ION EXCHANGE CHROMATOGRAPHY

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ION EXCHANGECHROMATOGRAPHY
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Type Of Column Chromatography

• Gel permeation
• Ion Exchange
• Affinity
• Hydrophobic Interaction

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Basic Idea

• IEC is about interactions between positively and
  negatively charged molecules
• Ion exchange

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Advantages To IEC

• High resolution
• High capacity
• Generally preparative
• Does not alter proteins
• Used for protein purification

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Four Components To The Set-Up

• Matrix (bead, resin)
• Charged groups on resin
• Mobile Counter Ions
• Sample

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I Matrix Is Support

• Matrix, polymeric, porous beads
• Sepharose , Sephacel, Sephadex
• Packed (poured) into the column as a slurry

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II Charged Groups

• Charged groups- covalently bound to matrix
• DEAE is positively charged
• CM is negatively charged
• So, have, for example, DEAE Sephadex, type of ion
  exchange resin

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III Mobile CounterIons

• Ions that move in buffer solution
• Flow through the column and interact with charged
  groups on the beads
• Cl- for DEAE
• Na for CM

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Terminology TwoTypes Of IEC Resin

• Anion Exchangers
• DEAE is positively charged
• Binds anions (-) so DEAE is an anion exchange
  resin

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• Cation Exchangers
• CM is negatively charged
• Binds cations () so CM is a cation exchange
  resin

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IV Sample

• Sample interacts with charged groups on the beads
• Consider sample in more detail as describe the
  process of IEC

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Six Stages To IEC

• Set up column other equipment (plumbing)
• Prepare resin pour column equilibrate resin
• Load sample onto column --adsorption
• Wash unbound off column
• Desorption elution
• End desorption regenerate column (optional)

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Stage I Set Up Column

• Attach column to ringstand
• Attach pump (optional)
• Attach detector (optional)
• Attach strip chart recorder
• Attach fraction collector (optional)

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Stage II Equilibration

• Prepare resin
• Pour into column
• Ion exchanger is brought to starting state
  equilibrated
• The charged groups on the beads are associated
  with mobile ions from starting buffer
• We use 0.2 M NTM buffer has 0.2 M NaCl
• Cl- ions are associated with all the DEAE
  positive sites

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Stage III Adsorption Of Sample

• Apply sample to the column --loading
• ß-galactosidase has a net negative charge and it
  will stick -- adsorb
• Some contaminants flow right through
• Other contaminants also stick to beads

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Stage IV Wash Off Unbound

• Wash with 0.2 M NTM
• Unbound materials wash off

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Stage V Elution

• Slowly increase salt concentration in buffer
  mobile phase from 0.2 M to eventually 0.5 M
• We will use a continuous gradient method
• The most weakly bound solutes fall off (elute)
  first
• Eventually the chloride ions displace
  ß-galactosidase and it also elutes
• As material comes off the column, collect it in
  fractions
• 0.5 mL in small tubes

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Stage VI End And Regenerate

• Can elute rest of solutes with high ionic
  strength buffer
• Regenerate the column with original buffer, in
  our case, 0.2 M NTM (optional)

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What MakesProteins Adsorb And Desorb From The
Column?

• Proteins have charges because their amino acids
  are charged
• At a given pH, different proteins have different
  charges, because they are composed of different
  amino acids

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Charge On Proteins

• Overall net charge on a protein is determined by
  the R groups of its amino acids
• Some amino acids have R group with positive
  charge, others negative charge, others no charge

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• In theory, IEC can separate proteins that differ
  by only one charged group

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Charge Distribution

• Charge distribution influences adsorption
• How charges are distributed on the folded protein
• Some amino acids may not be available to interact
  with environment

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pH Of Environment Is Crucial

• pH affects stability of proteins
• pH of environment affects charge on a protein
• pH is controlled with the buffer chosen

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More About pHAnd Protein Charge

• pI - Isoelectric point
• Definition The pH at which a protein carries no
  net charge

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pI Is Important

• First, pI for a protein of interest determines
  whether use cation or an anion resin

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Isoelectric Point
pl
Use anion exchange
Net charge on protein
pH
2
4
6
8
Use cation exchange
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pI

• Also, usually dont want to use a pH where the
  protein has no charge at all because it will
  never stick to the beads
• So, for example, pI is 6.2, may want to use a
  buffer at 7.2.

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Capacity OfColumn

• Measure of ability to adsorb sample
• Based on charge or equivalents
• Depends on molecular weight of proteins because
  greater MW usually means more charged groups
• Approximation is often used
• 0.5-1 g protein for 10 mLs gel matrix