3. CENTRIFUGATION
DEFINITION
∞ Centrifugation is a process which
involves the application of
the centripetal force for
the sedimentation of heterogeneous
mixtures with a centrifuge, and is
used in industrial and laboratory
settings. This process is used to
separate two immiscible substances.
More-dense components of the
mixture migrate away from the axis
of the centrifuge, while less-dense
components of the mixture migrate
towards the axis.
4. CENTRIFUGATION
APPARATUS USED
∞ A centrifuge is a piece of equipment that puts an object
in rotation around a fixed axis (spins it in a circle), applying
a potentially strong force perpendicular to the axis of spin
(outward). The centrifuge works using the sedimentation
principle, where the centripetal acceleration causes denser
substances and particles to move outward in the radial
direction. At the same time, objects that are less dense are
displaced and move to the center. In a laboratory
centrifuge that uses sample tubes, the radial acceleration
causes denser particles to settle to the bottom of the tube,
while low-density substances rise to the top.
6. CHROMATOGRAPHY
DEFINITION
∞ Chromatography is the collective term for a set of laboratory
techniques for the separation of mixtures.
∞ Chromatography may be preparative or analytical.
∞ The purpose of preparative chromatography is to separate the
components of a mixture for more advanced use (and is thus a form
of purification).
∞ one of the most useful analytical techniques chemists have at their
disposal, helpful in everything from identifying biological materials
to finding clues at crime scenes.
∞The moving substance is called the mobile phase and the substance
that stays put is the stationary phase. As the mobile phase moves, it
separates out into its components on the stationary phase.
8. CHROMATOGRAPHY: TYPES
PAPER
CHROMATOGRAPHY
∞ This is the "spot of ink on paper"
experiment you often do in school (also the
effect we described at the start when you
get your papers wet). Typically you put a
spot of ink near one edge of some filter
paper and then hang the paper vertically
with its lower edge (nearest the spot) dipped
in a solvent such as alcohol or water.
Capillary action makes the solvent travel up
the paper, where it meets and dissolves the
ink. The dissolved ink (the mobile phase)
slowly travels up the paper (the stationary
phase and separates out into different
components. Sometimes these are colored;
sometimes you have to color them by
adding other substances (called developers
or developing fluids) that help you with
identification.
9. CHROMATOGRAPHY: TYPES
COLUMN
CHROMATOGRAPHY
∞ Instead of paper, the stationary phase is a
vertical glass jar (the column) packed with a
highly adsorbent solid, such as crystals of
silica or silica gel, or a solid coated with a
liquid. The mobile phase is pumped at high
pressure through the column and splits into
its components, which are then removed and
analyzed. In liquid-column chromatography,
the mixture being studied is placed at one
end of the column and an extra added
substance called an eluant is poured in to
help it travel through. Thin-film
chromatography is a variation of this
technique in which the "column" is actually
a film of glass, plastic, or metal coated with a
very thin layer of adsorbent material
10. CHROMATOGRAPHY: TYPES
GAS CHROMATOGRAPHY
∞ is a largely automated type of chemical
analysis you can do with a sophisticated
piece of laboratory equipment called, not
surprisingly, a gas chromatograph
machine.
∞ First, a tiny sample of the mixture of
substances being studied is placed in a
syringe and injected into the machine. The
components of the mixture are heated and
instantly vaporize. Next, we add a carrier
(the eluant), which is simply a neutral gas
such as hydrogen or helium, designed to
help the gases in our sample move through
the column.
11. CHROMATOGRAPHY: TYPES
GAS CHROMATOGRAPHY
∞ In this case, the column is a thin glass or metal tube usually
filled with a liquid that has a high boiling point (or sometimes a
gel or an adsorbent solid). As the mixture travels through the
column, it's adsorbed and separates out into its components.
Each component emerges in turn from the end of the column
and moves past an electronic detector (sometimes a mass
spectrometer), which identifies it and prints a peak on a chart.
The final chart has a series of peaks that correspond to all the
substances in the mixture. Gas chromatography is sometimes
called vapor-phase chromatography (VPC) or gas-liquid
partition chromatography (GLPC).
12. CHROMATOGRAPHY
TOOLS AND APPARATUS USED
▸The column is where the actual separation takes
place. It is usually a glass or metal of with sufficient
strength to handle pressure.
▸A packed bed column in compromised of a stationary
phase which is granular form and packed into the
column as homogenous bed. The stationary phase
complete fills the column.
▸An open tubular column’s stationary phase is a thin
film or layer on the column wall.
13. CHROMATOGRAPHY
THE MOBILE AND STATIONARY PHASES
The mobile phase is comprised of a solvent into
which the sample is injected. The solvent and sample
flow through the column together; thus the mobile
phase is often referred to as the "carrier fluid." The
stationary phase is the material in the column for
which the components to be separated have varying
affinities. The materials which comprise the mobile
and stationary phases vary depending on the general
type of chromatographic process being performed.
14. CHROMATOGRAPHY
HOW DOES IT WORK?
Think of chromatography as a race and you'll find it's
much simpler than it sounds. Waiting on the starting line,
you've got a mixture of chemicals in some unidentified
liquid or gas, just like a load of runners all mixed up and
bunched together. When a race starts, runners soon
spread out because they have different abilities. In exactly
the same way, chemicals in something like a moving
liquid mixture spread out because they travel at different
speeds over a stationary solid. The key thing to remember
is that chromatography is a surface effect.
15. CHROMATOGRAPHY
HOW DOES IT WORK?
For chromatography to work effectively, we obviously need the
components of the mobile phase to separate out as much as possible
as they move past the stationary phase. That's why the stationary
phase is often something with a large surface area, such as a sheet of
filter paper, a solid made of finely divided particles, a liquid deposited
on the surface of a solid, or some other highly adsorbent material.
The mixture is dissolved in a fluid called the mobile phase, which
carries it through a structure holding another material called the
stationary phase. Different rates of migration cause the various
constituents of the mixture to travel at different speeds, causing them
to separate.
17. GEL ELECTROPHORESIS
DEFINITION
∞ Gel electrophoresis is a method for separation
and analysis of macromolecules
(DNA, RNA and proteins) and their fragments,
based on their size and charge. It is used in
clinical chemistry to separate proteins by charge
and/or size (IEF agarose, essentially size
independent) and in biochemistry and molecular
biology to separate a mixed population
of DNA and RNA fragments by length, to
estimate the size of DNA and RNA fragments or
to separate proteins by charge.
∞ Nucleic acid molecules are separated by
applying an electric field to move the negatively
charged molecules through a matrix of
18. GEL ELECTROPHORESIS
PHYSICAL BASIS
In simple terms, electrophoresis is a
process which enables the sorting of
molecules based on size. Using an electric
field, molecules (such as DNA) can be
made to move through a gel made
of agar or polyacrylamide. The electric
field consists of a negative charge at one
end which pushes the molecules through
the gel, and a positive charge at the other
end that pulls the molecules through the
gel. The molecules being sorted are
dispensed into a well in the gel material.
The gel is placed in an electrophoresis
19. GEL ELECTROPHORESIS
HISTORY
• 1930s – first reports of the use of sucrose for gel electrophoresis
• 1955 – introduction of starch gels, mediocre separation
• 1959 – introduction of acrylamide gels; disc electrophoresis (Ornstein and Davis); accurate
control of parameters such as pore size and stability; and (Raymond and Weintraub)
• 1966 – agar gels
• 1969 – introduction of denaturing agents especially SDS separation of protein subunit
(Weber and Osborn)
• 1970 – Laemmli separated 28 components of T4 phage using a stacking gel and SDS
• 1972 – agarose gels with ethidium bromide stain
• 1975 – 2-dimensional gels (O’Farrell); isoelectric focusing then SDS gel electrophoresis
• 1977 – sequencing gels
• 1983 – pulsed field gel electrophoresis enables separation of large DNA molecules
• 1983 – introduction of capillary electrophoresis
• 2004 – standardized time of polymerization of acrylamide gels enables clean and
predictable separation of native proteins
21. GEL ELECTROPHORESIS: TYPES OF GEL
AGAROSE
Agarose gels are made from the
natural polysaccharide polymers extracted
from seaweed. Agarose gels are easily cast
and handled compared to other matrices,
because the gel setting is a physical rather
than chemical change. Samples are also
easily recovered. After the experiment is
finished, the resulting gel can be stored in a
plastic bag in a refrigerator.
Agarose gels do not have a uniform pore
size, but are optimal for electrophoresis of
proteins that are larger than 200
22. GEL ELECTROPHORESIS: TYPES OF GEL
POLYACRYLAMIDE
Polyacrylamide gel electrophoresis (PAGE) is
used for separating proteins ranging in size from
5 to 2,000 kDa due to the uniform pore size
provided by the polyacrylamide gel. Pore size is
controlled by modulating the concentrations of
acrylamide and bis-acrylamide powder used in
creating a gel. Care must be used when creating
this type of gel, as acrylamide is a potent
neurotoxin in its liquid and powdered forms.
Traditional DNA sequencing techniques such
as Maxam-Gilbert or Sanger methods used
polyacrylamide gels to separate DNA fragments
differing by a single base-pair in length so the
sequence could be read. Most modern DNA
23. GEL ELECTROPHORESIS: TYPES OF GEL
STARCH
Partially hydrolysed potato
starch makes for another non-
toxic medium for protein
electrophoresis. The gels are
slightly more opaque than
acrylamide or agarose. Non-
denatured proteins can be
separated according to charge
and size. They are visualised
using Napthal Black or Amido
24. GEL ELECTROPHORESIS
GEL CONDITIONS
∞ Denaturing gels are run under conditions that disrupt the natural
structure of the analyte, causing it to unfold into a linear chain. Thus, the
mobility of each macromolecule depends only on its linear length and its
mass-to-charge ratio. Thus, the secondary, tertiary, and quaternary levels
of biomolecular structure are disrupted, leaving only the primary structure
to be analyzed.
∞ Native gels are run in non-denaturing conditions, so that the analyte's
natural structure is maintained. This allows the physical size of the folded
or assembled complex to affect the mobility, allowing for analysis of all
four levels of the biomolecular structure. For biological samples,
25. GEL ELECTROPHORESIS
PROCESS
Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain
the pH at a relatively constant value. There are a number of buffers used for electrophoresis.
The most common being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE).
After the electrophoresis is complete, the molecules in the gel can be stained to make them
visible. DNA may be visualized using ethidium bromide which, when intercalated into DNA,
fluoresce under ultraviolet light, while protein may be visualised using silver stain or
Coomassie Brilliant Blue dye.
After separation, an additional separation method may then be used, such as isoelectric
focusing or SDS-PAGE. The gel will then be physically cut, and the protein complexes
extracted from each portion separately. Each extract may then be analysed, such as by
peptide mass fingerprinting or de novo peptide sequencing after in-gel digestion. This can
provide a great deal of information about the identities of the proteins in a complex.
