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10006995B.qxp 6/13/2007 9:00 AM Page III Table of Contents Section 1 ...Introduction ........................................1 Section 2 Connecting to Bio-Rad’s Low-Pressure Chromatography Instruments ......................................24 Section 3 Connecting to Other Liquid Chromatography Systems ..................9 3.1 Connecting to BioLogic DuoFlow™ Systems .................9 3.2 HPLC Systems ......................10 3.3 FPLC Systems .......................
10006995B.qxp 6/13/2007 9:00 AM Page IV Section 7 Ordering Information.........................17 Section 8 References .......................................
10006995B.qxp 6/13/2007 9:00 AM Page 1 Section 1 Introduction Bio-Scale Mini cartridges have a patent-pending double-wall design that provides extra durability and allows easy, reliable runs with aqueous buffers commonly used for protein purification. The polypropylene luer fittings and internal sealing surfaces assure leak-free operation at pressures up to 45 psi. Bio-Scale Mini cartridges are convenient, disposable, and supplied ready for use.
10006995B.qxp 6/13/2007 9:00 AM Page 2 • Luer fittings for convenient connection to any chromatography system Bio-Scale Mini Bio-Gel P-6 cartridges are packed with Bio-Gel® P-6 gel. This gel consists of hydrophilic spherical polyacrylamide beads designed for the purification of proteins, nucleic acids, viruses, plasmids, and other macromolecules. The Bio-Gel P-6 beads are designed to provide high-efficiency desalting or buffer exchange at low backpressure.
10006995B.qxp 6/13/2007 9:00 AM Page 3 Table 1. Bio-Scale Mini Bio-Gel P-6 cartridge specifications. Size 10 and 50 ml bed volume Dimensions 10 ml: 80 mm length x 12.6 mm inner diameter 50 ml: 80 mm length x 28.2 mm inner diameter Maximum pressure tolerance Recommended flow rates: at ambient temperature (18–24°C) at cold room temperature (4–8°C) 10 ml: 45 psi 50 ml: 40 psi 10 ml: 0.5–4.0 ml/min (24–192 cm/hr) 50 ml: 2.5–20.0 ml/min (24–192 cm/hr) 10 ml: 0.5–2.0 ml/min (24–96 cm/hr) 50 ml: 2.5–10.
10006995B.qxp 6/13/2007 9:00 AM Page 4 Section 2 Connecting to Bio-Rad’s Low-Pressure Chromatography Instruments Bio-Scale Mini cartridges are ideal for use with Bio-Rad's BioLogic™ LP chromatography system, Econo™ gradient pump, the patented* Model EP-1 Econo™ pump, and all low-pressure chromatography instruments. Bio-Scale Mini cartridges can be conveniently connected directly to the system using the luer fittings on the cartridge. 1. Install 1.6 mm inner diameter (ID) tubing in the pumphead.
10006995B.qxp 6/13/2007 9:00 AM Page 5 (Use orange lock rings and medium size barb fittings with 1.6 mm tubing.) Platen pressure Platen pressure screw screw Lock-ring Lock-ring Tubing Tubing Luer fittimg Luer fitting See detail See detail 1 4 7 rm Ala C 2 5 8 0 3 6 9 . Fig. 1. BioLogic LP system setup.
006995B.qxp 2. 6/13/2007 9:00 AM Page 6 To apply samples efficiently, install 1.6 mm ID tubing from the pump to the MV-6 sample inject valve (if available). If using the MV-6 sample inject valve, turn the knob counterclockwise as far as it will go so it will now correspond to the printed diagram on the valve (see Figure 2).
10006995B.qxp 6/13/2007 9:00 AM Page 7 3. Connect the inlet of the cartridge to the male luer fitting on the MV-6 sample inject valve (see Figure 2). If not using the MV-6 sample inject valve, connect a barb to male luer fitting on the 1.6 mm ID tubing, then connect to the top of the female luer on the Bio-Scale Mini cartridge. For optimum performance, a cartridge should be mounted vertically with the arrow on the cartridge pointing downward (see Figure 3). 4. Connect the cartridge outlet to the 1.
10006995B.qxp 6/13/2007 9:00 AM Fig. 3. Cartridge and fittings.
10006995B.qxp 6/13/2007 9:00 AM Page 9 Section 3 Connecting to Other Liquid Chromatography Systems Bio-Scale Mini cartridges can be connected to any liquid chromotography system, provieded that the maximum pressure limits for the 10 ml (3 bar, 45 psi, or 300 kPa) or for the 50 ml (2.6 bar, 40 psi, or 260 kPa) cartridges are not exceeded. It is recommended that the system pressure limit be set according to the cartridge pressure limit.
10006995B.qxp 6/13/2007 9:00 AM Page 10 Fig. 4. Luer to 1/4-28 adaptor. 3.2 HPLC Systems The luer to 10-32 adaptor fittings kit (catalog #732-0112), provides fittings necessary to connect the Bio-Scale Mini cartridge to nut and ferrule type fittings found on most HPLC systems. Alternatively, the cartridge can be connected to HPLC systems via a low dead-volume 1/16 inch union with a new piece of stainless steel tubing attached to the union. Simply slip a short length of the 0.
10006995B.qxp 6/13/2007 9:00 AM Page 11 3.3 FPLC Systems The luer to M6 adaptor fittings kit (catalog #732-0111) provides fittings necessary to connect the Bio-Scale Mini cartridge to the M6 fittings found on FPLC or related systems. Alternatively, connection can be made by using one GE Healthcare Union luer lock female to M6 female fitting (GE 18-1027-12) and one female luer lock to M6 male fitting (Upchurch P-686 or GE 18-1027-62).
10006995B.qxp 6/13/2007 9:00 AM Page 12 Section 4 Preparing a Cartridge For Use Bio-Scale Mini Bio-Gel P-6 cartridges contain 20 mM Bis-Tris, pH 6.5 + 0.05% sodium azide as the storage solution. The fully hydrated support is ready to use after equilibrating the cartridge in the buffer of choice. To perform a buffer exchange, connect the cartridge to a liquid chromatography system or peristaltic pump and condition it as instructed below: 1. Set the pump flow rate for the 10 ml cartridge to 4.
10006995B.qxp 6/13/2007 9:00 AM Page 13 3. Equilibrate the cartridge with 2.2 column volumes of degassed equilibration buffer: 6 minutes at the room temperature flow rate setting or 12 minutes at the cold room temperature setting. 4. Reduce the flow rate to that which will be used in the buffer exchange or desalting cycle. 4.1 Sample Preparation If the sample contains insoluble material, filter it with a 0.45 µm filter before applying it to the cartridge.
10006995B.qxp 6/13/2007 9:00 AM Page 14 Section 5 Desalting and Buffer Exchange Samples can be desalted or the sample buffer exchanged by connecting the cartridge to a liquid chromatography system. The void volume for the 10 ml Bio-Scale Mini Bio-Gel P-6 Desalting cartridge is 3.6 ml and for the 50 ml Bio-Scale Mini Bio-Gel P-6 Desalting cartridge is 18 ml.
10006995B.qxp 6/13/2007 9:00 AM Page 15 5.1 With a Liquid Chromatography System 1. 2. 3. 4. Set the flow rate to the desired flow. a. 10 ml cartridge: 0.5–4.0 ml/min at room temperature, 0.5–2.0 ml/min at cold room temperature. b. 50 ml cartridge: 2.5–20 ml/min at room temperature, 2.5–10 ml/min at cold room temperature. Wash the prepared cartridge with degassed buffer as indicated below. a. 10 ml cartridge: 10 ml b.
10006995B.qxp 6/13/2007 9:00 AM Page 16 Section 6 Technical Assistance For additional information and technical assistance, contact your local Bio-Rad representative as listed on the back cover of our catalog, or in the US, call Technical Support at 1-800-4BIORAD.
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10006995B.qxp 6/13/2007 9:00 AM Page 19 Section 8 References Gagnon P, Avoiding Instrument Associated Variation in Purification Scale-up and Scale-down, BioPharm, 10 (3) 42–45, 1997 Harris ELV and Angal S, Protein Purification Methods, A Practical Approach, IRL Press, Oxford, 1989 Scopes RK, Protein Purification, Principles and Practice, (2nd Edition), Springer-Verlag, New York, 1987 Snyder LR and Kirkland JJ, Introduction to Modern Liquid Chromatography, (2nd Edition), John Wiley & Sons, Inc.
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10006995B.qxp 6/13/2007 9:00 AM Page 21 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
