King Saud UniversityRiyadh

Saudi Arabia

Dr. Gihan GawishDr. Gihan GawishAssistant ProfessorAssistant 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

clarifiedclarifiedclarifiedclarified

nonclarifiednonclarifiednonclarifiednonclarified

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. They are very high flow rate (120 ml min cm )

2. Great capacity3. Resistance to almost all solvents

4. Ability to be heated to high temperature for rapid drying5. Low cost

-1-1 -2-2

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 transfers to change the media of growing bacteria quickly.

Disadvantage:

Nitrocellulose filters contain both wetting agents and glycerol

which must be washed out before use.

Many bacteria adsorb to nitrocellulose( approximately 2 x 10

cells/cm )

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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.

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.

The use of these binding properties is best seen

by the following examples:

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

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