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CHAPTER 8
BIOCHEMICAL TECHNIQUES
1
• Technology Oriented • Business Driven • Sustainable Development • Environmental Friendly
Biochemical techniques
Biomolecules can be isolated and purified by applying
different techniques (based on various chemical and
physical properties) such as:
i) Techniques based on size and weight of the
molecules are centrifugation, gel filtration and
osmotic pressure
ii) Techniques based on polarity and charge are ion
exchange chromatography, electrophoresis,
isoelectric focusing, hydrophobic interaction, normal
and reversed‐phase chromatography 2
Biochemical techniques
iii)Techniques based on spectroscopy include

colorimetry, UV‐visible spectrophotometry,
fluorescence spectroscopy, refracAon index and
mass spectrometry
iv) Techniques based on solubility are the precipitation
of molecules with salts and organic solvents
3
Techniques based on molecular weight & size

(Centrifugation)
• Centrifugation; separating particles from a solution
according to their sedimentaAon rate (depends on factors
like size, shape, density, medium viscosity and centrifugal
force (speed)
• Analytical centrifugation; measuring the physical
properties of the sedimentation particles such as
sedimentation coefficient or molecular weight (analytical
ultracentrifugation)
• Preparative centrifugation; to isolate specific particles,
which can be reused (rate zonal, differential and isopycnic
centrifugation) 100

(Centrifugation)
Ultracentrifugation is carried out at

speed faster than 30,000 rpm. High‐
speed centrifugaAon is at speeds
between 10,000 and 30,000 rpm. Low‐
speed centrifugation is at speeds below
10,000 rpm 101

(Centrifugation)
Differential centrifugation 102


(Centrifugation)
Rate zonal centrifugation; the
sample is applied in a thin zone
at the top of the tube on a
density gradient. Under
centrifugation force, the protein
will begin sedimenting through
the gradient in separate zone
according to their size, shape,
and density or the
sendimentation coefficient
103

(Centrifugation)
Isopycnic centrifugation; the
density gradient column
encompasses the whole
range of density of the
sample particles (sample is
uniformly mixed). Under
centrifugation force,
particles can be separated
A.sample is evenly distributed throughout the into zones solely on the
centrifuge tube basis of their buoyant
B. after centrifugation protein migrate to their
isopycnic densities density differences. 104

(Gel-filtration)
• Gel filtraAon chromatography; separaAon based on
size
• The staAonary phase consists of porous beads with a
well‐defined range of pore sizes
• Small proteins can fit inside all the pores in the beads
and are said to be included
• Proteins that are too large to fit inside any of the
pores are said to be excluded
9

(Gel-filtration)
10

(Osmotic pressure)
• Osmotic pressure; is the pressure which needs to

be applied to a solution to prevent the inward flow
of solvent molecule across a semipermeable
membrane
• OsmoAc pressure of a solution depends on the
concentration of a solute and the temperature of
the solution
11

(Osmotic pressure)
12
Techniques based on charge (Ion-exchange

chromatography)
• Ion‐exchange chromatography; separaAng of almost any
type of charged molecule, from large proteins to small
nucleotides and amino acids
• Anion‐exchange chromatography; separating negatively
charged proteins (anions). Stationary phase has positively
charged groups , which bind negative sites on the protein
• Cation‐exchange chromatography; separating positively
charged proteins (caAons). Stationary phase has negatively
charged groups , which bind positive sites on the protein
13
Techniques based on charge (Ion-exchange

chromatography)
110
Techniques
based on charge
(Ion-exchange
chromatography)
111
Techniques based on charge (Electrophoresis)

• Electrophoresis; migraton of charged molecules such as
proteins in an electrical field is based on size, shape and
charge
• Most use a supporting media such as starch, paper,
polyacrylamide or agarose
112
Techniques based on charge (Electrophoresis)
Preparation of SDS-PAGE (sodium

dodecyl sulfate polyacrylamide gel Picture of an SDS-PAGE. The
electrophoresis molecular marker is in the left 113
Techniques based on charge (Isoelectric focusing)
• Isoelectric focusing; separating proteins according to their

isoelectric pH by carrying out electrophoresis in a gel
containing a pH gradient
• Upon application of a current, the protein will move toward
either the anode or cathode (depending on its charge) until
it encounters that part of the gel, which corresponds to its
isoelectric point at which point it will have no net charge
and will, therefore, stop migrating
18
19
Isoelectric focusing employs a pH

gradient extending the length of an
electrophoresis gel. A protein stops
migrating when it enters the zone
in which the surrounding pH equals
its isoelectric point, pI. At any other
point in the gradient, the protein
acquires a charge which causes it
to migrate toward its pI (green and
blue arrows)
20
Techniques based on polarity (Hydrophobic-

interaction chromatography)
• HIC; molecules are separated according to their

hydrophobic interaction between the stationary phase and
mobile phase
• Protein and peptides differ from one another in their
hydrophobic properties and this difference forms the basis
of a HIC separation
• Salt solutions are ol en used to mediate the binding of
sample molecules to a hydrophilic matrix substituted with a
hydrophobic ligand
21
Techniques based on polarity (Hydrophobic-

interaction chromatography)
22
Techniques based on polarity (Normal-phase

chromatography)
• Normal‐phase chromatography; the staAonary phase

is polar and the mobile phase is nonpolar (based on
adsorpAon to a staAonary surface chemistry and by
polarity)
• AdsorpAon strengths increase with increased analyte
polarity, and the interacAon between the polar analyte
and the polar staAonary phase (relaAve to the mobile
phase) increases the eluAon Ame.
119
Techniques based on polarity (Normal-phase

chromatography)

is polar and the mobile phase is nonpolar (based on
adsorpAon to a staAonary surface chemistry and by
polarity)
• AdsorpAon strengths increase with increased analyte
polarity, and the interacAon between the polar analyte
and the polar staAonary phase (relaAve to the mobile
phase) increases the eluAon Ame.
129
Techniques based on polarity (Reversed-phase

chromatography)

is nonpolar and the mobile phase is polar
• The retenAon Ame can be increased by adding more
water to the mobile phase; thereby making the affinity of
the hydrophobic analyte for the hydrophobic staAonary
phase stronger relaAve to the now more hydrophilic
mobile phase
25
Techniques based on polarity
Stationary Phase Particle

Stationary Phases
Silica CN C8 C18 (ODS) Chromatographic
Polarity Spectrum
Mobile Phase
Mobile Phases
Water Alcohol Acetonitrile THF Hexane
Chromatographic
Polarity Spectrum
Salt Alcohols Ether Aromatic Fluorinate Compound/Analyte

Sample Analytes Chromatographic
Acids Ketones Halogenated Aliphatic
Polarity Spectrum
26
Techniques based on polarity
Normal-Phase Chromatography
Reversed-Phase Chromatography
27
Techniques based on spectroscopy
• Spectroscopy; the use of absorpAon, emission or

scarering of electromagneAc radiaAon by marer to
qualitaAvely or quanAtaAvely study (atoms, molecules,
atomic or molecular ions or solids)
• AdsorpVon; molecules absorb a parAcular
electromagneAc radiaAon of the spectrum which is
followed by a transiAon of electrons from a lower
energy level to a higher level with transfer of energy
from the radiaAon to the absorber, atom, molecule or
solid 124
• Emission; Atoms or molecules absorbing electromagneAc

radiaAon are excited to high energy levels and can decay to
lower levels by emisng radiaAon (emission) in the form of
heat or light. If no radiaAon is emired, the transiAon from
higher to lower energy levels is called nonradiaVve decay.
• Scaeering; RedirecAon of light due to its interacAon with
marer. Scarering may or may not occur with a transfer of
energy (i.e., the scarered radiaAon might or might not have
a slightly different wavelength compared to the light
incident on the sample)
29
• Technique of spectrophometry is generally used for the

qualitaAve and quanAtaAve esAmaAon of biomolecules
such as proteins, sugars, carbohydrates, amino acids, nucleic
acids, vitamins and etc.
30

(Electromagnetic radiation)
• ElectromagneAc radiaAon; transverse energy wave that is
composed of an oscillaAng electric field component, E, and
an oscillaAng magneAc field component, M
• The electric and magneAc fields are orthogonal to each
other, and orthogonal to the direcAon of propagaAon of the
wave
• A wave is described by wavelength, λ, which is the physical
length of one complete oscillaAon, and the frequency, ν,
which is the number of oscillaAons per second.
31

(Electromagnetic radiation)
Schematic representation of an electromagnetic wave

32
Techniques based on spectroscopy (Visible

spectrum)
• The visible region of the electromagneAc radiaAon is

approximately 400 to 750 nm.
• The short wavelength cutoff is due to absorpAon by
the lens of the eye and the long wavelength cutoff is
due to the decrease in sensiAvity of the
photoreceptors in the reAna for longer wavelengths
33
Techniques based on spectroscopy (Visible

spectrum)
The visible spectrum of electromagnetic radiation 130

Techniques based on spectroscopy (Colorimetry)
• Colorimetry; the interacAon of light with colored

soluAons (as light passes through a colored soluAon,
some of the wavelengths of light will be absorbed by
the soluAon)
• The amount of light absorbed by the soluAon will be
directly proporAonal to the concentraAon of the light‐
absorbing molecules present in the soluAon
• The technique is based on the Beer‐Lawbert law
35
Techniques based on spectroscopy (Colorimetry)
Colorimeter
36
Techniques based on spectroscopy (UV-visible

spectrophotometer)
• UV‐visible spectrophotometer; to measure the
amount of light that a sample absorbs (measuring the
intensity of light reaching a detector)
• The beam of light consists of a stream of photons,
when a photon encounters an analyte molecule, there
is a chance the analyte will absorb the photon
• The technique is based on the Beer‐Lawbert law
• It is possible to provide the absorpAon spectra of the
compound 133
Techniques based on spectroscopy (UV-visible

spectrophotometer)
38
Techniques based on spectroscopy (Beer-Lambert

law)
• Beer‐Lambert law is the linear relaAonship between
absorbance and concentraAon of an absorbing
species
• The absorbance of a soluAon at a given wavelength is
directly proporAonal to the path length or the
thickness of the absorbing medium and the
concentraAon of the absorbing species of the
molecules in the soluAon
39
Techniques based on spectroscopy (Beer-Lambert

law)
A = lc
I 
A = log  o 
 I1 
Absorption of light by a sample

Beer-Lambert law is applicable in all
spectroscopic techniques, which involve the
adsorption and emission of electromagnetic
radiations
40
Techniques based on spectroscopy (Fluorescence

spectroscopy)
• Fluorescence spectroscopy; type of electromagneAc

spectroscopy which analyzes fluorescence from a sample
(it involves using a beam of light, usually ulraviolet light,
that excites the electrons in molecules of certain
compounds and causes them to emit light of a lower
energy)
• The emission intensity of an emisng substance is linearly
proporAonal to low analyte concentraAon, and useful for
quanAtaAng the emisng species of molecule 41
Techniques based on spectroscopy (Fluorescence

spectroscopy)
42
Techniques based on spectroscopy (Refractive-

index)
• RI; medium is the raAo of the velocity of light through
a vacuum to the velocity of light through the medium
• As light moves from a medium water or glass into
soluAon containing biomolecule, it may change its
propagaAon direcAon in proporAon to the change in
refracAve index
43
Techniques based on spectroscopy (Refractive-

index)
140
Techniques based on spectroscopy (Mass

spectrometry)
• MS; an analyAcal technique that measures the mass to charge
raAo of charged biomolecules
• A substance is bombarded with an electron beam having
sufficient energy to fragment the molecule
• The posiAve fragments, which are produced (caAons and radical
caAons) are accelerated in a vacuum through a magneAc field
and are sorted on the basis of mass‐to‐charge raAo
• Since the bulk of the ions produced in the mass spectrometer
carry a unit posiAve charge, the value m/e is equivalent to the
molecular weight of the fragment
45
Techniques based on spectroscopy (Mass

spectrometry)
Schematic representation of a
mass spectrometer Mass spectrum of toluene
46
Techniques based on spectroscopy (Other

methods)
• AAS (Atomic absorpAon

spectrophometry)
• AES (Atomic emission spectroscopy)
• Spectroradiometry
• NMR (Nuclear magneAc resonance)
• X‐ray crystallography
47
Combination techniques
High performance liquid
chromatography (HPLC)
Chromatography
column
UV‐light/
fluorescence/RI/
MS detector
144
Combination techniques
Gas chromatography (GC)
49
Techniques based on solubility (Salt precipitation)

• This is a very simple and widely used technique to isolate
proteins and enzymes from other macromolecules such as
carbohydrates and nucleic acids
• The solubility of protein in an aqueous medium is
dependent on i. ionic strength, ii. pH of the soluAon, iii.
nature of the solvent, iv. other dissolved compounds (i.e.,
urea) and v. temperature
• A change in one (or all) of these factors can cause a
protein to “drop out” of soluAon (denaturing)
50
Techniques based on solubility (Salt precipitation)

• It dissolves to give ions that become hydrated by
hydrogen bonding and it disturbs the interacAon of water
molecules with the protein molecules (expose the
hydrophobic patches of protein and results in the
hydrophobic interacAons between protein molecules)
• It leads to the aggregaAon of protein molecules and their
precipitaAon.
• The salt‐precipitated proteins (denatured) can be
redissolved in a buffer and the acAvity can be restored
51
Techniques based on solubility (Precipitation with

organic solvent)
• In the presence of organic solvents, the dielectric
constant of the soluAon is lowered and that causes the
increased arracAon between the oppositely charged
amino acid residues present on the surfaces of the
protein molecules
• This results in the coagulaAon and formaAon protein
aggregates ensuing in the precipitaAon (depend on ionic
strength of the soluAon, organic solvent used, and the
temperature)
52
Techniques based on solubility (Precipitation with

organic solvent)
• Examples of organic solvent; acetone and PEG (polymer
polyethylene glycol)
53
The end….
Thank you
54

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CHAPTER 8

iii)Techniques based on spectroscopy include

Techniques based on molecular weight & size

Techniques based on molecular weight & size

Ultracentrifugation is carried out at

Techniques based on molecular weight & size

Differential centrifugation 102

Techniques based on molecular weight & size

Techniques based on molecular weight & size

Techniques based on molecular weight & size

Techniques based on molecular weight & size

Techniques based on molecular weight & size

• Osmotic pressure; is the pressure which needs to

Techniques based on molecular weight & size

Techniques based on charge (Ion-exchange

Techniques based on charge (Ion-exchange

Techniques based on charge (Electrophoresis)

Techniques based on charge (Electrophoresis)

Preparation of SDS-PAGE (sodium

Techniques based on charge (Isoelectric focusing)

• Isoelectric focusing; separating proteins according to their

Techniques based on charge (Isoelectric focusing)

Techniques based on charge (Isoelectric focusing)

Isoelectric focusing employs a pH

Techniques based on polarity (Hydrophobic-

• HIC; molecules are separated according to their

Techniques based on polarity (Hydrophobic-

Techniques based on polarity (Normal-phase

• Normal‐phase chromatography; the staAonary phase

Techniques based on polarity (Normal-phase

• Normal‐phase chromatography; the staAonary phase

Techniques based on polarity (Reversed-phase

• Normal‐phase chromatography; the staAonary phase

Techniques based on polarity

Stationary Phase Particle

Salt Alcohols Ether Aromatic Fluorinate Compound/Analyte

Techniques based on polarity

Techniques based on spectroscopy

• Spectroscopy; the use of absorpAon, emission or

Techniques based on spectroscopy

• Emission; Atoms or molecules absorbing electromagneAc

Techniques based on spectroscopy

• Technique of spectrophometry is generally used for the

Techniques based on spectroscopy

Techniques based on spectroscopy

Schematic representation of an electromagnetic wave

Techniques based on spectroscopy (Visible

• The visible region of the electromagneAc radiaAon is

Techniques based on spectroscopy (Visible

The visible spectrum of electromagnetic radiation 130

Techniques based on spectroscopy (Colorimetry)

• Colorimetry; the interacAon of light with colored

Techniques based on spectroscopy (Colorimetry)

Techniques based on spectroscopy (UV-visible

Techniques based on spectroscopy (UV-visible

Techniques based on spectroscopy (Beer-Lambert

Techniques based on spectroscopy (Beer-Lambert

Absorption of light by a sample

Techniques based on spectroscopy (Fluorescence

• Fluorescence spectroscopy; type of electromagneAc

Techniques based on spectroscopy (Fluorescence

Techniques based on spectroscopy (Refractive-