Unit 10

Question 1

describe electrophoresis

Answer 1

separation technique based on the differential rates of migration of charged species in an electric field

Question 2

what molecules moves towards the cathode?

Answer 2

pos charged molecules move towards the cathode (neg electrode)

Question 3

what molecules moves towards the anode?

Answer 3

neg charged molecules move towards the anode (pos electrode)

Question 4

what does the direction and rate of migration depend on?

Answer 4

physical characteristics of the electrophoresis system and solute molecules

Question 5

what are 2 most important aspects of the solute molecules electrophoresis?

Answer 5

their size and charge

eg. larger molecules don’t diffuse through as well

Question 6

electrophoresis can be carried out in what two modes?

Answer 6

1. free solution (eg. capillary zone electrophoresis: CZE)

2. with the aid of a stabilizing or supporting medium (eg. paper, gel, etc…)

Question 7

what is the role of solvent reservoirs?

Answer 7

buffers to control pH of the system

Question 8

what kind of capillaries are used for capillary electrophoresis?

describe the properties

Answer 8

narrow bore capillaries

• made of fused or modified silica
• diameter: 10-200 um
• range of potentials: 5-30kV (pos or neg)

Question 9

what detectors are used by capillary electrophoresis?

Answer 9

same as HPLC detectors

• absorbance
• fluoresence
• mass spectrometry
• conductivity

Question 10

what happens when a charged particle is placed in an electric field

Answer 10

it will be accelerated and start to move towards the electrode with an opposite charge

Question 11

what is the electrophoretic velocity?

Answer 11

the rate of migration of the charged particle through an electric field

Question 12

what is the unit for electrophoretic mobility?

Answer 12

cm^2/V*2

Question 13

what is the unit for the applied field?

Answer 13

V/cm

Question 14

what is Mep?

Answer 14

charge density

Question 15

what are factors affecting mobility of a charged analyte?

Answer 15

• strength of applied electric field
• temp of the separation system
• pH of the separation buffer
• type of buffer ions and ionic strength of the separation buffer
• size and shape of analyte molecules
• charge of the analyte molecules

Question 16

what happens when a high voltage is applied across the fused-silica capillary with a buffer solution?

what is this called?

Answer 16

the buffer liquid starts to migrate towards the cathode

the rate of this migration is the elctroosmotic flow velocity

Question 17

what is Veo?

Answer 17

electroosmotic flow velocity

Question 18

what is the unit of Veo?

Answer 18

unit * cm/s

Question 19

what causes the elctroosmotic flow velocity?

Answer 19

the charge distribution at the silica/buffer interface and the creation of an electrical double layer

when a voltage is applied, cations in the diffuse portion of the double layer migrate in the direction of the cathode, carrying water with them

Question 20

when is the elctroosmotic flow more pronounced? why?

Answer 20

at high pH

there are more O- at the surface

Question 21

what is the equation for electroosmotic flow velocity?

Answer 21

Veo = Meo x E

Question 22

what is the direction of voltage of electroosmotic flow?

Answer 22

anode to cathode

Question 23

what are common buffers for capillary electrophoresis?

when is each of them used?

Answer 23

phosphate
acetate
borate

they are all used for different pH ranges

Question 24

what is Vtot?

Answer 24

1. the sum of electrophoretic velocity (Vep) of an ion and the electroosmotic velocity (Veo)
   Vtot = Vep + Veo

OR
2. Vtot = L/Tm
L = length of column from inlet to detector
Tm = total migration time

Question 25

how does the increase of electroosmotic flow affect the separation time?

Answer 25

reduces separation time

Question 26

at moderate pH values (pH >3), which is higher? a) Meo b) Mep what is the result of this?

Answer 26

Meo > Mep causes anions to migrate towards the cathode where the detector is

Question 27

at low pH values, describe the net migration.

Answer 27

Meo is weak and anions may never reach the detector unless the polarity of the instrument is reversed in order to change the location of the detector from the cathode to anode end

Question 28

what is an example of a small organic and inorganic ion?

Answer 28

malic acid

Question 29

what is an example of a small amino compound?

Answer 29

putrescine

Question 30

what is an example of a phenol, polyphenol or pigment?

Answer 30

chlorogenic acid

Question 31

negative charges of amino acids come from what functional groups?

Answer 31

carboxylic groups

Question 32

positive charges of amino acids come from what functional groups?

Answer 32

amine

Question 33

what is IEP? describe it

Answer 33

isoelectric point the pH at which net charge is zero

Question 34

at high pH, the net charge on proteins is... a) more positive b) more negative

Answer 34

higher pH = more negative