01 ion exchange chromatography
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Separation of molecules that involves twophases, the mobile phaseand thestationary phase.
Choice of mobile and stationary phasesdetermines extent of separation
Common criteria for separation:
> Polarity> Size
> Charge
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Where solute molecules are dissolved
Can be liquidor gaseous
Carries solute across the stationary phase
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Matrix where separation occurs
Interactions of molecules with stationary
phase reduces movement speedthrough matrix
Can be paperor column type
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Separates molecules based on charge
Solute is bound on the matrix due to
electrostatic interactions withimmobilized ionic groups
Binding is reversible
Became popular in biochemistry becauseit issimple, controllable, has highresolving powerand capacity
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Stationary phase: ion exchange resins
Mobile phase : buffer solution
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Protein Detection
Absorbance Bradford reagent
Regeneration
Washing column and re-equilibrating with buffer
Elution
Gradient: 0.1 M, 0.2 M, 0.3 M, 0.5 M, 1.0 M KCl in buffer
Loading Samples
Sample: 5 mg/ml each of invertase, albumin and casein
Column and Gel Preparation
Anionic: DEAE-cellulose Cationic: Dowex 50W
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Has ionic groups covalently bound onsurface
Attracts oppositely charged molecules Can be cationicor anionic
Synthetic resins are unsuitable for
biomolecules Cellulose, agarose, and dextran became
popular
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Source: Voet, Voet. Biochemistry4thed.
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Source: H. U. Khan. The Role of Ion Exchange Chromatography in Purification andCharacterization of Molecules
Stable below pI: anionic exchanger
Stable above pI: cationic exchanger
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Components must have same charge asmatrix
If not, it will take part in ion exchange pH also important (dependent of pKa)
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Source: D. Voet, J. Voet. Biochemistry4thedition
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-0.03
-0.02
-0.01
0
0.01
0.02
0.03
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Absorbance
[KCl], molL-1
Absorbance versus salt
concentration for DEAE-cellulose ~0.2 M KCl
Source of Error: pH of the
Buffer
InstrumentalError
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-0.03
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0 0.2 0.4 0.6 0.8 1 1.2
Absorbance
[KCl], molL-1
Absorbance vs salt
concentration ~0.3 M KCl
Source of Error: pH of the Buffer
InstrumentalError
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Dowex 50 fractions with Bradford reagent (start from left)
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Separation was not achieved using bothexchangers
Possible sources of error:
> pH of buffer
> Instrument
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Use only DEAE-cellulose for IEC of proteinsamples with pI below 7
Prepare reagents (esp. buffers) on theday of the experiment
Spectrophotometric assay must be done
on the same day