Electrophoresis Flashcards
What are colloids
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)
Explain colloid instability
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
Whag influence colloid stability
Concentration: if higher, they more likely to interact with each other
Do they like to stick together
What solution it’s in
What are the two types of stabilization for colloids which are barriers to floculations
Steric stabilization (stick polymer chain on it that pushes away other molecule)
Electrostatic stabilization
Explain why flocculation is favourable to happen
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
What are the sources of charge on surfaces
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
What is the equation for van der waals attractive force
Repulsive force
-A/12pir^2
On sheet
Explain the van der waal attractive force and repulsive force and the net energy in the one graph
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
Explain the flocculation curve
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
Explain the double layer model
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)
Explain the double layer plot
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
Why do we measure the zeta potential at ethe spliiping plane
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
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Why does the double layer plot go to zero at infinity
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
Do the surface and the zeta potential have the same sign
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
What are the compounds that let you change the charge on the surface
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
What is the purpose of reversing charges using PSS or PAH
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
At was potential is a particle /colloid stable
What does this mean
+/- 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
What is the effect of the pH on the zeta potential
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
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
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
What is the Debye length
What does it mean
At what point does it end
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
If the diffuse layer drops has its exponential decay very quickly what does this mean
Very slow decay
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
What is the effect of higher ionic strength of the diffuse layer
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
What is meant by shielding and why does it happen
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
When do we say something is fully shielded
When the potential reaches 1/e or 1/3 of the original potential value (at the surface)
What is the effect of higher valency ions on the Debye length
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
Describe the Debye length in polar and non polar media
Polar more ions , smaller Debye length, need less to shield
Non polar less ions , larger Debye cause need more to shield
Explain the implications of dilutions a sample and Whag you have to make sure
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
What is the equation for electric force
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
If F= ma what’s stopping something on a gel from just going really fast through the gel and flying off
There is resistance from the gel
So a force of drag
On sheet equation
What is electrophoretic mobility
How fast a particle moves under a particular electric field
Equation on sheet
In the electrophoretic mobility equation what does it mean if kappa a is»_space;1 and «1
What are the names of the approximations
Smoluchowski:
ka»_space; 1: the Henry function (f(kappa a) ) is 1.5
Huckel:
ka «_space;1: Henry function is 1.0
Which approximations are used for a non polar and polar media
Debye length = 1/kappa
So kappa= 1/ Debye length
Polar: need small Debye length, so kappa»_space;1, smoluchowski
Non polar: need large Debye length, so kappa «1, huckel
Explain the situations for the particle in each approximation
Explain what’s bad
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
Which approximation do we usually use and why
The schomulowski because using higher salt, not using non polar solvent
What is the other way to write electrophoretic mobility
Velocity of the particle / electric field
v/E
What affect would a greater charge of the particle have on the electrophoretic mobility
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
How would a higher gel concentration affect the electrophoretic mobility
Thicker gel, higher viscosity (denominator), decreases electrophoretic mobility , slower
Smaller velocity, smaller electrophoretic movility
Electrophoretic mobility equating and browning motion equations
On sheet
Explain the brownian motion equation
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
It’s the Brownian motion a distribution
Yes
How does Brownian motion relate to chromatography and gel electrophoresis
Can affect the distribution of our protien molecules in the gel (due to interacting with the solvent particles)
This leads diffusion (causing band broadening)
Explain the diffusion plot
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
How does the hydrodynamic radius relate to zeta potential
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
What is the stokes Einstein equation telling us
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)
What does stokes Einstein assume
Assume a spherical particle
But Not always spherical, sometimes unfold to look like they have a bigger radius sometimes and larger other times
Why do we cut the band out of the gel right after finish with is
If leave it a lot room temp, higher diffusion, it’ll keep broadening
Explain the stokes equation
The faster something moves through water the larger the drag is (Fd)
What assumption do we make for each of the equations we’ve talked about
That the particle is free to move wherever it wants (not true)
Moves at the same speed everywhere (not true)
Diff in laminate flow and turbulent flow
What do we assume for gels
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
Explain the problem with the gels having laminar flow
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
What is the advantage to gels being porous
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
What is isoelectric focusing
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
Using the FE (electrostatic force equation) and F drag equation explain what the electrostatic force is proportional to
What is the problem here
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
What are balanced forces
fv=qE
Friction times velocity = charge times electric field
Explain the free mobility of particles
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
Explain the free mobility of particles Ferguson plot
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
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Explain the free mobility of particles Ferguson plot when the charges are proportional to length
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
When doors diffusion happen
Always
Explain ion exchange columns
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
Explain sec columns
The smaller particles move slower because go into the resin cavities
Bigger particles move faster
Explain affinity columns
Ligand protien interaction
Use salt or competing ligand to wash out
