Protein Purification Flashcards
What are the surface properties of proteins
Why purify proteins
What is salting out
How does salting out work *
Proteins in solution are surrounded by water molecules that form a hydration shell, stabilizing them and keeping them dissolved. This hydration shell is essential for maintaining protein solubility because the water molecules interact with the charged and polar groups on the protein surface
When a salt (like ammonium sulfate or sodium chloride) is added to the solution, its ions compete with the protein molecules for water molecules. As the salt concentration increases, more water molecules surround the salt ions, reducing the availability of water to interact with the protein
This causes the proteins to aggregate, because their hydrophobic regions are exposed and tend to stick together.
With the reduction of the hydration shell and increased aggregation, the proteins become insoluble and precipitate out of the solution
What are the two types of protein chromatography
What is the general set up for protein chromatography
What are the characteristics of adsorption/desorption chromatography
What are the characteristics of adsorption/desorption chromatography
What is the languir isotherm + assumptions *
Mathematical model used to describe the adsorption of molecules onto a solid surface
Monolayer Adsorption: Adsorption occurs only at specific sites, forming a single layer of adsorbate (no multilayer adsorption).
Finite Number of Adsorption Sites: There is a limited number of identical adsorption sites on the surface.
No Interaction Between Adsorbed Molecules: The adsorbed molecules do not interact with each other, so the adsorption of one molecule does not affect the adsorption of another.
Equilibrium: The rate of adsorption is equal to the rate of desorption at equilibrium.
What is the original Langmuir isotherm
What is the derived Langmuir isotherm equation
What does alpha represent in the Langmuir isotherm
What affects alpha in the Langmuir isotherm*
In real systems, the adsorption surface is often not homogeneous. Some adsorption sites may have higher or lower affinity for the adsorbate than others If α<1, it implies that the adsorbent surface has heterogeneous adsorption sites with varying affinities, leading to non-ideal adsorption behavior
it is assumed that adsorbed molecules do not interact with each other. However, in many real systems, interactions between adsorbed molecules can occur
Multilayer of adsorbed molecules can occur
the pH and ionic strength of the solution, can affect the charge distribution on both the adsorbent surface and the adsorbate, influencing adsorption behavior.
At different temperatures, the affinity of the adsorbate for the adsorption sites might change, causing
α
α to shift
Why is the derived Langmuir isotherm not always accurate
What are the dynamic effects of adsorption/desorption
What is dispersion in adsorption/desorption chromatography*
dispersion refers to the broadening of a solute band as it moves through the chromatographic column. This broadening can result from various factors that cause the solute molecules to spread out over time, leading to a decrease in separation efficiency and resolution
In a packed column, solute molecules can travel through different paths because of the varying sizes and arrangements of the stationary phase particles. Some paths are shorter or more direct, while others are longer
Longitudinal diffusion- occurs along the length of the column as solute molecules diffuse from regions of higher concentration (the center of the band) to regions of lower concentration (the edges).
As solutes adsorb onto and desorb from the stationary phase, there may be a delay or lag due to the time it takes for the solute to transfer between phases
The speed at which the mobile phase flows through the column affects dispersion. At higher velocities, solute molecules have less time to diffuse longitudinally
What are the 2 main contributing factors to dispersion
What is turbulent flow is ad/desorption chromatography*
turbulent flow refers to a flow regime within the chromatographic column where the movement of the mobile phase (liquid or gas) is chaotic and irregular
How does diffusion contribute to dispersion in ad/desorption chromatography
How does turbulent flow contribute to dispersion in ad/desorption chromatography
What equation can be used to describe dispersion vs optimal flow rate
What are the characteristics of the equation for dispersion vs optimal flow
What is self sharpening in ad/desprption chromatography *
chromatographic peaks become narrower as they move through the column, rather than broadening due to dispersion
When the solute concentration in the leading edge of the peak is higher, molecules at the front are retained longer, causing them to slow down.
Conversely, the trailing edge of the peak, where the solute concentration is lower, experiences faster movement because fewer molecules are being adsorbed.
This differential retention causes the peak to sharpen naturally as it travels through the column, with the front being retarded and the tail catching up
In some cases, the diffusion of solute molecules between the mobile and stationary phases is slow enough that solute molecules at the front of the peak are held back while those at the rear catch up, leading to peak sharpening
What are the different types of ad/desorption chromatography
What is the characteristics of ion exchange chromatography*
Ion exchange chromatography (IEC) is a type of chromatography used to separate and purify ions and polar molecules based on their charge properties
The stationary phase in ion exchange chromatography consists of an insoluble matrix (resin) that carries charged functional groups
electrostatic interactions between the charged solutes and the oppositely charged groups on the resin. Molecules with a higher net charge (of opposite polarity to the resin) will bind more strongly to the stationary phase, while those with a lower charge will elute more quickly.
The strength of the interaction depends on:
The charge density of the molecule.
The pH of the mobile phase, which can influence the ionization of both the solute and the stationary phase.
The ionic strength of the mobile phase
The mobile phase in IEC is typically an aqueous buffer that maintains a specific pH and ionic strength.
pH Control: The pH of the mobile phase is crucial because it affects the charge of the solutes and the ion-exchange sites. By adjusting the pH, you can alter the ionization of the solutes, thus affecting their binding and elution.
Gradually increasing the salt concentration (ionic strength) in the mobile phase during elution helps to displace bound ions from the stationary phase. This process, known as salt gradient elution
What are the typical substituents used in ion exchange chromatography* what is their role
The primary role of the substituents is to provide sites for electrostatic interactions with the ions in the mobile phase. In cation exchange chromatography, negatively charged substituents attract and bind positively charged ions (cations), while in anion exchange chromatography, positively charged substituents attract and bind negatively charged ions (anions).
How does ion exchange chromatography work*
The sample containing a mixture of ions or charged molecules is introduced into the chromatography column. As the mobile phase flows through the column, the solutes begin to interact with the stationary phase
As the sample passes through the column, cations in the sample are attracted to the negatively charged sites of cation exchange resin, or anions are attracted to the positively charged sites of anion exchange resin.
The binding occurs through electrostatic interactions, where the charge of the solute ions attracts them to the oppositely charged groups on the resin.
Different ions have different affinities for the stationary phase, leading to varying retention times.
Stronger Interactions: Ions that bind more strongly to the resin will have longer retention times and will elute later.
Weaker Interactions: Ions with weaker interactions will elute first, as they are released from the resin more quickly
To separate bound ions effectively, the mobile phase’s ionic strength can be increased gradually (gradient elution) by adding more salt to the buffer. This competition for binding sites causes previously bound ions to be displaced and eluted from the column.
What happens to alpha in ion exchange chromatography *
What other factors should be taken into account from ion exchange chromatography
What is band sharpening*
Band sharpening occurs as a result of reduced diffusion and dispersion effects. Efficient separation and faster diffusion of molecules in a packed column help to produce narrower bands.
Well-packed columns with uniform particle size and distribution contribute to better band sharpening by minimizing voids and channeling.
Flow Rate: An optimal flow rate can promote band sharpening. Very high or very low flow rates can lead to band broadening instead.
Column Length: Shorter columns can enhance band sharpening as there is less opportunity for dispersion
What is chromatofocussing*
A type of ion exchange chromatography
used for the separation and purification of proteins, peptides, and other charged biomolecules based on their isoelectric points (pI)
The isoelectric point is the pH at which a molecule carries no net charge. At pH values below its pI, a protein is positively charged; above its pI, it becomes negatively charged
Anion exchange resin (+ve) with protein pI = 7 start at pH 9
• Start above the pI so we get binding
How do you create a pH gradient in chromatofocussing
What is HIC chromatography*
In HIC, the stationary phase (the resin) contains hydrophobic groups, such as phenyl, octyl, or butyl chains, that interact with the hydrophobic regions of proteins.
Proteins generally have both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions. Under specific conditions, hydrophobic interactions can dominate, causing proteins to bind to the stationary phase
HIC typically operates under high-salt conditions. The presence of salt in the mobile phase reduces the solubility of hydrophobic regions of proteins in water, promoting their interaction with the hydrophobic stationary phase
To elute bound proteins, the salt concentration is gradually decreased, or a buffer with lower ionic strength is introduced. This reduces the hydrophobic interactions, allowing the proteins to be released from the stationary phase
What are the characteristics of HIC chromatography
What is reverse phase chromatography*
Used particularly in the analysis of small molecules, peptides, and proteins. The method relies on the differences in hydrophobicity among the analytes to achieve separation
stationary phase is hydrophobic
hydrophobic surface of the stationary phase interacts with the hydrophobic regions of the analytes
mobile phase is usually a polar solvent, often water mixed with organic solvents
The polar mobile phase allows the hydrophobic analytes to be retained on the hydrophobic stationary phase while facilitating their separation.
more hydrophobic compounds tend to interact more strongly with the stationary phase and are retained longer in the column. Conversely, more polar compounds interact less with the stationary phase and elute faster.
As a result, the order of elution is typically based on the hydrophobicity of the analytes: less hydrophobic analytes elute first, followed by more hydrophobic ones
What ligands can be used in affinity chromatography
How does affinity chromatography using CNBr work
How does affinity chromatography using a thiol based system work*
Affinity chromatography using a thiol-based system is a specialized approach that takes advantage of the strong and specific interactions between thiol groups and certain ligands, such as maleimides or iodoacetates. This method is particularly useful for the purification of proteins and other biomolecules that contain reactive cysteine residues.
When a protein sample is applied to the column, any proteins containing exposed thiol groups can covalently bind to the ligands on the stationary phase. Non-target proteins, which do not have reactive thiols, will be washed away
To elute the bound protein, a reducing agent can be introduced to cleave the covalent bond between the thiol group and the ligand, or competitive elution can be performed by introducing a thiol-containing compound that competes with the bound protein for the ligand
How does elution work for affinity chromatography*
The column is washed with a binding buffer to remove unbound or weakly bound impurities.
The bound target analyte is eluted using a suitable elution buffer that disrupts the ligand-analyte interaction
What is gel filtration and how does it work *
used to separate molecules based on their size as they pass through a porous gel matrix
The stationary phase consists of gel beads with specific pore sizes, allowing smaller molecules to enter the pores while larger molecules cannot
The size of the pores in the gel beads determines the molecular weight range that can be effectively separated. The beads are typically made from materials like dextran, agarose, or polyacrylamide.
Molecules larger than the pore size are excluded from entering the beads and pass through the column more quickly, while smaller molecules can enter the pores and take longer to elute
The elution of the separated molecules is typically monitored using a detector that measures the absorbance at a specific wavelength (e.g., 280 nm for proteins).
The elution profile produces a series of peaks corresponding to different molecules, which can be collected as fractions
How do proteins flow through gel filtration columns
What is Kav
What is a purification table
What is the nature of the stationary phase in ion exchange chromatography
What are the limitations of affinity chromatography
