HiLoad 16/600 And 26/600 Superdex 30 Prep Grade HiLoad .
GE HealthcareLife SciencesInstruction 28-9920-17 ACHiLoad 16/600 and 26/600 Superdex 30 prep gradeHiLoad 16/600 and 26/600 Superdex 75 prep gradeHiLoad 16/600 and 26/600 Superdex 200 prep gradeIntroductionHiLoad 16/600 and 26/600 Superdex 30 prep grade, HiLoad 16/600 and26/600 Superdex 75 prep grade, and HiLoad 16/600 and 26/600Superdex 200 prep grade (pg) are prepacked XK columns designed forpreparative gel filtration.Superdex 30 pgKav0.7Superdex prep grade is a composite matrix of dextran and highly cross-linkedagarose. The steep selectivity of dextran and the high chemical and physicalstability of agarose enable high resolution separations. Steep selectivitycurves give unmatched resolution for biomolecules in the molecular weightrange up to 10 000 for Superdex 30 pg, 3 000 to 70 000 for Superdex 75 pg,and 10 000 to 600 000 for Superdex 200 pg (Fig 2).0.5The chromatography media combines high mechanical strength with highhydrophilicity, allowing high flow rates and minimal non-specific interactions.0.1Table 1: Contents of the delivery boxNo. supplied121110.40.30.2300100010 000 MrSuperdex 75 pg and Superdex 200 pgKavDextrans0.80.60.4g0p20 gp75ComponentTransport device1/16” male connectorsStop plugHiLoad columnInstructionsPEGpeptides0.60.2104105106107 MrSuperdex 75 pg and Superdex 200 pgGlobular proteinsKav0.80.60.4g0p20pg75InstructionsTransport 1/16” maleHiLoad 16/600 or 26/600 Superdex 30 pgdeviceconnectors, HiLoad 16/600 or 26/600 Superdex 75 pgstop-plugHiLoad 16/600 or 26/600 Superdex 200 pg0.2104Fig. 1 Package includes HiLoad column, transport device, two connectors,two stop plugs and instructions.105106 107 MrFig. 2 Selectivity curves from Superdex 30 pg, Superdex 75 pg andSuperdex 200 pg.
Instruction 28-9920-17 ACTable 2: HiLoad column characteristicsMatrixDextran covalently bound to highlycross-linked agaroseMean particle size34 μmSeparation range (Mr) 10 000 (Superdex 30 pg)Globular proteinsRecommended running conditionsFlow rate*Sample volume3 103 to 7 104 (Superdex 75 pg)SamplepreparationBuffer1 104 to 6 105 (Superdex 200 pg)Dextrans5 102 to 3 104 (Superdex 75 pg)*Column volumeSample volume†Recommended flow rateRecommended flow rate rangeTheoretical platesMaximum pressure over thepacked bed duringoperation, ΔpColumn hardwarepressure limit‡pH stability:long term and working rangeshort termStorage:- Superdex 30- Superdex 75- Superdex 200*†‡1 103 to 1 105 (Superdex 200 pg)120 to 124 ml (16/600)319 to 330 ml (26/600)Up to 5 ml (16/600)Up to 13 ml (26/600)30 cm/h at room temperature(1 ml/min for 16/600 or2.6 ml/min for 26/600)10 to 50 cm/h at room temperature(0.3 to 1.6 ml/min for 16/600 or0.9 to 4.4 ml/min for 26/600) 13 000 m-10.3 MPa, 3 bar, 42 psiRegeneration*Recommended flow rates are valid for H2O at 25ºC.Read “Optimizing” for information on how to optimize a separation.2Two CV of buffer, for example, 0.05 M sodium phosphate, 0.15 M NaCl,pH 7.2 at 50 cm/h (1.6 ml/min for 16/600 or 4.3 ml/min for 26/600).Note:0.5 MPa, 5 bar, 73 psi3 to 121 to 14- 0.2 M sodium acetate, 20% ethanolat room temperature- 0.2 M sodium acetate, 20% ethanolat room temperature- 20% ethanol at room temperatureThe surface of the medium is not directly visible at the bottom piece. Therefore, whencalculating the total column volume, calculate the height of the medium from thelowest part of the bottom piece to the surface of the medium/adapter.For HiLoad 16/600 deduct 30 mm, and for HiLoad 26/600 deduct 36 mm.Optimal sample volume depends on the complexity of the sample and the flow rate.If the sample contains substances with small differences in size, either decrease thesample volume, or decrease the flow rate (in very difficult cases, it may benecessary to decrease both).See “Adjusting pressure limits in chromatography system software”.30 cm/h (1 ml/min for 16/600 or2.6 ml/min for 26/600)0.5% to 4% of the CV (0.6 to 4.8 ml for 16/600 or1.6 to 12.8 ml for 26/600)Note: Sample volume is critical for the separation.Dissolve the sample in running buffer, filter through0.22 μm filter or centrifuge at 10 000 x g for 10 min0.05 M NaPO4, 0.15 M NaCl, pH 7.2 or select a bufferappropriate for the next purification step. To avoidpH dependent non-ionic interactions with thematrix, include at least 0.15 M salt in the buffer(or use a buffer with equivalent ionic strength).Regenerate the column after each run with one CVof running buffer at 30 cm/h (1 ml/min for 16/600or 2.6 ml/min for 26/600)When running under cold conditions or using buffer with high viscosity,adjust the flow rate so that the back pressure limit is not exceeded.Delivery and storageThe prepacked column is delivered in 0.2 M sodium acetate, 20% ethanol(Superdex 30 and Superdex 75) or 20% ethanol (Superdex 200). If the columnneeds to be stored for more than two days after use, wash the column withfour CV of distilled water, and then equilibrate with four CV of 0.2 M sodiumacetate, 20% ethanol or 20% ethanol only, depending on the media. Use thetransport device to prevent air from entering the column and destroying thecolumn packing. Connect the transport device to the capillary tubing at thecolumn outlet. Start the pump and fill the device up to approximately 50% ofthe total device volume.Daily useAll commonly used aqueous buffers, pH 3 to 12CleaningFirst time useConnecting the column1Before connecting the column to a chromatography system, start thepump to remove any air bubbles from the system, particularly in thetubing and valves.2Stop the pump.3Mount the column vertically, remove the stop plug and connect the inlettubing to the system “drop-to-drop”.4Remove the transport device and connect the column outlet tubing to, forexample, a monitor cell. Save the transport device for use when storingthe column. The column is now ready for use.Acetonitrile, up to 30%NaOH, up to 1 MEthanol, up to 70% (Superdex 30 pg)Ethanol, up to 24% (Superdex 75 pg and Superdex 200 pg)Acetic acid, up to 1 MIsopropanol, up to 30%Guanidine hydrochloride, up to 6 MUrea, up to 8 MHydrochloric acid, up to 0.1 M (Superdex 30 pg)AvoidUnfiltered solutionsEquilibrating the columnTip: Equilibrate the column a day before usage to save time.Ensure that an appropriate pressure limit has been set. Equilibrate the columnfor first time use, or after long-term storage as follows:12One column volume (CV) of low ionic strength buffer at 30 cm/h(1 ml/min for 16/600 or 2.6 ml/min for 26/600).Buffers and solvent resistanceDe-gas and filter all solutions through 0.22 μm filter to increase the columnlifetime. Buffers and solvents with high viscosity will affect the back pressureand flow rate.
Instruction 28-9920-17 ACChoosing a bufferColumn resolution is calculated as:Buffer composition does not directly affect the resolution. Select a buffer thatis compatible with the stability and activity of the protein to be purified. Bufferconcentration must be sufficient to maintain a buffering capacity and aconstant pH. Ionic strength should be at least 0.15 M NaCl in the buffer, toavoid non-specific ionic interactions with the matrix.2 ( V R2 – V R1 )R s ------------------------W b2 W b1where,VR1 Retention (elution) volume of the first peakVR2 Retention (elution) volume of the second peakWb1 Base width of the first peakWb2 Base width of the second peakVR and Wb in same units.xBed length: x - 30 mm (16/600)x - 36 mm (26/600)i.d. 16 mm or 26 mmRegular cleaningWash the column with one-half to one CV of 0.5 M NaOH at a flow rate of25 cm/h (0.8 ml/min for 16/600 or 2.2 ml/min for 26/600) to remove most ofthe non-specifically bound proteins from the chromatography medium.Fig. 3 Dimensions of the column.OptimizingPerform a first run as described in “Recommended running conditions”. If theresults obtained are unsatisfactory, consider the following:ActionDecrease flow rateDecrease sample volumeEffectImproved resolutionImproved resolutionResolution, RsFigures 4 and 5 demonstrate the influence of sample volume and flow rate onthe resolution.1.5After cleaning, immediately equilibrate the column with at least two CV ofbuffer. Further equilibration is necessary if the buffer contains detergents.Wait until the UV baseline stabilizes before starting a new purification.More rigorous cleaningWash the column at a flow rate of 25 cm/h (0.8 ml/min for 16/600 or2.2 ml/min for 26/600) at room temperature with the following solutions:1Four CV of 1 M NaOH (removes hydrophobic proteins or lipoproteins)followed by four CV of distilled water.2One-half CV of 30% isopropanol (removes lipids and very hydrophobicproteins), followed by two CV of distilled water.Before starting a new purification, equilibrate the column after cleaning withat least five CV of running buffer.1.0Changing the adapter net ring0.50012345Sample volume (% of CV)Column:Sample:HiLoad 16/600 Superdex 200 pgSolution of transferrin (Mr 81 000) andIgG (Mr 160 000) by equal weightSampleconcentration:Buffer:8 mg/ml50 mM NaPO4, 0.1 M NaCl, pH 7.2Flow rate:30 cm/h (1 ml/min)Fig. 4 Influence of sample volume on column resolution.Resolution, RsCleaning-In-Place (CIP)1.51.51.002040601Close the outlet tubing of the column with a stop plug, and mark the levelof the chromatography medium surface on the glass tube using acolored pen.2Slacken the adapter O-ring slightly by turning the black adjusting knobcounter-clockwise.Note: It should still seal against the glass wall but allow the adapter toslide. Unscrew the top piece from the column.3Connect the adapter to the pump and start pumping at a flow rate of30 cm/h (1 ml for 16/600 or 2.6 ml/min for 26/600). Allow the flow to pushthe adapter upwards.4When the glass tube is completely full, take out the adapter and stop thepump. The glass tube should be completely filled with liquid.a)5Change the adapter net ring.b)6To avoid any air bubbles under the net, inject 20% ethanol through theadapter using a syringe.7Insert the adapter into the column at an angle of 45 , avoiding airbubbles. Slide the plunger 1 to 2 cm down and tighten the O-ring. Removeexcess liquid completely before screwing the top piece onto the columnend piece.8Remove the syringe and slide down the adapter until it touches thechromatography medium surface. Tighten the O-ring and reconnect theinlet tubing to the system, avoiding air bubbles.0.50After following the cleaning procedures above, if the back pressure of thecolumn remains too high, change the net ring in the column adapter. Followthe instructions below thoroughly since column efficiency is easily impaired ifhandled without care. Use distilled water as a liquid. For an exploded view ofthe adapter, see Figure 8.80Flow rate (cm/h)Column:Sample:Sampleconcentration:Sample volume:Buffer:HiLoad 16/600 Superdex 30 pgIGF-1 containing monomers and dimersa) 1.25 mg/mlb) 5 mg/ml1 ml (0.8% of CV)50 mM sodium acetate, 0.1 M NaCl, pH 5.0Fig. 5 Influence of flow rate on the column resolution.3
Instruction 28-9920-17 AC910Remove the stop plug and start the pump. Increase the flow rate until themedium surface is approximately 3 mm above the pen mark. Stop thepump and close the outlet tubing with the stop plug again.Adjusting pressure limits in chromatographysystem softwareNote: This step requires a pump with high flow rate capacity up to apressure of 0.5 MPa (5 bar).Pressure generated by the flow, through a column, affects the packed bed, andthe column hardware, see Figure 7. Increased pressures might be generatedwhen running/using one or a combination of the following conditions:Disconnect the inlet tubing and slacken the adapter O-ring slightly byturning the adjusting knob counter-clockwise. Press the adapterdownwards up to the pen mark. Tighten the O-ring.Note: Do not loosen the O-ring too much as this will result inchromatography medium passing through the O-ring.11 High flow rates Buffers or sample with high viscosity Low temperature A flow restrictorNote: Exceeding the pressure limits (see Table 2) will damage the column.Reconnect the inlet tubing and avoid introducing air into the system.pre-column pressureTroubleshootingSymptomIncreased back pressureover the columnRemedyClean the column according to thesection “Cleaning-In-Place (CIP)”Loss of resolution and/ordecreased samplerecoveryClean the column according to thesection “Cleaning-In-Place (CIP)”Air bubbles in the columnReverse the direction of flow and pumpfive CV of degassed water through thecolumn at the same flow rate that wasused during the run.Space between adapter andmediumClose the outlet tubing with the stop plugand then disconnect the inlet tubing.Slacken the O-ring slightly by turning theadjusting knob counter-clockwise andpush or screw the adapter down until ittouches the medium surface. Tighten theO-ring. To maintain an airtight system,reconnect the inlet tubing immediately.Testing the column efficiencyAbsorbanceGE Healthcare Life Sciences packs columns to the highest standards and eachcolumn is thoroughly tested with respect to the number of theoretical platesper meter (N/m) (Fig 6).(Δp), pressure over thepacked bedpost-column pressureFig. 7 Pre-column and post-column measurements.ÄKTA avantThe system will automatically handle all pressure limits, which facilitates anoptimal functionality without any need of adjustments.ÄKTAexplorer , ÄKTApurifier , ÄKTAFPLC and othersystems with pressure sensor in the pumpTo obtain optimal functionality, the pressure limits in the software may beadjusted according to the following procedure:1Replace the column with a piece of tubing. Run the pump at themaximum intended flow rate. Note the pressure as total system pressure.2Disconnect the tubing and run the pump at the same flow rate used instep 1. Note that there will be a drip from the column valve. Note thepressure during this operation as measured pressure.3Calculate column pressure limit as a sum of total system pressure andΔp (pressure over the packed bed) (see Table 2).4Replace the column pressure limit in the software with the calculatedvalue.VRWhFig. 6 Column efficiency testSample:2% acetone in waterSample volume:200 μl (16/600) and 500 μl (26/600)Eluent:Distilled waterFlow rate:60 cm/h2.0 ml/min (16/600)5.3 ml/min (26/600)Temperature:Room temperature (25ºC)Column efficiency is calculated using the equation:2N/m 5.54 VR /LWhwhere,VR Peak retention (elution) volumeWh Peak width at half peak heightL Bed height (meter)VR and Wh have the same units.4Calculate post-column pressure as the difference between total systempressure and measured pressure.Column hardware pressure limit (see Table 2) must never exceed the sum ofpost-column pressure and Δp.Note:Repeat the procedure each time the parameters are changed.
Instruction 28-9920-17 ACOrdering information1 2 3123456789101112456789106 11 12Net ringSupport screenO-ringPlungerFerruleCapillary tubingInner shaftAdapter shaftTop end capAdjusting knobHiTrap/HiPrep, 1/16" male connector for ÄKTA designStop plugFig. 8 Exploded view of the XK column adapter used at the top of the HiLoadcolumn.Intended useHiLoad Superdex columns are intended for research use only, and shall not beused in any clinical or in vitro procedures for diagnostic purposes.ProductPack sizeCode No.HiLoad 16/600Superdex 30 prep grade1 x 120 ml28-9893-31HiLoad 26/600Superdex 30 prep grade1 x 320 ml28-9893-32HiLoad 16/600Superdex 75 prep grade1 x 120 ml28-9893-33HiLoad 26/600Superdex 75 prep grade1 x 320 ml28-9893-34HiLoad 16/600Superdex 200 prep grade1 x 120 ml28-9893-35HiLoad 26/600Superdex 200 prep grade1 x 320 ml28-9893-36AccessoriesNo. supplied Code No.Accessory kit XK 16*128-9899-78*Accessory kit XK 26128-9899-79Support screen XK 16519-0651-01Support screen XK 26518-9377-01Net ring (10 μm) XK 16518-8761-01Net ring (10 μm) XK 26518-8760-01O-ring XK 16519-0163-01O-ring XK 26528-9782-27Stop plug female, 1/16”511-0004-64HiTrap/HiPrep 1/16” male connector forÄKTA design828-4010-81Transport device118-1176-43*Accessory kits XK 16 and XK 26 are suitable for repacking purposes and contain:2 support screens, 5 net rings, 2 O-rings, 2 stop plugs, 10 HiTrap/HiPrep 1/16” maleconnectors for ÄKTA design, and 1 tool for dismantling.Related literatureCode No.Gel Filtration Handbook, Principles and Methods18-1022-18Gel Filtration Columns and Media, Selection Guide18-1124-19Prepacked chromatography columns for ÄKTA systems,Selection Guide28-9317-785
For local office contact information, visitwww.gelifesciences.com/contactGE Healthcare Bio-Sciences ABBjörkgatan 30751 84 cationGE, imagination at work and GE monogram are trademarks of General Electric Company.ÄKTA, ÄKTAexplorer, ÄKTAFPLC, ÄKTApurifier, HiLoad and Superdex are trademarks of GE Healthcarecompanies. 2001-2011 General Electric Company—All rights reserved.First published Apr. 2001.All goods and services are sold subject to the terms and conditions of sale of the company within GEHealthcare which supplies them. A copy of these terms and conditions is available on request. Contactyour local GE Healthcare representative for the most current information.GE Healthcare UK LtdAmersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UKGE Healthcare Bio-Sciences Corp800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USAGE Healthcare Europe GmbHMunzinger Strasse 5, D-79111 Freiburg, GermanyGE Healthcare Japan CorporationSanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan28-9920-17 AC 12/2011
Instruction 28-9920-17 AC GE Healthcare Life Sciences HiLoad 16/600 and 26/600 Superdex 30 prep grade HiLoad 16/600 and 26/600 Superdex 75 prep grade
603.05 Welded Wire Fabric 600-3 604 CONSTRUCTION JOINTS 600-4 605 DEPOSITING THE CONCRETE 600-4 606 CONSOLIDATING CONCRETE 600-4 607 CURING 600-5 607.01 General 600-5 607.02 Cold Weather Requirements 600-6 608 REMOVAL OF FORMS 600-6 609 CONCRETE SURFACE FINISHES 600-6 609.01 Filling and Repairing Bolt Holes 600-8
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