CIRCULAR DICHROISM SPECTROSCOPY

Circular Dichroism (CD) is an absorption spectroscopy method based on the differential

absorption of left and right circularly polarized light. Optically active chiral molecules
will preferentially absorb one direction of the circularly polarized light. The difference
in absorption of the left and right circularly polarized light can be measured and
quantified.

**Chirality is an important geometric property relating to a molecule's symmetry. A chiral

molecule is non-superimposable with its mirror image, and has a "handedness"

For detailed note on this topic following references are suitable:

(a)
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Sup
plemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/
Circular_Dichroism#:~:text=Circular%20Dichroism%20(CD)%20is%20an,of%20the%20circularly%20p
olarized%20light.

(b) https://www.edmundoptics.in/knowledge-center/application-notes/optics/introduction-to-
polarization/#:~:text=Linear%20polarization%3A%20the%20electric%20field,phase%20difference%2
0of%20%CF%80%2F2. (for light polarization concept)

(c) http://electron9.phys.utk.edu/optics421/modules/m7/polarization.htm (for right/left circularly

polarized light concept)

Applications:

For biologically important molecules, such as proteins, nucleic acids, carbohydrates, and
lipids, CD spectroscopy is the technique of choice to investigate and characterize
structural molecular behaviour in solution.

For proteins, made of L-amino acid residues, CD is advantageously used to monitor,

characterize, and estimate the protein secondary structure in the far-UV region (180–
250 nm).

Another important use of CD is to probe qualitatively and quantitatively binding

interactions in a solution of biomolecules without the need of immobilizing and labeling
them.
Circular dichroism (CD) spectroscopy is routinely used in the biopharmaceutical
industry to study the effects of manufacturing, formulation, and storage conditions on
protein conformation and stability.

MASS SPECTROMETRY

Mass spectrometry (MS) is an analytical technique used to ascertain the composition of

chemical substances by accurately measuring their molecular masses. In MS analysis,
the molecules of interest are vaporized and ionized, and the mass-to-charge (m/z)
ratios of molecular ions are determined. For instance, lately, MS has evolved into a
highly sensitive, accurate, and powerful tool for rapid qualitative and semi-quantitative
characterization of the peptides and proteins present in samples ranging from
individual cells, tissue sections, and biological fluids to entire organisms.

• Principles of Mass Spectrometric Analysis and Instrumentation

MS analysis consists of two essential steps: (1) a process to vaporize and ionize analytes
of interest from a sample, followed by (2) a means to measure the masses of the
resulting ions.

FIGURE 1: Two ionization techniques commonly used in biological mass spectrometry: (A)
electrospray ionization (ESI); (B) matrix-assisted laser desorption/ionization (MALDI). As
shown in (A), ESI is an ionization technique that uses high voltage at a capillary tip to disperse
and spray solvated analytes from the capillary. The spray produces multiply charged ions that
are transferred directly into a mass analyzer. As shown in (B), MALDI is a technique that uses a
laser to irradiate analytes co-crystallized with a matrix that absorbs the laser energy and forms
singly charged gaseous analyte ions.
The most effective ionization of proteins is achieved by soft ionization methods such as
electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
The ionization of biological samples by either ESI or MALDI is effective as these two
approaches limit molecular fragmentation so that larger molecules can be studied.
Briefly, in MALDI a liquid or solid biological sample is co-crystallized with a large excess
of ultraviolet (UV)-absorbing matrix; illumination of the matrix-sample crystals by a UV
laser results in a microexplosion that produces primarily singly charged gaseous
analyte ions. In ESI, electric potential is applied to a capillary filled with a liquid sample.
This generates an electrospray consisting of small liquid droplets containing the sample
that are desolvated to form multiply charged gas-phase ions.

Following ionization of the sample is achieved (either via ESI/MALDI/Chemical

ionization (CI)), the most important consequence of the fragmentation process is that it
generates a characteristic, reproducible mass spectrum that serves as a spectrometric
‘fingerprint’ of the analyte. Mass spectra are generally recorded in the form of a line
diagram (bar graph).

The Mass Analyzer is the part of the mass spectrometer that subjects the ions to a
series of electric or magnetic fields in order to allow determination of the m/z of the
ions. In fact, the mass analyzer of the mass spectrometer separates ionized atoms and
molecules according to their mass-to-charge ratio, m/z. The maximum practical
resolving power, R, of a mass spectrometer is an important instrumental characteristic.
It is effectively a measure of the instrument’s ability to separate ions.

As different mass analyzers offer different advantages therefore a clear idea about them
would be handy. The following are five common mass analyzers that are used in various
MS studies:

✓ Quadrupole mass analyzers

✓ TOF (Time of Flight) analyzers, the ions are formed in the ionization source and
accelerated by an applied potential so that all ions have the same kinetic energy
before being introduced to the field-free region of the mass analyzer. Thus, ions
travel at different velocities based on their kinetic energy. The flight time that
each ion spends travelling in the field-free region is converted to an m/z value.
The TOF instrument has the ability to measure ions over a wide mass range.
✓ Ion trap instruments
 ✓ FT-ICR (also known as FTMS)
✓ Orbitrap mass analyzers

▪ The Nature of Mass Spectra

A mass spectrum will usually be presented as a vertical bar graph, in which each bar
represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar
indicates the relative abundance of the ion. The most intense ion is assigned an
abundance of 100, and it is referred to as the base peak. Most of the ions formed in a
mass spectrometer have a single charge, so the m/z value is equivalent to mass itself.
Modern mass spectrometers easily distinguish (resolve) ions differing by only a single
atomic mass unit (amu), and thus provide completely accurate values for the molecular
mass of a compound. The highest-mass ion in a spectrum is normally considered to be
the molecular ion, and lower-mass ions are fragments from the molecular ion, assuming
the sample is a single pure compound.

FIGURE 2: Instrumentation of Mass spectrometry and steps involved in analyses

• Applications:

MS has wide range of applications in pharmaceutical (drug discovery,

pharmacokinetics, drug metabolism), clinical (neonatal screening, hemoglobin analysis,
drug testing), environmental (water quality, food contamination, pollutant
determination), geological (oil composition, hydrocarbon fraction determination in
petroleum industry), metallurgy (determination of rare earth metals and metals at ppq
(parts per quadrillion)), sports (dope test of drugs in athletes), forensic (poison and
drug metabolite determination) and biotechnology (proteins, peptide analysis) like
fields.

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INFRARED SPECTROSCOPY (IRS)

Infrared spectroscopy (IRS) is one of the most important analytic techniques that
provide qualitative and quantitative information about a test sample in a fast, cost-
effective and nondestructive way, which does not require the use of polluting chemicals,
and can be carried out even by minimally trained personnel. This makes IRS a potent
tool for the quality control/assurance in a variety of different (industrial) settings and
suitable for applications under steady process conditions, that is, on-line.

In IRS, a test sample is irradiated with infrared radiation and the transmitted or
reflected radiation is measured. Infrared (IR) spectra are obtained by plotting the
measured absorbance against the corresponding wavelengths. IR radiation triggers
transitions in the vibrational states of the sample molecules. Each functional group of a
molecule shows characteristic IR absorption at specific wavelengths. The IR region of
the electromagnetic spectrum can be divided into three portions: The far IR (FIR, 400–
10 cm-1) has the lower energy and induces rotational transitions in the molecules. The
mid-IR (MIR, 4000–400 cm-1) induces fundamental vibrational transitions in the
molecules. The near-IR region (NIR, 14000–4000 cm-1) induces transitions over two
(first overtone), three (second overtone), or higher energy levels. In general, the
absorption bands in the MIR region are more intense than those in the NIR region.
Besides the chemical composition of the test sample, the IR spectral characteristics also
depend on the test sample light scattering properties and thus to its microstructure.

Chemical information can be extracted from an IR spectrum from the bands position,
intensity, and shape. In pure component systems or very simple mixtures, the band
position can be easily related to the molecular structure of a component in the mixture,
whereas the band intensity is related to its concentrations through the law of Lambert–
Beer.
 • Instrumentation

Conventional IR instruments consist of a light source, accessory for the sample

preparation, a monochromator, a detector, and optical components (lens, collimators,
beam splitters, etc.). A schematic description of a typical dispersive IR instrument
withdouble-beam operation is given in Figure 3. Radiation produced by a broadband
source is split into two by a beam splitter. One radiation is sent to the sample and the
other to a reference. The radiation transmitted (or reflected) by the sample and the
reference is then dispersed by a monochromator into component frequencies and
collected by the detector, which compares the two radiations and generates an electrical
signal, which is converted to an IR spectrum. The radiation source is commonly an inert
solid electrically heated to 1000–1800°C. Based on the type of monochromator, IR
spectrophotometers can be classified as filter instruments, scanning monochromator
instruments (a grating or prism is used to separate the single wavelengths), or FT
spectrophotometers. (FT=Fourier Transform)

FIGURE 3: Schematic representation of a typical conventional IR instrument in

transmittance mode and double-beam operation.
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