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INTRODUCTION TO FILTRATION

INTRODUCTION TO FILTRATION. PRINCIPLES. Common separation method based on simple principle: Materials smaller than a certain size pass through porous filter Larger do not. EXAMPLES. Spaghetti through colander Coffer through coffee filter. Gases also: Car filter Furnace filter.

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INTRODUCTION TO FILTRATION

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  1. INTRODUCTION TO FILTRATION

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

  3. EXAMPLES • Spaghetti through colander • Coffer through coffee filter

  4. Gases also: • Car filter • Furnace filter

  5. TERMINOLOGY • What passes though = filtrate • What is caught on filter = retentate • Sometimes want filtrate • Coffee • Sometimes want retentate • spaghetti

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

  7. IN THE LAB • Traditionally, place filter in a funnel for support • Pour through liquids

  8. To speed up, could add a vacuum to the flask • Simple systems like this are still used

  9. 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

  10. FOUR COMPONENTS ALWAYS PRESENT • Filter • A support (such as funnel) • A vessel to receive the filtrate • A driving force, such as gravity or vacuum

  11. 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

  12. 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

  13. MACROFILTERS • Relatively inexpensive • Made of: • Sand • Paper • Glass • Cloth • Also called depth filters

  14. LAB MACROFILTERS • Usually cellulose (paper) or glass • Glass • faster flow • more compatible with chemicals • more consistent • more expensive

  15. 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

  16. 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

  17. 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%).

  18. 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.

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

  20. 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

  21. 0.45 - 0.80μm, used for general particle removal • 1.0, or 2.5 or 5.0 μm, for “coarse” particles

  22. 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.

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

  24. 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.

  25. 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.

  26. In industry, HEPA filters may be used to filter the air in entire rooms to protect products from contaminants. • Called “clean room”.

  27. 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.

  28. 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

  29. 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.

  30. The applications of ultrafiltration can be classified as either fractionation, concentration, or desalting.

  31. FRACTIONATION • The separation of larger particles from smaller ones. • For example, proteins that are significantly different in size can be separated from one another

  32. 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.

  33. 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.

  34. 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.

  35. 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.

  36. 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.

  37. 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.

  38. 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.

  39. 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.

  40. 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.

  41. 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.

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

  43. 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.

  44. 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.

  45. IN LAB • Filtration requires a force. • Gravity and vacuum filtration are conventional in lab.

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