Electrophoresis

Question 1

2 types of electrical methods of analysis

Answer 1

electrophoresis and electrochemistry

Question 2

process of electrophoresis

Answer 2

the separation of analytes using an electric field

Question 3

individual components in electrophoresis can be…

Answer 3

characterised, quantified and isolated

Question 4

why is electrophoresis different to chromatography?

Answer 4

because there’s no mobile phase. only the charged species in the solutions move

Question 5

if they are free to move, applying a voltage by a pair of electrodes produces…

Answer 5

movement of the charged molecules

Question 6

what are they free to move in?

Answer 6

in solution or porous gel

Question 7

different molecules move at…

Answer 7

different rates

Question 8

why is it a powerful technique?

Answer 8

because we can adjust the charge of many biological molecules by adjusting the pH

Question 9

Acidic biological compounds properties

Answer 9

low pH. examples: -COOH, -NH3+. Protonated. Positive isoelectric point.

Question 10

Basic biological compounds properties

Answer 10

high pH. Examples: -COO^- and -NH2. Deprotonated. Negative isoelectric point.

Question 11

Factors that affect rate of movement (relating to size)

Answer 11

smaller molecules mover faster than larger ones

Question 12

Factors affecting rate of movement (relating to shape)

Answer 12

shapes of molecules are important

Question 13

factors affecting rate of movement (relating to charge)

Answer 13

the more highly charged molecules move faster than less charged ones

Question 14

factors affecting the rate of movement (relating to voltage)

Answer 14

the greater the voltage the greater the movement

Question 15

acids

Answer 15

molecules that can donate a hydrogen ion

Question 16

bases

Answer 16

molecules that can accept a hydrogen ion

Question 17

how can you control the pH of molecules so they can move in electrophoresis?

Answer 17

protonate them to become an acid or deprotonate them to become a base

Question 18

what’s the isoelectric point?

Answer 18

pH where charge is zero.

Question 19

supporting mediums

Answer 19

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.

Question 20

Different types of gels used as supporting mediums

Answer 20

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.

Question 21

Applications of electrophoresis are..

Answer 21

limited to charge molecules so we can control acids and bases using appropriate buffers.

Question 22

what molecules can we use electrophoresis for?

Answer 22

nucleic acids - can deprotonate them
proteins - can protonate and deprotonate to become acids and bases because of their side chains

Question 23

what can we learn from separating molecules?

Answer 23

to work out what they are and what they’re doing.

Question 24

why are nucleic acids easier to deal with in electrophoresis?

Answer 24

because they have a more predictable structure inc a negative phosphate group. charge per unit length is always the same.

Question 25

nucleic acids - the repeating negative charges repel so…

Answer 25

the molecules adopt a long thin shape

Question 26

what do all nucleic acid fragments have in common?

Answer 26

they have the same charge and shape

Question 27

variable to test nucleic acids in electrophoresis?

Answer 27

how long/size the chain is.

Question 28

small sized nucleic acid fragments move..

Answer 28

faster than longer sized fragments

Question 29

what applications can electrophoresis sort DNA fragments out?

Answer 29

DNA sequencing - can tell you what the fragments do, DNA profiling - compare and recognise.

Question 30

proteins have different…

Answer 30

sizes, shapes and charges

Question 31

how we deal with proteins in electrophoresis?

Answer 31

measure the molecular masses of proteins by SDS PAGE (sodium dodecyl sulfate polyacrylamide gel)

Question 32

What is sodium dodecyl sulfate?

Answer 32

a detergent. C12 H25 SO3.

Question 33

what happens if we introduce lots of sodium dodecyl sulfate with poteins?

Answer 33

SDS will stick to protein molecule - so protein is swamped with negative charge. so proteins will all have negative charge. which denatures protein. and the negative charge will repel each other and make the shape long and thing. makes them the same charge and shape.

Question 34

how long the chain is in other words…

Answer 34

the molecular mass of the protein

Question 35

hwo to work out molecular mass of protein?

Answer 35

by plotting a calibration graph. natural log (ln) of molar mass (on y-axis) against distance travelled in electrophoresis gel.

Question 36

calibration graph creates

Answer 36

straight line going downwards

Question 37

how can we use the calibration graph of known masses of proteins?

Answer 37

can work out molecular mass of unknown masses by comparing to graph of known molecular mass

Question 38

how can you sue isoelectric focussing to separate protein?

Answer 38

molecules will stop when their charge is zero (neutral) and therefore rate of movement is irrelevent.

Question 39

across the gel there’s a difference in…

Answer 39

electrical field gradient - negative to positive
pH - high to low (basic to acidic)

Question 40

negative molecules will move to…

Answer 40

positive side of gel & vice versa (and becomes protonated)

Question 41

when does molecule stop in gel

Answer 41

when the molecule becomes neutral and therefore not affected by the electric field