Download 1-1-Membrane Filtration and Dialysis (Nitrocellulose, Glass and

King Saud University
Riyadh
Saudi Arabia
Dr. Gihan Gawish
Assistant Professor
Gihan Gawish.Dr

A common operation in chemistry and biochemistry is the
separation of one substance from another.

Filters were of fine cloth (e.g., cheesecloth) is sometimes still
used for preliminary clarification of tissues extracts.


Cheesecloth
porous paper
cellulose
acetate, nitrocellulose or fiberglass matrix.
Nitrocellulose have adsorptive properties
which enable them to bind particular
macromolecules like proteins.



The pores of the filter are not circular but
irregularly shaped and account for roughly
80% of the surface area.
The range of maximum particles size passed
is from 0.01 m to 14 m.
The most commonly used filter has
a pore size of approximately 0.45
m.
Gihan Gawish.Dr

Solve : Because of both the small pore size
and the surface tension, liquids don’t easily
pass through these filters with gravity as the
driving force so that pressure or suction is
usually employed.

One of the most common laboratory procedure for
clarifying solutions and trapping precipitates
(With certain filters, it is possible to trap bacteria or
fungi (> 0.3 m), but not viruses (> 0.02 m).

Simple assembly: Buchner Funnels or simple
funnels equipped with filter, and subjected to
vacuum generated by water flow or pump.

Variety of possible filters: Asbestos, glass fibers,
cellulose, nitrocellulose, or polycarbonate filters
nonclarified
nonclarified
clarified
clarified



Slow (slide no. 6)
Nitrocellulose is soluble in a variety of solvents
and attacked by certain chemicals.
Manufacturers supply similar membrane filters
of cellulose acetate, nylon, Teflon and poly
vinyl chloride for use in certain solvents
systems.

They consist of a network of fine glass fibers.
They are relatively thick- approximately 0.25mm

2-1 Advantages:

1.
2.
3.
4.
5.
-1
-2
They are very high flow rate (120 ml min cm )
Great capacity
Resistance to almost all solvents
Ability to be heated to high temperature for rapid drying
Low cost
1.
There is not an absolute pore size as there is with a
membrane filters.
2.
Sometimes tiny pieces of glass fiber are found in the
filtrate
3.
A small number of porosities are available.
3-1 Clarifications of solutions:
It is necessary to remove particular matter from a liquid,
such as bacteria, dust, and fine precipitates (chemical and
biological applications).
3-1-1 Disadvantage of clarification using nitrocellulose
Some proteins, viruses, and bacteriophages adsorb to nitrocellulose so that the filter
must be pretreated in some way.
3-1-2 Disadvantage of clarification using fiberglass
If the solution is to be clarified for special studies such as measurement of
absorbance, fluorescence( if light scattering is to be done), tiny glass particles from
fiberglass enter the filters.
3-2-1 For counting macromolecular radioactivity, it is
almost
always necessary to separate radioactive macromolecules
from small radioactive molecules.
3-2-2 For liquid scintillation counting, fiberglass should be used
because it produces slightly higher counting efficiency, and it
can be dried at high temperature.
media
to
transfers
change the media of growing bacteria quickly.
Disadvantage:

Nitrocellulose filters contain both wetting agents and glycerol
which must be washed out before use.
6

Many bacteria adsorb to nitrocellulose( approximately 2 x 10
2
cells/cm )

Nitrocellulose filters bind proteins and single-
stranded DNA under certain conditions.

This binds can be used as a bases for a large
number of enzymatic and physicochemical
assays and as a means of purifications of
various materials.
 The use of these binding properties is best seen by the following
examples:
1.
Purification of covalently closed, circular DNA
2.
Assay of messenger RNA
3.
Purification of mRNA from mammalian cells
4.
Assay of complementary single stranded DNA
5.
Assay of proteins that bind to double stranded DNA
6.
Assay of specific aminoacyl t-RNA synthetase
7.
Elucidation of the genetic code by membrane binding
8.
Detection of antigen antibody interactions by membrane binding.
3-4-1. Purification of covalently closed, circular
DNA:

Some strains of bacteria and some animal cells contain
circular double stranded DNA molecules.

Usually circular DNA is just a small fraction of the total DNA
in an organism or cell.
See Physical
Biochemistry book
(Application to
Biochemistry and
Molecular Biology),
David Freifelder
page;150-Fig. 7-3
1- The DNA sample containing both double stranded linear DNA
and circular DNA is denaturated by treatment with alkali (linear
and circular DNAs are double stranded).
The hydrogen bonds in the DNA are broken and the two
strands are separated
2. The denaturated DNA is neutralized and incubated at a slightly high
temperature, The coiled circular DNA rapidly renature (reform) but the
separated single strands from linear DNA don’t
3. The mixture is filtered through a nitrocellulose filter at moderate ionic
strength.
4. The double stranded circles pass through the filter but the single strands bind
to the filter.
So, the pure circular DNA will be in the filtrate.

Antibodies are proteins and therefore bind to membrane
filters.

Many antigens are small organic molecules that don’t bind to
nitrocellulose filters.

Antigen bind to nitrocellulose only when it makes a complex
with antibody( Ag-Ab)
Polycarbonate filters are different from nitrocellulose and fiberglass
filters in a number of way:
1.
They are made of a continuous sheet of very thin polycarbonate
containing cylindrical holes
2.
They are thin; very flexible, transparent
3.
No need for pre washing (they haven’t detergents and softening
agents)
4.
The adsorption very poor (It is useful for sterilizing proteins solutions)

They have special use in cell biology and clinical testing
because they are flexible and transparent (no need to
transfer material from the filter to a microscope slide).

In clinical testing the poly carbonate membranes are useful
for collecting and concentrating cancer cells from body fluids
such as urine and cerebrospinal