Capillary Electrophoresis Flashcards

1
Q

what is electrophoresis?

A

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

cations move toward cathode, anions move towards anode

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2
Q

In what direction do cations and anions move in electrophoresis?

A

cations move toward cathode, anions move towards anode

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3
Q

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

A
cathode = negative
anode = positive
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4
Q

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

A

physical characteristics of electrophoresis system

solute molecule characteristics (size, charge)

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5
Q

migration describes ____ and ____ of the molecule’s movement

A

direction

rate

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6
Q

describe the general design/features of a capillary electrophoresis system

A

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)

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7
Q

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

A

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

absorbance, fluorescence, MS, conductivity…

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8
Q

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

A

accelerates towards electrode with opposite charge

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9
Q

What is ‘Vep?’

A
electrophoretic velocity (cm/s)
(rate of migration of particle in E field)
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10
Q

What does ‘Uep’ stand for

A

electrophoretic mobility (cm/sV)

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11
Q

The electrophoretic mobility is proportional to:

A

the charge density (q/rs)

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12
Q

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

A
strength of E field
temperature
pH of separation buffer
buffer ion type/ionic strength
size/shape of analyte molecules
charge of analyte molecules
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13
Q

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

A

buffer liquid starts to migrate towards cathode

Veo; electroosmotic flow velocity

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14
Q

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

A

true

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15
Q

What causes the electroosmotic flow velocity?

A

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

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16
Q

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

A

high pH

many O- charge at surface

17
Q

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

A

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

18
Q

What is the net charge within a CE capillary?

A

neutral (cancels out)

19
Q

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

A

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

20
Q

What CE buffer can be used at pH 9?

A

borate

pH range 8.14 - 10.14

21
Q

What CE buffer can be used at the lowest pH?

A

phosphate

pH 1.14-3.14

22
Q

What CE buffer is useful at a neutral pH?

A

phosphate

6.2 -8.2

23
Q

What are some zwitterionic buffers for CE?

A
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)
24
Q

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

A

Vtot

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

25
Q

Vtot can be expressed as: (3)

A

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

26
Q

how does electroosmotic flow affect separation time?

A

increasing Veo -> decrease separation time

27
Q

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

A
Utot = Tr (total retention time)
Ueo = Tm (dead time)

Utot-Ueo corresponds to adjusted retention time (Tr-Tm)

28
Q

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

A

identical directions (toward cathode)

opposite (Uep flows toward anode; negative flow)

29
Q

What has ‘negative flow’ in CE?

A

anions electrophoretic flow

30
Q

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

A

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)

31
Q

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

A

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

32
Q

What food chemicals can be separated with CE?

A
small organic/inorganic ions
small amino compounds (AA, biogenic amines, heterocyclic amines)
peptides/proteins
phenols/polyphenols/pigment
carbs
vitamins
pesticides
antibiotics
33
Q

What detector types are used with CE?

A

Spectrophotometry (absorption, fluorescence, thermal lens, raman, chemiluminescence)

MS

EC (potentiometry, amperometry)

34
Q

what allows for the separation of AA in CE?

A

charges on functional groups (positive, negative, etc)

35
Q

AA functional groups with neg charges: ______

AA functional groups with + charges: ______

A

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

36
Q

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

A

decreases (change from + to negative)

37
Q

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

A

isoelectric point (specific pH)