A collection of notes for the subject 'Modern Pharmaceutical Analysis (MPA)'

A collection of notes for the
subject
‘MODERN PHARMACEUTICAL
ANALYSIS (MPA)’
As per the syllabus for M. Pharm 1st
Semester, prescribed by Rajiv Gandhi
University of Health Sciences, Karnataka,
India.
Prepared by-
L. Sanathoiba Singha
M. Pharm, Ph. Analysis
1st Semester
Karnataka College of Pharmacy
Bangalore-64.

1
CONTENTS
Content Page no.
UV-Vis Spectroscopy 2
IR Spectroscopy 3-14
NMR Spectroscopy 15-28
Mass Spectrometry 29-45
Affinity Chromatography 46-48
Electrophoresis 49-61
X-Ray Crystallography 62-67
Immunological Assay 68-70
Note: I have not been able to include some chapters/topics prescribed in the syllabus due to lack
of time. Fortunately, the excluded topics are the ones whose reference materials are easily
obtained from relevant sources.

2
UV-VISIBLE SPECTROSCOPY
CHOICE OF SOLVENTS:

5
MODES OF VIBRATION: (see Ravi Sankar’s book )
SAMPLE HANDLING:

9
3. Sampling: (see previous pages)

14
FACTORS:
APPLICATIONS:
(see book)

15
CHAPTER-2
NMR SPECTROSCOPY
NMR spectroscopy is the study of spin changes at the nuclear level when a radiofrequency
energy is absorbed in the presence of magnetic field.
Quantum numbers and their role in NMR:

21
SPIN-SPIN COUPLING:
The spins of neighbouring groups of nuclei in molecule are said to be coupled if their spin states
mutually interact. The interactions, which involve electrons in the intervening bonds, result in
small variations in the effective magnetic fields experienced by one group of nuclei due to different
orientations of the spin angular momenta and magnetic moments of those in the neighbouring
group or groups, and vice versa. These lead to the splitting of the resonance signal into two or
more components that are shifted slightly upfield and downfield respectively from the position in
the absence of coupling, the probabilities of each being roughly the same because the permitted
nuclear spin energy levels are almost equally populated. Thus, the resonance signals for two single
adjacent nuclei with substantially different chemical shifts are each split into two component peaks
of equal intensity.

25
NUCLEAR MAGNETIC DOUBLE RESONANCE / SPIN SPIN DECOUPLING:
FT-NMR OR PULSED NMR:
In NMR, the radiofrequency energy can be introduced either by continuous wave (CW) scanning
of the frequency range or by pulsing the entire range of frequencies with a single burst of
radiofrequency energy. The two methods result in two distinct classes of NMR spectrometers viz.
CW NMR spectrometers and FT or pulsed NMR spectrometers.
In Fourier transform (FT) or pulse NMR studies, an instrument with a 2.1-Tesla magnetic field
uses a short (1 to 10 μ sec) bursts of 90MHz energy to accomplish. The source is turned on and
off very quickly, generating a pulse similar to that shown below.
According to a variation of the Heisenberg Uncertainity Principle, even though the frequency of
the oscillator generating this pulse is set to 90MHz, if the duration of the pulse is very short, the
frequency content of the pulse is uncertain because the oscillator was not on long enough to
establish a solid fundamental frequency. Therefore, the pulse actually contains a range of

26
frequencies centred around the fundamental frequency. This range of frequencies is great enough
to excite all of the distinct types of hydrogens in the molecule at once with this single burst of
energy.
When the pulse is discontinued, the excited nuclei begin to lose their excitation energy and return
to their original spin state or relax. As each excited nucleus relaxes, it emits electromagnetic
radiation. Since the molecule contains many different nuclei, many different frequencies of
electromagnetic radiation are emitted simultaneously. This emission is called a free induction
decay (FID) signal. The intensity of the FID decays with time as all of the nuclei eventually lose
their excitation. The FID is a superimposed combination of all the frequencies emitted and can be
quite complex. The individual frequencies due to different nuclei are extracted by using a computer
and a mathematical method called Fourier transform (FT) analysis.
Therefore, the FID is the superimposition of many different frequencies, each of which could have
a different decay rate. The FT analysis will separate each of the individual components of this
signal and convert them to frequencies. The FT analysis breaks the FID into its separate sine or
cosine wave components. This procedure is too complex to be carried out by eye or by hand and
it requires a computer. Pulsed FT NMR spectrometers have computers built into them that not only
can work up the data by this method but can control all of the settings of the instrument.
Fig. The appearance of the FID when the decay is removed.
are added together.

31
INSTRUMENTATION:
A block diagram of a mass spectrometer is shown in Figure 2. It is operated under a vacuum of
10-4 to 10-7 Nm-2 as the presence of air would swamp the mass spectra of samples, and damage
the ion source and detector.

34
(v) Time of flight:
IONIZATION TECHNIQUES or DIFFERENT TYPES OF IONIZATION:

38
MALDI:
Matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser
energy absorbing matrix to create ions from large molecules with minimal fragmentation. It is
similar in character to electrospray ionization (ESI) in that both techniques are relatively soft (low
fragmentation) ways of obtaining ions of large molecules in the gas phase, though MALDI
typically produces far fewer multi-charged ions.
MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix
material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering
ablation and desorption of the sample and matrix material. Finally, the analyte molecules are
ionized by being protonated or deprotonated in the hot plume of ablated gases, and then they can
be accelerated into whichever mass spectrometer is used to analyse them.
The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-
dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (α-CHCA,
alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB).[15] A solution of one of
these molecules is made, often in a mixture of highly purified water and an organic solvent such
as acetonitrile (ACN) or ethanol. A counter ion source such as Trifluoroacetic acid (TFA) is usually
added to generate the [M+H] ions.
The identification of suitable matrix compounds is determined to some extent by trial and error,
but they are based on some specific molecular design considerations. They are of a fairly low
molecular weight (to allow easy vaporization), but are large enough (with a low enough vapor
pressure) not to evaporate during sample preparation or while standing in the mass spectrometer.
They are often acidic, therefore act as a proton source to encourage ionization of the analyte.
The matrix solution is mixed with the analyte (e.g. protein-sample). A mixture of water and organic
solvent allows both hydrophobic and water-soluble (hydrophilic) molecules to dissolve into the
solution. This solution is spotted onto a MALDI plate (usually a metal plate designed for this
purpose). The solvents vaporize, leaving only the recrystallized matrix, but now with analyte
molecules embedded into MALDI crystals. The matrix and the analyte are said to be co-
crystallized. Co-crystallization is a key issue in selecting a proper matrix to obtain a good quality
mass spectrum of the analyte of interest.
MALDI techniques typically employ the use of UV lasers such as nitrogen lasers. The most
common mass analyzer paired with MALDI is the time of flight (TOF) mass spectrometer. MALDI
produces ions in short bursts due to the use of a pulsed laser, and produces a wide variety of ion
masses that require a detector with a broad mass range.
APCI:
Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass
spectrometry which utilizes gas-phase ion-molecule reactions at atmospheric pressure (105 Pa),
commonly coupled with high-performance liquid chromatography (HPLC). APCI is a soft
ionization method similar to chemical ionization where primary ions are produced on a solvent
spray. The main usage of APCI is for polar and relatively less polar thermally stable compounds
with molecular weight less than 1500 Da. The application of APCI with HPLC has gained a large

39
popularity in trace analysis detection such as steroids, pesticides and also in pharmacology for
drug metabolites.
A typical APCI usually consists of three main parts: a nebulizer probe which can be heated to 350-
500°C, an ionization region with a corona discharge needle, and an ion-transfer region under
intermediate pressure. The analyte in solution is introduced from a direct inlet probe or a liquid
chromatography (LC) eluate into a pneumatic nebulizer with a flow rate 0.2–2.0mL/min. In the
heated nebulizer, the analyte coaxially flows with nebulizer N2 gas to produce a mist of fine
droplets. By the combination effects of heat and gas flow, the emerged mist is converted into a gas
stream. Once the gas stream arrives in the ionization region under atmospheric pressure, molecules
are ionized at corona discharge which is 2 to 3 kV potential different to the exit counter-electrode.
Sample ions then pass through a small orifice skimmer into the ion-transfer region. Ions may be
transported through additional skimmer or ion-focusing lenses into a mass analyzer for subsequent
mass analysis.
ESI:
Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an
electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful
in producing ions from macromolecules because it overcomes the propensity of these molecules
to fragment when ionized. ESI is different from other atmospheric pressure ionization processes
(e.g. matrix-assisted laser desorption/ionization (MALDI)) since it may produce multiple-charged
ions, effectively extending the mass range of the analyser to accommodate the kDa-MDa orders
of magnitude observed in proteins and their associated polypeptide fragments.
Mass spectrometry using ESI is called electrospray ionization mass spectrometry (ESI-MS) or,
less commonly, electrospray mass spectrometry (ES-MS). ESI is a so-called 'soft ionization'
technique, since there is very little fragmentation. This can be advantageous in the sense that the
molecular ion (or more accurately a pseudo molecular ion) is always observed, however very little
structural information can be gained from the simple mass spectrum obtained. This disadvantage
can be overcome by coupling ESI with tandem mass spectrometry (ESI-MS/MS). Another
important advantage of ESI is that solution-phase information can be retained into the gas-phase.
APPI (Atmospheric Pressure Photoionization):
All mass spectrometers require the molecules to be in the gas phase and charged (ionized either
positive or negative). In this technique, UV light photons are used to ionize sample molecules. The
technique works well with nonpolar or low polarity compounds not efficiently ionized by other
ionization sources.

40
First the sample (analyte) is mixed with a solvent. Depending on the type used, the solvent could
increase the number of ions that are formed.
The liquid solution is then vaporized with the help of a nebulizing gas such as nitrogen, then enters
an ionization chamber at atmospheric pressure. There, the mixture of solvent and sample molecules
is exposed to ultraviolet light from a krypton lamp. The photons emitted from this lamp have a
specific energy level (10 electron volts, or eV) that is just right for this process: high enough to
ionize the target molecules, but not high enough to ionize air and other unwanted molecules. So
only the analyte molecules proceed to the mass spectrometer to be measured.
First, we’ll look at what happens when just the solvent and analyte molecules are exposed to the
UV light. Then we will look at the slightly more complicated, and much more typical, scenario in
which a dopant (a kind of additive) is introduced into the mixture.
Once they are exposed to the UV light, the analyte molecules are ionized in two ways:
Direct APPI-
A minority of them will be ionized directly by the UV light (Photoionization)
M + hν → M+•
+ e-
Dopant Assisted APPI-
The dopant ion can donate a proton to the analyte molecule. The result is an ionized
sample molecule. Toluene is commonly used as a dopant.
D++
+ M → [M+H]+
+ [D-H]+

41
MASS FRAGMENTATION AND ITS RULES:
Fragmentation is the dissociation of energetically unstable molecular ions formed from passing
the molecules in the ionization chamber of a mass spectrometer. The fragments of a molecule
cause a pattern in the mass spectrum used to determine structural information of the molecule.

43
METASTABLE IONS:
Stable ions form the conventional mass spectrum. Some of the ions, however do not break down
before reaching the ion collector of a mass spectrometer. These are called as metastable ions. They
appear as broad peaks called metastable ion peaks.
Fragment of a parent ion will give rise to a new ion (daughter) plus either a neutral molecule or a
radical.
M1
+
→ M2
+
+ non charged particle
An intermediate situation is possible; M1
+
may decompose to M2
+
while being accelerated. The
resultant daughter ion M2
+
will not be recorded at either M1 or M2 but at apposition M+
as a rather
broad poorly focused peak. Such an ion is called as metastable ion.
Metastable ions have lower kinetic energy than normal ions and metastable peaks are smaller than
the M1 and M2 peaks and also broader.

44
Significance of metastable ions-
 Metastable ions are useful in helping to establish fragments routes.
 Metastable ion peak can also be used to distinguish between fragmentation process,
which occur in few microseconds.
ISOTOPIC PEAKS:

46
Chapter-4
AFFINITY CHROMATOGRAPHY:
According to the International Union of Pure and Applied Chemistry (IUPAC), affinity
chromatography is defined as a liquid chromatographic technique that makes use of a "biological
interaction" for the separation and analysis of specific analytes within a sample. For example, a
protein that binds metals such as nickel can be purified in the presence of other non-specific
proteins using a resin containing immobilized nickel. A protein can bind nickel because of the
presence of amino acids such as histidine positioned in a specific manner, which contains
imidazole functionality that can co-ordinate nickel.
The biomolecule of interest interacts reversibly with a specific ligand bound to a matrix allowing
for a specific binding on the matrix in the presence of other contaminants and later elution of the
bound biomolecule. Using this method the biomolecule can be purified in a single step, with
efficient recovery and high purity.
PRINCIPLE:
There are certain specific requirements for an affinity chromatography that must be met. These
requirements are as follows:
 A biospecific ligand that can be covalently attached to a chromatography matrix.
 The bound ligand must be able to bind the target biomolecule specifically.
 The binding between the ligand and target molecule must be reversible to allow the target
molecules to be removed in an active form.
The biological interactions involve mostly non covalent interactions between the reactive groups
of molecule targeted for purification and ligand with a dissociation constant Kd.
Where, A is assumed as molecule targeted and B as ligand and AB is the complex formed
between them. Kd varies between 10-3
to 10-7
M for affinity binding.
The principle of affinity chromatography is that the stationary phase consists of a support medium
(e.g. cellulose beads) on which the substrate (or sometimes a coenzyme) has been bound
covalently, in such a way that the reactive groups that are essential for enzyme binding are exposed.
As the crude mixture of proteins is passed through the chromatography column, proteins with
binding site for the immobilized substrate will bind to the stationary phase, while all other proteins
will be eluted in the void volume of the column.
Some of the examples of types of interactions utilized in the affinity chromatographic
purification include:
 Antigen : antibody
 Enzyme : substrate analogue

47
 Binding protein: Ligand
 Receptor : ligand
 Lectin : polysaccharide, glycoprotein
 Nucleic acid : complementary base sequence
 Hormone, vitamin : receptor, carrier protein.
 Glutathione : glutathione-S-transferase or GST fusion proteins.
 Metal ions : Poly (His) fusion proteins, native proteins with histidine or cysteine on their
surfaces.
PROCEDURE:
The steps involved in a typical affinity chromatographic separation are as follows:
i. The ligand is first covalently coupled to a matrix, such as agarose beads.
ii. The matrix is poured into a column.
iii. An impure mixture containing biomoleule of interest is loaded on the affinity column.
iv. Biomolecules sieve through matrix of affinity beads and interact with affinity ligand.
Molecules that do not bind to ligand elute from the column.
v. Wash off contaminant molecules that bind to ligand loosely.
vi. Elute proteins that bind tightly to ligand and collect purified protein of interest using
either a biospeciifc or nonspecific elution methods:
a. Biospecific – An inhibitor is added to the mobile phase (free ligand). Free ligand
will compete for the solute.
b. Nonspecific – A reagent is added that denatures the solute. Once denatured, the
solute is released from the ligand.
APPLICATIONS:
 The technique was developed for purification of enzymes but now affinity chromatography
is used for various other purposes like purification of nucleotides, nucleic acid,
immunoglobulin, membrane receptors etc.
 Immunoglobulin purification (antibody immobilization)-Antibodies can also be
immobilized by adsorbing them onto secondary ligands. Alternatively, antibodies can be
directly adsorbed onto a protein A or protein G support due to the specific interaction of
antibodies with protein A and G. Immobilized antibodies on the protein A or G support can
easily be replaced by using a strong eluent, regenerating the protein A/G, and re-applying
fresh antibodies. Generally, this method is used when a high capacity/high activity support
is needed.
 Recombinant tagged proteins- Purification of proteins can be easier and simpler if the
protein of interest is tagged with a known sequence commonly referred to as a tag. This
tag can range from a short sequence of amino acids to entire domains or even whole

48
proteins. Tags can act both as a marker for protein expression and to help facilitate protein
purification.
 GST tagged purification- Glutathione S-transferase (GST) is a 26 kDa protein (211 amino
acids) located in cytosole or mitochondria and present both in eukaryotes and prokaryotes.
Separation and purifcation of GST-tagged proteins is possible since the GST tag is capable
of binding its substrate, glutathione. The free glutathione replaces the immobilized
glutathione and releases the GST-tagged protein from the matrix allowing its elution from
the column.

49
Chapter- 5
ELECTROPHORESIS
PAPER ELECTROPHORESIS:
(refer Ravi Sankar page 28)
GEL ELECTROPHORESIS:
This technique involves the separation of molecules based on their size, in addition to the electrical
charge. Gel electrophoresis is the core technique for genetic analysis and purification of nucleic
acids for further studies. Nucleic acids are separated and displayed using various modifications of
gel electrophoresis and detection methods.
It is used in:
 Clinical chemistry to separate proteins by charge and/or size.
 Biochemistry and Molecular biology to separate DNA and RNA fragments by length, or
to separate proteins by charge.
Gel electrophoretic methods provide the highest resolution of all protein separation techniques.
PRINCIPLE-
Electrophoresis is the migration of charged particles or molecules in an electric field. This occurs
when the substances are in aqueous solution. The speed of migration is dependent on the applied
electric field strength and the charges of the molecules. Thus, differently charged molecules will
form individual zones while they migrate. In order to keep diffusion of the zones to a minimum,
electrophoresis is carried out in an anticonvective medium such as a viscous fluid or a gel matrix.
Therefore, the speed of migration is also dependent on the size of the molecules. In this way
fractionation of a mixture of substances is achieved with high resolution.
GEL TYPES-
There are two types of gel most typically used:
i. Agarose gel
ii. Polyacrylamide gel (PAGE).
Each type of gel is well-suited to different types and sizes of analyte.
i. Agarose gel-
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations
of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Higher percentages
requiring longer run times. Agarose gels have greater range of separation, and are therefore
used for DNA fragments of usually 50-20,000 bp in size.

50
Instrumentation of agarose gel-
Preparation and running of agarose gel-

51
Staining of the bands-
The bands are visualized with fluorescent dyes that are visible in UV light – ethidium bromide or
SYBR Green. SYBR Green is less mutagenic and more sensitive than ethidium bromide. The best
results and highest resolutions are obtained when the gels are stained after the run.
Recovery of DNA fragments from gels-
Several different procedures are used for the isolation of nucleic acids from agarose gels :
 Electroelution
 absorption to DEAE paper
 absorption to glass powder or resins
 digestion of agarose with enzymes.
For preparative electrophoresis, it is very important to use highly purified agarose that is free from
polymerase and other enzyme inhibitors. Since the advent of polymerase chain reaction (PCR)
technology, tiny amounts of DNA fragments can easily be amplified for further experiments.
ii. Polyacrylamide gel (PAGE)-
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains
is dictated by the concentration of acrylamide used, which is typically between 3.5 and
20%. It is used for separating proteins ranging in size from 5 to 2,000 kDa due to the
uniform pore size provided by the polyacrylamide gel. In contrast to agarose,
polyacrylamide gels are used extensively for separating and characterizing mixtures of
proteins.

52
Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be
handled with gloves due to the possible presence of free acrylamide. Acrylamide is a potent
neurotoxin and should be handled with care.
Preparation and running of polyacrylamide gel-
 Polyacrylamide gels are prepared by chemical copolymerization of acrylamide
monomers with a crosslinking reagent, usually N,Nʹ-methylenebisacrylamide.
 A clear transparent gel is obtained, which is chemically inert, mechanically stable
and without electroendosmosis.
 Polymerization of the acrylamide monomers and the cross-linker molecules occurs
in the presence of free radicals. These are provided by ammonium persulfate as
catalyst; tertiary amino groups, usually N, N, Nʹ, Nʹ-tetramethylethylenediamine
(TEMED), are required as accelerators.
 Because oxygen is a scavenger of free radicals, polymerization is performed in
closed cassettes.
 Sample application wells for vertical gels are formed at the upper edge of the gel
during polymerization with the help of an inserted comb (see Figure).
 Sample wells for flatbed gels are made by using self-adhesive tape glued onto one
of the glass plates.

53
 The samples are denatured just prior to loading the gel. Sample DNA may re-anneal if
denatured for an extended time before loading and may produce indeterminate
fragments.
 For electrophoresis in vertical systems, the complete gel cassettes are placed into the
buffer tanks; the gels are in direct contact with the electrode buffers.
 Gels for flatbed systems are polymerized on a film support and removed from the
cassette before use.
Detection of bands-
Silver Staining:
Ethidium bromide and SYBR Green staining are rarely used for polyacrylamide gels, because the
signals are weaker than in agarose gels.
The most sensitive staining for protein is silver staining. This involves soaking the gel in Ag NO3
which results in precipitation of metallic silver (Ag0) at the location of protein or DNA forming a
black deposit in a process similar to that used in black and white photography.
FACTORS AFFECTING GEL ELECTROPHORESIS:
 The higher the voltage/current, the faster the DNA migrates.
 High voltage causes a tremendously increase in buffer temperature and current in very
short time. The high amount of the heat and current built up in the process leads to the
melting of the gel. Therefore, it is highly recommended not exceed 5-8 V/cm and 75 mA
for standard size gels or 100 mA for minigels.
 Electrophoresis is performed in buffer solutions (Electrophoresis buffers TBE) to reduce
pH changes due to the electric field, which is important because the charge of DNA and
RNA depends on pH.
 Running for too long can exhaust the buffering capacity of the solution so it should be
changed from time to time.
APPLICATIONS OF GEL ELECTROPHORESIS:
i. Agarose gel electrophoresis technique is extensively used for investigating the DNA
cleavage efficiency of small molecules and as a useful method to investigate various
binding modes of small molecules to supercoiled DNA.
ii. It is also a useful method to investigate various binding modes of small molecules to
supercoiled DNA.
iii. Development of new metallonucleases as small molecular models for DNA cleavage
at physiological conditions. Since DNA cleavage is a biological necessity, these small
molecular models have provided much of our most accurate information about nucleic
acid binding specificity.
Examples of metallonucleases:
 [Cu(II)(hist)(tyr)]+

54
 [Cu(II)(phen)(his-leu)]+
CAPILLARY ELECTROPHORESIS:
Capillary electrophoresis (CE) is a family of related techniques that employ narrow-bore (20-
200 μm i.d.) capillaries to perform high efficiency separations of both large and small molecules.
These separations are facilitated by the use of high voltages, which may generate electro-osmotic
and electro-phoretic flow of buffer solutions and ionic species, respectively, within the capillary.
The properties of the separation and the ensuing electropherogram have characteristics resembling
a cross between traditional polyacrylamide gel electrophoresis (PAGE) and modern high
performance liquid chromatography (HPLC).
PRINCIPLE-
One of the fundamental processes that drive CE is electroosmosis. This phenomenon is a
consequence of the surface charge on the wall of the capillary. The fused silica capillaries that are
typically used for separations have ionizable silanol groups in contact with the buffer contained
within the capillary. Therefore, the inside walls of the capillary negatively charged because the
inside wall has silanol groups (SOx
-
). This means that the inner wall has a net negative charge.
The buffer solution in each reservoir has equal amounts of cations and anions, and the capillary
ends are each placed in a buffer reservoir. Each reservoir also has an electrode connected to the
power supply.
When the voltage is applied to the circuit, one electrode become net positive and the other net
negative. The (wall’s) immobile silanol anions pair with mobile buffer cations, forming a double
layer along the wall (wall-->buffer cations-->buffer anions-->bulk buffer solution). The remaining
buffer cations are attracted to the negative electrode, dragging the bulk buffer solution with them.
This is electroosmotic flow. For an uncoated capillary, the electroosmotic force (EOF) is toward
the negative electrode.
If the analyst wants the EOF (to flow) in the opposite direction then the capillary can be purchased
coated with a cationic surfactant, or one is added to the buffer, and the capillary walls will be
negatively charged and the electroosmotic flow will be reversed, that is, toward the positively
charge electrode. This might be chose based on a specific analyte separation. In the case below the
wall is uncoated, the wall is net negatively charged and the EOF is toward the negative electrode.

55
So everything injected into the buffer flows with the EOF. But, like the flow of analytes in a gas
chromatographic carrier gas, separation wouldn’t occur unless the analytes flow towards the
detector at different speeds. In GC this occurs because of interaction with the GC columns
stationary phase. In CE this occurs because analytes have different electrophoretic mobilities. In
the simplest approximation, electrophoretic mobility can be because of analyte charge and size.
Large, singly charged analytes will travel slower than small, singly charge analytes, and small,
doubly charged ions will travel faster than larger, doubly charged analytes, etc. In other forms of
CE separation is more complicated. The electrical potential also effects this process.
INSTRUMENTATION-
 The basic instrumental configuration for CE is relatively simple.
 The requirements are:
i. fused-silica capillary with an optical viewing window
ii. A controllable high voltage power supply
iii. Two electrode assemblies
iv. Two buffer reservoirs
v. An ultraviolet (UV) detector.
 The ends of the capillary are placed in the buffer reservoirs and the optical viewing
window is aligned with the detector.
 After filling the capillary with buffer, the sample can be introduced by dipping the end of
the capillary into the sample solution and elevating the immersed capillary a foot or so
above the detector-side buffer reservoir.
 Virtually all of the pre-1988 work in CE was carried out on homemade devices following
this basic configuration. While relatively easy to use for experimentation, these early
systems were inconvenient for routine analysis and too imprecise for quantitative analysis.
 A diagram of a modern instrument, the P/ACE™ 2000 Series, is illustrated in the figure
below. Compared to the early developmental instruments, this fully automated instrument
offers computer control of all operations, pressure and electrokinetic injection, an
autosampler and fraction collector.
 Automated methods development, precise temperature control, and an advanced heat
dissipation system. Automation is critical to CE since repeatable operation is required for
precise quantitative analysis.

56
 A fundamental term in chromatography is retention time. In electrophoresis, under ideal
conditions, nothing is retained, so the analogous term becomes migration time. The
migration time (tm) is the time it takes a solute to move from the beginning of the capillary
to the detector window.
FACTORS AFFECTING CE:
i. The Capillary Surface-
The inner surface of a capillary is an extremely important factor in CE. The inner wall
is in contact with the separation chemistry and the samples. As noted earlier, the
capillary wall is the site of the mechanism by which EOF is created.
ii. Surface modifications-
Capillaries perform best when they are “dedicated” to a specific type of buffer species.
This dedication of a capillary to one type of system is a relatively inexpensive way to
improve results.
iii. Separation buffers-
The significance of the capillary wall in controlling the process of separation in CE
cannot be overstated. The separation, however, takes place in the separation buffer. It
is here that the conditions are such that the differences in mobility can exist. Even the
best instrument system will not perform properly with a poorly prepared buffer.
iv. Significance of pH-
In CE it is extremely important to properly control pH since it affects analyte charge,
electroosmotic flow, and, by affecting current, heat production. Thus small changes in
pH tend to have greater impact in CE.
v. Additives-
Other reagents are frequently added to the buffer systems used in CE. The most
common are detergents, such as sodium dodecyl sulfate (SDS), viscosity modifiers,
such as linear polyacrylamide, organic solvents, such as acetonitrile, denaturants, such
as urea, or combinations of these additives.
The addition of detergent to a buffer used in CE can change dramatically the separation
properties of the system. Detergents can aid in solubilizing analytes and in reducing
analyte-wall interactions. They may also bind to the capillary wall, affecting the EOF.
vi. Temperature-
Temperature control is crucial to reproducible separations in CE. However,
temperature regulation is complicated by several factors.
First is that the passage of electrical current through the buffer-filled capillary results
in the production of heat. This self-heating effect is inherent in electrophoretic
separations and is called Joule heating. Thus temperature control in CE is as much a
task of removing heat as it is maintaining a constant temperature environment.
Second is that the temperature of the contents inside the capillary is difficult to measure.

57
APPLICATIONS-
i. It is very useful for the separation of proteins and peptides since complete resolution
can often be obtained for analytes differing by only one amino acid substituent.
ii. It is particularly important in tryptic mapping where mutations and post-translational
modifications must be detected.
iii. Other applications where CE may be useful include separation of inorganic anions and
cations such as those typically separated by ion chromatography.
iv. Small molecules such as pharmaceuticals can often be separated provided they are
charged.
ZONE ELECTROPHORESIS:
Capillary electrophoresis comprises a family of techniques that have dramatically different
operative and separative characteristics. The techniques are:
 Capillary zone electrophoresis
 Isoelectric focusing
 Capillary gel electrophoresis
 Isotachophoresis
 Micellar electrokinetic capillary chromatography
Therefore Zone Electrophoresis (ZE) or Capillary Zone Electrophoresis (CZE) is one of the
types of capillary electrophoresis.
Capillary zone electrophoresis (CZE), also known as free solution capillary electrophoresis,
is the simplest form of Capillary electrophoresis (CE). The separation mechanism is based on
differences in the charge-to-mass ratio. Fundamental to CZE are homogeneity of the buffer
solution and constant field strength throughout the length of the capillary.
PRINCIPLE-
The separation mechanism is based on differences in the charge-to-mass ratio. Following injection
and application of voltage, the components of a sample mixture separate into discrete zones as
shown in the figure.

58
The fundamental parameter, electrophoretic mobility, μep, can be approximated from Debeye-
Huckel-Henry theory.
Where,
q is the net charge
R is the Stokes radius, and
η is the viscosity.
The net charge is usually pH dependent. For example, within the pH range of 4-10, the net charge
on sodium is constant as is its mobility. Other species such as acetate or glutamate are negatively
charged within that pH range and thus have negative mobilities (they migrate towards the positive
electrode). At alkaline pH, their net migration will still be towards the negative electrode because
of the EOF. Zwitterions such as amino acids, proteins, and peptides exhibit charge reversal at their
pI’s (Isoelectric point) and, likewise, shifts in the direction of electrophoretic mobility.
Separations of both large and small molecules can be accomplished by CZE. Even small
molecules, where the charge-to-mass ratio differences may not be great, may still be separable.
INSTRUMENTATION-
(same as previous)
APPLICATIONS-
(same as previous)
ISOELECTRIC FOCUSING:
Capillary electrophoresis comprises a family of techniques that have dramatically different
operative and separative characteristics. The techniques are:
 Capillary zone electrophoresis
 Isoelectric focusing
 Capillary gel electrophoresis
 Isotachophoresis
 Micellar electrokinetic capillary chromatography
Therefore Isoelectric focusing (IEF) is one of the types of capillary electrophoresis.
The fundamental theory of isoelectric focusing (IEF) is that a molecule will migrate so long as it
is charged. Should it become neutral, it will stop migrating in the electric field. IEF is run in a pH
gradient where the pH is low at the anode and high at the cathode (Figure 7). The pH gradient is
generated with a series of zwitterionic chemicals known as carrier ampholytes.

59
PRINCIPLE-
Molecules that carry both positively and negatively charged groups exhibit, at a specific pH, an
equal number of positive and negative charges. At this pH, known as the isoelectric pH or pI, the
molecule, although charged, behaves as if it is neutral because its positive and negative charges
cancel each other. The molecule, therefore, has no tendency to migrate in an electrical field. In
isoelectric focusing, special reagents called ampholytes are used to create a pH gradient within the
capillary. These ampholytes are mixtures of buffers with a range of pKa values. In an electrical
field, ampholytes will arrange themselves in order of pKa; this gradient is trapped between a strong
acid and a strong base. Analytes introduced into this gradient will migrate to the point where the
pH of the gradient equals their pI. At this point the analyte, having no net charge, ceases to migrate.
It will remain at that position so long as the pH gradient is stable, typically as long as the voltage
is applied.
The pH of the anodic buffer must be lower than the pI of the most acidic ampholyte to prevent
migration into the analyte. Likewise, the catholyte must have a higher pH than the most basic
ampholyte.
INSTRUMENTATION AND WORKING-
(instrumentation similar to previous)
The three basic steps of IEF are:
i. Loading
ii. Focusing
iii. Mobilization.
i. Loading- The sample is mixed with the appropriate ampholytes to a final concentration
of 1-2% ampholytes. The mixture is loaded into the capillary either by pressure or
vacuum aspiration.
ii. Focusing- The buffer reservoirs are filled with sodium hydroxide (cathode) and
phosphoric acid (anode). Field strengths on the order of 500-700 V/cm are employed.
As the focusing proceeds, the current drops to less than 1 mA. Overfocusing can result
in precipitation due to protein aggregation at high localized concentrations. Dispersants
such as nonionic surfactants or organic modifiers such as glycerol or ethylene glycol
may minimize aggregation. These agents are mild and usually do not denature the
protein. Urea could also be used, but the protein will become denatured. Because of
precipitation problems, very hydrophobic proteins are not usually separated by IEF.
Gel-filled capillaries are sometimes useful for separating troublesome proteins.
iii. Mobilization- Mobilization can be accomplished in either the cathodic or anodic
direction. For cathodic mobilization, the cathode reservoir is filled with sodium

60
hydroxide/sodium chloride solution. In anodic mobilization, the sodium chloride is
added to the anode reservoir. The addition of salt alters the pH in the capillary when
the voltage is applied since the anions/cations compete with hydroxyl/hydronium ion
migration. As the pH is changed, both ampholytes and proteins are mobilized in the
direction of the reservoir with added salt. As mobilization proceeds, the current rises
as the saline ions migrate into the capillary. Detection is performed at 280 nm for
proteins since the ampholytes absorb strongly in the low UV range.
APPLICATIONS-
i. In addition to performing high resolution separations, IEF is useful for determining the
pI of a protein.
ii. IEF is particularly useful for separating:
 Immunoglobulins
 Hemoglobin variants and
 Post-translational modifications of recombinant proteins.
MOVING BOUNDARY ELECTROPHORESIS:
Moving boundary electrophoresis (MBE) was developed by Arne Tiselius in 1930. Tiselius was
awarded the 1948 Nobel Prize in chemistry for his work on the separation of colloids through
electrophoresis.
MBE is a technique for separation of chemical compounds by electrophoresis in a free solution.
PRINCIPLE-
The moving boundary electrophoresis apparatus includes a U-shaped cell filled with buffer
solution and electrodes immersed at its ends. The sample applied could be any mixture of charged
components such as a protein mixture. On applying voltage, the compounds will migrate to the
anode or cathode depending on their charges. The change in the refractive index at the boundary
of the separated compounds is detected using Schliren optics at both ends of the solution in the
cell.

61
Fig. MBE
Fig. Schlieren photography
APPLICATIONS-
 To study the homogenicity of a macromolecular system.
 Analysis of complex biological mixtures.

62
X-RAY CRYSTALLOGRAPHY:
X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal,
in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific
directions. By measuring the angles and intensities of these diffracted beams, a crystallographer
can produce a three-dimensional picture of the density of electrons within the crystal. From this
electron density, the mean positions of the atoms in the crystal can be determined, as well as their
chemical bonds, their disorder and various other information.
PRINCIPLE:
X-rays are electromagnetic radiation with wavelengths between about 0.02 Å and 100 Å. Because
X-rays have wavelengths similar to the size of atoms, they are useful to explore within crystals.
Since X-rays have a smaller wavelength than visible light, they have higher energy. With their
higher energy, X-rays can penetrate matter more easily than can visible light. Their ability to
penetrate matter depends on the density of the matter, and thus X-rays provide a powerful tool in
medicine for mapping internal structures of the human body (bones have higher density than tissue,
and thus are harder for X-rays to penetrate, fractures in bones have a different density than the
bone, thus fractures can be seen in X-ray pictures).
Bragg’s law- When X-rays are scattered from a crystal lattice, peaks of scattered intensity are
observed which correspond to the following conditions:
 The angle of incidence = Angle of scattering
 The pathlength difference is equal to an integer number of wavelength.

63
PRODUCTION OF X-RAYS:
X-rays are produced when high speed electrons collide with a metal target. High speed electrons
are generated from hot tungsten filament (cathode) and after applying a high accelerating voltage
(15-60kV) and are made to fall on metal target (anode) like copper/aluminium, molybdenum or
magnesium. Water is circulated as coolant o copper block containing desired target metal.

64
DIFFERENT X-RAY METHODS:
1. Laue Photographic Method-
A single small crystal is placed in the path of a narrow beam of X-rays from a tungsten
anticathode and the resulting diffracted beam is allowed to fall on a photographic plate.
When the photographic plate is developed, a characteristic pattern, known as Laue pattern
of spots is seen. From the positions of the spots of the distance of the photographic plate
from the crystal, 9 is calculated and the relative spacing between the planes is estimated.
Laue pattern can be used to orient crystals for solid-state experiments and to determine the
symmetry of single crystal. However, the significance of reflection intensities is uncertain
due to non-homogeneous nature of the incident X-rays.

65
2. Rotating Crystal Method-
The method developed by Schiebold (1919) and M. Polanyl (1921), is perhaps the most
widely used method in the study of crystal structure. In this method a beam of
homogeneous X-ray is allowed to penetrate a small crystal at right angles. The crystal being
rotated around an axis parallel to one of the crystal axes. During the rotation of the crystal
various planes come successively into suitable positions for diffraction to occur and the
corresponding spots are observed on a photographic plate.
In fig. 14, A and B show points on two successive lattice planes. For diffraction maxima
to occur the difference (BR) in the path of two diffracted rays must be equal to whole
number of wavelengths i.e., equal to nλ. Since the value of n depends on the angle θ (BAR),
series of directions of diffraction corresponding to increasing value of n is obtained on the
photographic plat. Horizontal lines are seen for all lattice planes having the same spacing
(AB) in the direction parallel to the axis of rotation. Such lines are referred to as layer lines.
If the X of incident X-rays is known and the distance from the crystal to the photographic
plate and vertical distance between the layer lines is determined, it is possible to calculate
9 and hence the spacing of the planes AB. A set of spots in a transverse direction called
row lines is also observed on the photograph which is used to deduce lattice spacings and
the size of the unit cell.
3. Oscillating Crystal Method-
In oscillation method the crystal is oscillated through an angle of 15° to 20°. But the number
of reflecting positions exposed to the incident X-rays is limited. The oscillations of the
crystal are synchronized with the movement of the cylindrical photographic film. The
position of a spot on the plate indicates the orientation of the crystal at which the spot was
formed.
4. Powder Crystal Method-
Powder method was devised independently by P. Debye and P. Scherrer (1916) and A. W.
Hill (1917). This method employs powdered samples in which the crystals are oriented in
all directions so that some of the crystals will be properly oriented for a observable
reflections. A narrow beam of monochromatic X-rays is allowed on the finely powdered
specimen. The diffracted rays are then passed on to a strip of film which almost complete
surrounds the specimen. The random orientation of crystals produces diffraction rings or

66
caves rather than spots. The method is commonly employed for identification purposes by
comparing the observed spacing of the axes produced on the film. Extensive files of
spacings from powder photographs are available for comparison. For a cubic crystal the
identification of lines in the powder photograph is relatively simple. Also the indexing of
lines in hexagonal, rhombohedral, tetrahedral etc. is not very complicated. However, in
crystals of lower symmetry a large number of lines are observed which cannot be accurately
identified.
TYPES OF CRYSTALS:

67
APPLICATIONS OF X-RAY DIFFRACTION:
i. Study of polymorphism in drugs.
ii. Measurement of the average space ‘d’ between layers of atoms.
iii. Determination of the orientation of a single crystal and crystal structure of unknown
material.
iv. Measurement of size, shape and internal stress of small crystalline regions.
v. Measurement of thickness of thin films and multi layers.
vi. Determination of each type in mixed crystals.
vii. Size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences
among various materials, especially minerals and alloys.
viii. Used to study many materials which form crystals like salts, metals, minerals,
semiconductors, as well as various inorganic, organic and biological molecules.

68
6. IMMUNOLOGICAL ASSAY
RADIOIMMUNOLOGY ASSAY (RIA) & ELISA:

70
BIOLUMINESCENCE ASSAYS:
Bioluminescence is the production and emission of light by a living organism. It is a form of
chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as
well as in some fungi, microorganisms including some bioluminescent bacteria and terrestrial
invertebrates such as fireflies.
Bioluminescence assays involve the use of the property of bioluminescence for measuring cell
proliferation, apoptosis, drug metabolism, kinase activity, etc.
PRINCIPLE:
Bioluminescence is a form of chemiluminescence where light energy is released by a chemical
reaction. This reaction involves a light-emitting pigment, the luciferin, and a luciferase, the
enzyme component. Because of the diversity of luciferin/luciferase combinations, there are very
few commonalities in the chemical mechanism.
For example, the firefly luciferin/luciferase reaction requires magnesium and ATP and produces
carbon dioxide (CO2), adenosine monophosphate (AMP) and pyrophosphate (PP) as waste
products. Other cofactors may be required for the reaction, such as calcium (Ca2+
) for the
photoprotein aequorin, or magnesium (Mg2+
) ions and ATP for the firefly luciferase. Generically,
this reaction could be described as:
Luciferase
Other cofactors
Oxyluciferin Light energyO2Luciferin
HARNESSING BIOLUMINESCENCE:
1. Food testing using ATP (Bioluminescence) Technology-
The test kit for this test contain firefly luciferase and luciferin which are used to detect the
presence of ATP, a compound that is found in all living cells; this includes any live microbes
like Salmonella or E. coli that might be present in food and food products.
The intenisty of the light produced from the reaction is detected by a luminometer.
The more the ATP, the brighter the light, so the intensity of luminescence reveals how many
bacteria are present. This test is able to detect even tiny amounts of microbial contamination,
using very senstitive instruments to measure light production. It requires mere minutes instead
of the days needed to detect contaminated food by growing bacterial cultures.
2. Bioluminescent imaging-
Fireflies have helped scientists develop real-time, noninvasive imaging to see what's happening
inside living organisms.
When LUC (luciferase) genes are used to label particular cell or tissue types, very sensitive
cameras can be used to detect their light inside the live animal.

71
Mice bearing tumors are tagged with LUC gene which express luciferase and are further
injected with luciferin. Researchers then use a sensitive camera system to view, without killing
the mice, the tumor and any effects of the different cancer agents.
Tumor cells can be grown in culture medium and then treated with different drugs. Using
luminescence-based tests to measure cell viability, those drugs most effective at killing tumor
cells can be quickly identified. This way, potential new chemotherapies for treating cancers
can be tested.
3. Luciferase (LUC) gene as reporter for the activity of other genes-
Here, the researchers splice the LUC gene together with a specific gene they want to study,
and then insert this spliced DNA into living cells.
Whenevr the spliced DNA gets transcribed, the cells will manufacture luciferase. When
luciferin is added, these cells will respond by lighting up. This technque has been used, for
instance, to find out exactlty when and where specific plant genes get turned on.
To learn about particular genes regulating plant growth, biologists have spliced the LUC gene
into different bits of plant DNA. When plants are sprayed or fed with luciferin-containing
water, the leaves will glow whenever LUC gene gets turned on. This allows researchers to
identify specific genes regulating plant growth at different times and locations.
Such reporter genes have also provided tools for studying diseases, for developing new
antibiotic drugs and for gaining new insights into many human metabolic disorders.
4. Development of new treatment for antibiotic-resistant tuberculosis-
To help discover new treatments for antibiotic-resistant tuberculosis, scientists have infected
mice with luciferase-labeled tuberculosis bacteria. They then treat the mice with various anti-
tuberculosis drugs and use bioluminescence imaging to monitor the bacteria inside.
5. Measurement of calcium changes inside cells- The photoprotein aequorin requires Ca2+, it
is often used to measure calcium changed indside cells.
6. Discovery of green protein (GFP)-
One of the most well-known developments to come out of bioluminescence research is the
discovery of the green fluorescent protein (GFP). While GFP is not a bioluminescent protein,
it serves as an accessory emitter by receiving energy from a luciferin-luciferase reaction and
re-emitting it as green light.
**********************************************************************

72
REFERENCES:
1. Hollas, J. M. Modern Spectroscopy. John Wiley & Sons, Inc. England. 2004 (4).
2. Silverstein, R. M, Webster, F. X. Spectrometric Identification of Organic Compounds. John
Wiley & Sons, Inc. England. Sixth Edition.
3. Douglas, A. S, West, D. M. Principles of Instrumental Analysis. Saunders College, USA. 1980.
4. Pavia, D. L, Lampman, G. M, Kriz, G. S. Introduction to Spectroscopy. Thomson Learning,
Inc. USA. 2001 (3).
5. Kasture, A. V, Mahadik, K. R, Wadodkar, S. G, More, H. N. A Textbook of Pharmaceutical
Analysis Instrumental Methods. Nirali Prakashan, Pune. 2008 (17).
6. Sankar, S. R. Textbook of Pharmaceutical Analysis. Rx Publications, Tirunelveli. 2016 (4).
7. Satyanarayana, U, Chakrapani, U. Biochemistry. Books and Allied Pvt. Ltd Kolkata. 2007 (3).
8. Kealey, D, Haines, P. J. Instant Notes Analytical Chemistry. BIOS Scientific Publishers Ltd.
UK. 2002.

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