CHEM SEP final 2 Flashcards
Moment Analysis (Method of Moments)
*
Every distribution function (Gaussian or otherwise) has a set
of statistical moments characterizing its position and shape.
Go through The different variations you can have with moment analysis
Continuous vs discrete (summation vs integral),
Raw vs central moments (raw just alues as is, central is value - mean (make the mean the origin so the population is deviations from the mean); in raw moment the origin is the center
Population vs sample: sample is value /numebr of samples , population is integration over the function
REVIEW GUIDE FOR WHAT THESE LOOK LIKE
What are the 4 sample moments
Mean, variance, Skewness, kurtosis
4 sample moments how to write equations for each
Review how to write equations for each
How to interpret skewness?
0 - gaussian, > 0 is fronting (right side) and < 0 is tailing (left side)
How to interpret kurtosis?
excess = kurtosis - 3; gaussian is 3 , smallest is 1 (makes it a rectangle) - higher you go the more narrow (a measure of how flat)
GC can analyze compounds that are ..?
Voltalie or can be derivitized to become volatile
Parts of a GC system
: a gas source with pressure
and flow regulators, an injector, a column in an oven, a detector, and a
computer for data acquisition.
So GC using a compressible fluid means what
As we get a pressure drop (poissons/darcys law) - we get a density change - which means the gas expands (via Boyles law) - so this causes an increase of linear local velocity and local flow
In GC what determines the change in rate of velocity from inlet vs outlet
Ratio of Pressure in/Pressure out - the higher - the steeper the change close to end of column
Know how to derive pressure in out equation equation
Difference in GC velocity van deemter vs experiemntal
in van deemter it is average ; in experimental its at the outlet
What are 3 things that need to be corrected for , for experimental GC average flow rate/veloctiy measurement
1) pressure gradient drop (j) (multiply it by V out to get V average)
2) Temperature correction (Tc/Tout) because outlet is a lower temp causing reduction in flow rate (because measure outside of GC with bubble flowmeter)
3) Bubble flowmeter causes gas to be saturated with water so need to account for addition of pressure from water vapor (Pout - Pw/ Pout)
2 ways to determine average velocity in GC
Multiply measured velocity out by j, pressure correction factor and temp correction factor OR just use retention time (L/rt))
How is volume corrected for in GC
Similarly since based off flow and we know flow is affected - need correction terms j for compressibility , NET parameters are tr- tm, Vr - vm
What is corrected and NET volume in GC
So volume usually got by tr * F HOWVER F is affected in GC (F at outlet vs average) - so we need to adjust it instead of V = t * F it’s now V = t *j *F and this is now called corrected volume NET volume is uncorrected volume - corrected volume
What is specific retention volume
Specific retention volume is commonly used in GC to permit
inter-comparisons of retention of an analyte i in columns
containing different weights of the same stationary phase at
the same column temperature . Calculated as NET volume / weight of stationary phase multiplied by 273/ column temp
What is specific retention volume related to in thermodyanmic terms
on top R * 273 on Botton inverse with MW of stationary phase, the activity coefficient of analyte I in stationary phase and Vapor pressure of pure I in standard state (analyte I(
How is GC from other chromatographic methods in terms of retention
Not based on partition or seperation but on vapor pressure and volatility because the gaseous mobile phase is a lot more than concentration fo analyte and has a much lower density so we assume it doesn’t interact with the solute (especially incomparison to the stationary phase) - so K is dependant on temperature here
How is chemical potential split for GC
So depends on uio which is the same but then the dilution of entropy terms which inis activity term * concentration so when we consider this in the mobile phase of GC - we assume ideal gas so y =1 and the concentration is just the PARTIAL PRESSURE of component I
so this simplifies to u = uio + RTln(Pi)
What is selectivity in GC sepeartion based off of and why
Vapor pressure of sample components (when we do u - uio + RTln(C *y) in an ideal gas this just turns into partial pressure of analyte ; AND
Solute solvent interactions with Stationary phase
What is yis dependant on
PiM - partial vapor pressure of analyte ,, inversely related to MOLE FRACTION of I in SP and PiO - vapor pressure of pure component I at column temp
Distribution coefficeint in GC
(moles in SP / Weight SP) / (moles MP / corrected mobile phase volume) (in other words MOLALITY / MOLARITY)
No how to derive thermodynamics of retention showing that Specific retention volume depends on yis and Pio (inverse related)
What is yis and what are its values
activity coefficient of i in stationary phase - measure of influence of stationary solvent on vapor liquid equilibrium of I
if y= 1 SEPERATION ONLY RELATED TO DIFFERENCE IN VAPOR PRESSURES
if y< 1 vapor pressure is lower than that of ideal solution I - MEANS STRONG SOLVATION in SP
if y > 1 vapor pressure is HIGHER thanideal solute means SP solvation IS WEAK
in GC how does concetration relate to yis
kept low to keep yis constant across run
What is yis 2 contributors
Yent - athermal activity - from excess entropy changes in forming the solution that is in excess for an ideal solution
Yint - the thermal activity - assoc with enthalpy change due to molecular interactions (dipoles, H bonding etc) - MAJOR CONTRIBUTOR (h = RTln(yint)
What is the trouton rule
If solutes are not too polar can estimate enthalpy of evaporation as 22* boiling point
For our specific retetnion time related to boiling point equation - which assumptions must be true
Ideal solution, not too polar analyte and also if not ideal solution h (enthalpy of solute interactions) needs to vary consistent with Hv (enthalpy of evap of pure solvent)
What are good qualities of a carrier gas
oxygen free, moisture free, no hydrocarbon impuriteis and chemically inert
common carrier gases
N2, He, H2, Ar, O2 and CO2
Factors in choosing GC mobile phase
detector response (eg H2 and O2 as burner), efficiency and speed, stability, hazards , availability/expense
How does viscosity function in GC temp preogramming
As we increase temp - viscosity increases and average linear velocity decreases
What gas is best for capillary GC
H2 - for diffusivity and broad working range
Types of GC column
PACKED, - high stable
WCOT, -liquid -large sample capacity
SCOT, -liquid -large (but less sample capacity)
PLOT -solid - for adsoprtion - stable - less applications -
(wall coated, support coated and porous layer
Capillary vs Packed bed GC
Capillary is much more efficient
What is phase ratio and how does it effect GC van deemter
so GC van deemter - ratio fo SP vs MP - affects C term mass transfer - if THIN film - smaller plate height can be operated at higher velocities - but THICKER filmes increase retention sample capacity etc
In thin film columns talk about van deemter, maxes and what it means for u opt and hmin
SO van deemter for GC is NO A term no eddy - so just B/v + C * v
and we largely care about Cm
So UOPT = ROOT( B/C)
and Hmin = 2ROOT(BC)
These terms (B and C) respectively are:
B: 2Dm/V
and Cm is k * v (d^2)/Dm
So for uopt
it simplifies to Dm / d * root (K term)
and Hmin simplifies to
d* root ( k term)
SO as k is above 5 it rapidly approaches 1 so really just d or diameter that determines this
Good GC Sp characteristics
unreactive with carrier gas and solute, low vapor pressure and viscosity, good coating, solubility, wide temp operating range
Common stationary phase types in GC
non polar - hydro carbon and perfluorocarbon
polar - ether and esters
specially (high temp and chiral
How to choose SP based on retention index
LSER - or Kamlet TAFT parameters -
looks at
R2 - molar refraction
Dipolarity/polarizability
hydrogen bond acidity/basicity
and gas liquid partition coefficient for n - hexadecane
Also has system constants based on MP, SP and temp
gives you k
KOVATS RETENTION INDEX explain
uses n -alkanes to make a retetnion series
basically you have adjusted retention time and z is the carbon number of the lakane eluting before and z_1 the alkane eluting right after and gives you an index n#
Gives you I
McREYNOLDS -phase constants explain
measures retention indices of 5 compounds as a guide to interactions of SP (Butanol, pentanone, nitropropane benzene and pyridine, and compares YOUR SP to SQUALENE (subtract from squalene calculated value)
Gives you I - I squalene - which indicatespolarity of SP
3 ways to pick SP for GC and differences
Mcreynoalds, -properties of specific molecules
Kovaks and
TAFT KAMLET - uses solvatochromic params
what are GC PLOT colmns used for
seperating hydrocarbons - molecular sieves
What is desired in GC detetor
Tempreature range and short response time independant of flow rate are the majors (then typical things, sensitive, stable, reliable etc)
TCD detector (thermal conductive) describe function
measures thermal conductivity - sees if gas cools a hot filament -
Has large linear range, non destructive, faster response
disadvantages - effected by impurities and low sensitivity
FID - describe
unviersal
based of hydrogen air flame - burn our organics they release ions and electrons and current of those ions detected (proportional to concentration)
Specific for combustible(so no response from non combustible background gases like water, CO2, Noble)
linear dynamic range - sensitive
Problem L destructive
NPD describe it
specific for Nitrogen or phosphorosu containing
alkali metal (rubidium or cesium) vapor ignites the hydrogen and forms cold plasma
– this formation is increased in the presence of nitrogen and phosphorus containing compounds
ADVANTGES:
environmental and drug testing, doesn’t detect common carrier gas or impurities
LOD - sensitive more than FID
DISADVANTAGE _ destructive
specific
ECD describe it
based on electron capture by electronegative atoms in a moelucle
we have electorns produce by radioactive source makes a current we detect - if something electornegative swings by - takes some e- messes up current
ADVANTAGE - real sensitive and specific
DISADVANTAGE:
narrow dynamic range
Beneftis of LC vs GC
sample just needs to be soluble - more compounds
get selectivity across SP and MP
most detectors non destructie
BUT LC - has worse band broadening
Know the knonx equation
VAN DEEMTER BUt
include the x term Av^1/3 instead of justeddy diffusion
Whats dominan tvan deemter force in LC
STAGNANT MOBILE PHASE _ mass stransfer for SP - 3050% of H
What is total plate height in LC from
eddy, londituginal, mass transfer AND EXTRA COLUMN BAND BRAODENING - tubes, detector plumbing etc
What materials are used to increase pH range LC
Al2O3, TiO2, and ZrO2
What is HPLC SP bound to
Si-OH covalent bond
Why are columns generally bound at pH ranges and what can extend the range
Siloxane - bond hydrolyzes below pH 2
-BULKY ISOBUTLYLGROUPS - can protect it
What LC SP has both polar and non polar retention
Cyano
General order of reverse phase retentivitiy
Cyano < C4< Phenyl< C8< C18
LC system general schematic
solvent reservoir - gelium source -> pump -> injecting -> filer -> pressure -> column -? detector
wHAT ARE THE TWO TYPES OF lc PUMPS and how do they work
DISPLACEMENT -
RECIPROCATING
How do displacement pumps work
Has a screw that as it screws down - mobile phase pushes up -
NO PULSE
but small capacity
How do reciprocating pumps work
Basically two seal check valves - ONE isopened and a piston pulls back drawing liquid in - then they switch bottom cosed - top is open - piston pushes down pushing liquid up
DISADVANTGE _ complicated, seal and valve maintenance - ALSo pulseS! - so typically have two in parallel that are out of sync (so the low pressure part of one is high pressure part of the other)
What are the two types of LC detectors
BULK PROPERT and analyte
kind of what they sound like
bulk property is like refractive index, di electric constant, conductivity
analyte is everything normal
HOW DO RI detectors work and benefits and cons
Universal detector based on refractive index of liquid leaving column
(snells law Nsin(theta) = N2sin(theta2)
primarily used for unknowns
expensive
low sensitivity
CANT BE USED WITH GRADIENT
UV vis equations
T - P/Po
Absorbance - -log(T)
A - ecl
UV vis absorbance detector designs
fixed wavelength, variable wavelength and photodiode array detector
fixed wavelength is on e(254), variable does multiple (requires a CONTINUUK SOURCE - tungsten lamp etc)
Photodiode array - can read all wavelengths at once(USES dispersion device and an array of detectors - - good for rapid LC
What is a flow cell detector (UV)
basically a cell that flows continuously and runs for a bit inline with beam as such INCREASES the path length by a lot making it a lot more sensitivite
Fluorescence detector benefits
SELECTIVE
HIGH SENSTIVITY
Cons - not everything fluoresces so may require dervitization
How does fluorescence detector work
So electrons can be in singlet state - paired or triplet state unpaired
When energized to S2 state - can relax down to S1via INTERNAL CONVERSION
and from S1 - can undergo intersystem crossing to go to T1
These are known as radiationless transitions
SO THEN FLUORESNENCE is when transition from S1 down to S0 (VERY quick)
ANd phosphorescence is when go from T1 down to S0 - longer time scale
Fluorescence or phosphorescence band comapred to the absorption?
Its higher (because lost energy - it’s like a mirror image shifted up (because longer wavelength))
T
What is stokes shift? and why
wavelenegth difference between the lowest energy peak of absorbance and the highest energy peak of emission (basically the difference between the two) - GETS RID OF BACKGROUND - stops excitation by the fluorescence ) makes more sensitive
How does UV geometry vary from fluoresence
UV can be 180 degree in line, FLuorescence is 90 degrees
What is the “Hook Effect”
IN fluroescence if concentration too high - falls off pretty quick due to get 2ndary absorption
Electrochemical Detector explain
detects ability of solution to be reduced or oxidized
Setup : working, counter and reference electrode
Reference is Ag AGCl - stable Electrochem potential
Amperometric detector stats
hgih sesntivity, convenience, broad
CONS: MP must be conductive, prone to interference and less repdouicble
Whats LC attribute of good seperation
k for all peaks between 0.5 and 20
resolution > 2..?
assyemtry between 0.9 and 1.5
How to optimize isocratic retention
choose column and organic solvent
optimize K (adjust % organic)
check for Low N (COLUMN DEPENDANT) (n = 3000*L/ DP)
adjust alpha by:
adjust solvent type
adjust column temp
adjust column type
optimize column dimensions to increase N
What is the solvent selectivity triangle
small set of 10 experiments to optimize mobile phase trying proportions of different solvents
the corners are H BONDING, DIPOLE DIPOLE and proton acceptor
How to decide if you should use gradient or isocratic
if DELTA t/tg > 0.25 iuse gradient - if less use isocratic
note Tg - gradient time and delta T = difference in retention between first and last peak
Explain dwell volume and dwell time and they role in reducing gradient time
SO dwell volume is the volumen between the point at which solvents are mixed and beginning of column and dwell time is the same thing but time
SO
dwell time = dwell volume/flow rate
this lets you know how long it takes a change in mobile phase solvent to reach column - IMPORTANT because when reducing your time - you want to know what %age things start eluting at and what %age they stop so you can only eute over that percentage
Ion exchanger - types of stationary phase supports(not active groups) and why
REsins - small molecule (highly crosslinked - means denser)
Gels - for biomolecules and proteins and nucleic acids
Inroganic - for harsh chemical conditions
What are the groups used for ion exchanges
For cation excchange use carboxylic acids or sulfonyl group (SO3-)
For anion - quaternary amine or ammonium hydroxide
Polarizing power - what is it
P = Z^2/r it’s essneitally how favored something is for ion exchange - its higher charge and smaller hydrated radius
How do you regenerate an ion exchange column
wash with something ike H+ (if it is attached to Na+ you can replace it
Whats ion exclusion chormatoraphy
Ion-exclusion chromatography is used to separate low molecular weight ions and
neutral substances by a combination of partition, adsorption and ion repulsion.
2. The stationary phase is a high capacity ion exchanger with same type of
immobilized ionic groups as the sample ions.
3. Donnan exclusion: same charge as the stationary phase repelled and not
allowed to enter the stagnant mobile phase, but the ions with opposite charges
or neutral do ente
applications of IEC
salt exchange , water purification
LC vs GC
More applicable because just need in solution vs volatile (especially for biologicals)
Lower temp - so good for thermally labile compounds
MP and SP both separate - more flexible
Typically non destructive
easier sample recovery
What is bonded SP and what are some normal phase SP’s
bonded to the Si-Oh - normal phase includes, amide, amino , cyano diol etc
Why does the efficiency (plate height) of liquid chromatography increases as the stationary-phase particle size is reduced?
For H - from SP - its (K term) * dp^2 * v / Dm
so as dp goes down H goes down;
2 types of (SEC, or gel filtration or gel permeation)
GROUP seperation (components seperated into major size groups
High res seperation/fracitonation - separated according to molecular size
Explain the basics of thermodynamic SEC (enthalpy vs entropy factors)
so usually u = H - TS ; and generalyl H predominates - it’s the interacts with the solution enthalpy of reacting; BUT in this case our ENTROPY change is what matters because we are in POROUS MEDIA (the pore diameter is on the same magnitude as the diameter of the parittioning species) - in fact we keep H the same because MP is kept constant
What is K in SEC
C in pores / C in bulk (so concentration of per unit volume of pore space vs concentration of bulk solution - so tends to just be volume ratio- ACCESIBLE VOLUME / TRUE VOLUME
What is K in SEC? do for cylinder
C in pores / C in bulk (so concentration of per unit volume of pore space vs concentration of bulk solution - so tends to just be volume ratio- ACCESSIBLE VOLUME / TRUE VOLUME (so in cylinder its (pi (r-thickness)^2 L ) / (pi r^2 l)
What is K in SEC for an approximation of a variety of pore shapes and what do things mean
K = (1- sa/2) ^2
so here dc which was diameter of a column) is replaced by 4/s where s is the WALL AREA OF THE CAPILLARY per unit volume of pore space
s is proportional to mean pore size (so for simple shapes (note bigger pore size means smaller S - K approaches one as S becomes huge)
What is the random plane model of pore space
rigid moelcules of complex shape - accounts for complex molecules and their project length along various axes
K = e^(-sL/2)
GPC vs GFC?
gel perpeation vs gel filtration (polymer vs biochem
SEC common stationary phases and considerations
Sephadex or bio gel -P
DIameter important - if really small can exclude those with large MW;; larger you go the more MW you can accommodate (but I guess smaller things pass through easier)
Total movile phase volume in SEC column
Vm = Vi + Vo
Vo is interstitial volume
Vi is pore volume
What is RETETNION volume in SEC - and what is it based on
Vr = Vo + K*Vi based on the idea that only part of the Vi is accessible to a solute
What is K in SEC in terms of volumes and what does it depend on
Vr - Vo / Vm - Vo
K is SIZE DEPENDANT
Doesn’t that simplify to KVi / Vi which equals K …lol
so it’s kind of like K but for the PORE space because you’re removing Vo which everyone has access to
Interpret K for SEC - what do various values mean eg 0, 1
so if K = 0 it’s all Vo - this is true for a large molecule that does not permeat the gel - NO retention
if K = 1 it’s the exact opposite - and the total volume is the same as accessible volume (so really small particles that freely flow through - no separation
SO we want something between the two (called selective permeation region
How to determine molecular mass from SEC?
Use calibration curve (log of MW) vs elution volume
How does pore size effect SEC
finer mass resolution, faster elution, narrower mass range
In CE - no factors- what ions go where
cations to cathode and anions to anode (electrons I guess go to cathode and cations follow:
What is electrophoretic mobility and what is the equation and contributing factors
So electrophoretic mobility relates our Electric field strength to velocity
(so EM * E = Velocity)
(from qE = fv so v = q/f * E)
it is = to q/f which if we put in stokes is
q / (6 * pi * viscosity * r)
SO ULTIMATELY or electrophoretic mobility is based off charge and inversely with size
Explain the diffuse double layer
rigid charged surface - with a cloude of oppositely charge dions around it -
INfluecned by ZETA POTENTIAL - which is the potential difference between the the slipping plane and bulk medium (the slipping plane is the ..2nd layer?
So as pH rises , charge and zeta increase - and this actually decreases the thickness of the double layer
Explain how diffuse double layer causes electroosmotic flow
walls are coated with Si - O- - that attracts cations to form an electrical double layer - there is a diffuse layer beyond that - they strength of which is determined by Debye Huckel; MAINLY though it;s about the cations and they are attracted to the cathode SO they move there and drag the bulk solution there as well WHICH RESULTS IN A PLUG FLOW!
Explain electroOSMOTIC mobility and the terms it relates to
the proprtion between the electrosmotic velocity and the applied field is the electrosomotic mobility
So electro osmotic velocity = EOM * E
and electroosmotic mobility = the dielectric constant of vacuum and buffer and zeta potential and inversely related to 4 pi viscosity
How can one manipulate the EOF
pH (via zeta), ionic strength, surfacant and surface modification
What is the coutner ion cloud and does it matter
an ion in solution will have counter ions surrounding it
Describe the apprent mobility of various ion types
comes down to electrophoretic mobility vs electrosomtic mobility
For cations its additive
for neutras its just electroosmotic mobility
and for anions its electrosomotic - electrophoretic
VAN DEEMTER FOR CE
No A term, No C term ALL B TERM - all longitudinal diffusion which means it stays pretty darn flat
Heigh in theoretical plates for CE
N - uapp * V / 2D
this assumes total capillary length and length from inlet to detector are similar and cancel out (so independent of column length theoretically)
dependant on VOLTAGE and diffusion coefficient (or solution
Resolution for CE
ROOT(N) /4 * delta(u app) / mean(u app)
which can be simplified to ROOT(N) /4 * (y -1)
seperaton factor is speed of faster species/ speed of slower species
Whats Joule heating, how does ti affect CE and how can we minimize it
Joule heating is the idea that the electric field can cause an uneven heating of our capillary - so ultimately for us that causes uneven heating and convection currents in our CE - BAD - causes band broadening
CAN limit by
1) LOWER current (so in this way a longer column allows for larger voltage which can increase N)
2)Using packed bed to prevent as much mixing
3)use more efficient cooling
Buffers in CE - what are some issues
The CURRENT densit y matters - need to consider buffer pH and concentration- so good means to change EOF (EOF decreased at low pH and increased at high pH) but be careful
How do the following effect our CE method
Electric field
IOnic strength
Organic modifiers
Surfactant
Temperature
Electric field - proprtional change in EOF but can cause joule heating
Ionic Strength - decreases Zeta and EOF as increases but can cause high current and joule heating
Organic mods - decrease Zeta and EOF -
Surfactant - adsorbs to capillary wall through hydrophobic or ionic interactions
How to deal with EOF flow change with pH change
Use a surface coating like polybrene + dextran sulfate - allows as the pH increases to maintain consistent flow
What does using a cationic surfactant do to CE
SO in general - a cationic surfacant will REUDCE flow - and an anionic one will increase FLOww - HOWEVER a large amount or surface modified with surfactant will create _ charge bilayer So the charge facing the ions is + which will THEN make an anion layer and moved towards the anode and reversing the flow)
WHAT ARE THE TWO WAYS OF INJECTING ON CE
Hydrodrynamic - use pressure
Electrokinetic - uses electric field to drive sample to capillary HOWEVER - there is a bias for highly mobile species then
What is ELECTROKINETIC SAMPLE STACKING
So in the capillary - there is consistent flow across BUT at the injection band - there actually is greater electric field here across it due to it being LOW conductivity - lower resistance than the buffer
THIS causes a natural separation - IN the band for cations to the cathode side and the anions to concentrate on the anode side of the band
which is good - increases signal intensity instead of being diffuse across the band?
How does an ABSORBANCE detector work
small volume - very much like UV - z shaped - read in the middle part of the Z- same as LC - not very good not very sensitivie
How does an LIFE detector work
Fluorscence again
LASER INDUCED - dynamic range good
CE-ECD
same thing?
4 CE detectors
ECD, MS, LF, Absorbance
WHAT is MEKC
Micellar electrokinetic chromatography -allows us to also separate based on lipophilicty and neutrals
Use a surfactant liek SDS to form micelles which are NEGATIVELY charged - move towards ANODE but EOF will ultimately bring it to the cathode BUT we are including our neutrals now into the separation (longer time in micelle - slower to elute)
Band broadening in MKEC
C term added back in mass transfer to maicelle BUT is fairly fast and modest
MKEC vs HPLC
SP change is a lot quicker - just put in a new surfactant
much higher column efficiency than HPLC
What is CEC
capillary electrochomratogrphy
-electrosomotically driven LC tehcnique
Uses a packed column get pumping effect BUT band is more plug like flow helps with band braodening
-so has a stationary phase and can do neutral solutes through a mix of PUMPING and PARTITION
DEscribe CEC stationary phase and mobile phase
will be a mix of normal (liek C18 - 90%) and the silica needed for stabilization of EOF
MP als a mix - have some ACN and instead of just water - a buffered solution
Describe slab gel electorphoresis
seperate biomolecules based on size - baseically DNA charged on backbone negative (consistent charge to size ratio) so electric field - head towards anode
ENTROPIC
3 DNA regimes! in electorphoresis
OGSTON SIEVING - in a sparse gel - the DNA can coil up and still get through
ENTROPIC TRAPPING - heterogenous gel - so DNA will coil up but need to deform to pass through and then maybe coil again
REPTATION- must completely stretch out to go
What are the major equations and terms relating to DNA movement in a gek
So we have U = free solution mobility
but the actually mobility is less SO
log(u) = log (U) -Kr*C
of which C is the fractional gel volume available
K is the retardation factor
U is our free solution mobility
and u is our actual mobility
What is a ferguson plot
It is log (u) vs gel %
and generally it goes down as gel % increases HOWEVER - as molecular weight increases K or retardation factor goes up so speed goes down at aFASTER rate
How do you use ferguson plots to idetnnfiy ogston sieving?
plot Log u AGAINST molecular weight - assuming standard K (which si the slope then - standard retardation factor)
How does mobility vary with M
PLOT is log U /u ) vs log (M)!As M goes up mobility goes down and we see these various phases starting with ogston sieving to reputation without stretching to reputation with stretching
aLONG THIS plot WWE have various dependence on E and M we want dependace on M and not E E should be constant
SO at large M - It becomes E dependant and not M
and middle lengths its M dependant and not E (what we want - I guess when it gets too big levels off - to the reputation with stretching region - at this point SIZE doesn’t matter because they all reptate the same way - its still just a thread kind of
SO I GUESS reputation with stretching is the more discerning part
WHAT IS CGE
capillary with porous gel for molecular sieving
needs to be a porous non cross linked
CGE concerns
EOF must be supressed
can use polyacrylamide, AGAROSE or methyl cellulose
used for DNA
what is CIEF
capillary isolectic focusing - basically pH gradient - proteins travel until reach pI point so stopped
What is the mobile phase used in IEC
so low strenght has few ions that could displace our sample but higher strength has MORE ions that would bing to the SP
What do IEC phases prefer among molecule traist
CHARGED and SMALL
P = Z^2 /r
but regardless of this ITS always concentration based so can use large concentration to regenerate
Things to watch out for in Vg calc
Delta Hs need to be in cals
R = 1.987
