left side of the spectrum
contains signals produced by X⸺H bonds (such as C⸺H, O⸺H, N⸺H), all of which stretch at high
wavenumber because hydrogen has the smallest mass
...
This
region contains all signals that arise from double bonds, triple bonds, and X⸺H bonds
...
This region generally contains many signals and is more difficult to analyze
...
As an illustration, compare the carbonyl groups (C=O bonds) in
the following two compounds:

The second compound is called an unsaturated, conjugated ketone
...
As shown, the carbonyl group of an unsaturated, conjugated ketone produces a
signal at lower wavenumber (1680 cm⸺1) than the carbonyl group of a saturated ketone (1720 cm⸺1)
...
Let’s begin with
the ketone:

Ketones have two significant resonance structures
...
This
means that the carbonyl group has some double-bond character and some single-bond character
...
In other words, does the carbonyl group have more double-bond character or more singlebond character? The second resonance structure exhibits charge separation as well as a carbon atom
(C+) that has less than an octet of electrons
...
Therefore, the carbonyl
group of a ketone has mostly double-bond character
...
That is, some bonds absorb IR radiation very efficiently, while other bonds are less efficient at
absorbing IR radiation
...
Recall that the dipole moment (μ) of a bond is defined by the following equation:
μ=e×d
where e is the strength of the partial charges (δ+ and δ⸺) and d is the distance separating them
...


As a bond vibrates, the distance between the partial charges is constantly changing, which means that the
strength of the dipole moment also changes with time
...
So as the dipole moment oscillates, the bond is essentially surrounded by an oscillating electric field,
which serves as an antenna (so to speak) for absorbing IR radiation
...

The efficiency of a bond at absorbing IR radiation therefore, depends on the strength of the dipole moment
...
Let’s first
analyze the carbonyl group (C=O bond)
...
The carbonyl group therefore has a
large dipole moment
...
One vinylic position is connected to alkyl groups,
while the other vinylic position is connected to hydrogen atoms
...

The oscillating electric filed associated with the carbonyl group is much stronger than the oscillating electric
field associated with the C=C bond (figure 1
...
therefore, the carbonyl group functions as a better antenna
for absorbing IR radiation
...
Carbonyl groups often produce the strongest signals in an IR spectrum, while C=C bonds
often produce fairly weak signals
...
One effect of H bonding is to
weaken the existing O⸺H bond
...
At any given
moment in time, the O⸺H bond in each molecule is weakened to a different extent
...
That is, some molecules are barely participating in H bonding, while others are participating
in H bonding to varying degrees
...

The shape of an OH signal is different when the alcohol is diluted in a solvent that cannot form
hydrogen bonds with the alcohol
...
The result is a narrow signal
...
The molecules that are not participating
in H bonding will give rise to a narrow signal, while the molecules participating in H bonding will
give rise to a broad signal
...
When O⸺H bonds do not participate in H bonding, they generally produce a signal
at approximately 3600 cm⸺1
...
When O⸺H bonds participate
in H bonding, they generally produce a broad signal between 3200 and 3600 cm ⸺1
...
Depending on the conditions, an alcohol will give a broad signal, or a
narrow signal, or both
...
This technique is often extremely helpful when performing a reaction in which a
functional group is transferred into a different group
...
In order to distinguish two compounds using IR spectroscopy, one must
know what to look for in the spectrum of the product
...
As a result, the object repels, and is
repelled by, the external magnetic field
...
Larger objects require a larger external magnetic field to induce levitation, but in theory, any object will
levitate if it is placed in a strong enough magnetic field
...
In 1977, researchers at Radboud University (in the Netherlands) demonstrated that
hazelnuts, strawberries, and even frogs will levitate in a magnetic field of 16 tesla
...
But in theory, stronger magnets would enable people to enjoy weightlessness
without having to travel to space
...
Indeed, the structure of a compound can often be determined
using NMR spectroscopy alone, although in practice, structural determination is generally accomplished through a
combination of techniques that includes NMR and IR spectroscopy and mass spectroscopy
...
A
wide variety of nuclei can be studied using NMR spectroscopy, including 1H, 13C, 15N, 19F, 31P
...
Analysis of an NMR spectrum provides information about how the
individual carbon and hydrogen atoms are connected to each other in a molecule
...

A nucleus with an odd number of protons and/or an odd number of neutrons possesses a quantum mechanical property
called nuclear spin, and it can be probed by an NMR spectrometer
...
Not that this property of spin does not refer to the actual
rotation of the proton
...
A spinning proton can be viewed as a rotating
sphere of charge, which generates a magnetic field, called a magnetic moment
...


Fig: (a) The magnetic moment of spinning proton
...

The nucleus of a 12C atom has an even number of protons and an even number of neutrons and therefore does not
possess this property
...

When the nucleus of a hydrogen atom (a proton) is subjected to an external magnetic field, the interaction between the
magnetic moment and the magnetic field is quantized, and the magnetic moment must align either with the field or
against the field (figure 15
...
The spin states are not equivalent in energy, and there is a quantifiable difference in energy
(ΔE) between them (figure 15
...

When a nucleus occupying the α spin state is subjected to electromagnetic radiation, an absorption can take place if the
energy of the proton is equivalent to the energy gap between the spin states
...
When a strong
magnetic field is employed, the frequency of radiation typically required for nuclear resonance falls in the radio wave
region of the electromagnetic spectrum (called radio frequency (rf) radiation)
...
Luckily, this is
not the case, as nuclei are surrounded by electrons
...
4)
...
All materials posses diamagnetic properties, because all materials contain electrons
...
Without this effect, all protons would absorb the same
frequency of rf radiation, and NMR spectroscopy would not provide us with any useful information
...
The proton is now subjected to two magnetic fields – the strong, external magnetic field and the weak induced

18

magnetic field established by the circulating electron density
...
The proton is said to be shielded by the electrons
...
Some protons are surrounded by more electron density and
are more shielded, while other protons are surrounded by less electron density and are less shielded or de-shielded
...
This allows us to probe the environment of each hydrogen
atom in a molecule
...
Protons, electrons and neutrons possess spin
...

2

Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axis
...
However, in
some atoms (such as 1H and 13C) the nucleus does possess an overall spin
...


If the number of neutrons and the number of protons are both even, then the nucleus has no spin
...


If the number of neutrons plus the number of protons is odd, then the nucleus has a half-integer spin (i
...
, ,

1 3
2 2

5

)

3
...
e
...


Asymetric atreching

Symetric streching

Rocking

Scissoring

Wagging
(Both bonds vibrating above and below the plane)

Twisting
One bond is above and the other
bond is below th eplane

What happened when a proton is placed in a magnetic field?
A nucleus with spin

1
2

will have two possible orientations
...
If a magnetic field is

applied, then the energy levels split
...
The lower energy level will
contain slightly more nuclei than the higher level
...
The frequency of radiation needed is determined by the difference in energy between the
energy levels
...
1 Gauss = 0
...


Number of nuclei in energy levels distribution: When the nucleus is in a magnetic field, the initial populations of the
energy levels are determined by thermodynamics, as described by the Boltzmann distribution
...
It is possible to excite these nuclei into the higher
level with electromagnetic radiation
...

Basic principle of NMR spectroscopy: When a proton is placed in a magnetic field, it starts precessing at a certain
frequency in the radio wave region and thus, will be capable of taking up one of the two orientations with respect to
the axis of the external field
...
It
comes back to the low energy aligned orientation (more stable) by losing energy
...
e
...
Stronger the field, higher frequency of the radiation is needed to flip the proton
to the higher energy state
...
If we irradiate the precessing nuclei with a beam of electromagnetic
radiation of desired frequency, the lower energy nuclei will absorb it and move to higher energy state
...
e
...
When this occurs, the nucleus and the radio frequency beam are
said to be in resonance
...

Nuclear magnetic resonance: If we irradiate the precessing nuclei with a beam of electromagnetic radiation of desired
frequency, the lower energy nuclei will absorb it and move to higher energy state
...
e
...
When this occurs, the nucleus and the radio frequency beam are said to be in resonance
...

Nuclear magnetic resonance spectrum: When protons (organic compound) placing in powerful magnetic field, the
proton will process at different frequencies
...
At some values of the magnetic field strength, the energy required
to flip the protons matches the energy of the radiation, absorption occurs and a signal is observed
...

Will all the NMR signal arise in the same place?
All protons do not absorb at the same applied field but absorption depends upon the magnetic field which a particular
proton feels
...
Thus, at a given radiofrequency, different protons (different sets
of equivalent protons) will require slightly different applied field strengths to produce the same effective field strengths
...

The number of signals at different applied field strengths are equal to the different sets of equivalent protons
...

This circulation causes a small magnetic field at the nucleus which opposes the externally applied field as a result of
the position of the NMR signal shifted
...

Chemical shift δ is usually expressed in parts per million (ppm) by frequency, because it is calculated from the equation
below:

Where

νsample is the absolute resonance frequency of the sample and
νreference is the absolute resonance frequency of a standard reference compound, measured in the same applied
magnetic field (B0)
Since, the number is usually expressed in hertz, and the denominator in megahertz, delta is expressed in ppm
...

The difference between the applied magnetic field and the field at the neclus is termed
the nucleus shielding
...
This means that the
applied field strength must be increased for the nucleus to absorb at its transition
frequency
...
Electrons in p-orbitals
have no spherical symmetry
...
This de-shielding is termed paramagnetic shift
...

Number of signals: The number of signals in NMR spectrum tell the number of different sets of equivalent protons in
a molecule
...
Example: Ethyl alcohol, CH 3⸺CH2⸺OH
...
The size or
the area of peaks tells the number of protons
...
We can take an example of ethyl alcohol, CH3⸺CH2⸺OH
...

Splitting of the signals: Each signal in a NMR spectrum represents one kind or one set of protons in a molecule
...

The splitting of an NMR signal is due to spin-spin coupling
...

Three protons triplet, two protons quarter and one proton singlet
...
Those
nuclei experiencing different environment or having different chemical shifts are non-equivalent
...
This effect shows up in the NMR spectrum
when the nuclei are non-equivalent
...
This effect is called spin-spin coupling or J coupling
...
There are two
of them, and each can have one of two possible orientations (aligned with or opposed against the applied field)
...
This has the effect of reducing the field
experienced by the methyl protons; therefore a slightly higher field is needed to bring them to resonance, resulting in
an upfield shift
...
The spins paired
in the direction of the field produce a downfield shift
...

Explain why the methylene peak is split into a quartet?
The effect of the methyl protons on the methylene protons is such that there are 8 possible spin combinations for the
three methyl protons:

Out of these eight groups, there are two groups of three magnetically equivalent combinations
...
The areas of the peaks in the quartet have the ratio of 1:3:3:1
...
e
...
Equivalent nuclei do not interpret with each other
...
The coupling
constant is not dependent on the applied field
...

Coupling constant, J value: The distance between the centers of the two adjacent peaks in a particular multiplets is
shown the value of J and is called coupling constant
...
NMR coupling refers to the effect of nuclei on each other in atoms within a
couple of bonds distance of each other in molecules
...
If we irradiate (decoupling) Hb with correct frequency energy then Ha will also appear as a
singlet
...

2
...

4
...

6
...


In biology: To study the biofluids, cells, perfused organs and bio-macromolecules, carbohydrates, proteins and
peptides
...

In physics and physical chemistry: To study high pressure diffusion, liquid crystals, liquid crystal solutions,
membranes, rigid solids
...

In chemistry: To determine the enantiometric purity, elucidate chemical structure of organic and inorganic
compounds
...

1
H is widely used for structure elucidation
...


To investigate the hydrogen bonding and ionization states of small organic compounds
...
g
...

9
...

10
...

• Tissue perfusion studies
• To detect tumors
C NMR spectroscopy: Carbon-13 (C-13) nuclear magnetic resonance (most commonly known as carbon-13 NMR
or 13C NMR or sometimes simply referred to as carbon NMR) is the application of nuclear magnetic resonance (NMR)
spectroscopy to carbon
...
As such 13C is an important tool in chemical structure
elucidation in organic chemistry
...
1%, because the main carbon isotope 12C is not detectable by NMR since it has 0 net spin
...

2
...

4
...


Total number of C atom in a molecule
...
e
...

Presence of benzene ring in a molecule
...

Presence of carbonyl carbon in a molecule
...
In contrast, mass
spectrometry is the study of the interaction between matter and energy source other than electromagnetic radiation
...

In a mass spectrometer, a compound is first vaporized and converted into ions, which are then separated and detected
...
These
electrons carry an extraordinary amount of energy, usually around 1600 kcal mol ⸺1, or 70 eV (electron volts)
...
This technique,
called electron impact ionization (EI), generates high-energy intermediate that is both a radical and a cation
...
The mass of the ejected electron is negligible compared to the mass of the molecule, so the mass of the
radical cation is essentially equivalent to the mass of the original molecule
...
The molecular ion is often very unstable and susceptible to fragmentation,
which generates two distinct fragments
...


24

In this way, the ionization process generates many different cations – the molecular ion as well as many different
carbocation fragments
...


The uncharged radical fragments are not deflected by the magnetic field and are therefore not detected by the mass
spectrometer
...
Smaller ions are deflected more than
larger ions, and ions with multiple charges are deflected more than ions with a charge of +1
...
The charge (z) on most ions is +1, and therefore, m/z is effectively
a measure of the mass (m) of each cation
...
The tallest peak in the spectrum is assigned a relative value of 100% and
is called the base peak
...
In the
case of methane (CH4)
...

In the mass spectra of larger compounds, it is very common for one of the fragments to produce the tallest peak
...

Mass spectroscopy is a powerful analytical technique used to:
•
•
•

Quantify known materials
Identify unknown compounds within a sample and
Elucidate the structure and chemical properties of different molecules

The complete process involves the conversion of the sample into gaseous ions, with or without fragmentation, which
are then characterized by their mass to change ratios (m/z) and relative abundances
...
In this technique, molecules are bombarded with a beam of energetic
electrons
...
Each kind
of ion has a particular ratio of mass to charge i
...
m/e ratio (value)
...

The molecular ion is called parent ion and usually designated as M+
...
The set of ions (fragment ions or daughter ions) are analyzed in such way that a signal is obtained for each
value of m/e that is represented
...
The largest peak in the structure is called the base peak
...
The intensity of the other
peaks is represented relative to the base peak
...
No two
compounds can have exactly similar mass spectra
...

To establish the structure of a new compound
...

It can give the molecular formula or it can reveal the presence of certain structural units in a molecule
...

Analyzer: For resolving the ions into their characteristic mass components according to their mass to charge
ratio
...


In addition, a sample introduction system is necessary to admit the sample to be studied to the ion source while
maintaining the high vacuum requirements (10⸺6 to 10⸺8 mm mercury) of the technique; and a computer is required
to control the instrument, acquire and manipulate data, and compare spectra of reference libraries
...

Application:
1
...
Biotechnology:
• Amino acid sequencing
• Analysis of macromolecules
3
...
Environmental
• Drug testing
• Water quality testing
• Contamination level measurements in food
5
...

6
...

Ionization methods
1
...

3
...

5
...

7
...

9
...

11
...

13
...

15
...


Chemical ionization: Chemical ionization uses a reagent ion to react with the analyte molecules to form ions
by either a proton or hydride transfer:
MH + C2H5+ → MH2+ + C2H4
MH + C2H5+ → M+ + C2H6
The reagent ions are produced by introducing a large excess of methane (relative to the analyte) into an electron
impact (EI) ion source
...
Plasma and glow discharge: A plasma is a hot, partially ionized gas that effectively excites and ionizes atoms
...
It is particularly effective at
sputtering and ionizing material from solid surfaces
...
Electron impact: An electron impact source uses an electron beam, usually generated from a tungsten
filament, to ionize gas phase atoms or molecules
...

4
...
A sample
solution is spread into the source chamber to form droplets
...
ESI is particularly useful
for large biological molecules that are difficult to vaporize or ionize
...
Fast atom bombardment (FAB): In FAB, a high energy beam of neutral atoms, typically Xe or Ar strikes a
solid sample causing desorption and ionization
...
FAB causes little fragmentation and usually gives a large molecular ion peak, making it
useful for molecular weight determination
...

6
...
High fields can be
created in an ion source by applying a high voltage between a cathode and an anode called a field emitter
...

7
...
The laser pulse accomplishes both vaporization and ionization of the
sample
...
Matrix associated laser desorption ionization (MALDI): MALDI is a LIMS method of vaporizing and
ionizing large biological molecules such as proteins or DNA fragments
...
A UV laser pulse ablates the matrix which carries some of the large
molecules into the gas phase in an ionized form so they can be extracted into a mass spectrometer
...
Plasma desorption ionization (PD): Decay of 252Cf (californium) produces two fission fragments that travel
in opposite directions
...
The other fragment
strikes a detector and triggers the start of data acquisition
...

10
...
Solid
samples must be vaporized by heating, sputtering or laser ablation
...
Secondary ionization (SIMS): A primary ion beam, such as 3He+, 16O+ or 40Ar+ is accelerated and focused into
the surface of a sample and sputters material into the gas phase
...
SIMS has the advantage that material
can be continually sputtered from a surface to determine analyte concentrations as a function of distance from
the original surface (depth profiling)
...
Spark source: A spark source ionizes analytes in solid samples by pulsing an electric current across two
electrodes
...

13
...
A sample is
deposited on a metal ribbon, such as Platinum or Rhenium and an electric current heat the metal to a high
temperature
...


Time of light (TOF): The time of light (TOF) analyzer uses an electric field to accelerate the ions through the same
potential, and then measures the time they take to reach the detector
...
Ions with a
lower mass will reach the detector first
...
The initial velocity is not
dependent on the mass of the ion what becomes a problem for TOF-MS
...
In this way, ions with the same m/z are going to arrive at different times in the detector
...

MALDI-TOF mass spectrometry: Matrix-associated laser desorption/ionization (MALDI) is an ionization technique
that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation
...

MALDI methodology is a three-step process:
First the sample is mixed with a suitable matrix material and applied to a metal plate
...

↓
Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated
gases, and can then be accelerated into mass spectrometer is used to analyze them
...

A solution of one of these molecules is made, often in a matrix of highly purified water and an organic solvent such as
acetonitrile (CAN) or ethanol
...
A good example of a matrix-solution would be 20 mg/mL sinapinic acid in ACN: water: TFA (50: 50:
0
...

Sample preparation: MALDI is the abbreviation of Matrix associated laser desorption/ionization
...

↓
The matrix absorbs the ultraviolet light (nitrogen laser light, wavelength 337 nm) and convers it to heat energy
...

TOF MS: TOF MS is the abbreviation for time of flight mass spectrometry
...
A potential difference V 0 between the sample slide and ground attracts
the ions in the direction shown in the diagram
...
As the potential difference V0 is constant with respect to all ions, ions with smaller m/z value
(lighter ions) and more highly charged ions move faster through the drift space until they reach the detector
...
The method of
mass spectrometry that exploits this phenomenon is called time of flight mass spectrometry
...
The method as initially introduced is technically simple and rapid
...
The dried preparations are exposed to laser pulses, resulting in energy transfer from the matrix to the nonvolatile analyte molecules, with desorption (removal) of analyte into the gas phase
...
The profile of biomarkers is then compared with profiles
of a collection of well-characterized organisms
...
Centrifugation
Centrifugation: Centrifugation is a technique used for the separation of particles from a solution according to their
size, shape, density, viscosity of medium and rotor speed
...
By applying a larger effective gravitational force to the mixture, like a centrifuge does,
the separation of the particles is accelerated
...
Centrifugation is making that
natural process much faster
...

2
...

4
...


Size and shape
...

Temperature or viscosity
Distance of particles displacement
Rotation speed
...
The particles are
usually cells, subcellular organelles or large molecules, all of which are referred to here as particles
...

1
...

3
...

5
...

7
...


Industrial scale centrifuges: An example is the cream separator found in dairies
...

Large centrifuges: Used to stimulate high gravity or acceleration environments
...

Gas centrifuges: Used for isotope separation, such as enrich nuclear fuel for fossil isotopes
...

Swimming head centrifuges
Continuous tubular centrifuges

Principle of sedimentation: In solution, particles whose density is higher than that of the solvent will sediment and
particles that are lighter will float at the top
...
If there is no
difference in density, the particles stay steady
...

The relative centrifuge force (RCF) is a dimensionless quantity given by the expression:
RCF =

𝑟𝜔2
𝑔

Where r is the rotational radius
ω is the angular velocity in radians per unit time
g is the gravitational force
Types of centrifuges:
1
...
They have maximum relative centrifugal force of 3000 – 7000 g
...


Large capacity refrigerated centrifuges: They have refrigerated rotor chamber and have capacity to change
rotor chambers for varying size
...

3
...

4
...
Preparative ultracentrifuge: It can produce relative centrifugal force of about 600,000 g and its
chamber is refrigerated, sealed and evacuated
...

b
...
The aim of this is to study
molecular interactions between macromolecules or to analyze their property
...

2
...

4
...


Analytical ultracentrifuge
Use small sample (less than 1 ml)
Built in optical system to analyze progress of
molecules during centrifugation
...

Used to precisely determine sedimentation
coefficient and MW weight of molecules
...


1
...

3
...

5
...

No optical read out
...

Used to estimate sedimentation coefficient
and MW
...


Types of centrifuge rotors:
1
...

3
...

2
...

4
...

6
...

8
...

Always run thin-walled, sealed tubes full in a fixed angle or vertical rotor
...

Avoid cleaning plastic tubes in automated dishwashers or glassware washes, which may produce excessively
hot temperatures
...

To prolong tube life and avoid breakage or collapse
...

2
...


4
...

6
...

Manufacturers of centrifuge should ensure effective lid locks
...
g
...

Older centrifuges where lid can be opened before the rotor has stopped running
...

This is to prevent aerosol formations arising from accidental spillage of sample which would contaminate the
rotor, centrifuge and possibly the whole laboratory
...


Separating chalk powder from water
...

3
...

5
...

7
...

9
...

Separating particles from an air-flow using cyclonic separation
...

Separation of urine components and blood components in forensic and research laboratories
...
g
...

Laboratory separations
...

Aeronautics and astronautics
...
Chromatographic Techniques
Chromatography: Chromatography is a technique or method for separating the components of a mixture on the basis
of the relative amounts of each solute distributed between a moving fluid stream (mobile phase) and stationary phase
...

Kinetic molecular motion continuously exchanges solute molecules between the two phases
...

The driving force for solute migration is the moving fluid, and the resistive force is the solute affinity for the stationary
phase; the combination of these forces, as manipulated by the analyst, produces the separation
...
Techniques by chromatographic bed shape:
1
...

2
...

• Paper chromatography
• Thin layer chromatography
3
...

B
...
Gas chromatography: The mobile phase is a gas
...
Liquid chromatography: The mobile phase is a liquid
...
Affinity chromatography:
1
...
Techniques by separation mechanism
1
...
Size exclusion chromatography
3
...
Special techniques:
1
...
Hydrophobic interaction chromatography
3
...
Simulated moving bed chromatography
5
...
Fast protein liquid chromatography
7
...
Periodic counter current chromatography
9
...
Aqueous normal phase chromatography
Applications of chromatography:
1
...

3
...


The purification of reaction mixtures in chemical synthesis
...

The analysis of complex sample mixtures such as those obtained in forensics (body fluids, paints etc
...


Gas chromatography: Gas chromatography is a type of chromatography used for separating and analyzing based on
boiling point (vapor pressure) differences of volatile compounds that can be vaporized without decomposition
...
Helium remains the most
commonly used carrier gas in about 90% of instruments although hydrogen is preferred for improved separations
...
The instrument used to
perform gas chromatography is called a gas chromatograph
...

2
...

4
...

6
...

8
...

10
...


Testing the purity of a particular substance by separating the different components of a mixture
...

Assuring the quality of products in the chemical industry
...

Analysis of hydrocarbons, fatty acids and carbohydrates
...

Quality management and certification
...

Analysis of main and trace components
...

Qualitative and quantitative analysis
...
This means that different compounds are adsorbed on an adsorbent at different degrees
...

1
...


Column chromatography
Thin layer chromatography

They are discussed below:
1
...
The most commonly used stationary phases are silica gel and alumina
...
It involves the separation of a mixture over a
column of adsorbent packed in a glass tube called chromatography column
...

The mixture adsorbed is placed on the top of the adsorbent column packed in the chromatography column
...
Depending upon the degree to which the
components are adsorbed, complete separation takes place
...

The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds
e
...
by analytical chromatography, UV absorption septra or fluorescence
...

Applications:
1
...

3
...

5
...

7
...


Analytical use: For analytical purposes, column chromatography is used to purify samples
...

Separation of components: The separation of cis-trans isomer is based on the steric factors
...

Chiral compounds can also be separated by using chiral silica gel
...

Separation of tautomeric mixtures
...


34

2
...
In this
case, the stationary phase is a glass plate of suitable size coated with a thin layer of stationary phase usually
silica gel or alumina
...

The solution of mixture to be separated is applied as a small spot about 2 cm above one end of the TLC plate
...
As the
mobile phase rises up the plate by capillary action, the components of the mixture move up along with the
solvent to different distances depending on their degree of adsorption and separation takes place
...

Retention factor (Rf): It is defined as the distance moved up or travelled by the component from the original
line to the distance travelled by the solvent from the original line
...
The retention
volume of a solute is that volume of mobile phase that passes through the column between the injection point
and the peak maximum
...
In TLC, the retention is measured as the retention factor Rf, the run
length of the compound divided by the run length of the eluent front
...

Applications:
1
...

3
...

5
...


It has been used for checking of the other separation procedures and purification processes
...

The main reasons for popularity of thin layer chromatography as an analytical and preparation
method are –
• It can be used for most of chemical compounds
...

For isolation and characterizing phytochemicals from plant extract
...

In quantitative analysis
...


2
...

4
...
The spots of
colorless components that are invisible to the eye but fluorescent in ultraviolet light can be detected
by putting the plate under ultraviolet light
...

Spraying an appropriate reagent on the TLC plate
...

Burn with acid solutions
...
An important partition
chromatography is paper chromatography
...
Mobile phase
is a solvent or a mixture of solvents
...
The solvent rises up the paper by capillary action and flows over the spot
...
The paper strip so developed is called
chromatogram
...
The spots of the separated colorless components may be observed either under UV light or
by the use of an appropriate spray reagent
...
The relative adsorption of each component of the mixture is expressed in terms of its Retardation factor (Rf)
(retention factor)
...
Alternatively, the solvent may be taken on the top in a container and be
allowed to come down, in which case it is termed as descending paper chromatography
...

2
...

4
...

Paper chromatography is ideally suited for rapid analysis of reaction mixture and so it is versatile tool in the
hand of organic chemists
...

It can be used to detect traces of pollutants in water food or in soil
...

Types: Depending on the substrate used i
...
stationary phase used, the HPLC is divided into the following types:
1
...

3
...

5
...

7
...
The stationary phase is polar and the mobile
phase is non-polar
...

Reverse phase HPLC: The mobile phase is polar and the stationary phase is non-polar or hydrophobic
...

Size exclusion HPLC: On the difference in molecular sizes, the separation of constituents will occur
...

The mobile phase used is aqueous buffer which will control pH and ionic strength
...

Partition chromatography: Analyte molecules partition between a liquid stationary phase and the eluent
...


Applications of HPLC:
1
...

3
...

5
...

Environmental applications: Monitoring of pollutants and detecting components of drinking water
...

Food and flavor applications: Sugar analysis in fruit juices, detecting polycyclic compounds in vegetables,
analysis of preservatives
...


Different units used in high performance liquid chromatography (HPLC):
•

Solvent reservoir: The contents of mobile phase are present in glass container
...
Depending on the composition of sample, the
polar and non-polar solvents will be varied
...
Maximum 250 kgfcm2 is the operating pressure of the pump
...

Sample injector: The injector can be solitary infusion or a computerized infusion framework
...
1 mL to 100 ml of volume
with high reproducibility and under high pressure
...
They are generally loaded with a
stationary phase with a molecule size of 3 μm to 10 μm
...
The temperature of the mobile phase and the column should be kept
consistent during investigation to keep precise retention time
...
Regularly utilized detectors are UV spectroscopy, fluorescence, mass
spectrometric and electrochemical
...
The PC coordinates the reaction of the indicator to every part and places it into
a chromatograph that is anything but difficult to interpret
...
Components of a mixture
may be interacting with the stationary phase based on charge, relative solubility or adsorption
...

Retention time: The retention is a measure of the speed at which a substance moves in a chromatographic system
...
In HPLC or GC, the retention is usually measured as the retention time, R t or tR, the time
between injection and detection
...
RT is used to identify a compound in a test sample
...
This peak width, W is based on the baseline intercepts of tangent lines to a Gaussian
peak, which is equivalent to the peak width at 13
...


•

Half peak height method: Width is calculated from the width at half the peak height (W0
...
Since, width can
be calculated easily by hand, it is the most widely used method
...
54 (

𝑤0⋅5

2

)

37

•

Area height method: Width is calculated from the peak area and height values
...

𝑡𝑟𝐻 2

N = 2π (

𝐴

)

Where,
A = area
H = height
tr = retention time
•

Exponentially modified Gaussian method: This method introduces parameters that accommodate the
asymmetry of peaks, and uses the peak width at 10% of the peak height (W 0
...
Furthermore, it cannot calculate the
width unless the peak is completely separated
...
1 = Width of first half of peak at 10% height
b0
...
Electrophoresis
The term literally means ‘migration with electricity’
...
The technique was first
developed by Arne Tiselius in 1930s for the study of serum proteins
...
Electrophoresis is defined as the migration of the charged particle
through a solution under the influence of an external electrical field
...
As an analytical tool, electrophoresis is simple, rapid
and highly sensitive
...

2
...


Complex mixtures can be separated
...

The relative mobility of individual molecules depends on several factors, the most important of which are:
• Net charge
• Charge-mass ratio
• Molecular shape and the temperature
• Porosity and viscosity of the matrix through which the molecules migrate

Principles of electrophoresis: If a mixture of electrically charged biomolecules is placed in an electric field of field
strength E, they will freely move towards the electrode of opposite charge
...
The velocity of movement
V of a charged molecule in an electric field depends on variables described by equation:
V=E

𝑞

(1)

𝑓

Where, f is the frictional coefficient
q is the net charge of the molecule and
Eq is the force that drives the molecules towards an electrode
...

Because the electrical field strength E may vary widely between different experimental formats, the electrophoretic
mobility μ of a sample is defined by
μ=

𝑉

(2)

𝐸

or, V = Eμ

(3)

So, from equation (1), we get
E

𝑞
𝑓

= Eμ

Or, μ =

𝑞
𝑓

(4)

That is, the ratio of net charge to frictional coefficient is the basis of migration of the biomolecules in electrophoresis
...

In general, molecules will move faster
•

As their net charge increases

39

•
•
•
•

If the strength of the electric field increases and
As f decreases
...

General procedure: General operations performed in conventional electrophoresis include:
1
...

3
...


Separation
Staining
Detection and
Quantification

In addition, several electrophoretic blotting techniques have been developed
...


2
...


Electrophoresis tank:
• Buffer chamber
• Electrodes
• Electrical connection to power
• Supply
• Space for keeping support medium
Power tank: To provide constant currents or constant voltage
...


Gel electrophoresis: Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA or proteins
according to molecular size
...
Separation is brought about through molecular sieving technique based on the
molecular size of the substance
...


A porous gel acts as a sieve by retarding or in some cases, by completely obstructing the movement of macromolecules
while allowing smaller molecules to migrate freely
...
Different types of gels which can be used are:
•
•

Agar and agarose gel, starch
Sephadex, polyacrylamide gels

Types: There are two types of gel electrophoresis: Agarose and Polyacrylamide
Although agarose and polyacrylamide differ greatly in their physical and chemical structures, they both make porous
gels
...
By preparing a gel with a restrictive pore size, the
operator can take advantage of molecular size differences among proteins or nucleic acids
...
Polyacrylamide, which makes a small pore gel, is used to separate most proteins and
small oligonucleotides
...


40

Agarose gel electrophoresis: DNA is negatively charged
...
An agarose gel is used to slow the movement of DNA and separate by size
...

Agarose gel: Agarose is a highly purified uncharged polysaccharide derived from agar
...
Agarose dissolves in water when added to
boiling liquid
...
Agarose gels are actually hydrocolloids and they are held together by the formation of weak hydrogen and
hydrophobic bonds
...

Procedure:
Preparation of agarose gel (melting, cooling and adding EtBr, mixing thoroughly)
↓
Pouring into casting tray with comb and allowing to solidification
↓
Running buffer is added, samples and marker are loaded
...

↓
DNA is viewed on UV light box and results are shown
...

2
...

4
...

6
...


Easy to prepare and small concentration of agar is required
...

Large quantities of proteins can be separated and recovered
...

It adsorbs proteins relatively less when compared to other medium
...

Recovery of protein is good; good method for preparatory purpose
...

2
...


Electro osmosis is high
...

Different sources and batches of agar tend to give different results and purification is often necessary
...

Difference between agarose gel and polyacrylamide gel:
1
...

3
...

5
...

Gel casted horizontally
...

2
...

4
...


6
...


Polyacrylamide gel
It is cross-linked polymer of acrylamide
...
Gels are made by free radical-induced polymerization of acrylamide and
N, N’-Methylenebisacrylamide
...

Polyacrylamide gel: It is prepared by polymerizing acryl amide monomers in the presence of methylene-bis-acrylamide
to cross like the monomers
...
It is thermostable, transparent, strong
and relatively chemically inert
...
Proteins are separated on
the basis of charge to mass ratio and molecular size; this phenomenon is called molecular sieving
...

Silver staining: The silver stain system is about 100 times more sensitive, detecting about 10 ng of proteins
...

Ethidium bromide, silver or Coomassie blue dye may be used for this process
...
If the analyte molecules fluoresce under UV light, a
photograph can be taken of the gel under UV lighting conditions
...

Staines used for the detection of DNA is ethidium bromide (limit 10 ng)
...

It is a highly fluorescent compound, so under UV light, the DNA will be visualized
...
Other influences on the rate of migration
through the gel matrix include the structure and
charge of the proteins
...

SDS is a detergent with a strong protein-denaturing
effect and binds to the protein backbone at a constant
molar ratio
...

Polyacrylamide forms a mesh-like matrix suitable for the separation of proteins of typical size
...
Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to
separate proteins based on their length in an easy, inexpensive and relatively accurate manner
...

Useful for separation and/or purification of
matrix of protiens
...


1
...

3
...

2
...


SDS PAGE
Separation is based upon the molecular weight
of proteins
...

Very useful for checking purity of protein
samples
...
In a discontinuous system, a non-restrictive large pore gel, called a stacking gel, is
layered on top of a separating gel
...
However, the continuous system is a little easier to set up
...

•
•
•

Above its isoelectric point, a protein has a net negative charge and migrates toward the anode in an electrical
field
...

At isoelectric pH, proteins or amino acids will not migrate in an electric field
...
Focusing occurs in two stages: First, the pH gradient gel is prepared using a mixture of chemicals
with different isoelectric point
...
When a protein reaches its isoelectric point in
the pH gradient, it carries a net charge of zero and will stop migration
...

↓
A protein mixture is placed in a well on the gel
...

↓
The anode of the column is connected to a reservoir containing an acidic solution like phosphoric acid and
cathode is connected to a reservoir containing alkaline solution like sodium hydroxide
...

↓
The valves are then closed and the current is switched on, causing the carrier ampholytes to migrate until they
reach the pH regions where they have no net charge
...

Advantages:
1
...


As spreading of bands is minimized due to application of the applied field and the pH gradient, high resolution
can be achieved
...
001 pH units can be separated
...
One dimensional electrophoresis is used
for most routine protein and nucleic acid separations
...


43

Two-dimensional gel electrophoresis is widely used to separate complex mixtures of proteins into may more
components that is not possible in conventional one-dimensional electrophoresis
...
In the first dimension, proteins are separated on the basis of electro-focusing by tube
gels
...
The analysis of
2D gels is more complex than that of one-dimensional gels because the components that show up as spots rather than
as bands
...
Capillary electrophoresis is a collection of wide range of separation techniques which involve
the application of high voltages across buffer filled capillaries to achieve separations
...

The rate at which the particle moves is directly proportional to the applied electric field; the greater the field strength,
the faster the mobility
...
If two ions are the same size, the one with
greater charge will move the fastest
...

Application of capillary electrophoresis: Capillary electrophoresis is a modern separation technique, with vast
acceptance in the academic and industrial communities
...
For this technique, genomic DNA must be amplified by PCR
...


Pulsed field gel electrophoresis: DNA in vivo is organized in extremely large single molecules which are arranged in
chromosomes
...
Sometimes it needs to study intact chromosomes or large
DNA fragments of chromosomes which exceed the resolution range of these systems
...

In pulsed field gel electrophoresis, the direction of the field is varied continuously during electrophoresis
...

Intact chromosomal DNA or large fragments of chromosomal DNA are obtained by first lysing cells by
treatment with a detergent or with enzymes such as lysozyme
...

↓
Large DNA molecules are then loaded on agarose gels and electrophoresis is initiated
...

↓
Pulses of electricity are passed through these electrodes
...

↓
It is thought that, during re-orientation, the helical structure is stretched and then compressed
...

The ratio of tr to tp is crucial for good separation in pulsed field electrophoresis
...
If tp is much larger than tr, then the
molecules will behave as in conventional agarose gel electrophoresis
...
1 and 1,
optimal separation on the basis of mass is achieved
...

2
...

4
...

In bacterial taxonomy: It has particular importance in bacterial taxonomy allowing the identification of
relationships between existing and novel strains of bacteria
...

Study on strand breaks: This technique can also be applied to studies on strand breaks in human chromosomes
as a result of exposure to toxic chemicals
...
Radioisotope Techniques
Radiation: Radiation is the emission or transmission of energy in the form of waves or particles through space or
through a material medium
...
Electromagnetic radiation
• Radio waves
• Microwaves
• Visible light
• X rays and
• Gamma radiation
2
...
Acoustic radiation
• Ultrasound
• Sound and
• Seismic waves (dependent on a physical transmission medium)
4
...

Electron: In 1897, while experimenting on cathode rays, Sir J
...
Thomson discovered electron
...
The mass of an electron is 9
...
Electron is negatively

⸺19

charged and the amount of charge on an electron is ⸺1
...
This is the smallest charge ever recorded,
hence this amount has been taken as one unit in atomic science
...
The
symbol for electron is e
...
The mass of a
proton is 1
...
In atomic mass scale it is equal to 1
...
So its mass is generally taken as one unit
...
So the charge of a proton is also taken as +1 unit
...
So it is also H+
...
The symbol of proton is P
...
In 1920, Rutherford first postulated the
existence of neutron
...
The mass of a neutron is 1
...
Neutrons reside inside the nucleus of an atom
...
The symbol of neutron is n
...
It is denoted by Z
...
Hence, the atomic
number of sodium is 11
...
It may be mentioned that an uncharged atom contains
also Z number of electrons
...
But protons reside in the nucleus and its number does
not change
...

It is denoted by A
...
Hence,
the atomic mass number of those sodium atoms is 11 + 12 = 23
...
For example, the masses of oxygen atoms in nature
is 16, 17 and 18 units
...
The atoms, which have the same number of protons, but whose
masses are different, are called isotopes of each other
...
They have same position in
the periodic table
...
Iso means same and topos means position
...
For example,
16
18
17
8𝑂 isotope has 8 neutrons, 8𝑂 has 9 neutrons and 8𝑂 has 10 neutrons
...

Isotopes of any element have same chemical and physical properties, only slight variation in melting and boiling points
may occur
...
Moreover, many isotopes
of all elements have been synthesized
...
Example: I-125, I-131
...
These may occur
naturally as in the case of radium or uranium or may be created artificially
...


2
...


Emits radioactive radiation which
• Have different penetrating ability with materials of different thickness and densities
• Kills cells
• Cause cell mutation
• Ionize molecules
Have the same chemical properties as non-radioactive isotopes of the same element
...


Creation of radioisotope: Scientists create artificial isotopes by bombarding stable atoms of an element with subatomic
particles in a nuclear reactor or in an atom smasher or cyclotron
...

Radiopharmaceutical: A radiopharmaceutical is a molecule that consists of a radioisotope tracer attached to a
pharmaceutical
...
The radioisotope attached to the targeting pharmaceutical will undergo decay and produce specific amounts of
radiation that can be used to diagnose or treat human diseases and injuries
...
Radioisotopes are an essential part of
radiopharmaceuticals
...
Industrial sources usually contain radioisotopes that emit γ or X rays
...
α and β particles, neutrons, X rays and γ rays are examples of ionizing radiation
...
In medicine
1
...

2
...

3
...

4
...

• Thrombosis: Sodium-24 is injected into the bloodstream to detect the position of blood clots
or thrombosis in the blood vessels
...

5
...

B
...
Study the effectiveness of fertilizers:

47

•

Nitrogen-15 or phosphorous-32 is added to soil water
...

2
...

3
...

• Better strain
• Higher resistance against diseases
...
Archaeology: Living animals and plants have a known proportion of carbon-14 in their tissues which remains
constant and decreases when they die
...

D
...
The α particles from polonium-210 is used to neutralize static charge in photographic plates and other
materials
...
Water can be made radioactive by dissolving some radioactive salt which contains sodium-24
...
The β particles emitted are detected by a Geiger-Muller tube
...
γ rays can be used to penetrate deep into weldings to detect faults
...
Tracers:
• Underground pipe leaks
...

• Detector is moved along the pipe
...

• The radioactive source will be a short half-life γ emitter
...
Thickness control:
• The manufacture of aluminum coil: β emitter is placed above the foil and a detector below it
...
The computer will send a signal to the roller to make the gap smaller or bigger
based on the count rate
...
7 × 1010 radioactive decay sec⸺1
In SI unit the curie has been replaced by Becquerel (Bq) where 1 Bq = 1 radioactive decay sec⸺1
= 2
...

1
...

3
...
e
...


Gaseous ionization detectors are radiation detection instruments used in radiation protection applications to measure
ionizing radiation
...

2
...

Particle having enough energy to ionize a gas molecule, the resulting electrons and ions cause a current flow
which can be measured
...


Ionization of gases: Gaseous ionization detectors are radiation detection instruments used in radiation
protection applications to measure ionizing radiation
...
Particle having enough energy to ionize a gas molecule, the resulting electrons and ions cause a current
flow which can be measured
...

2
...


Gieger Muller tubes

Advantages:
1
...


Will measure very high radiation rates
Sustained high radiation levels do not degrade fill gas
...

2
...

4
...
Excitation: Radioactive isotopes interact with matter in two ways, isolation and excitation
...
This process is known as
Scintillation
...
It consists of a scientillator which generates photons of light in response to incident
radiation, a sensitive photomultiplier tube which converts the light to an electrical signal, and the
necessary electronics to process the photomultiplier tube output
...
These are directed at the photomultiplier tube’s photocathode, which emits electrons by the
photoelectric effect
...
The scientillator must be in complete darkness so that visible light photons do not swamp
the individual photon events caused by incident ionizing radiation
...

2
...

4
...

Counting efficiencies are high
...

Highly automated
...

2
...

4
...


Cost of the instrument and cost per sample
...

Quenching
...

Phospholuminescence
...

2
...

4
...

6
...


Hand held radiation survey meters
...

Medical imaging
...

Border security
...

Oil well lodging
...
Autoradiography Autoradiography is the bioanalytical technique used to visualize the distribution of
radioactive labeled substance with radioisotope in a biological sample
...
It is a very
sensitive technique and is being used in a wide variety of biological experiments
...


49

Autoradiograph: An autoradiograph is an image on X-ray film or nuclear emulsion produced by the pattern
of decay emissions (e
...
beta particles or gamma rays) from a distribution of a radioactive substances
...
The film or emulsion is
apposed to the labeled tissue section to obtain the autoradiograph (also called an autoradiogram)
...

Method:
Living cells are briefly exposed to a pulse of a specific radioactive compound
↓
Samples are taken, fixed and processed for light or electron microscopy
↓
Left in the dark for days or weeks (while the radioisotope decays)
↓
The photographic emulsion is developed (as for conventional photography)
↓
Counter stained
↓
Alternatively, pre-staining of the entire block of tissue can be done
↓
It is not necessary to coverslip these slides
↓
The position of the silver grains in the sample is observed by light or electron microscopy
↓
These autoradiographs provide a permanent record
↓
Full details on the batch of emulsion used (dates, exposure time and conditions) should be kept for
each experiment
...

For example, micro-autoradiography was used to examine whether atrazine was being metabolized by the
hornwort plant or by epiphytic microorganisms in the biofilm layer surrounding the plant
...

2
...

4
...

6
...


To determine the tissue (or cell) localization of a radioactive substance
...

To find and investigate the various properties of DNA
...

To find the site and performance of targeted drugs
...


50

6
...
An array experiment makes use of common assay systems such as microplates of
standard blotting membranes
...

2
...

4
...

Disease diagnosis: Microarray helps researchers learn more about different disease such as heart diseases,
mental illness, infectious disease and especially the study of cancer
...
Pharmacogenomics is the
study of correlations between therapeutic responses to drugs and the genetic profiles of the patients
...
Toxicogenomics establishes correlation between responses to
toxicants and the changes in the genetic profiles of the cells exposed to such toxicants
...
Microfluidics is
used in a broad range of applications, from biology and medicine to chemistry and polymer science, because this
versatile platform enables rapid and precise repeatability of measurements and experiments on a relatively low-cost
laboratory platform
...

Applications:
1
...

3
...

5
...


Molecular diffusion coefficients
Fluid viscosity, pH
Chemical binding coefficients
Enzyme reaction kinetics
Capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry
...
Many of these applications have utility for clinical
diagnostics
...
They are used as a delivery system for drugs, genes and vaccines in therapeutics
...

↓
They recognize the liposomal drugs as a potential source of nutrient
↓
Cancer cells absorb liposomes loaded with anti-cancer drugs as a source of fat
↓
Once the anti-cancer drugs are released from liposome in the cell, the cancer cells are killed
...
The newly introduced genes
will encode proteins and correct the deficiencies that occur in genetic diseases
...
The initial
experiments on gene therapy are carried out in animals and then in humans
...

Approaches for gene therapy:

51

There are two approaches to achieve gene therapy:

1
...
These are the cells of an organism other than sperm or egg cells e
...
bone marrow cells, blood cells, skin
cells, intestinal cells
...
In essence, somatic cell gene therapy involves the insertion of a fully functional and expressible
gene into a target somatic cell to correct a genetic disease permanently
...
Germ cell gene therapy: The reproductive (sex) cells of an organism constitute germ cell line
...
For safety, ethical
and technical reasons, germ cell gene therapy is not being attempted at present
...
Therefore, somatic cell gene therapy is
preferred and extensively studied with an ultimate objective or correcting human diseases
...

Phase I trials with a small number (5-10) of human subjects to safety of the product
...

Phase III trials in large human samples for a final and comprehensive analysis of the safety and efficacy of the
product
...
There are several regulatory agencies whose permission must be sought
before undertaking any work related to gene therapy
...

A large number of genetic disorders and other diseases are currently at various stages of gene therapy trials
...

2
...

4
...
Germ line gene therapy: The goal of germ line gene therapy is to introduce transgenic cells into the germ line
as well as into the somatic cell population
...
Somatic cell gene therapy: The approach is to attempt to correct a disease phenotype by treating some somatic
cells in the affected person
...

Ex vivo gene therapy: This involves the transfer of genes in cultured cells (e
...
bone marrow cells)
which are then reintroduced into the patient
...

↓
Growth of the cells in culture
...

↓
Selection of the genetically corrected cells (stable transformation) and growth
...

The procedure basically involves the use of the patient’s own cells for culture and genetic correction, and
then their return back to the patient
...
Ex vivo gene therapy is efficient, only if the
therapeutic gene (remedial gene) is stably incorporated and continuously expressed
...

Selected examples of ex vivo gene therapy:

1
...
Therapy for familial hypercholesterolemia
3
...


In vivo gene therapy: The direct delivery of genes into the cells of a particular tissue is referred to as
in vivo gene therapy
...

↓
Inside the body, the inserted DNA is incorporated into the cells of the specific tissue it was
injected into
...

Parameters to success: The success of in vivo gene therapy mostly depends on the following
parameters:

1
...

2
...

3
...

Types of vectors or delivery methods:
A
...
Non-viral vectors
A
...
Adenovirus
2
...
Lentivirus
4
...
Parvovirus AAV
B
...

a
...
Physical:
• Gene gun
• Electroporation
• Magnetofection

53

Advantages of viral vectors over physical and chemical gene delivery system:

1
...

2
...

Limitations of viral vectors:

1
...

3
...

5
...


Acute immune response to viral vectors
...

Genes lost when the cell goes through mitosis cell division
...

Genes spliced at random into the genome could upset other genes
...


Applications of gene therapy:

1
...

3
...

5
...

7
...

9
...

Correcting cancer genes
...

Harnessing the immune response
...

Pro-drug gene therapy
...

Blocking viral genes e
...
HIV
...


Progress and prospects:
Progress:

1
...


2
...

3
...

4
...

Prospects:

1
...

2
...

3
...

Limitations and ethical issues:

1
...

3
...
This may cause inflammation and in severe cases, organ failure
...
If this happens,
healthy cells may be damaged, causing other illness or diseases such as cancer
...


54

4
...

Superovulation: Superovulation is the use of fertility drugs to cause an ovary to release two or more eggs in one cycle
rather than one egg, as usual for a typical menstrual cycle
...
However, it involves other risks like multiple pregnancies
...

Biomaterials: A biomaterial is any substance that has been engineered to interact with biological systems for a medical
purpose - either a therapeutic or a diagnostic one
...
The study of
biomaterials is called biomaterials science or biomaterials engineering
...

2
...

4
...


Lipid based gene vectors
Poly-L-Lysine based gene vectors
Polyethyleninmine based gene vectors
Polyamidoamine dendrimers based gene vectors
Chitosan based gene vectors

Drug delivery systems: Drug delivery systems are engineered technologies for the targeted delivery and/or controlled
release of therapeutic agents
...

2
...

4
...

6
...

8
...

Restrict drug distribution to target cells or tissues or organs and should have uniform capillary distribution
...

Drug release does not affect the drug action
...

Minimal drug leakage during transit
...

The preparation of the delivery system should be easy or reasonably simple, reproductive and cost effective
...

2
...

4
...

6
...

8
...

Toxicity is reduced by delivering a drug to its target site, thereby reducing harmful systemic effects
...

Avoidance of hepatic first pass metabolism
...

Dose is less compared to conventional drug delivery system
...

Selective targeting to infectious cells that compare to normal cells

---