SEPARATIONS: FILTRATION

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BASIC LABORATORY METHODS IN A REGULATED ENVIRONMENT

LECTURE OVERVIEW

• Principle of filtration and terminology
• Issues that can arise in filtration
• Classification of filters based on pore size
• Macrofilters
• Microfilters
• Ultrafilters
• HEPA filters
• Applications
• Fractionation
• Concentration
• Desalting
• Filtration systems at different scales
• Laboratory scale
• Filtration in bioprocessing

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LECTURE OVERVIEW

• Principle of filtration and terminology
• Issues that can arise in filtration
• Classification of filters based on pore size
• Macrofilters
• Microfilters
• Ultrafilters
• HEPA filters
• Applications
• Fractionation
• Concentration
• Desalting
• Filtration systems at different scales
• Laboratory scale
• Filtration in bioprocessing

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PRINCIPLE

• Common separation method based on simple principle:
• Materials smaller than a certain size pass through porous filter
• Larger do not

FAMILIAR EXAMPLES

• Spaghetti through colander
• Coffee through coffee filter

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MORE FAMILIAR EXAMPLES

• Gases also:
• Car filter
• Furnace filter

FILTRATION IN NATURE

• Water is cleared of particulates as it passes through sandy soil
• Kidneys are filters of unwanted metabolites in the kidneys

TERMINOLOGY

• What passes though = filtrate
• What is caught on filter = retentate
• Sometimes want filtrate
• For example, coffee
• Sometimes want retentate
• For example, spaghetti

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

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Trap prevents liquids from

contaminating vacuum lines

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MANY FILTRATION VARIATIONS

• Principle is simple, but there are many systems for different 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

QUESTION: WHAT ARE THE FOUR COMPONENTS OF THIS FILTER SYSTEM?

LECTURE OVERVIEW

• Principle of filtration and terminology
• Issues that can arise in filtration
• Classification of filters based on pore size
• Macrofilters
• Microfilters
• Ultrafilters
• HEPA filters
• Applications
• Fractionation
• Concentration
• Desalting
• Filtration systems at different scales
• Laboratory scale
• Filtration in bioprocessing

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PROBLEMS TO CONSIDER IN FILTRATION DESIGN

• Clogging, by particles, oils, films
• Sometimes “cure” by replacing filters
• Sometimes move liquid across filter
• Adsorption
• Some filters bind some materials strongly; can result in loss of desired substance
• Extraction is where materials from the filter dissolve into and contaminate the substance being filtered

FILTRATION ISSUES

• When reading catalogs, look for manufacturers’ notes about:
• Clogging
• Adsorption
• Absorption
• Extraction

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QUESTION: READ THIS CATALOG DESCRIPTION; WHICH ISSUE(S) IS ADDRESSED?

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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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ANSWER

• This filter minimizes adsorption of proteins to avoid their loss
• Note it is hydrophilic, which means it is intended for use with aqueous samples

LECTURE OVERVIEW

• Principle of filtration and terminology
• Issues that can arise in filtration
• Classification of filters based on pore size
• Macrofilters
• Microfilters
• Ultrafilters
• HEPA filters
• Applications
• Fractionation
• Concentration
• Desalting
• Filtration systems at different scales
• Laboratory scale
• Filtration in bioprocessing

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CLASSIFICATION OF FILTERS

• Macrofilters, retain materials 10 μm or larger
• Example, coffee filters
• Example, lab filter papers
• Microfilters, retain materials 0.01-25 μm
• Example, bacterial or whole cells separated from broth
• Ultrafilters, separate based on MW
• Example, large proteins separated from small ones
• Example, salts (small) separated from proteins (much larger)

Lseidman@madisoncollege.edu

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 vs qualitative grade:
• Important for some chemical analyses
• Quantitative papers leave low ash residue, qualitative leave a lot
• Hardened are good for vacuum filtration

MICROFILTERS -- MANY USES FOR BIOTECHNOLOGISTS

• Microfiltration separates particles in the range of about 0.01 μm to 10 μm
• Medium either liquid or gas.
• Filters are called membranes, so is also called membrane filtration
• Manufactured to have a particular pore size.
• Particles larger than rated size are retained on surface
• Smaller particles pass through

PORE SIZE MICROFILTERS

• Pore size (absolute) means 100% of particles above that size will be retained by the membrane under specified conditions

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• Pore Size (nominal) means particles of that size will be retained with an efficiency below 100 % (typically 90-98%).

APPLICATIONS MICROFILTERS

• Most important in lab might be to sterilize heat-sensitive materials
• Example, vitamins for media

STERILIZATION WITH MICROFILTERS

• 0.10 μm, recommended to remove Mycoplasma, a very small type of bacterium that can contaminate cell cultures

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• 0.22 μm, for routine sterilization

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• 0.45 μm, standard pore size for removing E. coli bacteria

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• 0.65 μm, to remove fungi and yeast

MICROFILTERS

• 0.45 - 0.80 μm, used for general particle removal

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• 1.0, or 2.5 or 5.0 μm, for “coarse” particles

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MICROFILTERS IN BIOPROCESSING

• Bioprocessing: air is often supplied to the vessel with cells; air provides agitation and oxygen.

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• Hydrophobic microfilters placed in the air stream remove contaminating particles and microorganisms; protect cells

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• Similarly, filters are attached to supply lines for carbon dioxide and air running to animal cell culture vessels.

MICROFILTERS IN BIOPROCESSING

• Filters also to protect the facility from the vessel contents.

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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.

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Lseidman@madisoncollege.edu

ULTRAFILTRATION

• Ultrafilters, membranes that separate materials based on 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

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• 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

MOLECULAR WEIGHT CUTOFF

• A membrane is less likely to retain a linear molecule than a coiled, spherical molecule of the same molecular weight.

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• The nature of the solvent, its pH, ionic strength, and temperature all affect the movement of solutes through membranes.

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• 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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APPLICATIONS ULTRAFILTRATION

• 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

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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 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 like those used for ultrafiltration.

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• Dialysis is based on differences in the concentrations of solutes between one side of the membrane and the other.

PRINCIPLE DIALYSIS

• 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

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• 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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Lseidman@madisoncollege.edu

EXAMPLE: DESALTING BY DIALYSIS

• During process of purifying a protein, it is common to precipitate the protein from solution by adding high concentrations of salt

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• Subsequent steps in the protein purification process require that the salt be removed; this is called desalting

DIALYSIS

• The sample is placed in a bag made of dialysis membrane.

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• The dialysis bag containing the sample is sealed at both ends and is suspended in a large volume of water or buffer solution.

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• Thus, the concentration of salts is much higher inside the bag than outside.

DIALYSIS

• Relatively large protein molecules cannot penetrate the pores of the dialysis membrane and remain inside the bag, salt molecules, including salt move through the membrane

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• Over time, salt molecules from inside the bag diffuse out through the dialysis membrane into the water or buffer solution

DIALYSIS

• Eventually, the concentration of salt inside the bag and outside the bag equalizes and the system reaches equilibrium

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• Salts are not completely removed the sample, but their concentration is much reduced

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• To further reduce the concentration of salt in the sample, the dialysis bag can be moved into fresh water or buffer and the process can be repeated

DIALYSIS

• Dialysis is relatively inexpensive (as compared to ultrafiltration), simple, and gentle

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• However, because dialysis relies on passive diffusion, it is a relatively slow process
• 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).

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• Reverse osmosis is important in water purification systems.

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• Water under pressure flows over a thin RO membrane.

REVERSE OSMOSIS

• The membrane allows water to pass through, but rejects 95 - 99% of impurities including:
• Viruses
• Particles
• Pyrogens,
• Microorganisms
• Colloids
• Dissolved organics
• Dissolved inorganic materials

REVERSE OSMOSIS

• 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.

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• 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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LECTURE OVERVIEW

• Principle of filtration and terminology
• Issues that can arise in filtration
• Classification of filters based on pore size
• Macrofilters
• Microfilters
• Ultrafilters
• HEPA filters
• Applications
• Fractionation
• Concentration
• Desalting
• Filtration systems at different scales
• Laboratory scale
• Filtration in bioprocessing

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HEPA FILTERS

• HEPA (High Efficiency Particulate Air) filters are used to remove particulates, including microorganisms, from air.

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• HEPA filters are manufactured to retain particles as small as

0.3 μm.

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• 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 important applications in the laboratory and in industry.

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• Used in laboratory biological safety hoods to protect products from contamination and/or personnel from exposure to hazardous substances.

USES OF HEPA FILTERS

• In industry, HEPA filters may be used to filter the air in entire rooms to protect products from contaminants.

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• Called “clean room”.

USES OF HEPA FILTERS

• It is possible to purchase HEPA filters to remove COVID 19 particles (or other undesirable substances) from rooms

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• Used in medical facilities

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• Used in homes

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• Note that viral particles individually are smaller than the mesh size of HEPA filters, but viruses are usually associated with droplets that do get trapped by filter

LECTURE OVERVIEW

• Principle of filtration and terminology
• Issues that can arise in filtration
• Classification of filters based on pore size
• Macrofilters
• Microfilters
• Ultrafilters
• HEPA filters
• Applications
• Fractionation
• Concentration
• Desalting
• Filtration systems at different scales
• Laboratory scale
• Filtration in bioprocessing

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SCALE

• Filtration principles of filtration are the same, whether the sample is 10 μL in the laboratory or 10,000 L in industry

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• But design of filtration systems depends on the scale.

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• 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

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• Gravity and vacuum filtration are conventional in lab

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• Centrifugation

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• Plunger (syringe)

Small, Lab Scale

Lseidman@madisoncollege.edu

SYRINGE FILTERS, VERY SMALL SCALE

• Another laboratory method to force samples through a filter is to use a syringe.

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• Sample is loaded into the syringe and the filter unit is mounted on the end.

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• Sample fluid is forced through the filter by depressing the plunger.

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• Syringe filtration units contain microfilters of varying pore sizes.

SYRINGE FILTERS, VERY SMALL SCALE

• Sterile syringe filter units are commonly used for sterilizing small volumes, such as milliliter solutions of an antibiotic for cell culture.

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• Very small syringe filter units are used for removing particulates from microliter volume samples prior to HPLC.

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Lseidman@madisoncollege.edu

SPIN FILTRATION

• Laboratory filtration system that uses centrifugal force

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• Spin filtration units contain ultrafilters, or sometimes microfilters, that are housed within a centrifuge tube

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• The sample is placed in the tube on top of the filter and the unit is spun in a centrifuge

LAB SCALE, USING CENTRIFUGATION FOR FILTRATION

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SPIN FILTRATION

• 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 are used to fractionate, concentrate, and desalt samples.
• Common in molecular biology to filter small volume samples of proteins, nucleic acids, antibodies, and viruses.

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LARGE SCALE, PROCESS FILTRATION

• Filtration of large volumes of liquids in:
• Biotechnology production facilities
• Pharmaceutical companies
• Food production facilities

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• For example, the first step in processing biotech product is to separate the cells from the liquid containing the product, “clarification”

CLARIFICATION

• Clarification often costly and challenging
• Thousands of liters of material
• Fragile protein product.

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• Filtration often preferred over centrifugation because filtration tends to be less expensive, gentler, and more convenient.

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PROCESS FILTRATION

• Systems must be capable of being cleaned and sterilized and their effectiveness must be validated

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• 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

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• The liquid to be filtered flows through the lumen of the fibers

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• 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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Lseidman@madisoncollege.edu

TANGENTIAL FLOW FILTRATION

• Membrane clogging is a major problem in industry

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• 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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• A good animation of this type of filtration

https://www.youtube.com/watch?v=LkoQX7U4eeo

https://www.freshwatersystems.com/blogs/blog/how-an-ultrafiltration-membrane-works

QUESTIONS

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.

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QUESTIONS CONT.

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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ANSWERS

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a.-c. ultrafiltration d. macrofiltration

e. microfiltration f. ultrafiltration

g. microfiltration h. microfiltration

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