Electrophoresis

Question 1

What are colloids

Answer 1

The are made of one phase dispersed into another phase (like liquid in liquid or solid in liquid)

We typically use liquid liquid, or solid in liquid (gel)

Question 2

Explain colloid instability

Answer 2

Colloids in solution are not stable, they flocculate (clump together) then coagulate, then sediment out of solution

Or it can sediment (drop to bottom of container), flocculate, then coagulate

Not good

Question 3

Whag influence colloid stability

Answer 3

Concentration: if higher, they more likely to interact with each other

Do they like to stick together

What solution it’s in

Question 4

What are the two types of stabilization for colloids which are barriers to floculations

Answer 4

Steric stabilization (stick polymer chain on it that pushes away other molecule)

Electrostatic stabilization

Question 5

Explain why flocculation is favourable to happen

Answer 5

Using hard sphere model

Van der walls forces pulls the molecules together at the lowest energy

The model says that if we leave the molecules together, they will start to stick together due to these attractive forces

This is the precursor to flocculation thencoagulation and precipitate

Question 6

What are the sources of charge on surfaces

Answer 6

Ph change: making a solution basic give a net negative charge on the surface groups

Silica: the silica changes charge depending on the pH of the solution (can go either way) , like running KOH in glass (basic) so net negative silica charge of the surface. Giving acid makes it acidic

Ionization of surface groups: ions like AgI, can solvate one ion and leave the other on the surface

Surfactants: can be cationic or anionic surfactants (like SDS), sick to the surface and carry a charge with it

Question 7

What is the equation for van der waals attractive force

Repulsive force

Answer 7

-A/12pir^2

On sheet

Question 8

Explain the van der waal attractive force and repulsive force and the net energy in the one graph

Answer 8

attractive force which makes the line go down in energy as molecules get closer

Repulsive force which makes them go higher in energy as they get closer

Then net energy hump is in between them which shows how there is an energy hill the molecules need to climb so they can eventually attract and flocculate

Question 9

Explain the flocculation curve

Answer 9

Particles far apart , no energy no interaction

Get close, start to have vander waals attractive forces, energy goes down

Then eventually you have repulsive force as they get too close, higher energy

If we get them close enough (for example higher concentration) Then they can cross over this high energy hump to follculate (crash out) and get closer together and to low energy

Eventually them stick together and turn into a clump that is incompressible

Question 10

Explain the double layer model

Answer 10

Surface: You have a negative charge particle in solution

Stern layer: Surrounding it is the tight bound positive charged counterions that move with the particle

Diffuse layer: has less tightly bound ions, can be postive or negative because now the particle looks postive, it’s all the ions outward of the stern layer

Inside diffuse layer the is the slipping plane

Slipping plane: the part where ions stop moving with the particle as it moves through the solution

this plane is the boundary between ions that are still moving with the particle and ions that are not and just stay in the solution (some point in the diffuse layer)

Question 11

Explain the double layer plot

Answer 11

P vs r plot for a positively charged particle

Surface potential: surface layer of the particle (0 r), the potential would be positive because it’s postive ion (

Stern potential: As we move outward from the surface and go to stern layer, the negative counterions start to drop the potential

Diffuse Layer: the potential decreases more due to shielding of the ions in the diffuse layer

Zeta potential: At some point in the diffuse layer we reach the slipping plane, potential measures here is the zeta potential

Then furthest away from the ion is close to zero because looks neutral

Question 12

Why do we measure the zeta potential at ethe spliiping plane

Answer 12

If try to move particle through the fluid is act as if it has a size due to the ions surrounding it

Measuring the zeta potential at the slipping plane lets us measure the size of the particle because at this point you can see the end point of the ions that contribute to its size

40:04 idk

Question 13

Why does the double layer plot go to zero at infinity

Answer 13

We’re so far away from the particle that there are a bunch of ions between where we measure and the particle

These in between ions position themselves around the particle based on what the charge on the particle looks like to them

This ultimately makes the potential neutralize and become zero

Question 14

Do the surface and the zeta potential have the same sign

Answer 14

No the sign of the zeta potential doesn’t tell you what the surface of the particle is

The zeta potential depends on the solution that the particle is in

If more polar, more ions, can change what the zeta potential looks like

There are ions close to the surface of the molecule that aren’t exactly associating with it but their charge affects what the zeta potential is

Just like how PAH and PSS affect what the charge looks like on the molecule

Question 15

What are the compounds that let you change the charge on the surface

Answer 15

PAH (postive charge)

PSS (negative charge)

If postive surface, add PSS, Makes surface negative

Can keep going with PAH to make it back to postive

Question 16

What is the purpose of reversing charges using PSS or PAH

Answer 16

If the surface was one third negatively charged and the rest neutral

You can still add PAH to make it look uniformly negative since it big and will cover regions that are neutral

Can turn something that is weakly charged into something that has a highly negative or postive charge

Question 17

At was potential is a particle /colloid stable

What does this mean

Answer 17

+/- 30mV

This mean if we measure the zeta and that is it’s value, there is enough of an energetic hump that the particles won’t easily flocculate and crash out

Question 18

What is the effect of the pH on the zeta potential

Answer 18

low ph, zeta potential positive, stable

High pH, zeta potential negative, stable

At the isoelectric point, the zeta potential is zero and no net charge pushing the particles apart, unstable because now not +/- 30 mV

This means there will be flocculation since no charge repelling the ions

Question 19

Can the isoelectric point of a particle be change and how

What could change shape of the zeta potential vs pH graph to start negative at low ph and get postive at high pH

Answer 19

Can be changed because of diff functional groups of the particle, can cause flocculation at lower ph or higher ph (so lower or higher isoelectric point)

Changing the sign at low ph would mean that having more h ions is causing the particle to be more negative , so not possible and weird

Question 20

What is the Debye length

What does it mean

At what point does it end

Answer 20

Lambda D = 1/kappa

Describes the length of the diffuse layer and the amount shielding of the particle

How far you have to go before the potential of the particle is shielded by the many counter ions in the diffuse layer and the potential becomes zero

Since the diffuse layer has exponential decay (never goes to zero) we say that the diffuse layer ends at a potential 1/e of its original value

So 1/3 of the diffuse layers original potential value

Question 21

If the diffuse layer drops has its exponential decay very quickly what does this mean

Very slow decay

Answer 21

Quick: say the diffuse layer is very thin, more ions, faster decrease because easier to drop the potential

Slow: diffuse layer very thick, less ions, , slower decrease because harder to drop the potential

Question 22

What is the effect of higher ionic strength of the diffuse layer

Answer 22

More ions in the diffuse layer, shielding is going to be very thin because that’s all you need to drop the potential to 1/e

If less ions (for example just water and not na+), the diffuse layer is bigger since you need more of them to shield and drop the potential to 1/e

Question 23

What is meant by shielding and why does it happen

Answer 23

If the particle is postive, the stern is negative, but ions in the diffuse layer look at that and try to make it more postive

Then the negative ions out look at, see a general postive charge, and configure themselves to make it more negative

This accumulation of opp charged ions makes it so that shielding of the particle happens and the potential looks to be neutral/zero very far away

Question 24

When do we say something is fully shielded

Answer 24

When the potential reaches 1/e or 1/3 of the original potential value (at the surface)

Question 25

What is the effect of higher valency ions on the Debye length

Answer 25

Ex. Fe3+ and fe2+ The higher valency ions is basically more charge meaning smaller Debye length since need less ion for shielding Sheilds it faster, gets to 1/e faster

Question 26

Describe the Debye length in polar and non polar media

Answer 26

Polar more ions , smaller Debye length, need less to shield Non polar less ions , larger Debye cause need more to shield

Question 27

Explain the implications of dilutions a sample and Whag you have to make sure

Answer 27

During dilution of something you want to preserve the pre-existing state of the surface of the particle Need the same pH, ionic concentration, surfactant/polymer concentration So you would centrifuge out the particles you want to dilute Then use the resulting solvent (with all the same properties) to dilute the particles

Question 28

What is the equation for electric force

Answer 28

FE= q (V/d) V is the potential difference across the electrodes d is the distance between electrodes (how far apart they are) q is the charge on the particle between the electrodes

Question 29

If F= ma what’s stopping something on a gel from just going really fast through the gel and flying off

Answer 29

There is resistance from the gel So a force of drag On sheet equation

Question 30

What is electrophoretic mobility

Answer 30

How fast a particle moves under a particular electric field Equation on sheet

Question 31

In the electrophoretic mobility equation what does it mean if kappa a is >>1 and <<1 What are the names of the approximations

Answer 31

Smoluchowski: ka >> 1: the Henry function (f(kappa a) ) is 1.5 Huckel: ka << 1: Henry function is 1.0

Question 32

Which approximations are used for a non polar and polar media

Answer 32

Debye length = 1/kappa So kappa= 1/ Debye length Polar: need small Debye length, so kappa >>1, smoluchowski Non polar: need large Debye length, so kappa <<1, huckel

Question 33

Explain the situations for the particle in each approximation Explain what’s bad

Answer 33

For smoluchowski, polar media, you need to have a large particle and a small Debye length Can get away with having a larger shielding layer as long as you have a larger particle For huckle, non polar media, you need to have a small particle and large Debye length If you have a small particle, to make this assumption hold you would need non polar solvent like benzene or hexene to get a larger Debye length If you have a very tiny particle in a low ion concentration, this is bad because doesn’t fit the smoluchowski approximation

Question 34

Which approximation do we usually use and why

Answer 34

The schomulowski because using higher salt, not using non polar solvent

Question 35

What is the other way to write electrophoretic mobility

Answer 35

Velocity of the particle / electric field v/E

Question 36

What affect would a greater charge of the particle have on the electrophoretic mobility

Answer 36

Using equation with zeta in it: The zeta value would increase because it’s related to the apparent charge on the particle Using v/E: the higher charge on the particle causes more force of it moving in gel electrophoresis, so higher velocity Each of these things causes higher electrophoretic mobility and the particle to move faster

Question 37

How would a higher gel concentration affect the electrophoretic mobility

Answer 37

Thicker gel, higher viscosity (denominator), decreases electrophoretic mobility , slower Smaller velocity, smaller electrophoretic movility

Question 38

Electrophoretic mobility equating and browning motion equations

Answer 38

On sheet

Question 39

Explain the brownian motion equation

Answer 39

have a particle in solution colliding with particles from the solvent to knock it around higher temp causes higher kinetic energy of solvent molecules, faster movement, high Vrms If the mass of the particle being knocked around increases, have low Vrms, slower particle

Question 40

It’s the Brownian motion a distribution

Answer 40

Yes

Question 41

How does Brownian motion relate to chromatography and gel electrophoresis

Answer 41

Can affect the distribution of our protien molecules in the gel (due to interacting with the solvent particles) This leads diffusion (causing band broadening)

Question 42

Explain the diffusion plot

Answer 42

There’s a greater probability of finding something further from where it started over time the peaks broaden due to diffusions due to being at room temp

Question 43

How does the hydrodynamic radius relate to zeta potential

Answer 43

R it’s related to the slipping plane When the particle moves through solution it move not as if it has its own radius but as if it has a larger one that includes all the solvent molecules

Question 44

What is the stokes Einstein equation telling us

Answer 44

Another way to tell diffusion BUT FOR PARTICLES THAT ARE FREELY DIFFUSING Fiset can Tell R (hydrodynamic radius) Can rearrange to get diffusion coefficient (D) which is the rate of diffusion Then if temp increases, D increases, so faster diffusion If viscosity increases, D decreases, slower diffusion If size increases, smaller D, slower diffusion (movement)

Question 45

What does stokes Einstein assume

Answer 45

Assume a spherical particle But Not always spherical, sometimes unfold to look like they have a bigger radius sometimes and larger other times

Question 46

Why do we cut the band out of the gel right after finish with is

Answer 46

If leave it a lot room temp, higher diffusion, it’ll keep broadening

Question 47

Explain the stokes equation

Answer 47

The faster something moves through water the larger the drag is (Fd)

Question 48

What assumption do we make for each of the equations we’ve talked about

Answer 48

That the particle is free to move wherever it wants (not true) Moves at the same speed everywhere (not true)

Question 49

Diff in laminate flow and turbulent flow What do we assume for gels

Answer 49

Laminar flow all water is following the same uniform path Turbulent flow the water is going in all different directions chaotic(like if filling water bottle) Assume Laminar flow

Question 50

Explain the problem with the gels having laminar flow

Answer 50

The velocity of the flow is different at the edges of the lane than at the centre , not same speed all the way across it This could lead to band broadening Problem with this is that the particles pile up at the edges, usually backflow happens near the walls to stop this

Question 51

What is the advantage to gels being porous

Answer 51

It’s allows for slow flow Suppress thermal convection: stops the solution from having motion due to heat transfer and things happening inside which cause it to move

Question 52

What is isoelectric focusing

Answer 52

Trying to separate the protiens by their isoelectric point Cathode on one end anode on the other to make a pH gradient (high pH at cathode (-), low pH at anode (+)) Every time there is a peak , that is where the protien is at its isoelectric point This tells us about the composition of the protien surface Can do as a 2D gel to split according to mass and isoelectric point

Question 53

Using the FE (electrostatic force equation) and F drag equation explain what the electrostatic force is proportional to What is the problem here

Answer 53

FE= qE So the electrostatic force is proportional to the charge in the particle (q), more charge more force, faster The drag is proportional to the size R, bigger size , more drag, slow In a protien during gel electrophoresis, we have constant charge per unit mass, meaning the bigger the mass, the more charge This is bad because if bigger mass it’s supposed to slow down (more drag) but also has more charge which is making it faster (more force) Can’t be both fast and slow at the same time, get nothing

Question 54

What are balanced forces

Answer 54

fv=qE Friction times velocity = charge times electric field

Question 55

Explain the free mobility of particles

Answer 55

The free mobility is independent of mass and charge If log gel concentration increase, electrophoretic mobility decrease If log zero percent gel, we only have the free mobility which is charge and mass independent

Question 56

Explain the free mobility of particles Ferguson plot

Answer 56

Log Ue (electrophoretic mobility) vs % gel Increase gel, decreasing mobility At 0% gel see the free mobility value Small particle high charge Small particle lowcharg Big particle high charge Big particle low charge The small and big particle high charge have similar free mobilities, but the high mass electrophoretic mobility drops off a lot more than the small one (charge independent, size dependent) This is because the gel introduced a new source of frictional force, making the movement size dependent The small particle high charge and small particle low charge, should have no net force on it at gel concentration of zero Big charge, big motion IDK33.42

Question 57

Explain the free mobility of particles Ferguson plot when the charges are proportional to length

Answer 57

In the free mobility situation they all reach the same electrophoretic mobility value Not getting good results from the electrophoresis Based on the size effect from the gel, they split and have diff slopes (same starting point) EMAIL AND ASK 60-65

Question 58

When doors diffusion happen

Answer 58

Always

Question 59

Explain ion exchange columns

Answer 59

The resin is a charged polycation or a charge poly anion There’s is an interaction between the molecules and the resin If negative resin, postive charge protiens move slowest and negative move fastest Changing pH can make protien bind and release it the column by changing its charge Higher Salt concentration also elutes , ions will compete with the particles on the column

Question 60

Explain sec columns

Answer 60

The smaller particles move slower because go into the resin cavities Bigger particles move faster

Question 61

Explain affinity columns

Answer 61

Ligand protien interaction Use salt or competing ligand to wash out