Chromatography Flashcards
What is chromatography?
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· laboratory technique used to separate mixtures of substances into their components.
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What properties of proteins do chromatography utilise ?
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· charge distribution
· molecular size
· solubility
· binding properties
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What is salting in?
Salt molecules stabilize protein molecules by decreasing the electrostatic energy between the protein molecules which increase the solubility of proteins.
What is salting out?
When the ionic strength of a protein solution is increased by adding salt, the solubility decreases, and protein precipitates.The salt molecules compete with the protein molecules in binding with water
Why do proteins associate with each other?
It is more energitically favourable
What is ion exchange chromatography?(6)
“separates larger amounts of materials than gel filtration2. stationary phase is polymer- matrix/resin attached with charged groups
3. Acidic groups of resin interact with positively charged proteins and are called cation exchangers.4. Cation exchangers bind to proteins/enzymes with positive charges5. If groups are basic in nature, they interact with negatively charged molecules and are called anion exchangers. 6. These will interact with negatively charged molecule”
Describe cation exchanger(5)
- Polymer beads can be positively charges
- Proteins bind to an ion exchanger with different affinities.
- Column is washed with buffer, to elute proteins based on relatively low affinities for the ion exchange resin.
- Elution can be determined by salt concentration of buffer and/or pH (stepwise elution or gradient elution).
- Proteins with more negative net charge move faster and elude earlier
What is gel filtration chromatography?(5)
“1. Widely used to purify biological molecules in complex samples e.g. blood
2.Smaller molecules enter the pores of particles that form the immobile phase so longer transit time than larger molecules
3. Column consists of porous beads of dextran or agarose: Sephadex and Sepharose are commonly used commercial preps. Typically 100 micrometres in diameter4. Large molecules elute first, before smaller molecules
5. generally used near the end of the purification process,
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What is elution volume?
“(Ve) is the volume of a solvent required to elute a given solute from the column. Can be used for IEX”
What is affinity chromatography?(3)
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1.Molecules that bind non-covalently.
2. Carries out a separation of components based on interactions between target protein and specific ligands
3. molecular technique which isolates antibodies, antigens, hormones, or other proteins by taking advantage of their binding affinity for their respective ligands
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What are the requirements of affinity?(4)
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• beads matrix e.g. agarose in column
- solution containing substance to be isolated
- ligand, a molecule that specifically binds to the protein of interest (target protein).
• a wash to elute non-bound. First wash must be of sufficient salt concentration, pH and temperature but not too extreme to cause the isolate to dissociate from the ligand
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What are the substances of affinity chromatography?
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• final wash with elution buffer containing a competitive ligand to elute the target protein. 2nd wash must elude the isolate so must be extreme in pH and temp to remove protein of interest
• solution of compound with higher affinity than the ligand(antigen) that was bound to the inert support
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What is high performance liquid chromatography?(4)
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- Highly improved form column chromatography.
- Solvent is forced under pressure of 400 atm through column- stationary phase. 3. Allows the use of smaller particle size for the column material, thus increasing surface area. 4. This allows much better separation of sample based on hydrophobicity”
Describe the steps of high performance liquid chromatography(6)
“1. Injection of a small volume of liquid sample into a tube packed with porous particles- stationary phase
2.individual molecules are transported and moved by liquid by gravity. The pump is to force the mobile phase through the liquid chromatography under specific flow rate in mm/min
3. Injector introduces liquid sample into flow stream of the mobile phase. Typical volume injected is 5-20 microlites. 4. Reservoir holds the solvent and a waste to collect waste. 5. Detector detects eludes. Most of the eludes do not have colours but are shown as colours on a computer software
6.Each compound eludes at a specific location
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What are the advantages and disadvantages of high performance liquid chromatograhy?
“Advantages
• Speed- fastest
- High resolution
- Sensitivity
- Reproducibility
- Accuracy
• Automation
Disadvantages
• Cost
• Complexity
• Coelution- sometimes components that have affinity for the stationary phase and elute at the same time so retention times are the same.
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