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Theory module: 10
DIALYSIS, ULTRA FILTRATION AND LYOPHILLIZATION
Dialysis:
Dialysis is a separation technique that facilitates the removal of small ions or during
purification of macromolecule by selective and passive diffusion through a semipermeable membrane. Cut off of semi- permeable membrane was decided based on
molecular weight of protein of interest. Dialysis was done with buffer, usually 200-500
times the volume of the sample, in which protein of interest was dissolved.
Sample molecules that are larger than the membrane- pores are retained on the sample
side of the membrane, but small molecules and buffer salts pass freely through the
membrane, reducing the concentration of those molecules in the sample. Changing the
dialysate buffer removes the small molecule that no longer in the sample and allows
more contaminants to diffuse into the dialysate. In this way the concentration within
the sample can be decreased to acceptable or negligible levels.
(a)
(b)
Figure 1. Principle of dialysis (a) before and after dialysis movement of molecules across
the membrane.
Dialysis works based on diffusion of molecules in solution higher to lower
concentration until equilibrium is reached. Movement of molecule from membrane to
liquid will be based on membrane cut off. Large molecules cannot pass through the
dialysis beg and will be retained inside the chamber. In contrast, small molecules will
freely diffuse across the membrane and obtain equilibrium across the entire solution
volume, effectively reducing the concentration of those small molecules with in the
sample.
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If dialysis is allowed to proceed to equilibrium before each change of dialysate buffer,
the substance retained by the membrane are purified by a factor equal to the ratio of
buffer volume to sample volume. For example, when dialyzing substances will be
decreased 200-fold when equilibrium is attained. Following two additional buffer
change of 200 ml each, the containment level in the sample will be reduced by a factor
of 8x106 (200x200x200). If the original sample contained 100 mM DTT, this would
potentially be decreased in the sample to approximately 12.5 nM following the three
complete cycles. If a further decrease in concentration is desired, the dialysis process
can be continued with additional volumes of dialysate.
Factors that affect the completeness of dialysis include
1. Dialysis buffer volume,
2. Buffer composition,
3. The number of Buffer changes,
4. Time,
5. Temperature and
6. Particle size vs. pore size.
Substances that are very much smaller than the pore size will reach equilibrium faster
than substances that are only slightly smaller than the pores.
Membrane molecular weight Cut-off
The MWCO determination for dialysis membrane determines the molecular weight cut
off (MWCO) of the molecule that can diffuse across it. Understanding the significance of
MWCO determination and how they characterized will allow selection of the best
membrane for a particular dialysis application. Proper selection of MWCO will prevent
loss of proteins of interest and ensure adequate removal of contaminants.
Protein concentration using dialysis membrane
Use of Hygroscopic substance. Many hygroscopic substances such as poly ethylene
glycol (as it is larger molecule than the pore size of the dialysis tubing) it will take on
water or buffer during the dialysis process due to osmotic pressure. To concentrate the
sample, dialysis membrane containing the sample is placed in a small plastic box
containing a solution of hygroscopic compound. To avoid contamination of the sample,
the hygroscopic compound must be composed of molecules that are (e.g., highmolecular weight polyethylene glycol). With this set-up, concentration occurs upon
diffusion of the water (osmosis) and other small molecules out of the sample and into
the hygroscopic solution.
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Forced dialysis: Vacuum is applied to a sample contained within a dialysis membrane;
this effectively "pulls" water, buffer salts and other low-MW compounds out of the
dialysis sample-chamber.
Diafiltration: It involves "pushing" samples through a dialysis membrane by
centrifugal force; this is the basis for protein concentrators, which have become popular
in recent years.
Dialysis in human body
In patients with failed kidney, dialysis is often used as a procedure for removing
undesirable solutes in the blood. During dialysis, concentration of the calcium,
potassium, and urea dialysate is kept low, thereby facilitating the target solutes in the
blood to diffuse across the semi-permeable membrane. It is very important to frequently
clean dialysate toavoidreaching concentration equilibrium, which could lead to a rising
concentration of unwanted solutes in the blood. In another case, solutes can also be
introduced into the blood. For example, bicarbonate ions are in high concentration in
the dialysate, which diffuse across the membrane. This is done to prevent metabolic
acidosis.
Filtration:
Filtration is a common separation method based on a simple principle: Particles
smaller than a certain size pass through a porous filter material; the filter traps particles
larger than a certain size.
The fluid and particles that pass through filters are called the filtrate or permeate.
Depending on the application, the sample typed, and the scale, filtration systems can
very greatly. Regardless of the simplicity or complexity of a filtration system, however,
the following four components are present:




The filter itself
A support for the filter (like the funnel)
A vessel to receive the filtrate
A driving force (such as gravity or vacuum) that drives the movement of
fluids and particles through the filter.
TYPES OF FILTERS
Filters are generally classified into three types according to the size of particles,
which they retain
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Macro filters or general filters (depth filters) are used for the separation of
particles in the order of 10μm or larger. A coffee filter and a common laboratory paper
filter are examples of macro filters.
Micro filters are typically used to separate particles whose sized range from
about 0.1μm to 10μm. Bacteria and whole cells fall in this size range.
Ultra filters are used to separate macromolecules on the basis of their molecular
weight. For example, large proteins can separated from smaller ones using ultra
filtration.
MACROFILTRATION:
General filters are inexpensive devices used to remove relatively large particles,
above about 10μm, from a liquid. These filters are made of materials such as paper,
glass fibers, sand, and cloth. General filters do not have pores or holes of a particular
size; rather they consist of a convoluted granular or fibrous matrix. Particles are trapped
both on the filter surface and within the sinuous matrix. Because these filters trap
particles throughout their depth, they are also called depth filters.
Macro filters used in the laboratory are usually made of cellulose (paper) or of
glass fibers. In practice, both types may be referred to as filter paper. Glass-fiber filters
consist of long strands of borosilicate glass that are formed under high heat conditions.
In contrast to filters made of paper, glass-fiber filters posses faster flow rate, are stable
under a wide range of temperatures, and are compatible with corrosive chemicals.
Manufactures make a variety of paper filters and glass-fiber filters. These filters
vary in the density of their mesh and therefore in the sizes of particles they tend to trap.
Filters that retain smaller particles tend to have slower flow rates than those that trap
only larger particles.
MICROFILTERATION:
Microfiltration typically separates particles in the range of about 0.1-10 μm from
a liquid or gas medium. Micro filtration filters are also called membranes filter and are
manufactured to have particular pore size. Particles larger than the rated size are
retained on the surface of the membrane and smaller particles pass through.
Microfiltration membranes can be manufactured from a variety of plastics, metals, and
ceramic materials as shown in Table.
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Table 1. COMMONLY USED FILTER MATERIALS
Filter material
Features
Cellulose
A filter material made from plant fibers. Because cellulose is a natural material, its
fibers are rough in texture and vary in size and shape. Compared to synthetic media,
these characteristics create a higher restriction to the flow of fluids.
Cellulose
-
Acetate
Cellulose-Regenerated
Cellulose
-
Nitrate
Teflon
Cellulose acetate membranes are not compatible
with organic solvents. They are
well suited for aqueous solutions. Recommended for use with proteins and protein
related samples.
Regenerated cellulose membranes are low in protein binding and have low levels of
extractable, while maintaining high chemical resistance. An ideal choice for HPLC
sample preparation, as well as filtration of aqueous biological samples and organic
solvents.
Also called nitrocellulose, a mixture of nitric esters of cellulose, and a highly flammable
compound that is the main ingredient of modern gunpowder. Nitrocellulose is a fluffy
white substance that retains some of the fibrous structure of untreated cellulose. It is
not stable to heat, and even carefully prepared samples will ignite on brief heating to
more than about 150º C (300º F). When nitrocellulose decomposes, it forms products
that catalyze further decomposition; this reaction, if not stopped in time, results in an
explosion.
Teflon membranes are compatible with all solvents, acids and bases, and have no
extractable.
Nylon
Nylon, comprising several grades of polyamides, is a general-purpose material in wide
use; it is tough and resistant and has good pressure ratings. Nylon membranes are
compatible with most solvents, both organic and aqueous. Use with strong acids, 70%
ethanol, and methylene chloride. DMF is not recommended.
Polytetrafluoroethelene
(PTFE)
PTFE is an insoluble compound that exhibits a high degree of chemical resistance and a
low coefficient of friction. It is sometimes marketed in proprietary classes of materials
such as Teflon.
Polyvinylidene Fluoride
(PVDF)
PVDF is a melt process able fluropolymer. It is similar in properties to other
fluropolymers, but has better strength and lower creep than the other members of this
family. PVDF has good wear resistance, and excellent chemical resistance. But does not
perform well at elevated temperatures.
Glass Fiber
Glass Fiber is an inorganic fiber that is completely incombustible. It has a high tensile
strength in relation to weight and dimensional stability. It does not stretch or shrink. It
will not absorb water, rot, and mildew, deteriorate or decay.
Applications of Micro filtration:



To check sterility of product.
To sterilize heat labile compounds such as vitamins, antibiotics ect.
To remove contaminated bacteria, yeast and fungi from solutions.
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
Membranes can be used in the quantitative separation or filtration of
suspended matter from liquids and gases.
HEPA Filters
High efficiency particulate air (HEPA) Filters is used to remove particulates,
including microorganisms, from air. HEPA filters are manufactured to retain particles
as small as 0.3 μm. Unlike micro filter membranes, however HEPA filters are depth
filters made of glass micro fibers that are formed into a flat sheet. The sheets are then
pleated to increase the overall surface area. The pleated sheets are separated and
supported by aluminum baffles.
HEPA filters have many applications both in the laboratory and in industry.
HEPA filters are used in laboratory biological safety hoods to protect products from
contamination and/or person from exposure to hazardous substances.
In animal care facilities, HEPA filters are used on the tops of animal cages to protect
experimental animals from infection with microorganisms.
In industry, HEPA filters may be used to filter the air in entire rooms to protect
products from contaminants.
ULTRAFILTRATION:
Ultrafiltration (UF) is type of pressure driven membrane in which pressure
differential across a semi permeable membrane is used to separate solvent and small
solute molecules from solution containing larger molecules. UF usually uses
membranes with MWCOS of 1 to 100kDa. The differential pressure is achieved by
vacuum, pressurized inert gas or centrifugal forces.
Molecular cut off value is the size of smallest particle that cannot penetrate the
membrane. MWCO values are not absolute because the degree to which a particular
solute is retained by an ultrafiltration membrane not entirely dependent on its
molecular weight. The shape of the solute, its association with water, and its charge also
affect its permeability through an ultrafiltraion membrane. For example, a membrane is
less likely to retain a linear molecule than a coiled, spherical molecule of the same
molecular weight. In addition, the nature of the solvent, its pH ionic strength, and
temperature, all affect the movement of solutes through membranes. By convention, if a
membrane is rated to have a MWCO of 10,000, this means that the membrane will retain
at least 90% of globular-shaped molecules whose molecular weight is 10 kDa or greater.
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Macromolecules
Feed
side
O
Porous
Membrane
Membrane
Support
Permeate
side
O
O
O
O
OO
O
O
O
Small ions and water
molecule
Figure 2: Principle of ultra-filtration under normal condition.
Application of Ultrafiltration
The application of Ultrafiltration can be classified as fractionation, concentration,
or desalting.
1. Fractionation. It is used for the separation of larger particles from smaller ones. For
example, proteins that are significantly different in size can be separated from one
another by ultra-filtration.
2. Concentration. In this process, solvent is forced through a filter while solute is
retained. The initial volume of the sample is thus reduced and the high molecular
weight species are concentrated above the filter. This technique is very useful to
concentrate sample after protein purification due to dilution of sample. Before
performing gel electrophoresis, the proteins must be concentrated because only a
very small volume can be applied to the gel.
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3. Desalting. In this process low molecular weight salt ions are removed from a
sample solution. Ultra filtration is a simple method to remove salts because they
readily penetrate the membranes, leaving the solutes of interest on the membrane
surface.
Ultra filters are also ideal for removal or exchange of sugars, non-aqueous
solvents, the separation of free from protein-bound ligands, the removal of materials of
low molecular weight, or the rapid change of ionic and/or pH environment.
Lyophilization
Lyophilization is a process which extracts the water from foods and other products so
that the foods or products remain stable and are easier to store at room temperature
(ambiant air temperature).
Lyophilization is based on a simple principle of sublimation, is the transition of a
substance from the solid to the vapour state, without first passing through an
intermediate liquid phase.
Lyophilization and freeze drying are terms that are used interchangeably depending on
the industry and location where the drying is taking place. Controlled freeze drying
keeps the product temperature low enough during the process to avoid changes in the
dried product appearance and characteristics. It is an excellent method for preserving a
wide variety of heat-sensitive materials such as proteins, microbes, pharmaceuticals,
tissues & plasma.
To extract water from foods, the process of lyophilization consists of
1.
2.
3.
4.
Freezing the food so that the water in the food become ice;
Under a vacuum, sublimating the ice directly into water vapour;
Drawing off the water vapour;
Once the ice is sublimated, the foods are freeze-dried and can be removed from
the machine.
Lyophilization is useful
1. For Preservation of temperature sensitive products for example biological origin,
such as enzymes, blood plasma, vaccines, etc.
2. To provide a practical solution for certain delivery problems, for example, the
packaging of constituents that cannot be mixed in the liquid state, but which are
solidified in successive stages and then freeze dried.
3. To concentrate the solute like purified proteins, enzymes, antibodies, vaccine
4. To improve storage life and improved marketing of the end product.
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