Chromatography Techniques Flashcards

Which chromatography technique is most commonly used to purify His-tagged recombinant proteins?

Answer: Immobilized metal affinity chromatography (IMAC)

IMAC uses a metal ion (e.g., Ni²⁺) chelated to the resin that binds specifically to the polyhistidine (His) tag on recombinant proteins.

In reverse-phase HPLC, the stationary phase is:

Answer: Nonpolar and hydrophobic

Reverse-phase HPLC uses a nonpolar, hydrophobic stationary phase (e.g., C18-bonded silica) that retains hydrophobic analytes.

What reagent is typically used to elute a protein bound to a Ni-NTA IMAC column?

Answer: Imidazole at high concentration

Imidazole competes with the histidine tag for binding to the Ni²⁺ ion, displacing the His-tagged protein and causing it to elute.

What does 'void volume' mean in the context of size exclusion chromatography?

Answer: The volume at which the largest excluded molecules elute

The void volume (V₀) is the volume of mobile phase outside the pores; molecules too large to enter the pores elute at this volume.

Hydrophobic interaction chromatography (HIC) typically uses which condition to bind proteins to the resin?

Answer: High salt concentration

HIC binding is promoted by high salt (kosmotropic salts like ammonium sulfate) which enhances hydrophobic interactions between the protein and the resin.

Which instrument component in an HPLC system is responsible for detecting separated analytes as they exit the column?

Answer: Detector

The detector (e.g., UV-Vis, fluorescence, or refractive index) monitors the column effluent and generates a signal proportional to analyte concentration.

In ion exchange chromatography, increasing the salt concentration in the elution buffer causes bound proteins to:

Answer: Elute from the resin

Increasing ionic strength introduces competing ions that displace the bound protein from the charged resin, causing it to elute.