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LC Flashcards

1
Q

factors of resolution

A

efficiency

selectivity

retention

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2
Q

band broadening factor/van Deemter equation

A
  1. eddy diffusion
  2. longitudal diffusion
  3. resistance to mass transit
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3
Q

LC instrumentation

A
  1. solvent reservoir
  2. pump
  3. injector
  4. column
  5. detector
  6. computer
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4
Q

Particle Size Advantages

A
  • decreases eddy diffusion and resistance to mass transit (A and C terms)
  • less band broadening
  • higher efficiency and resolution
  • larger range of optimal flow rates (smaller C)
  • shorter columns with same resultion
  • faster analysis with same resolution
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5
Q

particle size disadvantages

A
  • higher back pressures for given flow rate (increases faster than resolution)
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6
Q

Superficially porous (solid core) particles

A
  • solid core reduces diffusion into particles
  • lower eddy diffusion (A term)
  • less band broadening
  • higher efficiency and resolution
  • without higher pressures
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7
Q

Limitations of Silica

A

limited to pH 3-9

unreacted silanols cause unwanted retention and band broadening of polar analytes

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8
Q

Increasing Column Length Advantages

A
  • Increases interaction between phases
  • better separation/selectivity
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9
Q

Decreasing column Diameter Advantages

A
  • decreases path length variation
  • smaller eddy diffusion term
  • less band broadening
  • higher efficiency and resolution
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10
Q

Monolithic Column details

A
  • single interonnected network of fibres instead of particles
  • polymer or silica
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11
Q

Monolithic Column Advantages

A
  • Convection based flow (instead of diffusion)
  • less diffusion (B term)
  • less resistance to mass transit (C term)
  • lower back pressures
  • faster runtimes
  • less band broadening
  • larger range of optimal flow rates
  • can functionalise polymer
  • can produce monolithic column in any shape required
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12
Q

PLOT Columns Details

A

Porous Layer Open Tubular

hollow column

stationary phase coated surface

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13
Q

PLOT Advantages

A

Hollow means less pressure

can be longer

increased interaction between phases

less band broadening (eddy diffusion/A term)

potentially better separations

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14
Q

Eddy Diffusion Factors

A

column packing factor

particle diameter

is constant for a given column

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15
Q

Longitudal Diffusion Term Factors

A

obstruction factor of the column

diffusion coefficient of analyte in mobile phase

inversely proportional to flow rate

high at low flow rates smaller at large flow rates

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16
Q

Resistance to mass transit

A

particle diameter

flow rate

inversely proportional to diffusion coefficient

function of retention, k

higher at higher flow rates

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17
Q

Normal Phase Chromatography phases

A

Liquid mobile phase

solid/liquid stationary phase

non-polar/less polar mobile e.g hexane

polar stationary e.g. silica

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18
Q

Normal Phase Elution

A

non-polar analytes elute first

polar analytes are retained by polar column

higher polar mobile phases have higher elution strength

Competitive elution mechanism

best for polar organics

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19
Q

Reverse Phase Chromatography phases

A

Liquid mobile phase

solid stationary phase

polar mobile phase (water/acetonitrile)

non-polar/less polar stationary phase (C8/C18)

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20
Q

Reverse Phase Elution

A

Polar analytes elute first

Non-polar analytes retained

increasing organic modifier increases elution strength (decreases polarity)

solvophobic adsorption mechanism

best for non-polar analytes soluble in organics

21
Q

Reverse Phase Advantages

A

aqueous mobile phase means

  • can vary pH
  • can add additives to effect separations
22
Q

Solvent Properties

A

Dielectric Constant - measure of polarity

viscosity - affects back pressure

surface tension - energy to dissolve analyte

23
Q

Solvent on Separation

A
  1. affects retention and selectivities
  2. change order
  3. higher elution strength means faster elution shorter runtime
24
Q

Alternative Stationary Phases

A

Inorganic Oxides e.g alumina

polymers

graphite carbon

hybride silica/polymer

nano-diamonds

25
Transfer Rules
phase fractions of organic modifier for isoeluotropic mobile phases e.g ratio of methanol to acetonitrile
26
Gradient Elution
Increase elution strength of mobile phase over runtime by increasing organic modifier Advantages - shorter runtimes - less diffusion/band broadening of slower analytes - better selectivity - better efficiency
27
Temperature Effect
Changes solvent properties (decreases polarity, viscosity, ST) increases energy of molecules
28
Temperature Separtion
- decreased retention - decreased resistance to mass transit - higher diffusion (B term)
29
Temperature Advantages
- larger optimal flow rates - faster separations - lower back pressures - higher efficiency - doesn't affect selectivity - maintain separation but faster
30
Ion Suppression
use low pH (below pka) buffer to protonate analytes neutral molecules have stronger retention than ions in reverse phase better separation efficiency and resolution good for weak acids only may affect silica though
31
Ion Pair Model
add organic counter ion to mobile reacts with ionic analyte to form neutral ion pair the ion pair interacts more strongly with the column better separation good for strong acids etc
32
Dynamic Ion Exchange
Counter ion adsorbed to column react with ionic analytes binding them to column competitiviely eluted over time leading to separation
33
Detectors
UV/Visible Mass Spec Refractive Index Evaporative Light Scattering Potentiometry/Amperometric Conductivity Fluorescence have different detection limits
34
Ion Exchange Chromatography Details
MP: electrolyte with eluent ion and co ion SP: charged ion exchanger, oppositely charged to analyte Mechanism: competitive equilibrium between analyte and eluent ion for ion exchanger sites Analytes: inorganic ions, organic acids and bases, proteins/nucleic acids
35
Solvent characteristics
dipole interaction proton acceptor proton donor
36
IEC retention increases with
selectivity/equilibrium coefficient ion exchange capacity mass of startionary phase charge of analyte size of analyte
37
IEC retention decreases with
charge of eluent concetration of eluent volume of mobile phase
38
Reverse Phase phases
MP: polar (aqueous w/ organic) SP: non-polar (C18) mechanism: solvophobic
39
Reverse Phase analytes
organics, proteins/peptides, aromatics, organic acids and bases
40
Normal Phase phases
MP: less/non-polar e.g. hexane SP: polar e.g silica, amino, diol mechanism: competitive adsorption analytes: polar organics soluble in organics
41
HILIC details
MP: organic w/ %water SP: highly polar zwitterionic e.g. diols, aminos, silica Mechanism: partitioning, electrophilic Analytes: small highly polar organics e.g. sugars, nucleotides
42
Affinity details
Used in purification MP: bind/elute buffer SP: polymer/biopolymer solid support and receptor Mechanism: selective ligand/receptor interactions Analytes: proteins/dyes, enzymes/substrates
43
Size Exclusion Details
MP: solvent (aqueous/THF) SP: particle packed Mechanism: sieving/size exclusion, bigger molecules excluded from smaller channels, diffuse less, retained less Analytes: polymers, biopolymers, proteins
44
Ion Suppression
- exchange eluent counter ion e.g. Na+, with H+/OH- by ion exchange membrane - react with eluent ion to condense water
45
Suppressed Conductivity Benefits
- reduces baseline - direct detection - higher sensitivity - no climbing baseline with gradient - bigger peaks
46
Non suppressed conductivity details
if analyte conductivity less than eluent then indirect if analyte conductivity greater than eluent then direct sensitivity dependent on conductivity differences
47
Online Eluent Generator
Creates high purity eluent by electrolysis of water pulls counter ion from membrane allows for gradient elution
48
Ion Exchanger groups
cation exchangers: acids (COO-) anion exchangers: aminos (NR3+)

Analytical (7 decks)

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