Q1. What is a filter?

Name the different types

Ans1.

Q2. Define chemical shift.

ANS.2 The variation of the resonance frequency of a nucleus in nuclear magnetic resonance
(NMR) spectroscopy in consequence of its magnetic environment. 
 For 1H and 13C NMR the reference signal is usually that of tetramethylsilane (SiMe4). If a
resonance signal occurs at lower frequency or higher applied field than an arbitrarily selected
reference signal, it is said to be upfield, and if resonance occurs at higher frequency or lower
applied field, the signal is downfield. Resonance lines upfield from SiMe4 have positive, and
resonance lines downfield from SiMe4 have negative  -values.

Q3. Define coupling constant

Ans3. The separation between the peaks of a first-order multiplet produced as a result of spin-
orbit coupling.  The larger the value of J, the greater the coupling between the nuclei.  J is
usually measured in Hertz, and is not dependant on the operating frequency of the instrument.

Q4. What is shielding and de-shielding?

Ans.4. Shieldingiswhenthenucleusexperiencesaweakermagneticfieldaroundit.This canbe

causedbyotheratoms“gettingintheway”ofthenucleusandthemagnetic field,orthenucleusitself
havingalowspin‐flipenergy.Duetotheweakermagnetic fielditexperiences,anucleuswithmore
shieldingwillhavealowerppmand thereforelieontherightsideofthechemicalshiftscale.

Deshieldingiswhenthenucleusexperiencesahighermagneticfieldaroundit.This canbeduetoits
proximitytoastrongmagneticfieldorhavingitselfahighspin‐flip energy.Becauseofitshigh
magneticfield,amoredeshieldednucleuswillhavea higherppmandthereforelieontheleftsideofthe
chemicalshiftscale.

Q5. Discuss basic principle of DTA

Ans5. In this technique, the difference in temperature between the sample & a thermally inert
reference material is measured as a function of temperature (usually the sample temperature).
Any transiton that the sample undergoes results in liberation or absorption of energy by the
sample with a corresponding deviation of its temperature from that of reference.

Q6. Discuss basic principle of DSC

Ans6. In this technique, the sample & references materials are subjected to a precisely
programmed temperature change. When a thermal transition (a physical & chemical change
that results in the emission or absorption of heat) occurs in the sample, thermal energy is
added to either the sample or the reference container in order to maintain both the sample &
reference at the temperature. Because the energy transferred is exactly equivalent in
magnitude to the energy absorbed or evolved in the transition, the energy yields a direct
calorimetric measurement of the transition energy.

Q7. Discuss basic principle of liquid-liquid extraction.

Ans7. Liquid–liquid extraction (LLE) is based on the principle that a solute or an analyte can
distribute itself in a certain ratio between two immiscible solvents, usually water (aqueous
phase) and organic solvent(organic phase).  There is a net transfer of one or more species
from one liquid into another liquid phase, generally from aqueous to organic. The transfer is
driven by chemical potential, i.e. once the transfer is complete, the overall system of chemical
components that make up the solutes and the solvents are in a more stable configuration
(lower free energy). The solvent that is enriched in solute(s) is called extract.

Q8. Discuss basic principle of solid phase extraction.

Ans8. The basic principle of SPE is the partitioning of compounds between

two phases of solid and liquid and there must be greater affinity for the solid phase than for
the sample matrix. The compounds retained on the solid phase can be removed by eluting
solvent with a greater affinity for the analytes.

Q9. What is radio-immuno assay?

Ans9. Radioimmunoassay (RIA) is an in vitro assay that measures the presence of an antigen
with very high sensitivity. Basically any biological substance for which a specific antibody
exists can be measured, even in minute concentrations.
Radioimmunoassay requires a radioactively labeled hormone. A defined concentration of the
radioactive hormone is incubated with a known quantity of antibody to which it binds, and
then the free or unbound hormone is separated from that bound to the antibody by a suitable
method.

Q10. What is the basic difference between LCMS and GCMS?

Ans10. LCMS (Liquid Chromatography coupled to Mass Spectrometry) is applied mainly for
the analysis of thermally unstable molecules in complex samples. (Example, analysis
biological fluids)
GCMS (Gas Chromatography coupled to Mass Spectrometry) is applied mainly for the
analysis of volatile compounds in complex samples. (example: analysis of gasoline and
petroleum products)

Q11. Discuss basic principle of LCMS.

Ans11. The LC-MS technology involves use of an HPLC, wherein the individual components
in a mixture are first separated followed by ionization and separation of the ions on the basis
of their mass/charge ratio. The separated ions are then directed to a photo or electron
multiplier tube detector, which identifies and quantifies each ion. The ion source is an
important component in any MS analysis, as this basically aids in efficient generation of ions
for analysis. To ionize intact molecules, the ion source could be APCI (Atmospheric Pressure
Chemical Ionization), ESI (Electronspray Ionization), etc. to name a few popular ones. The
choice of ion source also depends on the chemical nature of the analyte of interest i.e. polar
or non-polar.

Q12. What is calibration point?

Ans12. Calibration means matching the standard value of the instrument to the master (True)
Instrument value.
A calibration point is the test information that is used to calibrate an asset. The amount
of calibration points that are required for a calibration process is determined by business
requirements. A calibration point defines the nominal input value and the desired output
value for an analog asset function.

Q13. Define calibration with example.

Ans13. Calibration is the act of ensuring that a method or instrument used in measurement
will produce accurate results.

There are two common calibration procedures: using a working curve, and the standard-
addition method. Both of these methods require one or more standards of known composition
to calibrate the measurement.

Q14. What is meant by X-ray diffraction?

Ans14. X-ray diffraction, a phenomenon in which the atoms of a crystal, by virtue of their
uniform spacing, cause an interference pattern of the waves present in an incident beam of X
rays. The atomic planes of the crystal act on the X rays in exactly the same manner as does a
uniformly ruled grating on a beam of light.

Q15. What are the various methods for production of X-Rays?

Ans15.

Q16. Discuss importance of vacuum in Mass instrumentation.

Ans16. The vacuum system maintained in the Mass instrument ensures that the charged
molecules do not interfere with the air molecules or to ensure that the ions can pass through
unhindered by molecules in the air.

Q17. What is metastable-ion? Give examples.

Ans17. An ion which is formed with sufficient excitation to dissociate spontaneously during
its flight from the ion source to the detector.

Q18. What do you mean by quassi molecular ion? Give example?

Ans18. A protonated molecule or an ion formed from a molecular ion by loss of a hydrogen
atom.
RIA:
Q1 WHAT IS RIA?
Ans 1. Radioimmunoassay (RIA) is an in vitro assay that measures the presence of an antigen
with very high sensitivity. Basically any biological substance for which a specific antibody
exists can be measured, even in minute concentrations.
Radioimmunoassay requires a radioactively labeled hormone. A defined concentration of the
radioactive hormone is incubated with a known quantity of antibody to which it binds, and
then the free or unbound hormone is separated from that bound to the antibody by a suitable
method.

Q2 EXPLAIN RIA WITH EXAMPLE

Ans 2. 
When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or
antibodies, the technique of detection of the antigen-antibody complex is called
radioimmunoassay (RIA). Radioimmunoassay (RIA) is an in vitro assay that measures the
presence of an antigen with very high sensitivity. RIA was first described in 1960 for the
measurement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the
Veterans Administration Hospital in New York.

The classical RIA methods are based on the principle of competitive binding. In this method,
an unlabeled antigen competes with a radiolabeled antigen for binding to an antibody with the
appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are
incubated with the corresponding antibody, the amount of free (not bound to antibody)
radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the
mixture.

Principle- 
It involves a combination of three principles.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or competitive displacement reaction. (It gives specificity)
3. Measurement of radio emission. (It gives sensitivity)

Immune reaction- 
When a foreign biological substance enters into the body bloodstream through a non-oral
route, the body recognizes the specific chemistry on the surface of foreign substance as
antigen and produces specific antibodies against the antigen so as nullify the effects and keep
the body safe. The antibodies are produced by the body’s immune system so, it is an immune
reaction. Here the antibodies or antigens bind move due to chemical influence. This is
different from principle of electrophoresis where proteins are separated due to charge.

Q3 PRINCIPLE AND PROCEDURE OF RIA

Ans 3. Principle-
- It involves the separation of a protein (from a mixture) using the specificity of antibody -
antigen binding and quantitation using radioactivity.
- At increasing concentrations of unlabeled antigen, an increasing amount of radioactive
antigen is displaced from the antibody molecules.

Radio immune assay method- 

The target antigen is labeled radioactively and bound to its specific antibodies (a limited and
known amount of the specific antibody has to be added). A sample, for example a blood-
serum, is then added in order to initiate a competitive reaction of the labeled antigens from
the preparation, and the unlabeled antigens from the serum-sample, with the specific
antibodies. The competition for the antibodies will release a certain amount of labeled
antigen. This amount is proportional to the ratio of labeled to unlabeled antigen. A binding
curve can then be generated which allows the amount of antigen in the patient's serum to be
derived.

Requirements- 
1. Specific antiserum to the antigen to be measured
2. Availability of a radioactive labeled form of the antigen
3. A method in which the antibody-bound tracer can be separated from the unbound tracer
4. An instrument to count radioactivity
Radioactivity- 125-I labels are usually applied although other isotopes such as C14 and H3
have also been used. Usually, high specific activity radio-labeled (125-I) antigen is prepared
by iodination of the pure antigen on its tyrosine residue(s) by chloramine-T or peroxidase
methods and then separating the radio-labeled antigen from free-isotope by gel-filtration or
HPLC. Other important components of RIA are the specific antibody against the antigen and
pure antigen for use as the standard or calibrator.
Separation techniques:
Double antibody, charcoal, cellulose, chromatography or solid phase techniques are applied
to separate bound and free radio-labeled antigen. Most frequently used is the double antibody
technique combined with polyethylene. The bound or free fraction is counted in a gamma
counter.
Concomitantly, a calibration or standard curve is generated with samples of known
concentrations of the unlabeled standards. The amount of antigen in an unknown samples can
be calculated from this curve.
Sensitivity:
The sensitivity can be improved by decreasing the amount of radioactively-labeled antigen
and/or antibody. The sensitivity can also be improved by the so-called disequilibrium
incubation. In this case radioactively labeled antigen is added after initial incubation of
antigen and antibody.
  Radioimmunoassay (RIA) is an in vitro assay that measures the presence of an
antigen with very high sensitivity. Basically, any biological substance for which a
specific antibody exists can be measured, even in minute concentrations.

 RIA Method:

The target antigen is labelled radioactively and bound to its specific antibodies (a limited and
known amount of the specific antibody has to be added). A sample, for example a blood-
serum, is then added in order to initiate a competitive reaction of the labelled antigens from
the preparation, and the unlabelled antigens from the serum-sample, with the specific
antibodies. The competition for the antibodies will release a certain amount of labelled
antigen. This amount is proportional to the ratio of labelled to unlabelled antigen. A binding
curve can then be generated which allows the amount of antigen in the patient's serum to be
derived.

That means that as the concentration of unlabelled antigen is increased, more of it binds to
the antibody, displacing the labelled variant. The bound antigens are then separated from the
unbound ones, and the radioactivity of the free antigens remaining in the supernatant is
measured. A binding curve can be generated using a known standard, which allows the
amount of antigens in the patient's serum to be derived. 

 Principle:

Basically, radioimmunoassay is based on three principles which give it high sensitivity.

1. A strong immune binding reaction: Antigen vs Antibody reaction

2. The competitive binding reaction which gives specificity
3. The radio emission gives the specificity

Uses of Radioimmunoassay (RIA):

RIA has revolutionized research and clinical practice specially in blood banking, diagnosis of

allergy and endocrinology. Furthermore it is used to:

1. The test can be used to determine very small quantities (e.g. nanogram) of antigens and

antibodies in the serum.

2. The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens.

3. Analyze nanomolar and picomolar concentrations of hormones in biological fluids.

Applications:

1.  Evaluation of enzyme immunoassay and radioimmunoassay methods for the

measurement of plasma oxytocin

 2. Significance of prostate specific antigen in prostate cancer patients and in non cancerous

prostatic disease patients.

3. Estradiol measurement in translational studies of breast cancer. Plasma estrogen

measurement with use of radioimmunoassay has been instrumental in the development of

aromatase inhibitors for endocrine therapy of postmenopausal breast cancer.

4. Refinement of Zalcitabine pharmaco kinetics.

 Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) is a powerful

analytical technique that combines the separating power of liquid chromatography
with the highly sensitive and selective mass analysis capability of triple quadrupole
mass spectrometry.

 Uses of LC-MS-MS:

 Trace analysis in:

1. Environmental samples (i.e. soil, water, sediment)
2. Pharmaceutical analyses (i.e. active ingredient or impurity analyses)
3. Biological samples (i.e. crops, livestock tissues, milk, eggs, blood, urine)
 Structure confirmation analyses
 Direct injection MS/MS (no LC separation)
 Analysis of compounds lacking UV chromophores
 Updating existing methodology for higher specificity or sensitivity

 Working of LC-MS-MS:

A sample solution containing analytes of interest are pumped through a stationary phase (LC
column) by a mobile phase flowing through at high pressure. Chemical interaction between
the components of the sample, the stationary phase and the mobile phase affects different
migration rates through the LC column affecting a separation. The wide variety of stationary
phase and mobile phase combinations allow for customizing a separation to suit many
complex solutions.

After elution from the LC column, the effluent is directed to the mass spectrometer. The mass
spectrometer for an LC/MS/MS system has an ionization source where the LC column
effluent is nebulized, desolvated and ionized creating charged particles. These charged
particles then migrate under high vacuum through a series of mass analyzers (quadrupole) by
applying electromagnetic fields. A specific mass/charge precursor ion (or parent ion) is
targeted to pass through the first quadrupole, excluding all other mass/charge ratio particles.
In the collision cell, the selected mass/charge ions are then fragmented into product ions (or
daughter ions) by collision with an inert gas. The third quadrupole is used to target specific
product ion fragments. The resulting isolated product ions are then quantified with an
electron multiplier. This transition of ions from the precursor to product ion (also referred to
as MS2) is highly specific to the structure of the compound of interest and therefore provides
a high degree of selectivity.

The strength of this technique lies in the separation power of LC for a wide range of
compounds combined with the capability of the MS to quantify compounds with a high
degree of sensitivity and selectivity based on the unique mass/charge (m/z) transitions of each
compound of interest.

HYPHENATED AND EXTRACTION

 Hyphenated technique: It is a combination or coupling of two different analytical

techniques with the help of proper interface.

 Hyphenation refers to the online combination of a separation technique and one or

more spectroscopic detection techniques.

 Advantages of hyphenated techniques:

1. Fast and accurate analysis
2. Higher degree of automation
3. Higher sample throughput
4. Better reproducibility
5. Reduction of contamination due to its closed system
6. Separation and quantification achieved at same time

SOLID PHASE EXTRACTION-

 Solid phase extraction consists of bringing a liquid or gaseous test sample in contact
with a solid phase, whereby the analyte is selectively adsorbed on the surface of the
solid phase.

 In modern solid phase extraction, the adsorbent is packed between two flitted disks in
polypropylene cartridge and liquid phases are passed through the cartridge either by
suction or by positive pressure.

 Basic steps in solid phase extraction:

1. Condition
2. Load sample
 3. Washing
4. Elution

Principle of SPE
The basic principle of SPE is the partitioning of compounds between
two phases of solid and liquid and there must be greater affinity for the solid
phase than for the sample matrix. The compounds retained on the solid phase can
be removed by eluting solvent with a greater affinity for the analytes.

 Solid phases used in solid phase extraction:

1. Activated charcoal
2. Alumina
3. Silica gel
4. Magnesium silicate (Florisil)
5. Styrene divinylbenzene (polymer)

 Extracts can be defined as preparations of crude drugs which contain all the
constituents which are soluble in solvent.

 Marc- Solid residue obtained after extraction

 Menstruum- Solvent used for the extraction
 Synonyms of solid phase extraction:
1. Liquid solid extraction
2. Column extraction
3. Digital chromatography
4. Bonded phase extraction
5. Selective adsorption technique

 Applications of solid phase extraction:

1. Impurity profiling of pharmaceuticals
2. Environmental applications
3. In food industry
4. Analysis of wines and other alcoholic preparations
5. Biological fluids
6. Hair analysis

LIQ-LIQ EXTRACTION
 Liquid-liquid extraction involves the separation of the constituents (solutes) of a
liquid solution by contact with another insoluble liquid. Solutes are separated based
on their different solubilities in different solvents. Separation is achieved when the
substances constituting the original solution is transferred from the original sol. to
other liquid solution.

The solution which is to be extracted is called the feed and the liquid with which the feed is
contacted is the solvent.
  LLE also known as solvent extraction and partitioning is a method to separate
compounds based on their relative solubility’s in two different immiscible liquids
usually water and an organic solvent.

 The solvent rich product of the operation is called the extract and the residual liquid
from which solutes has been removed is the raffinate.

 Purpose of LLE:
1. To separate closed boiling point mixture.
2. Mixture that cannot withstand high temperature of distillation.

 Choice of solvent in liquid liquid extraction: Factors to be considered:

1. Selectivity
2. Distribution coefficient
3. Insolubility of solvent
4. Interfacial tension
5. Chemical reactivity
6. Cost
7. Viscosity
8. Vapor pressure
9. Flammability
10. Toxicity
11. Density difference between liquid phases

GC-MS

 Gas chromatography mass spectrometry (GC/MS) is an instrumental technique,

comprising a gas chromatograph (GC) coupled to a mass spectrometer (MS), by
which complex mixtures of chemicals may be separated, identified and quantified.

 Advantages of gc-ms:
1. Ability to separate complex mixtures
2. Quantify analytes
3. Determine trace levels of organic contamination

 Working/Principle of gc-ms:
GC/MS begins with the gas chromatograph, where the sample is volatized. This effectively
vaporizes the sample (the gas phase) and separates its various components using a capillary
column packed with a stationary (solid) phase. The compounds are propelled by an inert
carrier gas such as argon, helium or nitrogen. As the components become separated, they
elute from the column at different times, which is generally referred to as their retention
times.

Once the components leave the GC column, they are ionized by the mass spectrometer using
electron or chemical ionization sources. Ionized molecules are then accelerated through the
instrument’s mass analyzer, which quite often is a quadrupole or ion trap. It is here that ions
are separated based on their different mass-to-charge (m/z) ratios.

The final steps of the process involve ion detection and analysis, with compound peaks
appearing as a function of their m/z ratios. Peak heights, meanwhile, are proportional to the
quantity of the corresponding compound. A complex sample will produce several different
peaks, and the final readout will be a mass spectrum. Using computer libraries of mass
spectra for different compounds, researchers can identify and quantitate unknown compounds
and analytes.

 Applications of gc-ms:
1. GC/MS is the analysis method of choice for smaller and volatile molecules such as
benzenes, alcohols and aromatics, and simple molecules such as steroids, fatty acids,
and hormones. It can also be applied towards the study of liquid, gaseous and solid
samples.
2. Assay of pharmaceuticals and purity and characterization of raw materials.
3. Sports anti-doping analysis
4. Medical diagnosis
5. Studies of drug metabolite