Seperation Method Lectures Flashcards
What are the 4 things to consider when designing a purification protocol?
(1) Expression level in starting material - the higher the level, the less material required and purer the final product.
(2) Minimize the number of steps and sample handling (buffer exchange, concentration) - speed and temperature are key to clean and active products.
(3) Start with coarse steps, end with polishing - every technique offers a balance between resolution, capacity, speed, recovery.
(4) Know your protein’s preferences - pH, ionic strength, temperature sensitivity, propensity to aggregate/denature, presence of metals or co-factors.
What are the 4 main steps for protein production and purification?
(1) Choice of expression system, affinity tag, sequence/cloine screening.
a) Bacteria – inexpensive, easy genetics, high levels of expression
b) Yeast, insect cells – eukaryotic systems may necessary for protein folding
c) Mammalian cell culture – expensive, low expression, most physiological
(2) Cell lysis / fractionation
a) Total cell lysates – easiest, highest yield
b) Membranes / organelles – enriched starting materials, lower yield
c) Choice of detergents for membrane proteins
(3) Coarse purification
a) Precipitations (salting in or out, organic solvents)
b) Heat shock
c) Centrifugation
(4) Chromatography
a) Affinity purification
b) Ion exchange / other resins
c) Size exclusion chromatography
How is differential solubility used to purify proteins, and what role do salts or organic solvents play in heat shock purification?
- Differential solubility uses salts or organic solvents to precipitate the target protein (solidify) or remove contaminants without denaturing them.
- Proteins are separated by gradually increasing the precipitant concentration. (Example: Red protein is removed by centrifugation at a low salt concentration, and green protein is collected at a higher concentration.)
- Protein solubility follows a bell-shaped curve, with maximum solubility at intermediate salt concentrations.
What is centrifugation used for in protein purification?
Centrifugation is a key method for separating biomolecules based on their size, shape, and density by spinning them at high speeds, causing heavier components to pellet at the bottom while lighter components remain in the supernatant.
What is the basic principle of chromatography?
Chromatography separates molecules in a mixture by distributing them between a stationary phase (where molecules are retained) and a mobile phase (which carries molecules along). Molecules that spend more time in the mobile phase move faster.
What are the two main theories of chromatography, and how do they differ?
(1) Plate Theory.
- Describes chromatography as a series of equilibrium partitions between the stationary and mobile phases.
- Partition coefficient (Kd) = [concentration in stationary phase]/[concentration in mobile phase]
- originally developed from studies on distillation.
- view column as divided into a number (N) of adjacent imaginary segments called theoretical plates.
- within each theoretical plate analytes (s) completely equilibrate between stationary phase and mobile phase.
- significant: greater separation occurs with greater number of theoretical plates (N).
- Resolution is proportional to the square root of the number of theoretical plates
in equation so increasing the number of theoretical plates four times increases
the resolution by a factor of 2.
(2) Rate Theory.
- Explains the dynamics of molecular binding and movement during seperation.
- Focuses on factors affecting separation efficiency, such as diffusion, flow rate, and resistance to mass transfers.
Key difference: Plate theory simplifies separation as static equilibrium steps, while rate theory incorporates the kinetics and dynamics of molecule movement.
What is the partition coefficient (Kd) in chromatography, and what does it represent?
- determines how quickly a compound moves through a column.
- high Kd: molecule spends more time in stationary phase -> moves slower.
- low Kd: molecule spends more time in mobile phase -> moves faster.
- Differences in Kd allow separation of molecules in a mixture.
- Kd = [Ss]/[Sm]
How does the solute band behave in an ideal column versus a real column in chromatography?
- Ideal column: The solute moves without changing shape, staying as a narrow band.
- Real column: The solute band widens due to spreading (dispersion) as it moves.
- The band width increases in proportion to the square root of the distance traveled along the column.
How is resolution defined in chromatography, and what are the two different methods used?
- Resolution in chromatography refers to the ability to distinguish between two peaks.
1) Peak width at half-height.
2) Width based on the tangent line at half-height.
Both methods are used to measure how clearly the peaks are separated, but they define resolution in slightly different ways.
How can the number of theoretical plates (N) in a column be estimated?
The number of theoretical plates (N) can be estimated experimentally from a chromatogram, using methods like the half-height method to analyze the chromatographic peak after elution.
How do column dimensions affect chromatography efficiency?
(1) Shorter columns (50 mm):
Faster analysis.
Ideal for quick screening or gradient analysis.
(2) Longer columns (150-250 mm):
Better resolution for complex mixtures.
Requires longer analysis time.
How does the particle size affect column performance in chromatography?
(1) Smaller particles (<5 µm):
More theoretical plates.
Better resolution.
(2) Larger particles (>50 µm):
Less back pressure.
Higher flow rates.
Easier to use, but lower resolution.
How do bead size and flow rate affect chromatography performance?
(1) Smaller beads (faster flow rates):
- Faster analysis, but lower resolution and higher pressure.
(2) Larger beads (slower flow rates):
- Lower pressure and better resolution but slower analysis.
What is the first important prediction of Plate Theory?
Band spreading - the width of bands increases as their retention time (tr) or retention volume increases (Vr).
What practical considerations affect peak symmetry in chromatography? (Plate Theory)
- Peak Tailing: Occurs when some sites on the stationary phase retain the solute more strongly, causing the peak to have a longer tail.
- Factors affecting peak shape and retention time: sample size, volume loaded, flow rate, solvent gradients.
What is ‘peak fronting’?
Peak fronting is the opposite of peak tailing, where the solute interacts more strongly with the stationary phase at the front of the peak, causing the peak to be skewed forward.
What is the difference between isocratic and gradient solvent elution in chromatography?
1) Isocratic elution: Uses a single solvent composition throughout the run.
2) Gradient elution: The solvent composition changes over time, usually involving a mix of two components.
What are the advantages of using gradient solvent elution?
1) Sharper peaks.
2) Improved resolution.
3) Faster run times.
What is an example of chromatography that uses a single solvent?
Size-exclusion chromatography uses a single solvent throughout the process.
What are the main components of a chromatography system, and what does each do in practice?
(1) Mobile Phase(s):
- The solvent or mixture of solvents that moves through the column, carrying the sample with it.
- Can be a single solvent (isocratic) or a mixture of solvents (gradient elution).
(2) Pump:
- Provides the necessary pressure to push the mobile phase through the column.
- It maintains a constant flow rate, which is essential for reproducible results.
(3) Injector:
- The component that introduces the sample into the chromatographic system.
- It needs to deliver the sample consistently and in a controlled manner.
(4) Column:
- The part of the system where separation occurs.
- It contains the stationary phase, which interacts with the sample and causes separation based on the different affinities of the components for the stationary and mobile phases.
(5) Detector:
- Detects the separated components as they elute from the column.
- Common types of detectors include UV absorbance, fluorescence, or mass spectrometry.
(6) Fraction Collector:
- Collects the separated components in individual containers as they exit the detector.
- This allows for further analysis or purification of the separated substances.
What is Gel Filtration (Size Exclusion) Chromatography?
- Low resolution chromatography technique.
- Used often as the last step in purification.
- Isocratic elution (constant mobile phase composition).
- Separates molecules based on size, with larger molecules eluting first.
- Resin has holes that allow small proteins to enter. Large proteins are excluded and move through column faster.
- property: protein size and shape.
- binding and elution: any buffer.
- proteins do not interact with resin.
What is Ion Exchange Chromatography?
- A high-resolution technique that separates proteins based on their charge.
- Anion exchange: Uses positively charged resin to attract and separate anions (negative proteins).
- Cation exchange: Uses negatively charged resin to separate cations (positive proteins).
- Second most common chromatography technique for proteins.
- property: protein charge
- binding: low ionic strength
- elution: high ionic strength (or pH change*)
- proteins interact with resin: sample volume not limiting concentrates sample.
What are Other Partition Chromatographies used in protein purification?
(1) Hydrophobic Interaction Chromatography:
Separates proteins based on their hydrophobicity.
Less common but useful in certain contexts.
Property: hydrophobicity
Binding: high ionic strength
Elution: low ionic strength
Proteins interact with resin: sample volume not limiting concentrates sample.
(2) Hydroxyapatite Chromatography:
Uses calcium-phosphate as stationary phase.
Not stable at acidic pH.
(3) Immobilized Metal Ion Chromatography:
Uses metal ions like Zn²⁺, Mn²⁺, or Ni²⁺ to bind proteins with histidine tags.
What is Affinity Chromatography, and when is it used?
- Most common and useful chromatography technique.
- Highly selective, used to purify recombinant proteins.
- Works by using a stationary phase that specifically binds the target protein based on its affinity for a ligand.
- property: unique to the protein of interest
- binding: affinity tag (GST, MBP, 6xHis), specific conditions
- elution: specific conditions (by adding free ligand/by competition)
- proteins bind to resin: sample volume not limiting concentrates sample
What are the advantages & limitations of Gel Filtration Purification Technique?
(1) Advantages:
- can be used to change buffers, good last step in protocols.
(2) Limitations:
- slow, poor resolution, small sample volume, dilutes sample.
What are other applications of Size Exclusion Chromatography?
(1) Protein Interactions (study how proteins interact with each other or other molecules, such as DNA.)
(2) Oligomeric States. Useful for determining oligomeric state (whether proteins are monomers, dimers, or larger complexes) based on their size during elution.
What are key points about Gel Filtration (Size Exclusion) Chromatography for purification? (Superdex 75 (smaller proteins) vs Superose 12 HR 10/30 (large biomolecules)
(1) Superdex 75
- Larger proteins elute first because they are excluded from entering the smaller pores in the resin.
- Doubling protein size causes about a 15% change in elution time.
- Best suited for small proteins.
(2) Superrose 12 HR 10/30
- Designed for large biomolecules like IL-2.
- The large holes in the resin make it fragile and susceptible to damage from high pressures.
- Useful for removing aggregates from proteins like IL-2.
What are key considerations when using Gel Filtration with high-pressure pumps?
(1) Pressure Considerations.
- Pay attention to both static pressure and hydrodynamic pressure drop (Dp).
- Higher flow rates lead to higher pressures on the resin and column.
(2) Pressure Limits.
- The column casing determines the maximum allowed static pressure.
- The resin determines the maximum pressure drop (Dp).
What are advantages and disadvantages of Ion Exchange Purification Technique?
(1) Advantage: High resolution, speed, capacity
(2) Limitations: choice of buffers, starting conditions
What are advantages of Hydrophobic Interactions - Purification Technique?
(1) Advantages:
- Good resolution, speed, capacity, lowers ionic strength
What are advantages and disadvantages of affinity chromatography - purification technique.
(1) Advantages: high resolution, speed, capacity
(2) Limitations: specific conditions
What are the most common affinity chromatography tags and their advantages/disadvantages?
(1) GST
- Resin: immobilized glutathione
- Large tag, good affinity, chaperone effect
- Large size can affect protein function.
(2) Oligo-histidine tag
- Resin: Immobilized metal (Ni²⁺ or Co²⁺)
- Small tag, moderate affinity, good for purification
- Some contaminants may remain.
(3) Sumo-his-tag
- Resin: Immobilized metal (Ni²⁺ or Co²⁺)
- Cleavage without leaving residues, chaperone effect
- Larger tag.
(4) Streptavidin
- Resin: Biotin resin
- Very high affinity & purity
- Difficult to unbind, tag and resin can be switched.
(5) GFP
- Resin: Anti-GFP antibody resin
- Easy visualization, chaperone effect
- Expensive resin.
(6) Antibody epitopes
- Resin: Antibodies coupled to resin
- Versatile
- Expensive resin (protein A and G used for IgG purification).
