INTRODUCTION TO
FILTRATION
PRINCIPLES

Common separation method based on simple
principle:


Materials smaller than a certain size pass through
porous filter
Larger do not
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EXAMPLES


Spaghetti through colander
Coffer through coffee filter
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
Gases also:
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Car filter
Furnace filter
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TERMINOLOGY



What passes though = filtrate
What is caught on filter = retentate
Sometimes want filtrate

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Coffee
Sometimes want retentate

spaghetti
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FILTRATION IN NATURE


Water is cleared of particulates as it passes
through sandy soil
Kidneys are filters of unwanted metabolites in
the kidneys
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IN THE LAB


Traditionally, place filter in a funnel for
support
Pour through liquids
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

To speed up, could add a vacuum to the flask
Simple systems like this are still used
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MANY VARIATIONS


Principle is still simple, but now are many
complex systems for many applications
Small scale, few microliters, to tens of
thousands of liters
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FOUR COMPONENTS ALWAYS
PRESENT


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Filter
A support (such as funnel)
A vessel to receive the filtrate
A driving force, such as gravity or vacuum
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PROBLEMS TO CONSIDER IN
DESIGN

Clogging, by particles, oils, films



Adsorption

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Sometimes “cure” by replacing filters
Sometimes move liquid across filter
Some filters bind some materials strongly
Absorption
Extraction of materials from filter
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CLASSIFICATION

Macrofilters, 10 micrometers or larger



Microfilters, 0.01-25 micrometers

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Coffee filters
Lab filter papers
Bacterial and whole cells
Ultrafilters, separate on the basis of MW

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Large proteins versus small ones
Salts from proteins
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MACROFILTERS

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Relatively inexpensive
Made of:


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

Sand
Paper
Glass
Cloth
Also called depth filters
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LAB MACROFILTERS


Usually cellulose (paper) or glass
Glass



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faster flow
more compatible with chemicals
more consistent
more expensive
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PAPER FILTER GRADES

Density of mesh

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Quantitative versus qualitative grade

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Affects rate of flow
Size of particles trapped
Amount of ash when burned
Important for some chemical analyses
Quantitative papers leave low ash residue, qualitative leave
a lot
Hardened are good for vacuum filtration
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MICROFILTERS



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Microfiltration separates particles in the range of
about 0.01 μm to 10 μm
Medium either a liquid or gas.
Filters are called membranes, so is membrane
filtration
Manufactured to have a particular pore size.


Particles larger than rated size are retained on surface
Smaller particles pass through
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

Pore size (absolute) means 100% of
particles above that size will be retained by
the membrane under specified conditions
Pore Size (nominal) means particles of that
size will be retained with an efficiency below
100 % (typically 90-98%).
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OTHER FACTORS IN
SELECTING MEMBRANE

Pore size most important, but also consider:

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Resistance to organic solvents
Binding properties
Surface smoothness
Extractables
Hydrophilic versus hydrophobic
Rate of flow
Etc.
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MATERIALS COMMONLY USED TO MAKE MICROFILT ERS
MATERIAL
FEATURES
EXAMPLES OF APPLICATIONS
NITROCELLULOSE
Hydrophilic.
Frequently used.
Readily binds single-stranded DNA,
RNA and proteins.
General purpose microbiology.
Sterility testing.
CELLULOSE ACETATE Hydrophilic.
Very low aqueous extractability.
Very low binding.
Not resistant to organic solvents.
Resistant to heat.
General filtration.
Sterilizing tissue culture media.
General sterilization.
NYLON
Hydrophilic.
Very low extractability.
Readily binds proteins.
Compatible with alcohols
and solvents frequently used in HPLC.
Filtering organic solvents.
Filtering samples before HPLC.
POLYSULFONE
Hydrophilic.
Low extractability.
Low protein binding.
Wide chemical compatibility.
Autoclavable.
Sterilizing tissue culture media.
Manufacturing ultrafiltration membranes.
PTFE
Hydrophobic.
Inert to most chemically aggressive
solvents, strong acids, and bases.
Expensive.
Ideal for filtering gases, air.
Filtering organic solvents.
PVDF
Must be rendered hydrophilic.
Highly resistant to solvents.
Very low binding properties.
Filtering samples before HPLC.
POLYPROPYLENE
Hydrophilic.
Low fiber release.
Resistant to many solvents.
Filtering samples before HPLC.
APPLICATIONS

Most important in lab might be to sterilize
heat-sensitive materials

Example, vitamins for media
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
0.10 μm, recommended to remove Mycoplasma, a
very small type of bacterium that can contaminate
cell cultures

0.22 μm, for routine sterilization

0.45 μm, standard pore size for removing E. coli
bacteria

0.65 μm, to remove fungi and yeast
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
0.45 - 0.80 μm, used for general particle
removal

1.0, or 2.5 or 5.0 μm, for “coarse” particles
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

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Bacterial fermentation: air is often supplied to the
fermentation vessel; provides agitation and oxygen.
Hydrophobic microfilters placed in the air stream
remove contaminating particles and
microorganisms; protect cells
Similarly, filters are attached to supply lines for
carbon dioxide and air running to animal cell culture
vessels.
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
Filters also to protect the facility from the
vessel contents.
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
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HEPA (High Efficiency Particulate Air) filters are
used to remove particulates, including
microorganisms, from air.
HEPA filters are manufactured to retain particles as
small as 0.3 μm.
HEPA filters are depth filters made of glass
microfibers,formed into a flat sheet.

Sheets are pleated to increase the overall surface area.
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USES OF HEPA FILTERS


HEPA filters have many applications in the
laboratory and in industry.
Used in laboratory biological safety hoods to
protect products from contamination and/or
personnel from exposure to hazardous
substances.
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

In industry, HEPA filters may be used to filter
the air in entire rooms to protect products
from contaminants.
Called “clean room”.
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ULTRAFILTRATION


Ultrafilters,membranes that separate
materials on the basis of molecular weight.
Ultrafiltration membranes have pore
diameters from 1-100 Angstroms

Can separate particles with MWs ranging from
about 1,000 to 1,000,000.
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MOLECULAR WEIGHT CUTOFF


MWCO is the lowest molecular weight solute that is
generally retained by the membrane.
MWCO values are not absolute because the degree
to which a particular solute is retained is not entirely
dependent on its molecular weight. Also important:

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The shape of the solute
Association with water
Charge
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
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A membrane is less likely to retain a linear
molecule than a coiled, spherical molecule of the
same molecular weight.
The nature of the solvent, its pH, ionic strength,
and temperature all affect the movement of
solutes through membranes.
If a membrane is rated to have a MWCO of
10,000, the membrane will retain at least 90 % of
globular-shaped molecules whose molecular
weight is 10,000 or greater.
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
The applications of ultrafiltration can be
classified as either fractionation,
concentration, or desalting.
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FRACTIONATION

The separation of larger particles from
smaller ones.

For example, proteins that are significantly
different in size can be separated from one
another
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CONCENTRATION



Solvent is forced through a filter.
Volume of the sample is thus reduced and the high
molecular weight species are concentrated above
the filter.
Example, gel electrophoresis is used to separate
and visualize proteins.


Before electrophoresis, the proteins must be concentrated
because only a very small volume can be applied to the
gel.
Ultrafiltration can be used for this purpose.
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DESALTING


Low molecular weight salt ions are removed
from a sample solution.
Ultrafiltration is a simple method to remove
salts since they readily penetrate the
membranes leaving the solutes of interest on
the membrane surface.
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DIALYSIS


Dialysis, and reverse osmosis are
separation processes that use membranes
similar to those used for ultrafiltration.
Dialysis is based on differences in the
concentrations of solutes between one side
of the membrane and the other.
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PRINCIPLE


Solute molecules that are small enough to pass
through the pores of the membrane will diffuse from
the side with a higher concentration to the side with
a lower concentration.
The distinctive feature of dialysis is that differences
in solute concentration provide the “driving force”;
does not require pumps or a vacuum to force
materials through the pores of the membrane.
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EXAMPLE: DESALTING


During the process of purifying a protein, it is
common to cause the protein to precipitate
from solution by adding high concentrations
of salt.
Subsequent steps in the protein purification
process require that the salt be removed; this
is called desalting.
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

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The sample is placed in a bag made of
dialysis membrane.
The dialysis bag containing the sample is
sealed at both ends and is suspended in a
large volume of water or buffer solution.
Thus, the concentration of salts is much
higher inside the bag than outside.
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

Relatively large protein molecules cannot penetrate
the pores of the dialysis membrane and so remain
inside the bag, but small molecules, including salt,
readily move through the membrane.
Over time, low molecular weight salt molecules from
inside the bag diffuse out through the dialysis
membrane into the water or buffer solution.
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

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Eventually, the concentration of salt inside the bag
and outside the bag equalizes and the system
reaches equilibrium.
Observe that the salts are not completely removed
the sample, but their concentration is much reduced.
In order to further reduce the concentration of salt in
the sample, the dialysis bag can be moved into
fresh water or buffer solution and the process can
be repeated.
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

Dialysis is relatively inexpensive (as
compared to ultrafiltration), simple, and
gentle.
However, because dialysis relies on passive
diffusion, it is a relatively slow process.

A number of special devices are available from
manufacturers to make dialysis more efficient and
convenient.
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REVERSE OSMOSIS



RO removes very low molecular weight
materials, including salts, from a liquid
(usually water).
Reverse osmosis is important in water
purification systems.
Water under pressure flows over a thin RO
membrane.
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
The membrane allows water to pass through, but
rejects 95 - 99% of impurities
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Viruses
Particles
Pyrogens,
Microorganisms
Colloids
Dissolved organics
Dissolved inorganic materials
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

The permeate will contain very low levels of
contaminants that are able to get by even an RO
membrane, but most types of contaminants are
greatly reduced.
An RO membrane retains materials based both on
their size and on ionic charge and it can retain
smaller solutes than an ultrafiltration membrane.
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SCALE


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Filtration principles of filtration are the same,
whether the sample is 10 μL in the laboratory or
10,000 L in industry
But design of filtration systems depends on the
scale.
The filter’s size and shape, the support of the filter,
the type of force used to move fluids through the
filter, and the vessels involved can vary greatly.
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IN LAB


Filtration requires a force.
Gravity and vacuum filtration are
conventional in lab.
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SYRINGE FILTERS

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Another laboratory method to force samples through
a filter is to use a syringe.
The sample in loaded into the syringe and the filter
unit is mounted on the end.
The sample fluid is forced through the filter by
depressing the plunger.
Syringe filtration units contain microfilters of varying
pore sizes.
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

Sterile syringe filter units are popular for
sterilizing small volumes of sample, such as
milliliter solutions of an antibiotic for cell
culture.
Very small syringe filter units are used for
removing particulates from microliter volume
samples prior to HPLC.
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SPIN FILTRATION


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Laboratory filtration system that uses
centrifugal force.
Spin filtration units contain ultrafilters, or
sometimes microfilters, that are housed
within a centrifuge tube.
The sample is placed in the tube on top of the
filter and the unit is spun in a centrifuge.
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
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During centrifugation, the liquid and smaller particles
are forced through the filter and are captured in the
bottom of the centrifuge tube.
Larger molecules remain behind on the surface of
the filter.
Spin filters can be used to fractionate, concentrate
and desalt samples.
Are often used in molecular biology to filter small
volume samples of proteins, nucleic acids,
antibodies, and viruses.
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PROCESS FILTRATION

Filtration of large volumes of liquids

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Biotechnology production facilities
Pharmaceutical companies
Food production facilities
For example, the first step in processing the
product is to separate the cells and cell
debris from the liquid containing the product,
“clarification”.
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
Clarification often costly and challenging



Thousands of liters of material
Fragile protein product.
Filtration is usually preferred over
centrifugation because filtration tends to be
less expensive, gentler, and more
convenient.
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INDUSTRIAL FILTRATION


Systems must be capable of being cleaned
and sterilized and their effectiveness must be
validated.
Maximize the surface area for filtration.


The simplest way to do this is by using large
sheets of filter membrane.
More sophisticated systems form the membranes
into tubes, spirals or pleats to maximize surface
area.
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HOLLOW FIBER
ULTRAFILTERS



Cylindrical cartridges packed with ultrafiltration
membranes formed into hollow fibers.
The liquid to be filtered flows through the lumen of
the fibers.
As molecules pass through the lumen, substances
smaller than the MWCO penetrate the membrane
while those that are larger are concentrated in the
center
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TANGENTIAL FLOW
FILTRATION


Membrane clogging is a major problem in
industry.
One method to reduce clogging is to use
tangential flow, or cross flow filtration
where the fluid to be filtered flows over the
surface of the filter as well as through the
filter.
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PROBLEMS



For each of the separations below, state
whether it will involve macrofiltration,
microfiltration, or ultrafiltration.
a. Purifying antibodies from a liquid medium.
b. Removing viruses from a vaccine.
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

c. Removing salts from a solution containing
DNA.
d. Sieving large particulates from water
before it is treated in a sewage treatment
plant.
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



e. Sterilizing cell culture media by removing
bacteria.
f. Removing pyrogens (fever causing agents) from a
drug product.
g. Harvesting mammalian cells from a fermenter.
h. Removing Mycoplasma from bovine serum
(which is often added to cell culture media).
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1.
a.-c. ultrafiltration
e. microfiltration
g. microfiltration
d. macrofiltration
f. ultrafiltration
h. microfiltration
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The specifications for three filters are
reproduced below. Match the filters with the
applications below.
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“READY SEPARATION” HOLLOW FIBER
FILTRATION SYSTEM
The “Ready Separation” System is a compact hollow
fiber filtration system capable of processing from 5
to 100 L. The system is sterilizable and can be
purchased with microfiltration membranes from 0.10.80 μm or with ultrafiltration membranes from 300
to 500,000 MWCO.
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FILTER TYPE XYZ
The XYZ unit contains a hydrophobic,
solvent-resistant, 0.2 μm membrane
designed for use as a sterilizing filter for
gases and liquids. The unit is a pyrogen-free,
sterile, single use device.
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FILTER TYPE ABC
The ABC filtration unit is a low protein-binding,
sterile filter for aqueous, proteinaceous substances.
It is intended for applications where minimal sample
loss is desired. It is a 0.22 μm filter, single use
product for use with syringes.
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a. Application: A fermentation process is being
designed in which microorganisms produce an
antibiotic which will be isolated from the broth. The
microorganisms generate carbon dioxide which is
vented from the fermenter via a plastic tube. On the
end of the tube is a filtration unit which keeps the
organisms from the outside air from contaminating
the fermenter. What type of filtration unit is used?
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b. An antibiotic solution needs to be added to
a cell culture fermenter. It is heat sensitive
and is therefore sterilized by filtration. Which
filter would be used?
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c. An organic solvent is to be used with
HPLC. It needs to be filtered to remove
particulates. Which filter would be used?
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d. A biotechnology company uses a cell culture
process to produce a valuable protein that is
being tested as a drug. The protein is
secreted by the cells into the culture medium.
Which type of filter might be used in the
process of isolating the protein product from
the cell culture medium?
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3.
a. XYZ
c. XYZ
b. ABC
d. “Ready Separation”
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