INTRODUCTION TO FILTRATION
INTRODUCTION TO FILTRATION. [email protected] PRINCIPLES Common separation method based on simple principle: Materials smaller than a certain.
Dec 30, 2015
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INTRODUCTION TO FILTRATION
PRINCIPLES
Common separation method based on simple principle: Materials smaller than a certain size pass through
porous filter Larger do not
TERMINOLOGY
What passes though = filtrate What is caught on filter = retentate Sometimes want filtrate
Coffee Sometimes want retentate
spaghetti
FILTRATION IN NATURE
Water is cleared of particulates as it passes through sandy soil
Kidneys are filters of unwanted metabolites in the kidneys
IN THE LAB
Traditionally, place filter in a funnel for support
Pour through liquids
To speed up, could add a vacuum to the flask Simple systems like this are still used
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
FOUR COMPONENTS ALWAYS PRESENT Filter A support (such as funnel) A vessel to receive the filtrate A driving force, such as gravity or vacuum
PROBLEMS TO CONSIDER IN DESIGN Clogging, by particles, oils, films
Sometimes “cure” by replacing filters Sometimes move liquid across filter
Adsorption Some filters bind some materials strongly
Absorption Extraction of materials from filter
CLASSIFICATION
Macrofilters, 10 micrometers or larger Coffee filters Lab filter papers
Microfilters, 0.01-25 micrometers Bacterial and whole cells
Ultrafilters, separate on the basis of MW Large proteins versus small ones Salts from proteins
MACROFILTERS
Relatively inexpensive Made of:
Sand Paper Glass Cloth
Also called depth filters
LAB MACROFILTERS
Usually cellulose (paper) or glass Glass
faster flow more compatible with chemicals more consistent more expensive
PAPER FILTER GRADES
Density of mesh Affects rate of flow Size of particles trapped
Quantitative versus qualitative grade 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
MICROFILTERS
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
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%).
OTHER FACTORS IN SELECTING MEMBRANE
Pore size most important, but also consider: Resistance to organic solvents Binding properties Surface smoothness Extractables Hydrophilic versus hydrophobic Rate of flow Etc.
MATERIALS COMMONLY USED TO MAKE MICROFILTERS
MATERIAL FEATURES EXAMPLES OF APPLICATIONS
NITROCELLULOSE Hydrophilic. General purpose microbiology.Frequently used. Sterility testing.Readily binds single-stranded DNA, RNA and proteins.
CELLULOSE ACETATE Hydrophilic. General filtration.Very low aqueous extractability. Sterilizing tissue culture media.Very low binding. General sterilization.Not resistant to organic solvents.Resistant to heat.
NYLON Hydrophilic. Filtering organic solvents.Very low extractability. Filtering samples before HPLC.Readily binds proteins.Compatible with alcoholsand solvents frequently used in HPLC.
POLYSULFONE Hydrophilic. Sterilizing tissue culture media.Low extractability. Manufacturing ultrafiltration membranes.Low protein binding.Wide chemical compatibility.Autoclavable.
PTFE Hydrophobic. Ideal for filtering gases, air. Inert to most chemically aggressive Filtering organic solvents.solvents, strong acids, and bases. Expensive.
PVDF Must be rendered hydrophilic. Filtering samples before HPLC. Highly resistant to solvents.Very low binding properties.
POLYPROPYLENE Hydrophilic. Filtering samples before HPLC.Low fiber release. Resistant to many solvents.
APPLICATIONS
Most important in lab might be to sterilize heat-sensitive materials Example, vitamins for media
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
0.45 - 0.80 μm, used for general particle removal
1.0, or 2.5 or 5.0 μm, for “coarse” particles
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.
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.
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.
In industry, HEPA filters may be used to filter the air in entire rooms to protect products from contaminants.
Called “clean room”.
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.
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: The shape of the solute Association with water Charge
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.
The applications of ultrafiltration can be classified as either fractionation, concentration, or desalting.
FRACTIONATION
The separation of larger particles from smaller ones. For example, proteins that are significantly
different in size can be separated from one another
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The membrane allows water to pass through, but rejects 95 - 99% of impurities Viruses Particles Pyrogens, Microorganisms Colloids Dissolved organics Dissolved inorganic materials
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.
SCALE
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.
IN LAB
Filtration requires a force. Gravity and vacuum filtration are
conventional in lab.
SYRINGE FILTERS
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.
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.
SPIN FILTRATION
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.
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.
PROCESS FILTRATION
Filtration of large volumes of liquids 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”.
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.
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.
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
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.
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.
c. Removing salts from a solution containing DNA.
d. Sieving large particulates from water before it is treated in a sewage treatment plant.
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).
1. a.-c. ultrafiltration d. macrofiltratione. microfiltration f. ultrafiltration
g. microfiltration h. microfiltration
The specifications for three filters are reproduced below. Match the filters with the applications below.
“READY SEPARATION” HOLLOW FIBER FILTRATION SYSTEMThe “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 0.1-0.80 μm or with ultrafiltration membranes from 300 to 500,000 MWCO.
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.
FILTER TYPE ABCThe 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.
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?
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?
c. An organic solvent is to be used with HPLC. It needs to be filtered to remove particulates. Which filter would be used?
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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