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 considerations are made 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 is tailing in ad/desorption chromatography and how can it occur
Tailing in adsorption/desorption chromatography refers to the distortion of a chromatographic peak, where the peak has a long, drawn-out tail on the trailing side
Some solute molecules may interact too strongly with the stationary phase, leading to prolonged retention and slow desorption. This causes the trailing part of the peak to stretch out.
If the stationary phase is overloaded with solute molecules, the high concentration can saturate the adsorption sites. As a result, some solutes are forced to bind to less accessible or lower-affinity sites, leading to slower desorption and tailing of the peak.
Mass transfer refers to the movement of solute molecules between the mobile phase and stationary phase. If the exchange is slow, solutes may lag behind the bulk flow of the mobile phase, contributing to tailing. This can happen when the stationary phase has a porous structure or if the solute diffuses slowly into and out of the pores
In liquid chromatography, if the mobile phase pH is not properly controlled, solutes may become partially ionized, causing variable retention