IonExchange Chromatography

Ion-exchange chromatography separates biomolecules on the basis of charge characteristics. Charged groups on the surface of a protein interact with oppositely charged groups immobilized on the ionexchange medium. This unit provides thorough explanations of the theory behind the technology, troubleshooting tips for making the procedure work best for you, and detailed listings of column matrices and separation properties

TABLE OF CONTENTS

Unit Introduction Strategic Planning Basic Protocol 1: Batch Adsorption and Step-Gradient Elution with Increasing Salt Concentration Alternate Protocol: pH-Based Step-Gradient Elution Basic Protocol 2: Column Chromatography with Linear Gradient Elution Support Protocol 1: Test Tube Pilot Experiment to Determine Starting Conditions for IonExchange Chromatography Support Protocol 2: Measurement of Dynamic (Column) Capacity and Breakthrough Capacity of Ion-Exchange Columns Support Protocol 3: Gradient-Formation Techniques Support Protocol 4: Cleaning and Regeneration of Ion-Exchange Media Support Protocol 5: Storage of Ion-Exchange Media Commentary Bibliography

Figures Tables

Basic Protocol 1: Batch Adsorption and Step-Gradient Elution with Increasing Salt Concentration
Materials

QAE Sephadex A-25 (Amersham Pharmacia Biotech) or equivalent anion-exchange gel Binding buffer: 20 mM TrisCl, pH 7.5 (or other buffer as determined empirically; see Support Protocol 1) Protein sample to be purified Wash buffer: 20 mM TrisCl (pH 7.5)/100 mM NaCl (or other buffer/salt solution as determined empirically; see Support Protocol 1) Elution buffer: 20 mM TrisCl (pH 7.5)/350 mM NaCl (or other buffer/salt solution as determined empirically; see Support Protocol 1) Regeneration buffer: 20 mM TrisCl (pH 7.5)/2 M NaCl (also see Support Protocol 5) Boiling water bath (optional) 500-ml sintered-glass filter funnel, medium porosity Three 2000-ml side-arm flasks Conductivity meter

Basic Protocol 2: Column Chromatography with Linear Gradient Elution

Materials

Liquid chromatography system (FPLC or HPLC) Elution buffer: binding buffer (see Basic Protocol 1 and Support Protocol 1) containing 1 M NaCl Binding buffer (see Basic Protocol 1 and Support Protocol 1) RESOURCE Q chromatography column (1-ml packed bed volume; Amersham Pharmacia Biotech)

Protein sample to be purified Conductivity meter 0.22-m filter

Support Protocol 1: Test Tube Pilot Experiment to Determine Starting Conditions for Ion-Exchange Chromatography
Materials

20 mM piperazine, pH 5.0, 5.5, and 6.0 (prepare from 100 mM stock) 20 mM 1,3-bis[tris{hydroxymethyl}methylamino]propane (bis-Tris propane), pH 6.5 and 7.0 (prepare from 100 mM stock) 20 mM TrisCl, pH 7.5, 8.0, and 8.5 (APPENDIX 2; prepare from 100 mM stock) Q Sepharose Fast Flow (50% slurry in 20% ethanol; Amersham Pharmacia Biotech) or equivalent anion exchange resin in appropriate buffer Protein sample to be purified, containing known quantity of target and total protein 4 M NaCl 15-ml test tubes Centrifuge with rotor accommodating 15-ml test tubes (optional)

Support Protocol 2: Measurement of Dynamic (Column) Capacity and Breakthrough Capacity of Ion-Exchange Columns
Additional Materials (also see Basic Protocol 2) Ion-exchange gel of unknown capacity, and column Elution buffer capable of eluting target protein in single step of salt concentration or pH (e.g., 2 M NaCl; see Support Protocol 1)

Figure 10.10.1 Net charge of a protein as a function of pH, showing the pH ranges in which protein is bound to anion or cation exchangers. The pH range over which the protein is stable may be only a small fraction of the binding range; this must also be taken into consideration when choosing an ion-exchange medium

Liquid column chromatography system with gradient maker.

Results of a typical test tube pilot experiment for selecting initial conditions for anion-exchange chromatography. Series of tubes with protein and ion-exchange medium at (A) varying pH (to select binding and elution pH); (B) constant pH, varying NaCl concentration (to select binding and elution salt concentrations); (C) constant pH and NaCl concentration, varying protein concentration (to determine available capacity). The binding pH here will be 7.5; the elution pH will be 5.0; the binding salt concentration will be 0.15 M; the elution salt concentration will be >0.3 M; and the available capacity will be >50 mg/ml.

Typical chromatogram for determining the capacity of an ion-exchange column. The volume of eluant (x) at 50% full-scale deflection is used to calculate the breakthrough capacity (QB50

Gradient mixer for forming gradients of pH and salt concentration used during ion-exchange chromatography. The apparatus shown is an Amersham Pharmacia Biotech Gradient Mixer GM-1, which can be used for preparing gradients up to 500 ml. A linear gradient of salt concentration is produced by filling the reservoirs with buffers at the same pH and different salt concentration. The mixing chamber contains the lower-salt-concentration (binding) buffer, and the other chamber contains the higher-salt-concentration (elution) buffer. A continuous linear or nonlinear pH gradient may be produced by filling the reservoirs with buffers at the same salt concentration and different pH. A gradient of increasing pH is used for cation-exchange separations, and a gradient of decreasing pH is used for anion-exchange separations. The buffer chambers are joined by a channel controlled by a valve, and outflow from the mixing chamber is controlled by another valve.

Peristaltic pump accommodating three pumping channels for continuous gradient formation.

Construction of a chromatographic titration curve. (A) Six chromatographic separations (a to f) of a sample containing proteins A, B, and C are carried out at six different operating pHs using a linear gradient of increasing salt concentration. (B) The elution ionic strengths for components A, B, and C (y axis) are plotted against the operating pH (x axis) for experiments a to f. Note that the salt concentration in mM NaCl (y axis) is ascending in both directions. The optimal resolution with respect to component A occurs below pH 5 with cation exchange and above pH 9 with anion exchange.

Chromatogram representing typical results of stages of an ion-exchange column chromatography experiment. A linear gradient of 0% to 100% elution buffer is used as in Basic Protocol 2. The broken line superimposed on the chromatogram represents the composition of the elution gradient (100% binding buffer to equilibrate column and remove unbound sample components, followed by a linear gradient from 100% binding buffer to 100% elution buffer to elute bound proteins and a final wash with 100% elution buffer). Vc represents the packed bed volume of the column.