Основы белковой хроматографии
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Solid phase particle sizes & resolution
Small beads give high back pressure but great resolution, good for analytical work and demanding small scale preps
Big beads give low back pressure but less resolution, good for preparative work in large columns
Life Science Group I Chromatography
Solid phase particle sizes & resolution
Small beads give high back pressure but great resolution, good for analytical work and demanding small scale preps
Blue trace – Beads size 10 µm
Big beads give low back pressure but less resolution, good for preparative work in large columns
Read trace: Beads size 50 µ
Life Science Group I Chromatography
Optimal Flow Rate
Van Deemter plot
–Heght Equivalent to a Theoretical Plate (HETP)
–Lower is better
–Should be optimized
to have optimal resolution
Life Science Group I Chromatography
Sample Mass Affects Resolution
A) 1 mg sample |
B) 50 mg sample |
A A B
B
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
0 |
1 |
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3 |
4 |
5 |
6 |
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Column Volumes |
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Column Volumes |
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Overloaded sample results in poorer separations and faster elution
Life Science Group I Chromatography
Gradient Impact on Separation
Linear
1 |
100% |
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2
3
%B
CV |
10 |
5% |
Gradient: Linear, 5%B to 100%B in 10 CV Flow rate: 2 mL/min
Step
1 |
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Step |
100% |
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3 |
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75% |
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2 |
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%B |
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Isocratic |
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20% |
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CV |
10 |
15 |
0% |
Gradient: Step 1 - 20%B for 10 CV Step 2 - 75%B for 5 CV
Flow rate: 2 mL/min
Life Science Group I Chromatography
Biological Targets of Purification
Biomolecules
Small Biomolecules
e.g. Antibiotics, Vitamins Organic acids, statins
Tetracycline antibiotics
Large Biomolecules
e.g. Proteins, Nucleic acids Enzymes, polysaccharides, inclusion bodies
Protein (Lysozyme)
Life Science Group I Chromatography
A typical protein macromolecule
Proteins are structured with areas of positive and negative charge as well as neutral areas which are determined by the amino acid prevalence and position
Combination of positive and negative regions create protein’s net charge
Net charge is used to determine best IEX method and can be influenced by pH
Life Science Group I Chromatography
Protein chromatography purification techniques
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Protein Properties |
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Separation Technique |
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• Charge and isoelectric point |
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Ion exchange chromatography (IEX) |
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• Cation exchange chromatography (CIEX) |
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• Anion exchange chromatography (AIEX) |
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• Size, molecular weight and |
• Size exclusion (SEC) - gel filtration (GF) |
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conformation |
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Specific ligand recognition |
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Affinity chromatography (AC) |
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• Immobilized metal ion affinity |
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chromatography (IMAC) |
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Hydrophobicity |
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Hydrophobic interaction chromatography (HIC) |
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Hydrophobicity |
• Reversed phase chromatography (RPC) |
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Isoelectric point |
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Chromatofocusing |
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Isoelectric point , charge and |
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Mixed mode |
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size |
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Life Science Group I Chromatography
Ion Exchange Chromatography
–Separation based on the charge of the protein
–Charged groups (anionic or cationic) are immobilized on to a matrix. Molecules of the opposite charge interacts with them.
–The adsorbed molecules are then eluted with gradient increasing ionic strength (salt) of eluent
–After IEX usually proteins are in solution of high salt concentration
–The most commonly used technique
Life Science Group I Chromatography
Ion Exchange Chromatography
Buffer pH/Protein pI
+
net
protein
charge
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H2N |
COOH |
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binds |
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denaturation |
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cation exchanger stability range |
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+H N |
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COOH |
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3 |
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2 |
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10 |
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pI |
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H N |
COO- |
pH |
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2 |
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stability range |
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binds |
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denaturation |
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anion exchanger |
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Life Science Group I Chromatography