UNIT - 01

Q1)What is Void Volume? How does this relate to Dead Time? Please use any
chromatography technique of your choice to explain with example.
The void volume is the volume of the mobile phase that surrounds the stationary
phase in a chromatographic column. It is the volume that elutes before any solute
interacts with the stationary phase.
2. Importance:
Understanding void volume is essential for calculating retention times and
optimizing separation conditions. It helps in determining the efficiency of the
separation process.
3. Calculation:
The void volume can be calculated using the dimensions of the column and the
porosity of the packing material. For a cylindrical column, the void volume can be
approximated using the formula: VO = π× r² × h
Applications
Void volume chromatography is commonly used in various fields such as
1. Analytical Chemistry:
It helps in the analysis of complex mixtures by providing a baseline understanding
of how quickly unretained substances pass through the system.
2. Biochemistry:
Used in size-exclusion chromatography (SEC) to separate biomolecules based on
size, where smaller molecules penetrate the pores of the stationary phase and
larger molecules are excluded from these pores. where r is the radius of the co
Examples of Techniques
1. Size-Exclusion Chromatography (SEC):
Utilizes void volume to separate molecules based on size. Larger molecules elute
first because they cannot enter the pores of the stationary phase.
2. High-Performance Liquid Chromatography (HPLC):In HPLC systems, knowing the
void volume is critical for method development and optimization to ensure
accurate retention time calculatio0
3. Gel Filtration Chromatography:
A specific type of SEC used in biochemistry to purify proteins and nucleic acids,
where the void volume is crucial for understanding how different sizes of
biomolecules behave during separation
Conclusion
In summary, void volume chromatography is a fundamental concept in
chromatographic techniques that plays a crucial role in the separation and analysis
of various compounds. Understanding and calculating void volume helps improve
the efficiency and effectiveness of chromatographic methods across a wide range
of scientific and industrial application stationary phase .
Q2: Can centrifugation be considered a type of Chromatography? Please explain
• Centrifugation is a process used to separate or concentrate materials suspended
in a liquid medium. The theoretical basis of this technique is the effect of gravity
on particles (including macromolecules) in suspension, Two particles of different
masses will settle in a tube at different rates in response to gravity.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
This method is a Favorite in many labs because of its efficiency using a liquid (or
solvent) mobile phase, that pushes the sample through the machine using a
high-pressure pump. Additionally, since the machines do most of the work, this
method requires the least amount of training.
Uses for This Method: High-performance liquid chromatography is best for any
scenarios that require separating non-volatile mixtures. Industries that prefer this
method include:
Environmental Forensics Biotechnology
Chemical Research Pharmaceutical Beverage Research
Industrial
THIN-LAYER CHROMATOGRAPHY
With this method, lab technicians use a silica gel or alumina as its stationary phase
and coat it onto a thin glass or plastic plate. On the other hand, the mobile phase
is like high-performance liquid chromatography, in that it uses either a single
solvent or a mixture of solvents.
Uses for This Method: Thin-layer chromatography is best suited for smaller
experiments that require analyzing liquid or solvent compounds, the number of
components, or the purity of compounds in a mixture. The most common
industries that use this method include:
Food Chemistry Toxicology Agriculture (mostly for pesticide analysis)
Pharmaceuticals
PAPER CHROMATOGRAPHY
The mobile phase again is a solvent or mixture of solvents, and the stationary
is-you guessed it-paper. However, it's not any piece of paper, it's a very absorbent
piece that allows for liquid to pass through the pores.
Uses for This Method: Paper chromatography is most commonly used in forensics,
as it helps forensic scientists identify DNA from fingerprints. But this method is
useful in the following industries and for the following uses as well:

Food Coloring Plant Pigment Reaction

Monitoring
Q3)What is Gas Chromatography (GC)? What type of analytes are separate using
GC?
GAS CHROMATOGRAPHY
This method is one of the most common in the chromatography world. Like all
other forms of chromatography, it has a mobile phase and a stationary phase. In
this case, the mobile phase is an inert gas, and the stationary phase is a column
within the machine.
Uses for This Method: Gas chromatography is ideal in any scenario that needs to
separate volatile mixtures. Industries that commonly use this method of
chromatography include:
• Pharmaceuticals Industrial Medicine
Food and Beverage • Agriculture Cosmetics and Cleaning
Environmental
Applications of Gas Chromatography
•G.C is capable of separating, detecting & partially characterizing the organic
compounds, particularly when present in small quantities.
Qualitative Analysis-by comparing the retention time or volume of
the sample to the standard/by collecting the individual components as they
emerge from the chromatograph and subsequently identifying these compounds
by other method
Quantitative Analysis area under a single component elution peak is proportional
to the quantity of the detected component/response factor of the detectors.
• Used for analysis of drugs & their metabolites.
Checking the purity of a compound.
1.3. Gas Chromatography Separation

Separation by GC occurs within the column. The sample containing multiple

compounds is injected into the column together with the mobile phase. (In GC,
the mobile phase is a gas referred to as the carrier gas. He is frequently used.)
Both the sample and the mobile phase travel through the column, but the rate of
progression within the column differs depending on the compound. Accordingly,
differences arise in the times at which the respective compounds arrive at the
column outlet. As a result, a separation between each compound occurs.
The row of peaks drawn when the electrical signals output from the GC detector
are plotted on the vertical axis and the elapsed time after sample injection is
plotted on the horizontal axis is called a chromatogram.
The components passing through the column are transported by the mobile phase
(gas phase) while being partitioned from and adsorbed into the stationary phase
(liquid phase and solid phase).
Q4: Please define Gel Filtration Chromatography and how this technique helps
separate molecules based on their molecular size.
Gel filtration (GF) chromatography
separates proteins solely on the basis of molecular size. Separation is achieved
using a porous matrix to which the molecules, for steric reasons, have different
degrees of access--i.e., smaller molecules have greater access and larger
molecules are excluded from the matrix. Hence, proteins are eluted from the GF
column in decreasing order of size. This unit describes the experimental theory
behind gel filtration and contains many useful tables listing the properties and
characteristics of currently available matrices.
Gel permeation chromatography Gel permeation chromatography (sizexclusion
chromatography) can be used on humic acids or fulvic acids in water and is
frequently applied to the analysis of fatty samples such as fish. Gel permeation
chromatography is a very effective method based on differences in molecular
mass. Large molecules are excluded from the pores of the gels and are eluted first.
This technique can easily be automated. However, after gel permeation
chromatography for fractionation, additional liquid-solid columchromatography is
still necessary. Thus, there is little advantage in using gel permeation
chromatography for the cleanup of less
Q5:Please describe the detection system used in High Performance Liquid
Chromatography
Types of detectors used in HPLC
In order to quantify and identify the compounds separated in the column, HPLC
analysis requires a detector to monitor the compounds themselves. The sort of
detector used depends on what is being detected. It's crucial to think about the
type of sample you'll be analysing before choosing a detector. On some detectors,
certain sample types will not operate or will have lesser sensitivity. Some
detectors destroy the sample to detect it.
The following are some significant aspects to look for in HPLC detectors (1):
• Each molecule in the sample mixture must be detected by the detector.
• Variations in the system's flow rate, temperature, mobile phase composition,
and pressure should not be affected by the detector.
• It should be unaffected by the solvent or mobile phase employed in the
experiment.
• It should be able to detect both high and low solute concentrations.
• It should respond to linear concentrations in a linear manner.
This should result in outcomes that are repeatable and consistent.
• In isocratic and gradient elution, it should have no effect on the mobile phase
composition response.
Types of detectors
There are two categories: specific and bulk property detectors (2)
Specific HPLC Detectors
1. Mass Spectroscopic Detectors
The sensitivity and selectivity of a mass spectroscopy detector are both high. The
detection is based on electric field-induced molecular fragmentation, and the
separation is based on the mass to charge ratio of fragmented molecules . This is a
destructive detector.
2. UV/VIS HPLC Detector
The ultraviolet (UV) or visible (VIS) detector is the most common in HPLC because
it provides good stability, is simple to operate, and has a high sensitivity (1). Fixed
wavelength detectors, variable wavelength detectors, and diode array detectors
are the three types of UV/VIS detectors.
3. Photodiode Array (PDA) Detectors PDA is a UV detector and because it has
numerous individual diodes, resolution elements, or pixels, it can detect several
wavelengths at the same time (1). In a UV/VIS dispersive spectrophotometer, this
is a typical ideal detector for the full spectrum (1).
4. Fluorescence Detectors
Fluorescence detectors have great sensitivity for specific groups of components
(1). A certain wavelength is used to stimulate the component atoms, which then
release a light signal (1). The intensity of the radiated light is utilised to determine
the component concentration (1). Fluorescent absorbance can be found in a
variety of natural materials, medicines, petroleum products, and clinical samples
(1).
Bulk Property HPLC Detectors
1. Electrical Conductivity Detectors Conductivity detectors are bulk property
detectors since they are used to determine conductivity, and of their main
features is high-sensitivity detection of charged species and surfactants (1). They
detect electrical resistance, and the observed result is proportional to the ion
concentration in the solution (1).
2. Refractive Index Detectors (RID) A refractive index detector measures the
analyte's refractive index in relation to the solvent. The deflection and reflection
of light in solution are the two concepts that it relies on (1). There are different
RID detectors such as the thermal lens detector, the Christiansen effect detector,
the dielectric constant detector, and the interferometer (1).
3. Electrochemical Detectors
The electrochemical detector is used to identify compounds that are undergoing
oxidation-reduction reactions and to quantify the electric currents generated as a
result of these events (1). Because the voltage required for the
oxidation-reduction reaction varies depending on the molecule, the
electrochemical detector has a high sensitivity and selectivity (1). This is a
destructive detector.
4. Light Scattering Detectors
The dispersed light emitted by the eluent is detected using a light scattering
detector (1). It is used for molecules with a significant molecular weight, for
instance, surfactants, lipids, and sugar (1). There are two types of light scattering
detectors, known as low angle laser light scattering detector and multiple angle
laser light scattering detector (1). This is a destructive detector.
5. Charged Aerosol Detectors
The Charged Aerosol Detector (CAD) is a detector that is used in HPLC to measure
the amount of chemicals in a sample by producing charged aerosol particles that
are then detected using an electrometer (3). It's widely used to analyse
substances that don't have a chromophore and can't be detected using typical
UV/Vis methods (3).
Charged aerosol detectors measure different analytes in the areas of
pharmaceuticals (large and small molecules), biomolecules, foods and beverages,
specialty chemicals, and polymers (3).
At the Bio-Analysis Centre, our HPLC services use the Shimadzu Nexera XR system
(Fig.1), and, at the moment, we have PDA and RID
Q6:What is a fraction collector? How would you use this in a Chromatographic
separation?
Chromatography Fraction Collector
The fraction collector is typically a rotating rack that can be filled with test tubes
or similar containers. It allows samples to be collected in fixed volumes, or can be
controlled to direct specific fractions detected as peaks of protein concentration,
into separate containers.
Many systems include various optional components. A filter may be added
between the mixer and column to minimize clogging. In large FPLC columns the
sample may be loaded into the column directly using a small peristaltic pump
rather than an injection loop. When the buffer contains dissolved gas, bubbles
may form as pressure drops where the buffer exits the column; these bubbles
create artifacts if they pass through the flow cells. This may be prevented by
degassing the buffers, e.g. with a degasser, or by adding a flow restrictor
downstream of the flow cells to maintain a pressure of 1-5 bar in the eluant line.
Fractionation, or the separation of a mixture of molecules into different parts or
"fractions,” helps scientists understand and/or use a complex solution. Collecting
and isolating individual fractions from a mixture can enable many downstream
applications as specific molecules can be separated based on physical and
chemical properties (ex: size, charge, polarity, etc.).
Applications that Use Fraction Collection
Fraction collection requires a flow managed by gravity, pump, or pressurization.
Fraction collectors are often integrated with chromatographic
purification/separation systems (HPLC, LC/MS, or flash chromatography).
Molecules are separated, and analytes of interest are collected as they flow out of
the stationary phase. These applications include:
1. Purifying molecules of interest. When purifying proteins, peptides,
oligonucleotides, or other molecules, fractionation separates molecules of interest
from side reaction products such as modified forms of the proteins, truncated
oligonucleotides, lipids, and other small molecules. Collected fractions are then
analyzed to identify those that contain the molecule of interest for further
processes in the workflow (ex: evaporation).
2. Removing specific contaminant(s). For residue analysis from environmental,
food, or tissue samples, gel permeation chromatography (GPC) is used to separate
contaminants that may interfere with residue analysis. As the solution exits the
GPC column, only the fraction containing the family of analytes of interest is
isolated for further analysis. The contaminants that can harm the analytical
equipment are discarded.
Q7: What is Immobilized metal affinity chromatography (IMAC)?
Immobilized Metal Affinity Chromatography
Metal-Chelate Affinity Chromatography (MCAC), also known as Immobilized Metal
Affinity Chromatography (IMAC), was first successfully demonstrated in 1975 by
Porath and collaborators for human serum proteins.

Principle
• Transition metal ions are used, electron pair acceptors.
e.g. Cu2+, Ni2+, Zn2+, Co2+, Fe3+
Co-ordination between immobilized metal and electron donor from protein
surface.
Electron donors (N,S,O) present in chelating compound attached chromatographic
support forms metal chelates, which can be monodentate or multidentate
Denticity: No. of donor groups in a single ligand that bind to central atom in
coordination complex.
Remaining metals sites are occupied by water molecules and are exchanged by
electron donor from protein
Immobilized metal affinity
chromatography Immobilized metal affinity chromatography (IMAC) is a
specialized variant of affinity chromatography where the proteins or peptides are
separated according to their affinity for metal ions that have been immobilized by
chelation to an insoluble matrix. At pH values around neutral, the amino acids
histidine, tryptophan, and cysteine form complexes with the chelated metal ions
(e.g., Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, and Fe2+). They can then be eluted by
reducing the pH, increasing the mobile phase ionic strength, or adding
ethylenediaminetetraacetic acid (0.05 M) to the mobile phase. This technique is
especially suited for purifying recombinant proteins as poly-histidine fusions and
for membrane proteins and protein aggregates where detergents or
high-ionic-strength buffers are required.
Q8: Can one use isocratic elution with Ion-Exchange chromatography? Please
explain
. • Chromatography is the separation of a mixture of compounds into its individual
components based on their relative interactions with an inert matrix.
• Ion exchange chromatography (or ion chromatography) is a process that allows
the separation of ions and polar molecules based on their affinity to ion
exchangers.
The principle of separation is thus by reversible exchange of ions between the
target ions present in the sample solution to the ions present on ion exchangers.
In this process two types of exchangers i.e., cationic and anionic exchangers can
be used.
1. Cationic exchangers possess negatively charged group, and these will attract
positively charged cations. These exchangers are also called "Acidic ion exchange"
materials, because their negative charges result from the ionization of acidic
group.
2. Anionic exchangers have positively charged groups that will attract negatively
charged anions. These are also called "Basic ion exchange" materials.
Ion exchange chromatography is most often performed in the form of column
chromatography. However, there are also thin-layer chromatographic methods
that work basically based on the principle of ion exchange.
Cation exchange chromatography is a form of ion exchange chromatography (IEX),
which is used to separate molecules based on their net surface charge. Cation
exchange chromatography, more specifically, uses a negatively charged ion
exchange resin with an affinity for molecules having net positive surface charges.
Cation exchange chromatography is used both for preparative and analytical
purposes and can separate a large range of molecules from amino acids and
nucleotides to large proteins. Here, we focus on the preparative cation exchange
chromatography of proteins.
Q9: What is silanol in the context of a glass column? Why is this a potential source
of issue in Chromatography?
A silanol is a functional group in silicon chemistry with the connectivity Si-O-H. It
is related to the hydroxy functional group (C-O-H) found in all alcohols. Silanols are
often invoked as intermediates in organosilicon chemistry and silicate mineralogy.
[1] If a silanol contains one or more organic residues, it is an organ.

Q10: How is Thin Layer Chromatography superior to Paper Chromatography

Thin layer chromatography (TLC) is similar to paper chromatography but instead of
paper, the stationary phase is a thin layer of an inert substance (eg silica)
supported on a flat, unreactive surface (eg a glass plate).
TLC has some advantages over paper chromatography. For example:
• the mobile phase moves more quickly through the stationary phase
• the mobile phase moves more evenly through the stationary phase
• there is a range of absorbencies for the stationary phase
TLC tends to produce more useful chromatograms than paper chromatography,
which show greater separation of the components in the mixture - and are
therefore easier to analyse.
The distance a sample travels can depend on the size or the polarity of the
molecules involved. Larger molecules take longer to move up the chromatography
paper or TLC plate, whereas smaller molecules are more mobile.
Likewise, the polarity of the molecules can affect how far the spots travel,
depending on the type of solvent used. Polar molecules will be more strongly
attracted to polar solvents, and so would move further if a polar solvent was used
as opposed to a non-polar solvent.
The distance that spots move can be compared to the overall distance the solvent
has moved and comparisons and measurements made.
 UNIT - 02

Q1:Please describe the steps and reagents used to cast a) Agarose Gel, b)
Polyacrylamide Gel (Native PAGE), c) Sodium-dodecyle Sulphate Polyacrylamide
Gel (SDS-PAGE). In what situations are you likely to use each kind of gel? What is
sample denaturation?
• Agarose gel electrophorresis is a method to separate DNA or RNA molecules by
size.
• This is achieved by moving negatively charged nucleic acid molecules through an
agarose matrix with an electric field (electrophoresis).
Shorter molecules move faster and migrate faster than longer ones.
Polyacrylamide Gel Electrophoresis (PAGE)
Electrophoresis through agarose or polyacrylamide gels is a standard method used
to separate, identify and purify biopolymers, since both these gels are porous in
nature.
• Polyacrylamide gels are chemically cross-linked gels formed by the
polymerization of acrylamide with a cross-linking agent, usually
N,N'-methylenebisacrylamide.
The reaction is a free radical polymerization, usually carried out with ammonium
persulfate as the initiator and N,N,N',N'-tetramethylethylendiamine (TEMED) as
the catalyst.
• Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in
biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to
separate biological macromolecules, usually proteins or nucleic acids, according to
their electrophoretic mobility.
The most commonly used form of polyacrylamide gel electrophoresis is the
Sodium dodecyl suplhate Polyacrylamide gel electrophoresis (SDS-PAGE) used
mostly for the separation of proteins. Sodium Dodecyl Sulphate-Polyacrylamide
gel electrophoresis
The main objective of this experiment is to determine the relative mobility of the
separated protein bands of each sample obtained from SDS-PAGE gel, followed by
western blotting. Besides, to derive the molecular weight of each separated
protein bands based on the protein marker standards of known molecular weight
used to plot the standard curve. In addition, to determine the presence and
molecular weight of bovine serum albumin (BSA).
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a
commonly used laboratory technique to separate protein molecules on a porous
matrix based on their charges and molecular weight (MW). Polyacrylamide is
known as the most frequently used support matrix in running SDS-PAGE due to its
characteristic of small pore size which is able to separate out majority of the
proteins

Q2: What are Ampholytes? How do they relate to Zwitter ions in solution
Ampholytes ( Amphotrics )
They have very low irritation potential and are almost completely non- stinging to
the eyes.
Can be formulated in many types of shampoos.
Should avoid low pH when formulating because the amphotric become positively
charged since this can result in increased irritation.

Ampholytes are amphoteric molecules that contain both acidic and basic
functional groups. For example, an amino acid H2N-RCH-CO₂H has both a basic
group -NH2 and an acidic group -COOH, and exists as several structures in
chemical equilibrium:
H2N-RCH-CO₂H + H2O H2N-RCH-COO- + H3O+ H3N+-RCH-COOH + OH-
HN-RCH-COO- + H₂O
In approximately neutral aqueous solution (pH = 7), the basic amino group is
mostly protonated and the carboxylic acid is mostly deprotonated, so that the
predominant species is the zwitterion H3N-RCH-COO". The pH at which the
average charge is zero is known as the molecule's isoelectric point. Ampholytes
are used to establish a stable pH gradient for use in isoelectric focus. Metal oxides
which react with both acids as well as bases to produce salts and water are known
Metal as amphoteric oxides. Many metals (such as zinc, tin, lead, aluminium, and
beryllium) form amphoteric oxides or hydroxides. Aluminium oxide (Al2O3) is an
example of an amphoteric oxide. Amphoterism depends on the oxidation states of
the oxide. Amphoteric oxides include lead(II) oxide and zinc oxide

Q3:What is pulsed-field gel electrophoresis (PFGE)? Is it possible to use

periodically changing electric fields to conduct capillary and paper
electrophoresis? Please explain.
PULSED-FIELD GEL ELECTROPHORESIS
Pulsed field gel electrophoresis is a technique used for the separation of large
DNA molecules by applying to a gel matrix an electric field that periodically
changes direction.
While in general small fragments can find their way through the gel matrix more
easily than large DNA fragments, a threshold length exists above 30-50 kb where
all large fragments will run at the same rate, and appear in a gel as a single large
diffuse band. However, with periodic changing of field direction, the various
lengths of DNA react to the change at differing rates. Over the course of time with
the consistent changing of directions, each band will begin to separate more and
more even at very large lengths. Thus separation of very large DNA pieces using
PFGE is made possible.
The voltage is periodically switched among three directions; one that runs through
the central axis of the gel and two that run at an angle of 60 degrees either side.
The pulse times are equal for each direction resulting in a net forward migration
of the DNA.
This procedure takes longer than normal gel electrophoresis due to the size of the
20 fragments being resolved and the fact that the DNA does not move in a straight
line through the gel.
Q4:How does a pH electrode/meter work
Working Principle of pH Meter
A pH meter is used to determine the acidity or alkalinity of the solution. pH is the
concentration of hydrogen ions in the solution. A solution containing more H+ ions
remains acidic while the solution containing more OH ions remains alkaline. pH
value of solutions ranges from 1 to 14.
The solution having pH value 1 will be the highly acidie and with pH value 14 will
be highly basic. The acidity and alkalinity of any solution depend upon the
concentration of hydrogen ions (H) and hydroxyl ions (OH) respectively. A neutral
solution as pure water has pH 7.
pH meter is used to determine the pH of different solutions in pharmaceuticals. It
is more accurate method then the pH strip. A pH meter contains a pH probe that
passes the electrical signals to the pH meter and pH meter displays the pH value
of the solution.
Q5: Please define Isoelectric point. How does this relate to the technique of
Isoelectric focusing?
ISOELECTRIC POINT
• The ph. at which net charge on protein becomes zero.
Proteins are separated in a pli gradient according to their isoelectric points.
Proteins are amphoteric molecules with acidic and basic buffering groups (side
chain).
Proteins are positively charged in solutions at pH values below pl and migrate
towards cathode.
Proteins are negatively charged in solutions at pH values above pl and migrate
towards anode.
Iso-electric focussing
IEF is predicated on the fact that the charge on amino acid side- groups is
dependent on the pH of the surrounding solution. Proteins will migrate to a point
in the pH gradient corresponding with an isoelectric point or pH (pl) where there
is no net charge.
Q6: Please name any 5 buffer systems and their operational pH
pH Buffering Systems and Solutions
Making a pH Buffer Solution
"A buffer solution is water mixed with a chemical to give it special properties in
regards to pH (acidity). The chemical, known as a buffer agent, resists pH changes
when exposed to acids and bases when properly mixed in a solution. This property
makes it extremely useful in protecting sensitive equipment, dealing with
chemical accidents, and even in balancing the internal processes of "living things."
A great example of a buffered solution is your blood. It is the combination or
presence of carbonic acid (H2CO3) and bicarbonate (HCO3-) in the blood that
keeps the pH of the blood in the proper range (Source).
five buffer systems and their operational pH:
• Citric acid - Na2HPO4 Buffer: pH 2.6- 7.6
• Citric acid – Sodium Citrate Buffer: pH 3.0-6.2
Sodium Acetate - Acetic Acid Buffer: pH 3.7-5.6
• Na2HPO4 – NaH2PO4 Buffer: pH 5.8- - 8.0 at 25 °C
Imidazole (glyoxaline) – HCI Buffer: pH 6.2-7.8 at 25 °C
Q7:Using a viral vector system, you have introduced an exogenous gene into a cell
system of your choice. How would you use Electrophoresis to identify if your
recombinant protein is properly expressed?
Viral vectors
The viral vector can deliver nucleic acid into the target cells with high efficiency. In
conjugation with viral vectors miRNAscan also be carried into tumor cells.
Different types of viral vectors areused as a delivery system such as (i)
adenoviruses, (ii) adeno-associated viruses, (iii) retroviruses, (iv) lentiviruses, and
(v) hybrid vectors. In hepatoma cancer cells, miRNA replacement therapy was
utilized to deliver miR- 26a to the cells. A self-complementary adeno- associated
vector was utilized to clone the nucleic acid of miR-26a, which was further
conjugated with a green fluorescent protein. This conjugated molecule was
administrated to a tumor-bearing animal Another research group used a
lentivirus-encoded miRNA delivery system. The study utilized lentivirus-encoded
miR-15-16 in the xenograft prostate cancer mice model. One week after
treatment, the cancer cell growth was found arrested. While tumor treated with
an empty viral vector showed no significant growth change
Recombinant protein expression in bacteria requires the insertion of a DNA
fragment (open reading frame, ORF) into an expression vector, routinely a plasmid
vector and the transferral of this vector into bacterial cells (transformation). The
cells are then cultured and induced to express the desired protein. The cells are
harvested by centrifugation, samples prepared and proteins detected by
polyacrylamide gel electrophoresis and subsequent staining of the gel with
Coomassie Brilliant Blue or silver stain or by immunoblotting. Protein expression
in bacteria is highly scalable and can be adjusted from solid growth (colony
expression), to conical flasks for liquid cultures (up to 3 1), to fermentation
reaction chambers (often more than 100 l volume).
Q8: What is 2D Electrophoresis? In what situations would one use this technique
Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for
proteomics work. It is used for separation and fractionation of complex protein
mixtures from biological samples. 2-DE separates proteins depending on two
different steps: the first one is called isoelectric focusing (IEF) which separates
proteins according to isoelectric points (pl); the second step is SDS-
polyacrylamide gel electrophoresis (SDS- PAGE) which separates proteins based on
the molecular weights (relative molecular weight, Mr). Thus, thousands of
proteins can be separated, and the information about IEF and molecular weights
can be obtained. 2-DE is a widely used method for protein analysis with the ability
to separate thousands of proteins at one time. It can provide direct visual
information of changes in protein/post- translational modifications (PTMs)
abundance. It can also be used to other analysis, such as whole proteome
analysis, detection of biomarkers, drug discovery, and so on. There are several
Q9:Why is it important to place the support medium (gel) in a buffer tank?
Buffer Tank
A buffer tank is a storage tank used on the cold user side of an air-conditioning
system. The tank is used as storage to cover peak loads or in situations when a
surge in demand exceeds the capacity of the cooling system.
The system can be the secondary side of a traditional compressor-driven system
or a free-cooling system, where perhaps cooling only occurs at night. The buffer
tank is a container in which the cooled medium can be stored.
A buffer tank is typically used when there is a variable cooling requirement. In
such applications the tank is used as storage to cover peak loads or in situations
when a surge in demand exceeds the capacity of the cooling system. A buffer
tank's working principle is to store excess heat or cooling energy and release it
when needed:
Heating systems
A buffer tank stores excess heat generated by a system and releases it when
needed. This helps maintain a consistent temperature, saves energy, and provides
instant hot water. Buffer tanks are often used with heat pumps to prevent the
heat pump from short cycling, which can reduce efficiency and compressor life.
Cooling systems
A buffer tank stores cooled medium to cover peak loads or when demand exceeds
the cooling system's capacity. This reduces the number of times the cooling
system starts, which can decrease energy consumption and wear on the
compressor.
Q10: Please write out the steps of performing a DNA separation on Agarose. How
can the pore size be controlled by the experimenter?
The steps for performing a DNA separation on agarose are:
1. Prepare the gel: Measure the amount of agarose powder needed for the
desired gel percentage, usually between 0.7% and 2%. Mix the agarose powder
with a running buffer, microwave until dissolved, and let it cool. Pour the agarose
into a gel tray with a comb in place. Let the gel solidify.
2. Prepare the DNA samples: Add loading dye to the DNA samples.
3. Load the gel: Load the DNA into the wells in the gel.
4. Run the gel: Connect the gel box leads to the power supply, and turn it on. Set
the voltage and time for the run. Run the gel until the dye has migrated to the
desired distance.
5. Stain and visualize: Transfer the gel to a membrane and use a digital imaging
system to visualize the DNA.
6. Extract the DNA: Cut out the desired DNA fragments from the gel using a clean,
sterile razor blade. This process is called elution.
DNA fragments separate by size in the gel, with shorter fragments migrating faster
than longer fragments. The distance traveled by a DNA fragment is inversely
proportional to the log of its molecular weight. To estimate the size of the DNA
samples, include a reference sample with known nucleic acid sizes in the gel.
 UNIT - 03

Q1:Please define the principle of UV-Visible Spectroscopy. What components

would one need for such detection of biomolecules/biosensors
UV-Vis spectroscopy is based on the interaction between light and matter. When
light passes through or is absorbed by a molecule, it can cause the molecule to
vibrate. The wavelength of light that is most strongly absorbed by a molecule is
called the absorption maximum. By measuring the absorbance of light at different
wavelengths, it is possible to identify and characterize molecules.
• The principle of operation for a spectrophotometer is that the wavelength of
light is inversely proportional to the size of its aperture.
• This means that shorter wavelengths will pass through a smaller aperture than
longer wavelengths.
• The monochromator in a UV-Vis spectrophotometer is a disk with a series of slits
that can be adjusted to select the desired wavelength
Yes, UV-visible spectroscopy (UV- Vis) is a technique used to detect biomolecules
and biosensors:
Biomolecules
UV-Vis spectroscopy can identify biomolecules by using absorptions pectroscopy
to characterize molecules in the ultraviolet and visible wavelength ranges. When
biomolecules absorb UV/Vis light, their chromophores become excited and emit
characteristic spectra that help
identify the specific biomolecules. For example, UV-Vis spectroscopy can identify
phytochemicals by using maximum absorption (max) values.
Biosensors
Uv-vis Spectrophotometry can be used to detect proteins with a biosensor
scaffold medium, such as a cellulose dialysis membrane.
Q2: What is Fluorescence and how is it different from Phosphorescence?
Fluorescence is a type of luminescence, occurs in gas, liquid or solid chemical
systems.
Fluorescence's energy is supplied by the electromagnetic radiation, usually the
ultraviolet light.
Fluorescence is an optical phenomenon in which a molecule absorbs a
high-energy photon, and re-emits it as a lower-energy (longer-wavelength)
photon, the energy difference between the absorbed and emitted photons ending
up as molecular vibrations (heat). Usually the absorbed photon is in the
ultraviolet, and the emitted light (luminescence) is in the visible range.
Fluorescence and phosphorescence are both processes that involve a substance
absorbing light and then emitting light of a longer wavelength. The main
difference between the two is the time it takes for the light to be emitted:
Fluorescence
Occurs when a substance emits light almost immediately after absorbing it.
Fluorescence is usually only visible when the light source is continuously on.
Phosphorescence
Occurs when a substance stores the absorbed light energy and releases it over
time, resulting in an afterglow.Phosphorescence can last from a few seconds to
hours, depending on the material.
Q3: What is Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a
strong constant magnetic field are disturbed by a weak oscillating magnetic field
(in the near field[1]) and respond by producing an electromagnetic signal with a
frequency characteristic of the magnetic field at the nucleus. This process occurs
near resonance, when the oscillation frequency matches the intrinsic frequency of
the nuclei, which depends on the strength of the static magnetic field, the
chemical environment, and the magnetic properties of the isotope involved; in
practical applications with static magnetic fields up to ca.
20 tesla, the frequency is similar to VHF and UHF television broadcasts (60-1000
MHz). NMR results from specific magnetic properties of certain atomic nuclei.
High-resolution nuclear magnetic resonance spectroscopy is widely used to
determine the structure of organic molecules in solution and study molecular
physics and crystals as well as non-crystalline materials. NMR is also routinely
used in advanced medical imaging techniques, such as in magnetic resonance
imaging (MRI). The original application of NMR to condensed matter physics is
nowadays mostly devoted to strongly correlated electron systems. It reveals large
many-body couplings by fast broadband detection and should not be confused
with solid state NMR, which aims at removing the effect of the same couplings by
Magic Angle Spinning techniques.
Applications
NMR is extensively used in medicine in the form of magnetic resonance imaging.
NMR is widely used in organic chemistry and industrially mainly for analysis of
chemicals. The technique is also used to measure the ratio between water and fat
in foods, monitor the flow of corrosive fluids in pipes, or to study molecular
structures such as catalysts.
The application of nuclear magnetic resonance best known to the general public is
magnetic resonance imaging for medical diagnosis and magnetic resonance
microscopy in research settings. However, it is also widely used in biochemical
studies, notably in NMR spectroscopy such as proton NMR, carbon-13 NMR,
deuterium NMR and phosphorus-31 NMR. Biochemical information can also be
obtained from living tissue (e.g. human brain tumors) with the technique known
as in vivo magnetic resonance spectroscopy or chemical shift NMR microscopy
Q4:What is the principle of Gas Chromatography - Mass Spectroscopy (GCMS)?
What type of samples can be analyzed using this technique?
The principle of gas chromatography- mass spectrometry (GC-MS) is to separate
chemical mixtures and identify their components at a molecular level:
Gas chromatography (GC)
Separates a mixture's volatile components by heating the sample into a vapor. The
sample is injected into the GC inlet and carried through a column by a carrier gas,
such as helium. The compounds separate as they interact with the column's
coating and the carrier gas.
Mass spectrometry (MS)
Identifies and measures the vaporized compounds separated in the GC. The MS
ionizes the compounds and separates them based on their mass-to-charge ratios.
Gas chromatography-mass spectrometry (GC-MS) is an analytical method that
combines the features of gas-chromatography and mass spectrometry to identify
different substances within a test sample. Applications of GC-MS include drug
detection, fire investigation, environmental analysis, explosives investigation, food
and flavor analysis, and identification of unknown samples, including that of
material samples obtained from planet Mars during probe missions as early as the
1970s. GC-MS can also be used in airport security to detect substances in luggage
or on human beings. Additionally, it can identify trace elements in materials that
were previously thought to have disintegrated beyond identification. Like liquid
chromatography-mass spectrometry, it allows analysis and detection even of tiny
amounts of a substance.
Applications
Environmental monitoring and cleanup
GC-MS is becoming the tool of choice for tracking organic pollutants in the
environment. The cost of GC-MS equipment has decreased significantly, and the
reliability has increased at the same time, which has contributed to its increased
adoption in environmental studies.
Criminal forensics
GC-MS can analyze the particles from a human body in order to help link a
criminal to a crime. The analysis of fire debris using GC-MS is well established,and
there is even an established American Society for Testing and Materials (ASTM)
standard for fire debris analysis. GCMS/MS is especially useful here as samples
often contain very complex matrices, and results used in court need to be highly
accurate.

Q5:Please list out all the differences between Visible and Infrared (IR)
Spectroscopy.
Visible and infrared spectroscopy differ in the following ways: @
Wavelength range
Visible spectroscopy uses wavelengths from 400 to 800 nanometers (nm), while
infrared (IR) spectroscopy uses wavelengths from 700 to 1000 nm.
Energy
IR spectroscopy uses a longer, lower- energy wavelength range than visible
spectroscopy.
Technique
IR spectroscopy measures the light energy required to start molecular vibrations
in a sample, while UV-Vis spectroscopy can use aqueous solutions at very low
concentrations.
Quantitative vs qualitative
IR spectroscopy is largely qualitative, while UV-Vis spectroscopy can be highly
quantitative.
Applications
IR spectroscopy is particularly useful for chemical identification, while UV-Vis
molecular absorption is routinely used for drug testing and the analysis of
narcotics.Both visible and infrared radiation are part of the electromagnetic
radiation spectrum, which is separated into seven different categories based on
their wavelengths.

Q6: Please describe Beer-Lambert’s law and how it relates to Spectroscopy

The Beer-Lambert law, also known as Beer's law, describes the relationship
between the attenuation of light through a substance and the properties of that
substance. It states that the amount of radiation absorbed by a sample is directly
proportional to the product of its molar absorptivity, path length, and
concentration.
The Beer-Lambert law is used in chemical analysis to determine the concentration
of chemical species that absorb light. It allows the concentration of a solution to
be calculated by measuring its absorbance.
The Beer-Lambert law is also known as the extinction law, and is used in The
Beer-Lambert law is the basis of quantitative spectroscopy and is used in
spectrophotometry to measure the concentration of a substance in a solution:
Explanation
The Beer-Lambert law states that the amount of light absorbed by a solution is
directly proportional to the concentration of the substance in the solution, the
path length of the light through the solution, and the molar absorptivity of the
substance.understanding attenuation in physical optics.
Equation
The Beer-Lambert law is expressed as the equation A = ɛbc, where:
• A: is the absorbance of light by the substance
• ε: is the molar absorptivity of the substance
• b: is the path length of the light through the solution @
• c: is the concentration of the substance in the solution
Application
The Beer-Lambert law allows the concentration of a substance in a solution to be
calculated by measuring the absorbance of the solution. This is done by creating a
calibration curve of absorbance versus concentration from a series of solutions
with known concentrations.
Q7:What is Circular Dichroism (CD)? What types of molecules may be analyzed
using CD? How is chirality important to CD?
Circular Dichroism (CD) spectroscopy is a form of light absorption spectroscopy
which is used in determining molecular behaviour in a broad spectrum of
perturbation by measuring the difference in absorbance of right-circularly
polarized (RPR) and left-circularly polarized (LCP) light.
Circular dichroism (CD) spectroscopy is the technique of choice to study chiral
molecules in solution, in particular biologically important molecules such as
proteins, nucleic acids, carbohydrates, and therapeutic drugs.
Circular dichroism (CD) spectroscopy can be used to analyze many types of
molecules, including proteins, nucleic acids, carbohydrates, and
therapeutic drugs:
• Proteins: CD can determine the secondary structure of proteins, such as a-helix,
ẞ-sheets, turns, and random structures. It can also be used to study
protein-folding, stability, and ligand binding.
Nucleic acids: CD can be used to study nucleic acids.
• Carbohydrates: CD can be used to study polysaccharides. @
• Therapeutic drugs: CD can be used to study therapeutic drugs.
Application of CD
A primary use is in analysing the secondary structure or conformation of
macromolecules, particularly proteins as secondary structure is sensitive to its
environment, temperature or pH, circular dichroism can be used to observe how
secondary structure changes with environmental conditions or on interaction with
other molecules. Structural, kinetic and thermodynamic information about
macromolecules can be derived from circular dichroism spectroscopy.
Q8: What is a biosensor? Please describe the principle, design, and any 2
applications
Principle of a Biosensor
The desired biological material (usually a specific enzyme) is immobilized by
conventional methods (physical or membrane entrapment, non-covalent or
covalent binding). This immobilized biological material is in intimate contact with
the transducer. The analyte binds to the biological material to form a bound
analyte which in turn produces the electronic response that can be measured.
In some instances, the analyte is converted to a product which may be associated
with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer
can convert the product linked changes into electrical signals which can be
amplified and measured.
The principle of a biosensor is to measure the effects of a substance on a
biological element, and then convert that response into a measurable signal:
1. Biological element
A biological material, such as an enzyme, antibody, or nucleic acid, that interacts
with the analyte.
2. Transducer
A physicochemical device that converts the biological response into a measurable
signal, such as light, heat, pH, charge, or mass change.
3. Electronics
Amplifies and converts the signal from analog to digital.
4. Display
Quantifies the processed signal and generates an output, such as a numeric,
graphic, tabular, or image.
Biosensor design. A successful biosensor is composed of two main components,
mainly a biological receptor or sensor element and a transducer.
A signal processing unit that usually contains a display or printer is normally used
in conjunction to a biosensor as depicted
Applications of biosensors:
• Biosensors provide the following advantages over lab-based equipment:
• Size is small.
• Cost-effective
• Quick outcomes
• Very simple to use
• In the food industry:
• Biosensors have been widely employed in the food sector for quality assurance
and control.
• Applications in the agricultural field, such as crop cultivation and food
processing, are among them.
• Quality control is still an important aspect of food manufacturing since it ensures
that healthy food has a long shelf life and conforms with standards.

Q9: X-Ray Crystallography and Mass Spectrometry are both techniques used to
study the structure of matter/molecules, but they have different strengths and
weaknesses. Please describe.
X-ray crystallography and mass spectrometry are different techniques used to
analyze the structure of materials and molecules:
X-ray crystallography
Determines the atomic and molecular structure of a crystal by measuring how X-
rays diffract through the crystal. This technique can provide information about the
positions of atoms, chemical bonds, and crystallographic disorder. X-ray
crystallography is the primary method for characterizing the atomic structure of
materials.
Mass spectrometry
Provides chemical identity data with higher precision than X-ray crystallography.
Mass spectrometry can provide low-resolution data on interactions and proximal
distances between proteins. It can also be used to identify the stoichiometry of
membrane proteins complexed with ligand bindings. However, mass spectrometry
has some disadvantages, such as not being able to identify hydrocarbons that
produce similar ions.

X-ray crystallography and mass spectrometry can be used in combination to

analyze the structure of materials and molecules. For example, mass spectrometry
can be used to provide higher precision data when there is uncertainty about a
covalently-linked feature in an X-ray crystallography electron density map.
X-ray crystallography and mass spectrometry are both techniques used in
scientific research, but they have some strengths and weaknesses:
X-ray crystallography
• Strengths: A central source of experimental structural biology data, used to
discover new drugs and reveal the structure and function of biological molecules.
• Weaknesses: Requires a crystalline sample, which can be difficult to obtain for
large molecules and membrane proteins. X-ray crystallography also produces a
static three-dimensional analysis and can be limited for biological samples.
Mass spectrometry
• Strengths: Electrospray lonization Mass spectrometry (ESI-MS) can be useful
when NMR is difficult, such as with paramagnetic metals.
• Weaknesses: Poor signal intensity can make it difficult to identify or quantify
target compounds.

Q10: Infrared (IR) and Raman Spectroscopy are both vibrational spectroscopy
techniques, yet they describe slightly different properties of matter. Please explain
Infrared (IR) and Raman spectroscopy are both vibrational spectroscopy
techniques that provide information about how molecules vibrate when
interacting with light. However, they differ in several ways, including: @
Light source
IR spectroscopy uses infrared light, while Raman spectroscopy uses light in the
near-infrared (NIR), visible, or sometimes UV regions.
Interaction with light
In IR spectroscopy, the molecule absorbs infrared light and is excited to a higher
vibrational energy level. In Raman spectroscopy, the molecule is irradiated and
excited to a virtual energy state, then relaxes to a different vibrational state,
emitting a photon.
Molecular vibrations
IR spectroscopy relies on a change in dipole moment, while Raman spectroscopy
relies on a change in polarizability.
Information provided
IR spectroscopy provides a "fingerprint region" of the spectrum that is highly
characteristic of the bonding of atoms. Raman spectroscopy provides information
about intra- and intermolecular vibrations.
Applications
IR spectroscopy is used to study solution- phase supersaturation, while Raman
spectroscopy is used to analyze solid crystal forms in solution.