1. INTRODUCTION TO FILTRATION

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

3. lseidman@matcmadison.edu EXAMPLES Spaghetti through colander Coffer through coffee filter

4. lseidman@matcmadison.edu Gases also:  Car filter  Furnace filter

5. lseidman@matcmadison.edu TERMINOLOGY What passes though = filtrate What is caught on filter = retentate Sometimes want filtrate  Coffee Sometimes want retentate  spaghetti

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

7. lseidman@matcmadison.edu IN THE LAB Traditionally, place filter in a funnel for support Pour through liquids

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

10. lseidman@matcmadison.edu 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

11. lseidman@matcmadison.edu FOUR COMPONENTS ALWAYS PRESENT Filter A support (such as funnel) A vessel to receive the filtrate A driving force, such as gravity or vacuum

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13. 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

14. lseidman@matcmadison.edu 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

16. lseidman@matcmadison.edu MACROFILTERS Relatively inexpensive Made of:  Sand  Paper  Glass  Cloth Also called depth filters

17. lseidman@matcmadison.edu LAB MACROFILTERS Usually cellulose (paper) or glass Glass  faster flow  more compatible with chemicals  more consistent  more expensive

18. lseidman@matcmadison.edu 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

19. lseidman@matcmadison.edu 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

20. lseidman@matcmadison.edu 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%).

21. lseidman@matcmadison.edu 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.

23. lseidman@matcmadison.edu APPLICATIONS Most important in lab might be to sterilize heat-sensitive materials  Example, vitamins for media

24. lseidman@matcmadison.edu 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

25. lseidman@matcmadison.edu 0.45 - 0.80 μm, used for general particle removal 1.0, or 2.5 or 5.0 μm, for “coarse” particles

26. lseidman@matcmadison.edu 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.

27. lseidman@matcmadison.edu Filters also to protect the facility from the vessel contents.

28. lseidman@matcmadison.edu 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.

29. lseidman@matcmadison.edu 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.

30. lseidman@matcmadison.edu In industry, HEPA filters may be used to filter the air in entire rooms to protect products from contaminants. Called “clean room”.

31. lseidman@matcmadison.edu 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.

32. lseidman@matcmadison.edu 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

33. lseidman@matcmadison.edu  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.

34. lseidman@matcmadison.edu The applications of ultrafiltration can be classified as either fractionation, concentration, or desalting.

35. lseidman@matcmadison.edu FRACTIONATION The separation of larger particles from smaller ones.  For example, proteins that are significantly different in size can be separated from one another

36. lseidman@matcmadison.edu 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.

37. lseidman@matcmadison.edu 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.

38. lseidman@matcmadison.edu 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.

39. lseidman@matcmadison.edu 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.

41. lseidman@matcmadison.edu 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.

42. lseidman@matcmadison.edu 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.

43. lseidman@matcmadison.edu 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.

44. lseidman@matcmadison.edu 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.

45. lseidman@matcmadison.edu 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.

46. lseidman@matcmadison.edu 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.

47. lseidman@matcmadison.edu The membrane allows water to pass through, but rejects 95 - 99% of impurities  Viruses  Particles  Pyrogens,  Microorganisms  Colloids  Dissolved organics  Dissolved inorganic materials

48. lseidman@matcmadison.edu  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.

49. lseidman@matcmadison.edu 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.

50. lseidman@matcmadison.edu IN LAB Filtration requires a force. Gravity and vacuum filtration are conventional in lab.

52. lseidman@matcmadison.edu 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.

53. lseidman@matcmadison.edu 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.

55. lseidman@matcmadison.edu 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.

57. lseidman@matcmadison.edu 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.

58. lseidman@matcmadison.edu 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”.

59. lseidman@matcmadison.edu 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.

60. lseidman@matcmadison.edu 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.

61. lseidman@matcmadison.edu 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

63. lseidman@matcmadison.edu 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.

64. lseidman@matcmadison.edu 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.

65. lseidman@matcmadison.edu c. Removing salts from a solution containing DNA. d. Sieving large particulates from water before it is treated in a sewage treatment plant.

66. lseidman@matcmadison.edu 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).

67. lseidman@matcmadison.edu 1. a.-c. ultrafiltration d. macrofiltration e. microfiltration f. ultrafiltration g. microfiltration h. microfiltration

68. lseidman@matcmadison.edu The specifications for three filters are reproduced below. Match the filters with the applications below.

69. lseidman@matcmadison.edu “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 0.1- 0.80 μm or with ultrafiltration membranes from 300 to 500,000 MWCO.

70. lseidman@matcmadison.edu 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.

71. lseidman@matcmadison.edu 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.

72. lseidman@matcmadison.edu 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?

73. lseidman@matcmadison.edu 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?

74. lseidman@matcmadison.edu c. An organic solvent is to be used with HPLC. It needs to be filtered to remove particulates. Which filter would be used?

75. lseidman@matcmadison.edu 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?

76. lseidman@matcmadison.edu 3. a. XYZb. ABC c. XYZ d. “Ready Separation”