Column Chromatography Flashcards
(29 cards)
What is ion-exchange chromatography?
A type of adsorption chromatography used to separate molecules based on their charge.
What determines the charge of biological molecules such as proteins?
The composition of the molecule itself and the surrounding pH.
What are the two functional groups present in all amino acids?
- Amino group (NH2)
- Carboxyl group (COOH)
What happens to the amino group in an acidic environment?
It accepts a proton to form the cationic NH3+.
What happens to the carboxyl group in a basic environment?
It donates a proton to form the anionic COO-.
What part of a protein can become ionised?
Only the terminal amino and carboxyl groups.
What influences the overall net charge of a protein?
The number and type of ionisable amino acid side chain groups at a particular pH.
What is the isoelectric point (pI)?
The pH at which the overall net charge of the protein is zero.
What is an ion exchanger in ion-exchange chromatography?
The stationary phase that possesses either a permanent positive or negative charge.
What does an anion exchanger do?
Holds a positive charge and binds to negatively charged ions (anions).
What does a cation exchanger do?
Holds a negative charge and binds to positively charged ions (cations).
How do proteins bind to the ion exchanger?
Proteins of the opposite charge to the ion exchanger will bind, varying in strength based on their net charge.
What can be adjusted to interfere with the electrostatic interaction between the ion exchanger and proteins?
The salt gradient or pH of the mobile phase buffer.
What is the result of adjusting the salt gradient or pH in ion-exchange chromatography?
Desorption of the protein from the exchanger, permitting separation and elution.
What types of substances can ion-exchange chromatography be used to purify? .
Antibiotics
Vitamins
Hormones
Alkaloids
What is the defined pore size range of beads called?
Fractionation range
The fractionation range determines which molecules can access the pores.
What happens to molecules that exceed the exclusion limit of the beads?
They are excluded from the matrix
Larger molecules cannot fit into the bead pores.
In what order are proteins eluted from a size exclusion/gel filtration column?
Decreasing order of size
Larger proteins elute first, followed by smaller ones.
What are the materials called that are used as stationary phases to bind charged compounds?
Ion exchangers
Ion exchangers can be cationic or anionic.
What do cation exchangers bind?
Cations
Cation exchangers are negatively charged.
What do anion exchangers bind?
Anions
Anion exchangers are positively charged.
What competes with positively charged proteins for access to the ion exchanger?
Positively charged salt ions (Na+)
The buffer contains these ions.
