Electrophoresis Flashcards
2 types of electrical methods of analysis
electrophoresis and electrochemistry
process of electrophoresis
the separation of analytes using an electric field
individual components in electrophoresis can be…
characterised, quantified and isolated
why is electrophoresis different to chromatography?
because there’s no mobile phase. only the charged species in the solutions move
if they are free to move, applying a voltage by a pair of electrodes produces…
movement of the charged molecules
what are they free to move in?
in solution or porous gel
different molecules move at…
different rates
why is it a powerful technique?
because we can adjust the charge of many biological molecules by adjusting the pH
Acidic biological compounds properties
low pH. examples: -COOH, -NH3+. Protonated. Positive isoelectric point.
Basic biological compounds properties
high pH. Examples: -COO^- and -NH2. Deprotonated. Negative isoelectric point.
Factors that affect rate of movement (relating to size)
smaller molecules mover faster than larger ones
Factors affecting rate of movement (relating to shape)
shapes of molecules are important
factors affecting rate of movement (relating to charge)
the more highly charged molecules move faster than less charged ones
factors affecting the rate of movement (relating to voltage)
the greater the voltage the greater the movement
acids
molecules that can donate a hydrogen ion
bases
molecules that can accept a hydrogen ion
how can you control the pH of molecules so they can move in electrophoresis?
protonate them to become an acid or deprotonate them to become a base
what’s the isoelectric point?
pH where charge is zero.
supporting mediums
paper (cellulose) - simple level
gels - 90% water but 3D framework of molecules acts as support. molecules can sit in compartments in gel and won’t move until we switch on power supply.
Different types of gels used as supporting mediums
agarose - for nucleic acids, which can grow stuff.
polyacrylamide - for protein electrophoresis. polymer that is made up of a carbon backbone, with alternate amide groups on one side.
Applications of electrophoresis are..
limited to charge molecules so we can control acids and bases using appropriate buffers.
what molecules can we use electrophoresis for?
nucleic acids - can deprotonate them
proteins - can protonate and deprotonate to become acids and bases because of their side chains
what can we learn from separating molecules?
to work out what they are and what they’re doing.
why are nucleic acids easier to deal with in electrophoresis?
because they have a more predictable structure inc a negative phosphate group. charge per unit length is always the same.
