阴离子柱说明书

HiPrep 16/10 Phenyl FF (high sub),○

HiPrep 16/10 Phenyl FF (low sub),○○HiPrep 16/10 Butyl FF, and○HiPrep 16/10 Octyl FF

○○○○HiPrep 16/10 Phenyl FF (high sub), HiPrep 16/10 Phenyl FF○(low sub), HiPrep 16/10 Butyl FF, and HiPrep 16/10 Octyl FF○are prepacked, ready to use columns for hydrophobic

○interaction chromatography (HIC). The columns provide fast,○preparative separations of proteins and other biomolecules○based on their hydrophobic interaction with hydrophobic○groups attached to the uncharged gel. See table below for○column characteristics.

○○○Column data

○Matrix

6% highly cross-linked spherical○agarose (Phenyl)

○4% highly cross-linked spherical○agrose (Butyl, Octyl)Mean particle size90 µm○Bed volume20 ml○Bed height100 mm○i.d.

16 mm

○Column compositionMedical grade polypropylen○Connectors

M6

Recommended flow rate12–10 ml/min (60–300 cm/h)○Maximum flow rate1

10 ml/min (300 cm/h)○Maximum pressure over the

0.15 MPa, 1.5 bar, 22 psi

○packed bed during operation, Dp2○HiPrep column hardware0.5 MPa, 5 bar, 73 psi○pressure limit2

○Phenyl○PhenylButylOctyl(high sub)○(low sub)Hydrophobic ligandPhenyl○PhenylButylOctylLigand densityw

(µmol/ml gel)40○20

505pH stability○short term

2–14○2–142–142–14long term and working range3–13○3–133–133–13Storage

20%○20%20%20%ethanol

○ethanol

ethanol

ethanol

1

Water at room temperature. Flow rate is determined by v ○ h £ 10 ml/minwhere v = flow rate and h = viscosity.

○2

Many chromatography systems are equipped with pressure gauges to

○measure the pressure at a particular point in the system, usually just afterthe pumps. The pressure measured here is the sum of the pre-column

○pressure, the pressure drop over the gel bed, and the post-column pressure.○It is always higher than the pressure drop over the bed alone. We recommend○keeping the pressure drop over the bed below 1.5 bar. Setting the upperlimit of your pressure gauge to 1.5 bar will ensure the pump shuts down○before the gel is overpressured.

○If necessary, post-column pressure of up to 3.5 bar can be added to the○limit without exceeding the column hardware limit. To determine post-○column pressure, proceed as follows:

○To avoid breaking the column, the post-column pressure must never exceed 3.5 bar.○1.Connect a piece of tubing in place of the column.

○2.

Run the pump at the maximum flow you intend to use for chromatography.○Use a buffer with the same viscosity as you intend to use for○chromatography. Note the back pressure as total pressure.

○3.Disconnect the tubing and run at the same flow rate used in step 2.Note this back pressure as pre-column pressure.

○4.

Calculate the post-column pressure as total pressure minus pre-column○pressure.

○○If the post-column pressure is higher than 3.5 bar, take steps to reduce it(shorten tubing, clear clogged tubing, or change flow restrictors) and perform○steps 1–4 again until the post-column pressure is below 3.5 bar. When the○post-column pressure is satisfactory, add the post-column pressure to 1.5 bar○and set this as the upper pressure limit on the chromatography system.

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阴离子柱说明书

○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○

Table 1 lists volatile buffers used in cases where the purifiedsubstance has to be freeze-dried.

Table 1. Volatile buffer systems.

Trouble shooting

Symptom

Remedy

Increased backpressureReverse the flow direction and pump 100 ml elution

buffer at a over the column flow rate of 5 ml/min atroom temperature through the column. Return to

normal flow direction and run 100 ml buffer at a flowrate of 5 ml/min through the column. If backpressureis not decreased reverse the flow direction again andfollow the rigorous cleaning instructions.

Loss of resolutionFollow the procedure described in the sectionand/or decreased“More rigorous cleaning”.sample recoveryAir in the columnReverse the flow direction and pump 100 ml of well

de-gassed start buffer through the column at a flowrate of 5 ml/min at room temperature.

○○○○○○○○○○○○○○○○○○○○○○○○○○○○○

Ordering information

Designation

HiPrep 16/10 Phenyl FF (high sub)HiPrep 16/10 Phenyl FF (low sub)HiPrep 16/10 Butyl FFHiPrep 16/10 Octyl FFCompanion productsHiPrep 26/10 DesaltingHiTrap HIC Selection KitHiTrap Phenyl FF (high sub)HiTrap Phenyl FF (high sub)HiTrap Phenyl FF (low sub)HiTrap Phenyl FF (low sub)HiTrap Phenyl HPHiTrap Phenyl HPHiTrap Octyl FFHiTrap Octyl FFHiTrap Butyl FFHiTrap Butyl FFAccessories

Tubing connectors:

Union 1/16" female/M6 male*Transport syringe

Handbook, Hydrophobic Interaction

Chromatography Principles & Methods* 2 units included in HiPrep package

61

18-1112-5718-1017-6118-1020-90

1 (53 ml)5 x 1 ml5 x 1 ml5 x 5 ml5 x 1 ml5 x 5 ml5 x 1 ml5 x 5 ml5 x 1 ml5 x 5 ml5 x 1 ml5 x 5 ml

17-5087-0117-1349-0117-1355-0117-5193-0117-1353-0117-5194-0117-1351-0117-5195-0117-1359-0117-5196-0117-1357-0117-5197-01

No. per pack1 (20 ml)1 (20 ml)1 (20 ml)1 (20 ml)

Code No.17-5095-0117-5094-0117-5096-0117-5097-01

Optimization

Perform your first run according to “Try these conditions first”.If the obtained results are unsatisfactory, consider the following:

Action

Change pH/buffer salt

Effect

Column performance control

We recommend checking column performance at regularintervals. The function of the column can be checked asdescribed in Figure 1.

Changing the ionic strengthSmaller sample volumeLower flow rateShallower gradientChange to a medium witha different ligand

Selectivity change, weaker/stronger

binding. Increasing the salting-out effectstrengthens the hydrophobic interactions,whereas decreasing it weakens them

Selectivity change, weaker/stronger bindingImproved resolutionImproved resolution

Improved resolution, but broader peaksand decreased concentration in fractionsSelectivity change

For more information, please refer to the handbook

“Hydrophobic interaction chromatography. Principles andMethods” from Amersham Biosciences.

Cleaning in place (CIP)

www

Regular cleaning

Regenerate the column after each run by rinsing it with 100 mldistilled water at a flow rate of 5 ml/min at room temperature toelute material still bound to the column.

Re-equilibrate the column with at least 100 ml start buffer at aflow rate of 5 ml/min at room temperature until the UV baselineand pH/conductivity values are stable.

More rigorous cleaning

Reverse the flow direction and run the following sequence ofsolutions at a flow rate of 5 ml/min at room temperature:1.80 ml of a 1 M NaOH solution (removes precipitated

proteins, hydrophobically bound proteins, and lipoproteinsfrom the column) followed by 80 ml distilled water.2.80 ml of 70% ethanol or 30% isopropanol (removes

proteins, lipoproteins, and lipids that are strongly

hydrophobically bound to the column) followed by 60 mldistilled water.

After cleaning, equilibrate the column with approximately100 ml start buffer at a flow rate of 5 ml/min at roomtemperature before use.

DO NOT OPEN THE COLUMN!

to order:

Figure 1. Typical chromatogram from a function test of (a) HiPrep 16/10 PhenylFF (high sub), (b) HiPrep 16/10 Phenyl FF (low sub), (c) HiPrep 16/10 Butyl FFand (d) HiPrep 16/10 Octyl FF.

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Trademarks

HiPrep, HiTrap and ÄKTA are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciencesgroup which supplies them. A copy of these terms and conditions is available on request

Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place, Little Chalfont Buckinghamshire HP7 9NA, England Amersham Biosciences Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA Amersham Biosciences Europe GmbH Munzinger Strasse 9, D-79111 Freiburg GermanyAmersham Biosciences K.K. Sanken Building, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, JapanS(c) Amersham Biosciences UK Limited 2001 - All rights reserved

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Produced by Wikströms, Sweden 1010069, 01.2001

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