Ultrafree -CL Centrifugal Filter Units. Single-Use Only

Ultrafree -CL Centrifugal Filter Units Single-Use Only

Notice The information in this document is subject to change without notice and should not be construed as a commitment by Millipore Corporation. Millipore Corporation assumes no responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Millipore Corporation be liable for incidental or consequential damages in connection with or arising from the use of this manual. Copyright 1999, Millipore Corporation or an affiliated company. Millipore, Durapore, and Ultrafree are registered trademarks of Millipore Corporation or an affiliated company. Biomax is a trademark of Millipore Corporation or an affiliated company. Cello-Solve is a registered trademark of Union Carbide Chemicals & Plastics Technology Corporation. Freon is a registered trademark of E. I. du Pont de Nemours and Company. Tyvek is a registered trademark of E.I. du Pont de Nemours and Company. P17956, Rev C, 9/99

Contents Introduction... 1 Membrane Types and Applications... 2 Table: % Protein Recovered from Retentate @ 25 C... 4 Guidelines for Use... 7 How to Use the Ultrafree-CL Centrifugal Filter Unit... 14 Specifications... 15 Ordering Information... 16 Technical Assistance... 18 Standard Warranty... 19

Introduction Millipore Ultrafree-CL centrifugal filter units are disposable, single-use devices used to process aqueous biological solutions in the 0.5 to 2 milliliter (ml) range. Ultrafree-CL units can be used in standard, variable-speed centrifuges with fixed-angle rotors capable of holding 15 ml tubes. A complete device consists of the following components: Filter cup Filtrate collection tube with cap Ultrafree-CL units are available in a range of microporous and ultrafiltration membranes.

2 Ultrafree-CL Centrifugal Filter Units Membrane Types and Applications Ultrafree-CL units are available with ultrafilters and microporous filters. Ultrafilters Biomax TM high-flux polysulfone ultrafiltration series Low-binding, regenerated cellulose, Ultracel PL ultrafiltration series Polysulfone PT ultrafiltration series Description Highest flow rate characteristics for fast sample preparation of complex protein samples. Available in 5,000, 10,000, 30,000, 50,000, and 100,000 nominal molecular weight limit (NMWL). Exhibits the lowest binding for amino acids, most drugs, peptides, proteins, and nucleic acids. Available in 5,000, 10,000, and 30,000 NMWL. Millipore's original polysulfone UF membrane. Available in 10,000; 30,000; 100,000; and 300,000 NMWL.

http://www.millipore.com/ultrafree 3 Membrane Types and Applications, continued Microporous Filters Low-binding Durapore microporous series PTFE microporous series Description Available in 0.1, 0.22, 0.45, 0.65, and 5.0 micron (µm) sizes. Available in 0.2 and 0.5 µm sizes. Application Protein concentration or dialysis Clarification, particulate removal before instrument analysis (HPLC, MS, IC, GC) Protein removal before instrument analysis Fermentation broth screening (to remove interfering high molecular weight solutes) Serum or plasma concentrations Membrane 5,000; 10,000; 30,000; 50,000; 100,000 NMWL 0.1, 0.2, 0.45 µm Durapore 10,000 NMWL 10,000 or 30,000 NMWL 10,000 or 30,000 NMWL

4 Ultrafree-CL Centrifugal Filter Units % Protein Recovered from Retentate @ 25 C u Biomax High-flux Polysulfone Retentate MW 5,000 10,000 30,000 50,000 100,000 NMWL NMWL NMWL NMWL NMWL Vitamin B-12 (0.2 mg/ml) 1.3 kd 5 10% 5 10% 0 5% 0 5% 0 5% Bovine Insulin (0.25 mg/ml) 6 kd 85 90% 60 70% 45 55% 10 20% 5 10% Lysozyme (1 mg/ml) 14 kd 90 100% 80 90% 60 70% 40 50% 5 10% Bovine Serum Albumin (1 mg/ml) 67 kd 95 100% 95 100% 95 100% 90 100% 20 30% Goat IgG (1 mg/ml) 156 kd 95-100% 95 100% 95 100% 95 100% 90 100% u Passage of proteins with a molecular weight lower than the membrane rating may vary with g-force. In general, a lower g-force will favor increased passage.

http://www.millipore.com/ultrafree 5 % Protein Recovered from Retentate @ 25 C u Regenerated Cellulose, PL Series Retentate MW PLCC 5,000 PLGC 10,000 PLTK 30,000 NMWL NMWL NMWL Vitamin B-12 (0.2 mg/ml) 1.3 kd 15 20% 10 15% 5 10% Bovine Insulin (0.25 mg/ml) 6 kd 90 95% 80 90% 55 65% Lysozyme (1 mg/ml) 14 kd 95 100% 90 95% 60 70% Bovine Serum Albumin (1 mg/ml) 67 kd 95 100% 95 100% 95 100% Goat IgG (1 mg/ml) 156 kd 95 100% 95 100% 95 100% u Passage of proteins with a molecular weight lower than the membrane rating may vary with g-force. In general, a lower g-force will favor increased passage.

6 Ultrafree-CL Centrifugal Filter Units % Protein Recovered from Retentate @ 25 C u Polysulfone, PT Series Retentate MW PTGC PTTK PTHK PTMK 10,000 30,000 100,000 300,000 NMWL NMWL NMWL NMWL Vitamin B-12 (0.2 mg/ml) 1.3 kd 5 10% 0 5% 0 5% 0 5% Bovine Insulin (0.25 mg/ml) 6 kd 80 90% 55 65% 5 10% 0 5% Lysozyme (1 mg/ml) 14 kd 90 95% 60 70% 5 10% 0 5% Bovine Serum Albumin (1 mg/ml) 67 kd 95 100% 90 100% 10 20% 0 5% Goat IgG (1 mg/ml) 156 kd 95 100% 95 100% 90 100% 35 45% u Passage of proteins with a molecular weight lower than the membrane rating may vary with g-force. In general, a lower g-force will favor increased passage.

http://www.millipore.com/ultrafree 7 Guidelines for Use Follow these guidelines to optimize the performance of Ultrafree-CL units. Protein Binding While different membrane materials can nonspecifically bind proteins to varying degrees, many factors can influence protein binding, including ph, buffer strength, contact time, surface tension of the solution, solute concentration, and individual protein character. In general, when ultrafiltration membranes are soaked in protein solutions, regenerated cellulose membranes are the lowest binding, Biomax high-flux polysulfone membranes are somewhat higher binding, and PT series original polysulfone membranes are the highest binding. However, because Biomax membranes have such high flow rates when used in a centrifugal mode and therefore protein contact with the membrane is so brief, protein recoveries with Ultrafree-CL centrifugal filters containing Biomax membranes are often just as high (>95%) as those with regenerated cellulose. For protein concentration or desalination applications, we recommend comparing the Biomax membrane to PL and PT type filters.

8 Ultrafree-CL Centrifugal Filter Units Extractables All ultrafiltration membranes contain a humectant. A humectant ensures that the membrane retains enough moisture to maintain its structure. All of the ultrafiltration membranes used in Ultrafree-CL devices contain glycerin as their humectant. The exception is the 10,000 NMWL regenerated cellulose membrane, which contains triethylene glycol. If these humectants interfere with instrument-based analysis of a sample, flush the Ultrafree-CL device with Milli- Q-system water immediately before use (contact Technical Service for details). Chemical Compatibility with Ultrafree-CL Membranes Durapore Polysulfone/Biomax Regenerated cellulose Acetic Acid, glacial Acetic Acid, 5% Boric Acid Hydrochloric Acid (conc.) Hydrofluoric Acid Nitric Acid (conc.) Sulphuric Acid KEY: Recommended, Not recommended, Limited use: testing is recommended

http://www.millipore.com/ultrafree 9 Chemical Compatibility with Ultrafree-CL Membranes, continued Durapore Polysulfone/Biomax Regenerated cellulose Ammonium Hydroxide (6N) Sodium Hydroxide (conc.) Acetone Acetonitrile Amyl Acetate Amyl Alcohol Benzene Benzyl Alcohol (1%) Butyl Alcohol Carbon Tetrachloride Cello-Solve (ethyl) Chloroform Cyclohexanone Dimethylacetamide Dimethylformamide Dioxane KEY: Recommended, Not recommended, Limited use: testing is recommended

10 Ultrafree-CL Centrifugal Filter Units Chemical Compatibility with Ultrafree-CL Membranes, continued Durapore Polysulfone/Biomax Regenerated cellulose DMSO Ethyl Alcohol Ethers Ethyl Acetate Ethylene Glycol Formaldehyde Freon TF or PCA Gasoline Glycerine (glycerol) Hexane Hydrogen Peroxide (3%) Hypo (photo) Isobutyl Alcohol Isopropyl Acetate Isopropyl Alcohol Kerosene KEY: Recommended, Not recommended, Limited use: testing is recommended

http://www.millipore.com/ultrafree 11 Chemical Compatibility with Ultrafree-CL Membranes, continued Durapore Polysulfone/Biomax Regenerated cellulose MEK MIBK Nitrobenzene Paraldehyde Ozone (10 ppm in water) Pet Base Oils Pentane Perchloroethylene Petroleum Ether Phenol (5.0%) Pyridine Silicone Oils Toluene Trichloroethylene/ethane THF Xylene KEY: Recommended, Not recommended, Limited use: testing is recommended

12 Ultrafree-CL Centrifugal Filter Units Flow Rate Flow rate is affected by several parameters, including the NMWL, solute concentration, viscosity, centrifugal force, and temperature. For maximum recovery in protein concentration or buffer exchange applications, select a UF membrane with a NMWL at least three times lower than the molecular weight of your target molecule. Expect longer spin times for starting solutions with very high solids. Flow rates are approximately 1.5 times slower when the device is operated at 4ºC than when operated at 25ºC. Viscous solutions, such as 50% glycerol, will take up to five times longer to concentrate than very dilute solutions. Biomax PB UF membranes, particularly those in the 5,000 NMWL and 10,000 NMWL Ultrafree-CL centrifugal filter units, have exceptionally high flow rates when compared to membranes of the same NMWL, and will typically flow at 2 the flowtime of the PL membranes. A PL-10 membrane will require 60 minutes to concentrate a 2 ml 0.25 mg/ml Cytochrome C sample at 30.

http://www.millipore.com/ultrafree 13 Flow Rate Comparison (0.25 mg/ml BSA in PBS [ph=7.4]) u 5,000 NMWL Membranes at 5,000 g 10,000 NMWL Membranes at 5,000 g 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 PB (5K) PL (5K) 0 15 30 45 60 75 90 105 Time (min) jj u All tests were performed at 25 C. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 PB (10K) PL (10K) PT (10K) 0 5 10 15 20 25 30 Time (min) jj

14 Ultrafree-CL Centrifugal Filter Units How to Use the Ultrafree-CL Centrifugal Filter Unit This section provides steps for general use. General Use 1. Remove the top cap from the filtrate collection tube. Then pour or pipette your sample into the filter cup. Hold the Ultrafree-CL vertically, with the filter cup opening facing up. 2. Replace the top cap onto the filtrate collection tube. Then insert the Ultrafree-CL filter into the centrifuge fixed-angle rotor head. 3. Centrifuge the sample until required retentate or filtrate volume is achieved. Filter Type Typical Spin Time Microporous 1 4 minutes Ultrafiltration* 30 60 minutes * There is no deadstop with ultrafiltration devices and concentrate volume must be monitored carefully to prevent running to dryness.

http://www.millipore.com/ultrafree 15 Specifications Materials Molded polypropylene filter cup and microcentrifuge tube for filtrate collection, silicone rubber O-ring in filter cup Dimensions Filtrate collection tube 76.5 mm length, 16.2 mm inside diameter Filter cup 41 mm length, 11.8 mm inside diameter Capacity Filter cup 2.0 ml Collection tube 5 ml Hold-up volume 0.03 ml Effective filtration area 0.8 cm2 Maximum temperature 50 C (122 F) Microcentrifuge rotors Centrifuge with fixed-angle rotors capable of holding 15 ml microcentrifuge tubes Maximum g Force Rating 5,000 g (ultrafiltration devices) 12,000 g (microporous devices)

16 Ultrafree-CL Centrifugal Filter Units Ordering Information Ultrafree-CL filters are dry and nonsterile. Each contains a preassembled filter with collection tube and cap. Biomax UF Membranes 25/box 5,000 NMWL Biomax-5 high-flux UFC4 BCC 25 10,000 NMWL Biomax-10 high-flux UFC4 BGC 25 30,000 NMWL Biomax-30 high-flux UFC4 BTK 25 50,000 NMWL Biomax-50 high-flux UFC4 BQK 25 100,000 NMWL Biomax-100 high-flux UFC4 BHK 25 Regenerated Cellulose UF Membranes 25/box 5,000 NMWL regenerated cellulose UFC4 LCC 25 10,000 NMWL regenerated cellulose UFC4 LGC 25 30,000 NMWL regenerated cellulose UFC4 LTK 25

http://www.millipore.com/ultrafree 17 Ordering Information, continued Polysulfone UF Membranes 25/box 300,000 NMWL polysulfone UFC4 TMK 25 Microporous Membranes 25/box 100/box Presterile* 50/box 0.1 µm pore size Durapore PVDF UFC4 0VV 25 UFC4 0VV 00 0.22 µm pore size Durapore PVDF UFC4 0GV 25 UFC4 0GV 00 UFC4 0GV 0S 0.45 µm pore size Durapore PVDF UFC4 0HV 25 UFC4 0HV 00 UFC4 0HV 0S 0.65 µm pore size Durapore PVDF UFC4 0DV 25 5.0 µm pore size Durapore PVDF UFC4 0SV 25 0.2 µm pore size PTFE UFC4 0LG 25 0.5 µm pore size PTFE UFC4 0LH 25 * Presterile packages consist of 10 devices in each of five ETO-sterilized Tyvek pouches.

18 Ultrafree-CL Centrifugal Filter Units Technical Assistance Call the office in your country to get additional product information, technical assistance, or to order parts. In the U.S., call 1-800-MILLIPORE (800-645-5476). Elsewhere, see our laboratory products catalogue for the office nearest you. To receive the current edition of our catalogue, contact Technical Service at the Millipore office closest to you. You can also look us up on the Internet at our World Wide Web site (http://www.millipore.com). Up-todate information on the Ultrafree product line can be found at http://www.millipore.com/ultrafree. Our e-mail address is techservice@millipore.com.

http://www.millipore.com/ultrafree 19 Standard Warranty Millipore Corporation ("Millipore") warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products. MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications and descriptions of Millipore products appearing in Millipore's published catalogues and product literature may not be altered except by express written agreement signed by an officer of Millipore. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and if given, should not be relied upon. In the event of a breach of the foregoing warranty, Millipore's sole obligation shall be to repair or replace, at its option, the applicable product or part thereof, provided the customer notifies Millipore promptly of any such breach. If after exercising reasonable efforts, Millipore is unable to repair or replace the product or part, then Millipore shall refund to the customer all monies paid for such applicable product or part. MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL OR ANY OTHER INDIRECT DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY CUSTOMER FROM THE USE OF ITS PRODUCTS.