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Applications of Spectroscopy:

1. DNA, RNA, Protein measurement+ Identification

2. Turbidity measurement (O.D.)
3. To probe consistency and conformational structure of biological molecules.
4. An understanding of the properties of electromagnetic radiation and its
interaction with matter leads to an appreciation of the variety of types of
spectra and, consequently, different spectroscopic techniques and their
applications to the solution of biological problems.
5. To measure concentration & turbidity of solution.
6. To detect the presence of the sample.
7. Deals with the study of the interactions between particles like protons,
electrons, and ions.
8. Polarity, Non polarity, Ionic strength, Affinity Chromatography separate
solutes and they are detected by Spectroscopy.
9. Mass Spectroscopy is used to measure Molecular Weight, identify protein
and measure protein.
10.NMR is used to reveal side chains also.

Spectroscopic techniques employ light to interact with matter and thus probe
certain features of a sample to learn about its consistency or structure.

Or, Spectroscopy is a field of science, where interaction between matter and

electromagnetic radiation are studied to probe/detect certain features of the
samples.

Or, The study of the emission and absorption of light and other radiations by
matter is known as spectroscopy.

Electromagnetic Radiation:

• Electromagnetic radiation is composed of an electric (E vector) and a

perpendicular magnetic vector (M vector), each one oscillating in plane at
right angles to the direction of propagation.
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• The wavelength of light, and all other forms of electromagnetic radiation, is

related to the frequency by a relatively simple equation:

𝝑 = 𝒄/λ

Here, 𝜗 = Frequency of electromagnetic radiation

C = Speed of light (c= 2.998×108 ms-1)

λ= Wavelength of electromagnetic radiation

Fig. 12.1: Light is electromagnetic radiation and can be described as a wave

propagating transversally in space and time. The electric (E) and magnetic (M)
field vectors are directed perpendicular to each other. For UV/Vis, circular
dichroism and fluorescence spectroscopy, the electric field vector is of most
importance. For electron paramagnetic and nuclear magnetic resonance, the
emphasis is on the magnetic field vector.

The electromagnetic spectrum includes, from longest wavelength to shortest: radio

waves, microwaves, infrared, optical, ultraviolet, x-rays, and gamma-rays.
Electromagnetic radiation is produced whenever a charged particle, such as an
electron, changes its velocity—i.e., whenever it is accelerated or decelerated.
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Photon is the elementary particle responsible for electromagnetic phenomena. It

carries the electromagnetic radiation and has properties of a wave, as well as of a
particle, though having a mass of zero. As a particle, it interacts with matter by
transferring its energy E:

E = hc/𝝀 = h𝝑

Where h is the planck constant (h = 6.63 × 10−34 Js) and 𝜗 is the frequency of the
radiation as introduced above.

The wavelength λ is the spatial distance between two consecutive peaks (one
cycle) in the sinusoidal waveform and is measured in submultiples of meter,
usually in nanometers (nm).

The maximum length of the vector is called the amplitude.

The frequency 𝜗 of the electromagnetic radiation is the number of oscillations

made by the wave within the timeframe of 1 s.
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Spectrometer

• A spectrometer is typically used to measure wavelengths of electromagnetic

radiation (light) that has interacted with a sample. Incident light can
be reflected off, absorbed by, or transmitted through a sample; the way
the incident light changes during the interaction with the sample is
characteristic of the sample. A spectrometer measures this change over a
range of incident wavelengths (or at a specific wavelength).

• Most common types of spectrometer are optical spectrometers, mass

spectrometers, nuclear magnetic resonance (NMR) spectrometers.

Figure: Optical spectrometer, nuclear magnetic resonance spectrometer (NMR),

mass spectrometer.

Uv-vis spectroscopy is an analytical technique that measures the amount of

discrete wavelengths of UV or visible light that are absorbed by or transmitted
through a sample in comparison to a reference or blank sample.

The electronic transitions in molecules can be classified according to the

participating molecular orbitals. From the four possible transitions (n→π ∗, π→π∗,
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n→𝜎∗, 𝜎→𝜎∗), only two can be elicited with light from the UV/vis spectrum for
some biological molecules: n→π ∗ and π→π∗. The

Figure: Energy scheme for molecular orbitals (not to scale). Arrows indicate
possible electronic transitions. The length of the arrows indicates the energy
required to be put into the system in order to enable the transition. Black arrows
depict transitions possible with energies from the UV/vis spectrum for some
biological molecules. The transitions shown by grey arrows require higher energies
(e.g. X-rays)

Chromophores

Molecular (sub-) structures responsible for interaction with electromagnetic

radiation are called chromophores. In proteins, there are 3 types of chromophores
relevant for UV/Vis spectroscopy:

• Peptide bonds (amide bond);

• Certain amino acid side chains (mainly tryptophan and tyrosine); and

• Certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in

heme)
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The presence of several conjugated double bonds in organic molecules results in an

extended 𝜋-system of electrons which lowers the energy of the 𝜋* orbital through
electron delocalization. In many cases, such systems possess π→π∗ transitions in
the UV/Vis range of the electromagnetic spectrum. Such molecules are very useful
tools in colorimetric applications in following table:

2,3 examples enough

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Chromophores in proteins

➢ The electronic transitions of the peptide bond occur in the far UV. The
intense peak at 190 nm, and the weaker one at 210–220 nm is due to the
π→π* and n→π* transitions
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➢ The most useful range for proteins is above 230 nm, where there are
absorptions from aromatic side chains.

➢ Proteins that contain prosthetic groups (e.g. haem, flavin, carotenoid) and
some metal–protein complexes, may have strong absorption bands in the
UV/Vis range.

➢ While a very weak absorption maximum of phenylalanine occurs at 257 nm,

tyrosine and tryptophan dominate the typical protein spectrum with their
absorption maxima at 274 nm and 280 nm, respectively.

Chromophores in genetic material

The absorption of UV light by nucleic acids arises from n→ π * and π→π∗

transitions of the purine (adenine, guanine) and pyrimidine (cytosine, thymine,
uracil) bases that occur between 260nm and 275 nm. The absorption spectra of the
bases in polymers are sensitive to pH and greatly influenced by electronic
interactions between bases.

Quantification of light absorption

***The chance for a photon to be absorbed by matter is given by an extinction

coefficient which itself is dependent on the wavelength λ of the photon. If light
with the intensity I0 passes through a sample with appropriate transparency and the
path length (thickness) d, the intensity I drops along the pathway in an exponential
manner.

The characteristic absorption parameter for the sample is the extinction coefficient
α, yielding the correlation I= I𝜊 𝑒 −𝛼𝑑 . The ratio T= I/I𝜊 is called transmission.

Biochemical samples usually comprise aqueous solutions, where the substance of

interest is present at a molar concentration c. Algebraic transformation of the
exponential correlation into an expression based on the decadic logarithm yields
the law of Beer–Lambert:
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where [d]=1 cm, [c]= 1 mol d𝑚−3, and [𝜀]= 1 dm3 mo𝑙−1 c𝑚−1. 𝜀 is the molar
absorption coefficient (also molar extinction coefficient) (Extinction coefficient, 𝛼
= 2.303 × c ×𝜀).

A is the absorbance of the sample, which is displayed on the spectrometer. This

law is valid for low concentrations only. Higher concentrations might lead to
association of molecules and therefore cause deviations from the ideal behavior.

Instrumentation

The instrument used in ultraviolet-visible spectroscopy is called a UV/vis

spectrophotometer.

• Types: single-beam and dual-beam

• Light source: tungsten filament bulb (visible light) and deuterium bulb (UV
light).

• Sample containers: Cuvettes (which can be of plastic /glass/ quartz)

UV/Vis spectrophotometers are usually dual-beam spectrometers where the first

channel contains the sample and the second channel holds the control (buffer) for
correction.

Since the emitted light consists of many different wavelengths, a monochromator,

consisting of either a prism or a rotating metal grid of high precision called grating,
is placed between the light source and the sample. If wavelengths are selected by
prisms or gratings, the technique is called spectrophotometry.
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A prism splits the incoming light into its components by refraction. Refraction
occurs because radiation of different wavelengths travels along different paths in
medium of higher density.

The measured absorbance is the difference between the two transmitted beams of
light recorded.

The use of dual-beam spectrophotometer:

 This technique is used to measure concentration of such molecule, which

can absorb light.
 DNA can be measure by using UV/Vis which absorb light at 260 nm.
 Can detect missing of absorbance due to solvent concentration

Application of UV/vis Spectroscopy:

1. Detection of impurities

It is one of the best methods for determination of impurities in organic molecules.

Additional peaks can be observed due to impurities in the sample and it can be
compared with that of standard raw material.
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By also measuring the absorbance at specific wavelength, the impurities can be

detected

2. Structure elucidation of organic compounds

It is useful in the structure elucidation of organic molecules, such as in detecting

the presence or absence of unsaturation, the presence of hetero atoms.

• UV absorption spectroscopy can be used for the quantitative determination

of compounds that absorb UV radiation.
• UV absorption spectroscopy can characterize those types of compounds
which absorbs UV radiation thus used in qualitative determination of
compounds. Identification is done by comparing the absorption spectrum
with the spectra of known compounds.

. • This technique is used to detect the presence or absence of functional group in

the compound. Absence of a band at particular wavelength regarded as an evidence
for absence of particular group.

3. Kinetics of reaction can also be studied using UV spectroscopy. The UV

radiation is passed through the reaction cell and the absorbance changes can
be observed.

4. Detection of drugs are determined if they are either in the form of raw
material or in the form of formulation.

5. Molecular weights of compounds can be measured spectrophotometrically

FLUORESCENCE SPECTROSCOPY
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Fluorescence spectroscopy, also known as fluorimetry or spectrofluorometry, is a

type of electromagnetic spectroscopy that analyzes fluorescence from a sample.

It involves using a beam of light, usually ultraviolet light, that excites the electrons
in molecules of certain compounds and causes them to emit light; typically, but not
necessarily, visible light.

Devices that measure fluorescence are called fluorometers.

Used for measuring compounds in a solution and it is easy method to perform.

Principle:

Fluorescence is an emission phenomenon where an energy transition from a higher

to a lower state is accompanied by radiation. Only molecules in their excited
forms are able to emit fluorescence; thus, they have to be brought into a state of
higher energy prior to the emission phenomenon.
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Fig. 12.8: Jablonski diagram. Shown are the electronic ground state (S0), two
excited singlet states (S1, S2) and a triplet state (T1). Vibrational levels (v) are
only illustrated exemplarily. Solid vertical lines indicate radiative transitions,
dotted lines show non-radiative transitions. The inset shows the relationship
between electron configurations, total spin number S and multiplicity M.

M= (+n)+ (-n)+ 1
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Instrumentation

Spectrofluorometer is the basic instrument. It contains a light source, 2

monochromators, a sample holder, 2 detectors polarization filter.

• Light source: Xenon, mercury vapor lamps etc.

• Monochromator: used for both the selection of exciting light and the analysis
of sample emission.
• Sample holder: sample contained in a cuvette or in a flow cell. Cuvettes may
be circular, square and must be constructed of a material that will transmit
both the incident and emitted light.
• The detector is at 90 degrees to the excitation beam.

For accurate result temperature controlled is required

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Fluorescence spectroscopy works most accurately at very low concentrations of

emitting fluorophores. UV/Vis spectroscopy, in contrast, is least accurate at such
low concentrations*. One major factor adding to the high sensitivity of
fluorescence applications is the spectral selectivity. Due to the Stokes shift, the
wavelength of the emitted light is different from that of the exciting light.
Another feature makes use of the fact that fluorescence is emitted in all directions.
By placing the detector perpendicular to the excitation pathway, the background of
the incident beam is reduced.
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Fig. 12.11 Structures of some extrinsic fluorophores. Fura-2 is a fluorescent

chelator for divalent and higher valent metal ions (Ca2+, Ba2+, Sr2+, Pb2+, La3+,
Mn2+, Ni2+, Cd2+).
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• Fluorescence spectroscopy is used in, among others,

biochemical, medical, and chemical research fields for
analyzing organic compounds.

• Fluorescence spectroscopy can be adopted to microscopic level

using microfluorimetry.

• There has also been a report of its use in differentiating

malignant skin tumors from benign

• Fluorescence can also be used to redirect photos.

• In analytical chemistry, fluorescence detectors are used with

HPLC.

• Easy and quick to perform analysis

Difference between uv/vis and fluorescence

UV/Vis spectroscopy Fluorescence spectroscopy

1. UV/Vis measure the absorption of light 1. Fluorescence measures the light

in particular range (200-400 nm).
2. The instrument used in ultraviolet-
visible spectroscopy is called a UV/vis
spectrophotometer.
emitted by a sample in this range after
absorbing light at a higher energy than
it is emitting
2. Devices that measure fluorescence
are called fluorometers.

3. Here, Tungsten filament bulb or 3. The light source used in this

Deuterium bulb. technique is Xenon lamp or Mercury
vapour lamp.
4. Light source perpendicular to sample. 4. Light source are in straight line.
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5. In detector, one monochromoter is 5. In detector, two monochromoters are

used. used.

6. Absorbance is measured as the 6. Whereas fluorescence is measured

difference in intensity between light
passing through the reference and the
sample.( less sensitive)
7. UV/Vis spectroscopy, is less accurate
at low concentrations.
directly without any reference beam.
(more sensitive)
Fluoroscence spectroscopy is more
accurate at low concentrations.

NMR

Basic principle of NMR

In NMR technique, the protons are placed in a strong magnetic field . As a
result the protons will have aligned or anti-aligned with the magnetic field
and will be differentiated into higher energy state (anti-aligned) and lower
energy state. (aligned)

The difference between the two states have a resonance energy.

when hit by the appropriate frequency, the lower energy state
protons will flip into higher energy state.

The protons that have reached into higher state of energy will flip back
into lower state of energy by emitting the exact energy. This emitted
energy will be detected by using a detector.
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• Nuclear Magnetic resonance spectroscopy, most commonly known as NMR

spectroscopy, is a spectroscopic technique to observe local magnetic fields
around atomic nuclei.

• Chemists use this technique to determine molecular structure.

• Medical practitioners employ magnetic resonance imaging (MRI), a

multidimensional NMR imaging technique, for diagnostic purposes.

• In microbiology, NMR allows the determination of metabolic changes in

organisms in response to external stimuli, through the identification and
quantification of metabolites.

Overall Procedure of NMR

Effect of external magnetic field on proton:

Protons with (+1/2) spin

• Will align with the external magnetic field.

• Will be at lower state of energy (Eα state)

• Will be more stable.

Protons with (-1/2)spin

• Will align against the external magnetic field

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• Will be at higher state of energy (Eβ state)

• Will be less stable.

Chemical shift or Resonance

• In a magnetic field, there are 2 energy states for a proton: a lower energy
state (Eα ) in which the protons aligned in the same direction as B0 (external
magnetic field) and a higher energy state (Eβ ) in which the protons are
aligned against B0.

• When an external energy(Radio frequency wave) source that matches the

energy difference between these two states is applied, energy is absorbed
causing the spin-up protons to transit to the higher energy level spin down
states.

• This transition of protons from Eα(lower energy state) state to Eβ state(higher

energy state) is called chemical shift or resonance.

Determination of Resonance frequency

• The radio frequency wave must contain the right frequency to cause the
proton to resonate (transition energy states) and this frequency is known as
resonance frequency.

• The difference between two energy states can be determined by the

following formula: ΔE= Eβ – Eα= 2µB0

• If the frequency is denoted as 𝝑, then,

E= h𝝑=2µB0

=>𝝑=(2µB0)/h

• Here, h= Planck´s constant (6.626× 10^-34m2kg/s)

• 𝝑=frequency (Hz).

• µ= magnetic permeability(H/m), Eβ= energy level at β state, Eα=energy

level at α state, Bo= external magnetic field.
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Effect of electron density on resonance or chemical shift (Bnet= B0-

Binduced)
Higher electron density around the proton

• Induced magnetic field (B induced ) by electrons is more thus

net magnetic field(B net) felt by proton is less.
• Requires higher magnetic field strength for resonance
• The absorption takes place at upfield (feel lower energy).
• The chemical shift value in delta scale is less.
Lower electron density around the proton

• Induced magnetic field (B induced) is less thus net magnetic field(B net) felt by
proton is more.

• Requires less magnetic field strength for resonance.

• The absorption takes place at downfield (feel higher energy).

• The chemical shift value in delta scale is more.

Effect of electron density on proton resonance

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NMR signal detection:

• Downfield means higher energy-left side of the spectrum( higher ppm)

• Upfield means lower energy –right side of the spectrum (lower ppm)

A neighboring electronegative atom pulls the electron density thus

exposing the nucleus to a strong magnetic field:
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Chemical structure of Tertiary-butyl-methyl ether:

Applications:
• NMR spectroscopy is a spectroscopy technique used by chemists and
biochemists to investigate the properties of organic molecules, although it is
applicable to any kind of sample that contains nuclei possessing spin.
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• For example, the nmr can quantitatively analyze mixtures containing known
compounds. NMR can either be used to match against spectral libraries or to
infer the basic structure directly for unknown compounds.

• Once the basic structure is known, nmr can be used to determine molecular
conformation in solutions as well as in studying physical properties at the
molecular level such as conformational exchange, phase changes, solubility,
and diffusion.

• MRI

Fluroscence resonance energy transfer (FRET):

• Fluorescence resonance energy transfer (FRET)* is a distance-dependent
physical process by which energy is transferred non-radiatively from an
excited molecular fluorophore (the donor) to another fluorophore (the
acceptor) by means of intermolecular long-range dipole–dipole coupling.

• The efficiency of this energy transfer is inversely proportional to the 6th

power of the distance between donor and acceptor (10-100 A0), making
FRET extremely sensitive to small changes in distance.

FRET∞ 𝟏/𝑹𝟔𝝄

Fret peptides:
▪ Enzyme substrates based on FRET (fluorescence resonance energy transfer)
are extremely sensitive tools for determining protease activity, especially
when there is a requirement for specific amino acids on both sides of the
cleavage site in the target protein or peptide.
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▪ A fluorescent group and a quencher group are attached to either end of the
peptide sequence containing the cleavage site. The peptide should be just
long enough to contain the minimum sequence requirements of the protease.
▪ After cleavage there is no FRET transfer. For FRET transfer they must be
remain in 100-110 nm.

Bioluminescence
Bioluminescence is the production of emitting light by a living organism. As the
result of a chemical reaction during which chemical energy is converted to light
energy.
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• The most commonly used enzyme is luciferase. But most importantly,

enzymes are not need to supply from outside. It remains as intrinsic
inside the system.

• The color of the light emitted depends on the source of enzyme and
varies between 560nm (greenish yellow) and 620nm (red)
wavelength.

• It is a highly sensitive method due to high quantum.

Bioluminescence resonance energy transfer (BRET) is a transfer of energy

between a luminescence donor and a fluorescence acceptor.

• Because BRET occurs when the distance between the donor and acceptor is
<10 nm, and its efficiency is inversely proportional to the 6th power of
distance, it has gained popularity as a proximity-based assay to monitor
protein–protein interactions and conformational rearrangements in live cells.

BRET∞ 𝟏/𝑹𝟔𝝄

• In such assays, one protein of interest is fused to a bioluminescent energy

donor (luciferases from Renilla reniformis or Oplophorus gracilirostris),
and the other protein is fused to a fluorescent energy acceptor (such as GFP
or YFP).

Donor+ Interested protein

Acceptor+ GFP/YFP

• Because the BRET donor does not require an external light source, it
does not lead to phototoxicity or autofluorescence.

• It therefore represents an interesting alternative to fluorescence-based

imaging such as FRET.
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Figure: Energy transfer between protein A & B. EYFP= enhanced Yellow

Fluorescent Protein

Difference between FRET & BRET

FRET BRET

1.FRET stands for fluroscence 1.BRET stands for bioluminescence

resonance energy transfer resonance energy transfer

2.FRET use fluorescent proteins as 2.BRET use bioluminescent proteins as the

the donor donor
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3.FRET requires external light 3.BRET does not require external light
source to excite the donor source to excite the donor

4.FRET is a process that shifts 4.BRET is a form of radiation free energy

energy from an electronically that can occur when two compatible optical
excited molecule(donor)to a probes, an energy donor and an energy
neighbouring molecule(acceptor) acceptor are brought into molecular
proximity

5.Applications: 5.Applications:

1.used for determining the 1.used to observe protein -protein

distance between two fluorophores. interaction in living cells especially in the
field of GPCR research.
2.used to detect and track
interactions between proteins. 2. May be used in the future to
identify new protein complexes in human.
3.widely used for the
detection of a target interaction in
biomolecular and cellular research

Luminometry:

Luminometry is the technique used to measure luminescence, which is the

emission of electromagnetic radiation in the energy range of visible light as a result
of a reaction. It is actually the process of measuring light.

• Luminometers are the instruments that do the measuring. They can be

fundamentally defined as machines that detect very low light levels.
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Applications:

• There are 3 main systems which are of frequent use as:

1. Firefly
2. Bacterial luminescence
3. Luminol chemiluminescence.

The principle and applications of each of these are described below:

a. Firefly luminescence and ATP measurement: Luciferase enzyme catalyzes

the following reaction in the presence of Mg2+:

Here, for each molecule of ATP reacting, 1 photon of intensity of 562 nm is

produced. This system is highly specific for ATP if all the reagents are pure. By
linking this reaction with various other reactions, it can be used to assay a number
of ATP specific enzymes and their substrates such as creatine kinase, creatine
phosphate etc.

B. Bacterial luminescence and coenzymes measurement: the coenzymes that can

be measured by this method are the NADH and NADPH. This system utilizes a
purified oxidoreductase obtained from the bacterium Benecka harveyi. The
reaction can be then coupled to bacterial luciferase as follows:
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Here, bacterial luciferase catalyzes the oxidation of aldehyde by oxygen in the

presence of FMN𝐻2 during which a photon of maximum intensity at 495nm is
produced.

• C. Luminol based chemiluminescent assays: Luminol is oxidized by

hydrogen peroxide at pH 10-11 if chromium, copper or iron compounds are
used as catalysts. Photons with maximum intensity at 430nm are produced.

Gold Nanoparticles (GNPs)

Bioluminescence application:
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Chemiluminescence application:

:
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MALDI-TOF mass spectrometry, MALDI-TOF

▪ Matrix-assisted laser desorption ionisation (MALDI) produces gas phase

protonated ions by excitation of the sample molecules from the energy of a
laser transferred via a UV light-absorbing matrix.

• The matrix is a conjugated organic compound (normally a weak organic

acid such as a derivative of cinnamic acid and dihydroxybenzoic acid) that is
intimately mixed with the sample.

• These are designed to maximally absorb light at the wavelength of the laser,
typically a nitrogen laser of 337 nm or a neodymium/yttrium-
aluminium-garnet (Nd-YAG) at 355 nm.
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• The sample (1–10 pmol m𝑚−3 ) is mixed with an excess of the matrix and
dried on to the target plate, where they co-crystallize on drying.

• Pulses of laser light of a few nanoseconds duration cause rapid excitation

and vaporization of the crystalline matrix and the subsequent ejection of
matrix and analyte ions into the gas phase.

• This generates a plume of matrix and analyte ions that are analyzed in a TOF
mass analyzer.

Table : Examples of MALDI matrix compounds

Compound Structure Application

𝛼-Cyano-4- Peptides < 10 𝑘𝐷𝑎

hydroxycinnamic acid
(glycopeptides)
(CHCA)

Sinapinic acid (3,5- Proteins >10 kDa

dimethoxy-4-
hydoxycinnamic acid)
(SA)

2,5-Dihydroxybenzoic Neutral
acid (DHB) (gentisic carbohydrates,
acid) synthetic polymers
(oligos)
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3-Hydroxypicolinic Oligonucleotides
acid

Figure: MALDI ionisation mechanism and MALDI–TOF sample plate(a) The

sample is mixed, in solution, with a ‘matrix’ – the organic acid in excess of the
analyte (in a ratio between 1000 : 1 to 10 000 : 1) and transferred to the MALDI
plate. An ultraviolet laser is directed to the sample (with a beam diameter of a few
micrometres) for desorption. The laser radiation of a few nanoseconds’ duration is
absorbed by the matrix molecules, causing rapid heating of the region around the
area of laser impact and electronic excitation of the matrix. The immediate region
of the sample explodes into the high vacuum of the mass spectrometer, creating
gas phase protonated molecules of both the acid and the analyte. The laser flash
ionises matrix molecules: neutrals (M) and matrix ions (𝑀𝐻)+ , (𝑀 − 𝐻)− and
sample neutral fragments (A). (b) A MALDI sample plate.
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❖ Advantage of MALDI

➢ The particular advantage of MALDI is the ability to produce large mass

ions, with high sensitivity.

➢ Ionization of nonvolatile molecules.

➢ It is a very soft ionisation method that does not produce abundant amounts
of fragmentation compared with some other ionisation methods. Since the
molecular ions are produced with little fragmentation, it is a valuable
technique for examining mixtures.

❖ Disadvantage:

➢ High cost and time consuming.

➢ Need pure isolate grown culture.

Time-of-flight (TOF)

 TOF is the best type of mass analyzer to coupled to MALDI, as this

technique has a virtually unlimited mass range
 Proteins and other macromolecules of 𝑀𝑟 greater than 400,000 have been
accurately measured. The principle of TOF is following:

 Measures time of flight at detector. Larger molecule require more time.

Atomic absorption spectroscopy

Atomic absorption spectroscopy (AAS) and atomic emission spectroscopy (AES)
is a spectro-analytical procedure for the quantitative determination of chemical
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elements using the absorption of optical radiation (light) by free atoms in the
gaseous state.

▪ Atomic absorption spectroscopy is based on absorption of light by free

metallic ions. It can analyze around 70 elements.

▪ It can measure the concentration of metal in a sample.

▪ For instrumentation, flame, non-flame, and graphite furnace are available in

atomic absorption instruments. Any atomic absorption spectroscopy
instrumentation has the following types of components,

1. Atomizer
2. Hollow cathode lamp
3. Monochromator
4. Detector
5. Recorder
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Principle:

• The technique uses basically the principle that free atoms (gas) generated in
an atomizer can absorb radiation at specific frequency.

• Atomic-absorption spectroscopy quantifies the absorption of ground state

atoms in the gaseous state.

• The atoms absorb ultraviolet or visible light and make transitions to higher
electronic energy levels. The analyte concentration is determined from the
amount of absorption.

• Concentration measurements are usually determined from a working curve

after calibrating the instrument with standards of known concentration.

• Atomic absorption is a very common technique for detecting metals and

metalloids in environmental samples.

Applications:

• Determination of even small amounts of metals (lead, mercury,

calcium, magnesium, etc) as follows:

• Environmental studies: drinking water, ocean water, soil

• Food industry.

• Pharmaceutical industry.