Capillary Electrophoresis

Question 1

what is electrophoresis?

Answer 1

separation technique based on different rates of migration of charged species in E field

cations move toward cathode, anions move towards anode

Question 2

In what direction do cations and anions move in electrophoresis?

Answer 2

cations move toward cathode, anions move towards anode

Question 3

The cathode has a ____ charge. The anode has a ____ charge.

Answer 3

cathode = negative
anode = positive

Question 4

Migration of molecules in the E field will depend upon factors relating to (2):

Answer 4

physical characteristics of electrophoresis system

solute molecule characteristics (size, charge)

Question 5

migration describes ____ and ____ of the molecule’s movement

Answer 5

direction

rate

Question 6

describe the general design/features of a capillary electrophoresis system

Answer 6

2 separate solvent reservoirs, connected to high-voltage power supply (1 is anode, 1 is cathode)

capillary runs between them, with detector in middle (collect data on molecule movement between reservoirs)

Question 7

What type of capillary is used for CE? What other detectors can this be used with?

Answer 7

narrow bore capillaries
made of fused silica (or modified silica)

absorbance, fluorescence, MS, conductivity…

Question 8

When a particle is placed in an E field, what happens?

Answer 8

accelerates towards electrode with opposite charge

Question 9

What is ‘Vep?’

Answer 9

electrophoretic velocity (cm/s)
(rate of migration of particle in E field)

Question 10

What does ‘Uep’ stand for

Answer 10

electrophoretic mobility (cm/sV)

Question 11

The electrophoretic mobility is proportional to:

Answer 11

the charge density (q/rs)

Question 12

What factors affect mobility of a charged analyte in CE? (6)

Answer 12

strength of E field
temperature
pH of separation buffer
buffer ion type/ionic strength
size/shape of analyte molecules
charge of analyte molecules

Question 13

What happens to the buffer liquid in CE system when a high voltage is applied across the fused silica capillary? What is this called?

Answer 13

buffer liquid starts to migrate towards cathode

Veo; electroosmotic flow velocity

Question 14

T/F: electroosmotic flow velocity and electrophoretic velocity have the same unit

Answer 14

true

Question 15

What causes the electroosmotic flow velocity?

Answer 15

charge distribution at silica/buffer interface + creation of electric ‘double layer’ (fixed layer + mobility/diffuse layer)

apply voltage -> cations in mobility layer migrate to cathode, carries water with them

Question 16

The electroosmotic flow velocity effect is more pronounced at _____. Why?

Answer 16

high pH

many O- charge at surface

Question 17

What causes the formation of the electric double layer in the capillary?

Answer 17

negative charged capillary walls attract cations (+ charges) from the buffer

some bind tightly -> fixed layer (but doesn’t fully neutralize the neg. charge)

some cations are still attracted towards the walls -> diffuse layer

Question 18

What is the net charge within a CE capillary?

Answer 18

neutral (cancels out)

Question 19

What are some common buffers for CE and their pH range?

Answer 19

phosphate (1.14-3.14)
acetate (3.76-5.76)
phosphate (6.2-8.2)
borate (8.14-10.14)

Question 20

What CE buffer can be used at pH 9?

Answer 20

borate

pH range 8.14 - 10.14

Question 21

What CE buffer can be used at the lowest pH?

Answer 21

phosphate

pH 1.14-3.14

Question 22

What CE buffer is useful at a neutral pH?

Answer 22

phosphate

6.2 -8.2

Question 23

What are some zwitterionic buffers for CE?

Answer 23

MES (5.15-7.15)
PIPES (5.8-7.8)
HEPES (6.55-8.55)
tricine (7.15-9.15)
Tris (7.3-9.3)

Question 24

What is the ‘total velocity’ of the analyte in CE?

Answer 24

Vtot

sum of electrophoretic velocity (Vep) + electroosmotic velocity (Veo)

Question 25

Vtot can be expressed as: (3)

Answer 25

Vep + Veo (Uep + Ueo) x E L/tm (total migration time)

Question 26

how does electroosmotic flow affect separation time?

Answer 26

increasing Veo -> decrease separation time

Question 27

What do Utot and Ueo values in CE correspond to in chromatography?

Answer 27

``` Utot = Tr (total retention time) Ueo = Tm (dead time) ``` Utot-Ueo corresponds to adjusted retention time (Tr-Tm)

Question 28

In cations, Uep and Ueo flow in ___ directions, while in anions the flow in _____ directions

Answer 28

identical directions (toward cathode) opposite (Uep flows toward anode; negative flow)

Question 29

What has 'negative flow' in CE?

Answer 29

anions electrophoretic flow

Question 30

How can anions still be detected, if the electrophoretic flow direction is away from the detector?

Answer 30

at pH>3, Ueo (electroosmotic flow) is higher than Uep; so overall NET MIGRATION will be towards cathode *or; reverse polarity of instrument (detector at anode instead)

Question 31

What happens to anions if the pH is low (<3)?

Answer 31

Ueo is weaker than Uep; so anions will never reach detector (unless polarity is reversed)

Question 32

What food chemicals can be separated with CE?

Answer 32

``` small organic/inorganic ions small amino compounds (AA, biogenic amines, heterocyclic amines) peptides/proteins phenols/polyphenols/pigment carbs vitamins pesticides antibiotics ```

Question 33

What detector types are used with CE?

Answer 33

Spectrophotometry (absorption, fluorescence, thermal lens, raman, chemiluminescence) MS EC (potentiometry, amperometry)

Question 34

what allows for the separation of AA in CE?

Answer 34

charges on functional groups (positive, negative, etc)

Question 35

AA functional groups with neg charges: ______ AA functional groups with + charges: ______

Answer 35

neg: carboxylic func. groups (ex: glutamic acid) positive: amine func. groups (ex: arginine)

Question 36

As pH increases, what happens to the net charge on proteins?

Answer 36

decreases (change from + to negative)

Question 37

When the protein net charge is neutral, this is called the ______

Answer 37

isoelectric point (specific pH)