Protein Purification Flashcards

Question

What would the elution profile for self sharpening in ad/desorption chromatography look like

Answer 1

At lower [total protein], protein is statistically more likely to bind so migration down the column slows relative to higher PT, thus we get a sharpening of the leading edge and an extended elution profile for the trailing edge.

Answer 2

Elution is the process of removing or washing out adsorbed solutes from the stationary phase in chromatography by passing a solvent or mobile phase through the column. As the mobile phase flows through the column, it carries the solutes with it, and different solutes are eluted (or "washed out") at different times based on their interactions with the stationary phase. In essence, elution is the phase of the chromatographic separation where the solutes are "freed" from the stationary phase and move out of the column, ultimately being detected as they emerge. Elution order refers to the sequence in which solutes exit the chromatographic column and are detected

Answer 3

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

Answer 4

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).

Answer 5

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.

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

In affinity chromatography, a specific ligand is covalently attached to the stationary phase (the resin or column material). This ligand has a strong affinity for the target biomolecule (the analyte) that you wish to purify. Once the target analyte is bound to the stationary phase, it can be eluted by introducing a buffer that disrupts the interaction between the ligand and the analyte. This can be achieved through: pH Change: Altering the pH can disrupt electrostatic interactions. Salt Concentration Change: Increasing the ionic strength can lead to the displacement of the analyte. Competitive Elution: Adding a free ligand that competes with the bound analyte can facilitate elution

Answer 11

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

Answer 12

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

Answer 13

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