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Chem Bio > CHEM SEP final 2 > Flashcards

CHEM SEP final 2 Flashcards

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1
Q

Moment Analysis (Method of Moments)
*

A

Every distribution function (Gaussian or otherwise) has a set
of statistical moments characterizing its position and shape.

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2
Q

Go through The different variations you can have with moment analysis

A

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

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3
Q

What are the 4 sample moments

A

Mean, variance, Skewness, kurtosis

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4
Q

4 sample moments how to write equations for each

A

Review how to write equations for each

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5
Q

How to interpret skewness?

A

0 - gaussian, > 0 is fronting (right side) and < 0 is tailing (left side)

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6
Q

How to interpret kurtosis?

A

excess = kurtosis - 3; gaussian is 3 , smallest is 1 (makes it a rectangle) - higher you go the more narrow (a measure of how flat)

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7
Q

GC can analyze compounds that are ..?

A

Voltalie or can be derivitized to become volatile

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8
Q

Parts of a GC system

A

: a gas source with pressure
and flow regulators, an injector, a column in an oven, a detector, and a
computer for data acquisition.

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9
Q

So GC using a compressible fluid means what

A

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

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10
Q

In GC what determines the change in rate of velocity from inlet vs outlet

A

Ratio of Pressure in/Pressure out - the higher - the steeper the change close to end of column

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11
Q

Know how to derive pressure in out equation equation

A
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12
Q

Difference in GC velocity van deemter vs experiemntal

A

in van deemter it is average ; in experimental its at the outlet

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13
Q

What are 3 things that need to be corrected for , for experimental GC average flow rate/veloctiy measurement

A

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)

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14
Q

2 ways to determine average velocity in GC

A

Multiply measured velocity out by j, pressure correction factor and temp correction factor OR just use retention time (L/rt))

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15
Q

How is volume corrected for in GC

A

Similarly since based off flow and we know flow is affected - need correction terms j for compressibility , NET parameters are tr- tm, Vr - vm

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16
Q

What is corrected and NET volume in GC

A

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

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17
Q

What is specific retention volume

A

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

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18
Q

What is specific retention volume related to in thermodyanmic terms

A

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(

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19
Q

How is GC from other chromatographic methods in terms of retention

A

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

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20
Q

How is chemical potential split for GC

A

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)

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21
Q

What is selectivity in GC sepeartion based off of and why

A

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

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22
Q

What is yis dependant on

A

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

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23
Q

Distribution coefficeint in GC

A

(moles in SP / Weight SP) / (moles MP / corrected mobile phase volume) (in other words MOLALITY / MOLARITY)

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24
Q

No how to derive thermodynamics of retention showing that Specific retention volume depends on yis and Pio (inverse related)

A
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25
Q

What is yis and what are its values

A

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

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26
Q

in GC how does concetration relate to yis

A

kept low to keep yis constant across run

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27
Q

What is yis 2 contributors

A

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)

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28
Q

What is the trouton rule

A

If solutes are not too polar can estimate enthalpy of evaporation as 22* boiling point

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29
Q

For our specific retetnion time related to boiling point equation - which assumptions must be true

A

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)

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30
Q

What are good qualities of a carrier gas

A

oxygen free, moisture free, no hydrocarbon impuriteis and chemically inert

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31
Q

common carrier gases

A

N2, He, H2, Ar, O2 and CO2

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32
Q

Factors in choosing GC mobile phase

A

detector response (eg H2 and O2 as burner), efficiency and speed, stability, hazards , availability/expense

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33
Q

How does viscosity function in GC temp preogramming

A

As we increase temp - viscosity increases and average linear velocity decreases

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34
Q

What gas is best for capillary GC

A

H2 - for diffusivity and broad working range

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35
Q

Types of GC column

A

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

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36
Q

Capillary vs Packed bed GC

A

Capillary is much more efficient

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37
Q

What is phase ratio and how does it effect GC van deemter

A

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

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38
Q

In thin film columns talk about van deemter, maxes and what it means for u opt and hmin

A

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

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39
Q

Good GC Sp characteristics

A

unreactive with carrier gas and solute, low vapor pressure and viscosity, good coating, solubility, wide temp operating range

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40
Q

Common stationary phase types in GC

A

non polar - hydro carbon and perfluorocarbon
polar - ether and esters
specially (high temp and chiral

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41
Q

How to choose SP based on retention index

A

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

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42
Q

KOVATS RETENTION INDEX explain

A

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

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43
Q

McREYNOLDS -phase constants explain

A

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

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44
Q

3 ways to pick SP for GC and differences

A

Mcreynoalds, -properties of specific molecules
Kovaks and
TAFT KAMLET - uses solvatochromic params

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45
Q

what are GC PLOT colmns used for

A

seperating hydrocarbons - molecular sieves

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46
Q

What is desired in GC detetor

A

Tempreature range and short response time independant of flow rate are the majors (then typical things, sensitive, stable, reliable etc)

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47
Q

TCD detector (thermal conductive) describe function

A

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

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48
Q

FID - describe

A

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

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49
Q

NPD describe it

A

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

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50
Q

ECD describe it

A

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

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51
Q

Beneftis of LC vs GC

A

sample just needs to be soluble - more compounds
get selectivity across SP and MP
most detectors non destructie
BUT LC - has worse band broadening

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52
Q

Know the knonx equation

A

VAN DEEMTER BUt
include the x term Av^1/3 instead of justeddy diffusion

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53
Q

Whats dominan tvan deemter force in LC

A

STAGNANT MOBILE PHASE _ mass stransfer for SP - 3050% of H

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54
Q

What is total plate height in LC from

A

eddy, londituginal, mass transfer AND EXTRA COLUMN BAND BRAODENING - tubes, detector plumbing etc

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55
Q

What materials are used to increase pH range LC

A

Al2O3, TiO2, and ZrO2

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56
Q

What is HPLC SP bound to

A

Si-OH covalent bond

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57
Q

Why are columns generally bound at pH ranges and what can extend the range

A

Siloxane - bond hydrolyzes below pH 2
-BULKY ISOBUTLYLGROUPS - can protect it

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58
Q

What LC SP has both polar and non polar retention

A

Cyano

59
Q

General order of reverse phase retentivitiy

A

Cyano < C4< Phenyl< C8< C18

60
Q

LC system general schematic

A

solvent reservoir - gelium source -> pump -> injecting -> filer -> pressure -> column -? detector

61
Q

wHAT ARE THE TWO TYPES OF lc PUMPS and how do they work

A

DISPLACEMENT -
RECIPROCATING

62
Q

How do displacement pumps work

A

Has a screw that as it screws down - mobile phase pushes up -
NO PULSE
but small capacity

63
Q

How do reciprocating pumps work

A

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)

64
Q

What are the two types of LC detectors

A

BULK PROPERT and analyte
kind of what they sound like
bulk property is like refractive index, di electric constant, conductivity
analyte is everything normal

65
Q

HOW DO RI detectors work and benefits and cons

A

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

66
Q

UV vis equations

A

T - P/Po
Absorbance - -log(T)
A - ecl

67
Q

UV vis absorbance detector designs

A

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

68
Q

What is a flow cell detector (UV)

A

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

69
Q

Fluorescence detector benefits

A

SELECTIVE
HIGH SENSTIVITY
Cons - not everything fluoresces so may require dervitization

70
Q

How does fluorescence detector work

A

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

71
Q

Fluorescence or phosphorescence band comapred to the absorption?

A

Its higher (because lost energy - it’s like a mirror image shifted up (because longer wavelength))
T

72
Q

What is stokes shift? and why

A

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

73
Q

How does UV geometry vary from fluoresence

A

UV can be 180 degree in line, FLuorescence is 90 degrees

74
Q

What is the “Hook Effect”

A

IN fluroescence if concentration too high - falls off pretty quick due to get 2ndary absorption

75
Q

Electrochemical Detector explain

A

detects ability of solution to be reduced or oxidized
Setup : working, counter and reference electrode
Reference is Ag AGCl - stable Electrochem potential

76
Q

Amperometric detector stats

A

hgih sesntivity, convenience, broad
CONS: MP must be conductive, prone to interference and less repdouicble

77
Q

Whats LC attribute of good seperation

A

k for all peaks between 0.5 and 20

resolution > 2..?
assyemtry between 0.9 and 1.5

78
Q

How to optimize isocratic retention

A

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

79
Q

What is the solvent selectivity triangle

A

small set of 10 experiments to optimize mobile phase trying proportions of different solvents
the corners are H BONDING, DIPOLE DIPOLE and proton acceptor

80
Q

How to decide if you should use gradient or isocratic

A

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

81
Q

Explain dwell volume and dwell time and they role in reducing gradient time

A

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

82
Q

Ion exchanger - types of stationary phase supports(not active groups) and why

A

REsins - small molecule (highly crosslinked - means denser)
Gels - for biomolecules and proteins and nucleic acids
Inroganic - for harsh chemical conditions

83
Q

What are the groups used for ion exchanges

A

For cation excchange use carboxylic acids or sulfonyl group (SO3-)
For anion - quaternary amine or ammonium hydroxide

84
Q

Polarizing power - what is it

A

P = Z^2/r it’s essneitally how favored something is for ion exchange - its higher charge and smaller hydrated radius

85
Q

How do you regenerate an ion exchange column

A

wash with something ike H+ (if it is attached to Na+ you can replace it

86
Q

Whats ion exclusion chormatoraphy

A

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

87
Q

applications of IEC

A

salt exchange , water purification

88
Q

LC vs GC

A

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

89
Q

What is bonded SP and what are some normal phase SP’s

A

bonded to the Si-Oh - normal phase includes, amide, amino , cyano diol etc

90
Q

Why does the efficiency (plate height) of liquid chromatography increases as the stationary-phase particle size is reduced?

A

For H - from SP - its (K term) * dp^2 * v / Dm
so as dp goes down H goes down;

91
Q

2 types of (SEC, or gel filtration or gel permeation)

A

GROUP seperation (components seperated into major size groups
High res seperation/fracitonation - separated according to molecular size

92
Q

Explain the basics of thermodynamic SEC (enthalpy vs entropy factors)

A

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

93
Q

What is K in SEC

A

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

94
Q

What is K in SEC? do for cylinder

A

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)

95
Q

What is K in SEC for an approximation of a variety of pore shapes and what do things mean

A

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)

96
Q

What is the random plane model of pore space

A

rigid moelcules of complex shape - accounts for complex molecules and their project length along various axes
K = e^(-sL/2)

97
Q

GPC vs GFC?

A

gel perpeation vs gel filtration (polymer vs biochem

98
Q

SEC common stationary phases and considerations

A

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)

99
Q

Total movile phase volume in SEC column

A

Vm = Vi + Vo
Vo is interstitial volume
Vi is pore volume

100
Q

What is RETETNION volume in SEC - and what is it based on

A

Vr = Vo + K*Vi based on the idea that only part of the Vi is accessible to a solute

101
Q

What is K in SEC in terms of volumes and what does it depend on

A

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

102
Q

Interpret K for SEC - what do various values mean eg 0, 1

A

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

103
Q

How to determine molecular mass from SEC?

A

Use calibration curve (log of MW) vs elution volume

104
Q

How does pore size effect SEC

A

finer mass resolution, faster elution, narrower mass range

105
Q

In CE - no factors- what ions go where

A

cations to cathode and anions to anode (electrons I guess go to cathode and cations follow:

106
Q

What is electrophoretic mobility and what is the equation and contributing factors

A

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

107
Q

Explain the diffuse double layer

A

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

108
Q

Explain how diffuse double layer causes electroosmotic flow

A

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!

109
Q

Explain electroOSMOTIC mobility and the terms it relates to

A

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

110
Q

How can one manipulate the EOF

A

pH (via zeta), ionic strength, surfacant and surface modification

111
Q

What is the coutner ion cloud and does it matter

A

an ion in solution will have counter ions surrounding it

112
Q

Describe the apprent mobility of various ion types

A

comes down to electrophoretic mobility vs electrosomtic mobility
For cations its additive
for neutras its just electroosmotic mobility
and for anions its electrosomotic - electrophoretic

113
Q

VAN DEEMTER FOR CE

A

No A term, No C term ALL B TERM - all longitudinal diffusion which means it stays pretty darn flat

114
Q

Heigh in theoretical plates for CE

A

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

115
Q

Resolution for CE

A

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

116
Q

Whats Joule heating, how does ti affect CE and how can we minimize it

A

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

117
Q

Buffers in CE - what are some issues

A

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

118
Q

How do the following effect our CE method
Electric field
IOnic strength
Organic modifiers
Surfactant
Temperature

A

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

119
Q

How to deal with EOF flow change with pH change

A

Use a surface coating like polybrene + dextran sulfate - allows as the pH increases to maintain consistent flow

120
Q

What does using a cationic surfactant do to CE

A

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)

121
Q

WHAT ARE THE TWO WAYS OF INJECTING ON CE

A

Hydrodrynamic - use pressure
Electrokinetic - uses electric field to drive sample to capillary HOWEVER - there is a bias for highly mobile species then

122
Q

What is ELECTROKINETIC SAMPLE STACKING

A

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?

123
Q

How does an ABSORBANCE detector work

A

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

124
Q

How does an LIFE detector work

A

Fluorscence again
LASER INDUCED - dynamic range good

125
Q

CE-ECD

A

same thing?

126
Q

4 CE detectors

A

ECD, MS, LF, Absorbance

127
Q

WHAT is MEKC

A

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)

128
Q

Band broadening in MKEC

A

C term added back in mass transfer to maicelle BUT is fairly fast and modest

129
Q

MKEC vs HPLC

A

SP change is a lot quicker - just put in a new surfactant
much higher column efficiency than HPLC

130
Q

What is CEC

A

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

131
Q

DEscribe CEC stationary phase and mobile phase

A

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

132
Q

Describe slab gel electorphoresis

A

seperate biomolecules based on size - baseically DNA charged on backbone negative (consistent charge to size ratio) so electric field - head towards anode
ENTROPIC

133
Q

3 DNA regimes! in electorphoresis

A

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

134
Q

What are the major equations and terms relating to DNA movement in a gek

A

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

135
Q

What is a ferguson plot

A

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

136
Q

How do you use ferguson plots to idetnnfiy ogston sieving?

A

plot Log u AGAINST molecular weight - assuming standard K (which si the slope then - standard retardation factor)

137
Q

How does mobility vary with M

A

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

138
Q

WHAT IS CGE

A

capillary with porous gel for molecular sieving
needs to be a porous non cross linked

139
Q

CGE concerns

A

EOF must be supressed
can use polyacrylamide, AGAROSE or methyl cellulose
used for DNA

140
Q

what is CIEF

A

capillary isolectic focusing - basically pH gradient - proteins travel until reach pI point so stopped

141
Q

What is the mobile phase used in IEC

A

so low strenght has few ions that could displace our sample but higher strength has MORE ions that would bing to the SP

142
Q

What do IEC phases prefer among molecule traist

A

CHARGED and SMALL
P = Z^2 /r
but regardless of this ITS always concentration based so can use large concentration to regenerate

143
Q

Things to watch out for in Vg calc

A

Delta Hs need to be in cals
R = 1.987

Chem Bio (17 decks)

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