Week 13 (Biochemical Methods III) Flashcards
How can we Separate molecules by charge or polarity?
- Ion exchange chromatography
- Isoelectric focussing
- Reversed-phase chromatography
- TLC
Chromatography theory
Molecule A has a high affinity for the stationary phase Molecule B has a high affinity for the mobile phase
This allows effective separation of the two molecules
Ion exchange matrix
Tiny little beads with charged groups attached charged groups attached:
- Carboxymethyl (CM) cellulose
- Diethylaminoethyl (DEAE) cellulose
- sulphonic acid cellulose
- trimethylaminoethyl cellulose
What is an anion?
negatively charged molecule
What is a cation?
positively charged molecule
What is an Anion exchange resin?
Has positively charged groups on the beads, so that it will bind anions
What is an cation exhchange resin?
Has negatively charged groups on the beads, so that it will bind cations
Strong exchangers:
Always ionised
Weak exchangers:
Only ionised over a narrow pH range
Cation exchange: stationary phase
Charged beads
Cation exchange: mobile phase
Liquid around the beads
Cation exchange: what will positively charged ions bind to?
-Positively charged molecules will bind to the negatively charged groups on the beads
—Move slowly down the column
Cation exchange: what will negitively charged ions bind to?
-Negatively charged (or neutral) molecules will not bind to the beads
—Move quickly down the column, with the flow of the water/buffer
Anion exchange: what will molecules with a negative charge bind to?
- Molecules with a negative charge will bind to the positively charged groups on the beads
- Move very slowly down the column
Anion exchange: what happens to positively charged or neutral molecules?
- Positively charged (or neutral) molecules will not bind to the beads
- Move quickly down the column with the flow of the water/buffer
What are some applications of ion exchange chromatography?
Proteins Peptides Small molecules (charged) DNA/RNA
What are some preparative and analytical applications of ion exchange chromatography?
Purification
How do you calculate the net charge of a molecule?
- A molecule may have more than one charged group
- The net charge is the overall charge on the molecule when you add all the individual charges together
- This will change at a different pH
- A molecule may have more than one charged group
Isoelectric pH
Isoelectric pH = pI = pH at which the net charge of a molecule is zero
- For a molecule to bind to an anion exchange resin (resin is positively charged) need the pH to be greater than the pI
- For a molecule to bind to a cation exchange resin (resin is negatively charged) need the pH to be lower than the pI
Net charge of a protein
- Proteins contain many charged amino acids on their surface
- Different proteins have a different combination of amino acids
Do different proteins have different pI’s?
Yes, even if they are very similar in size
What can be done to separate proteins?
Often several different proteins will bind to the resin use a gradient of pH or [NaCl] to elute them one at a time
What are the Advantages of ion exchange chromatography?
- Straightforward
- Good resolution
- Quick
Theory of electrophoresis
- A molecule with a net charge will move in an electric field
- The speed (velocity, v) at which the molecule moves depends upon; the strength of the electric field, E the net charge on the molecule, z the frictional coefficient, f (how much drag it experiences)
v = Ez/f
Isoelectric focussing
- A pH gradient is established in the gel by adding ampholytes
- Sample is loaded at one end, and an electric field reapplied
- Proteins migrate through the gel until they reach the pH that matches their pl By adding ampholytes & applying an electric field
2D gels
- Combine isoelectric focussing and SDS-PAGE This allows separation based on size and pI
- Electrophoresis in the first dimension, molecules are separated linearly according to their isoelectric point. In the second dimension, the molecules are then separated at 90 degrees from the first electropherogram according to molecular mass
- Separation of the proteins by isoelectric point is called isoelectric focusing (IEF). Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pH values other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel.
Polarity & hydrophobicity
Polar molecules contain electronegative atoms, hydrogen bonding groups or charged groups E.g. O-H and N-H
Reversed-phase chromatography: stationary phase
Hydrophobic beads
Reversed-phase chromatography: mobile phase
Polar liquid around the beads
Mobile phase: Polar solvents are often a mixture of:
water methanol acetonitrile
- Alter the ratio of these components in a gradient to elute molecules of differing polarity
- As you decrease the polarity of the mobile phase you elute more hydrophobic molecules
Reversed-phase HPLC: Applications
Very useful for separating drugs & metabolites
Most useful for small molecules
Chromatography, but not in a column: TLC
- Thin layer of silica gel on a glass plate
- Organic solvents Non-polar
eg. hexane:diethyl ether:acetic acid:ethanol
90 : 20 :2 : 3
Describe TLC: hydrophobic molecules
Hydrophobic molecules, move in the solvent (mobile phase)
Move a long distance up the plate
Describe TLC: hydrophilic molecules
Polar molecules stick to the silica plate (stationary phase) Move a short distance up the plate
Visualising TLC plates
- To visualise lipids on a TLC plate expose the plate to iodine fumes
- Iodine reacts with double bonds in fatty acids.
- Turn yellow/brown
Rf values (retention factor)
Rf value = A/B (Distance moved by sample from the line of addition to the top of the spot /Distance moved by solvent which is called the solvent front)
describe relative polarity
