LC Flashcards
factors of resolution
efficiency
selectivity
retention
band broadening factor/van Deemter equation
- eddy diffusion
- longitudal diffusion
- resistance to mass transit
LC instrumentation
- solvent reservoir
- pump
- injector
- column
- detector
- computer
Particle Size Advantages
- 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
particle size disadvantages
- higher back pressures for given flow rate (increases faster than resolution)
Superficially porous (solid core) particles
- solid core reduces diffusion into particles
- lower eddy diffusion (A term)
- less band broadening
- higher efficiency and resolution
- without higher pressures
Limitations of Silica
limited to pH 3-9
unreacted silanols cause unwanted retention and band broadening of polar analytes
Increasing Column Length Advantages
- Increases interaction between phases
- better separation/selectivity
Decreasing column Diameter Advantages
- decreases path length variation
- smaller eddy diffusion term
- less band broadening
- higher efficiency and resolution
Monolithic Column details
- single interonnected network of fibres instead of particles
- polymer or silica
Monolithic Column Advantages
- 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
PLOT Columns Details
Porous Layer Open Tubular
hollow column
stationary phase coated surface
PLOT Advantages
Hollow means less pressure
can be longer
increased interaction between phases
less band broadening (eddy diffusion/A term)
potentially better separations
Eddy Diffusion Factors
column packing factor
particle diameter
is constant for a given column
Longitudal Diffusion Term Factors
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
Resistance to mass transit
particle diameter
flow rate
inversely proportional to diffusion coefficient
function of retention, k
higher at higher flow rates
Normal Phase Chromatography phases
Liquid mobile phase
solid/liquid stationary phase
non-polar/less polar mobile e.g hexane
polar stationary e.g. silica
Normal Phase Elution
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
Reverse Phase Chromatography phases
Liquid mobile phase
solid stationary phase
polar mobile phase (water/acetonitrile)
non-polar/less polar stationary phase (C8/C18)
Reverse Phase Elution
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
Reverse Phase Advantages
aqueous mobile phase means
- can vary pH
- can add additives to effect separations
Solvent Properties
Dielectric Constant - measure of polarity
viscosity - affects back pressure
surface tension - energy to dissolve analyte
Solvent on Separation
- affects retention and selectivities
- change order
- higher elution strength means faster elution shorter runtime
Alternative Stationary Phases
Inorganic Oxides e.g alumina
polymers
graphite carbon
hybride silica/polymer
nano-diamonds
Transfer Rules
phase fractions of organic modifier for isoeluotropic mobile phases
e.g ratio of methanol to acetonitrile
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
Temperature Effect
Changes solvent properties (decreases polarity, viscosity, ST)
increases energy of molecules
Temperature Separtion
- decreased retention
- decreased resistance to mass transit
- higher diffusion (B term)
Temperature Advantages
- larger optimal flow rates
- faster separations
- lower back pressures
- higher efficiency
- doesn’t affect selectivity
- maintain separation but faster
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
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
Dynamic Ion Exchange
Counter ion adsorbed to column
react with ionic analytes binding them to column
competitiviely eluted over time leading to separation
Detectors
UV/Visible
Mass Spec
Refractive Index
Evaporative Light Scattering
Potentiometry/Amperometric
Conductivity
Fluorescence
have different detection limits
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
Solvent characteristics
dipole interaction
proton acceptor
proton donor
IEC retention increases with
selectivity/equilibrium coefficient
ion exchange capacity
mass of startionary phase
charge of analyte
size of analyte
IEC retention decreases with
charge of eluent
concetration of eluent
volume of mobile phase
Reverse Phase phases
MP: polar (aqueous w/ organic)
SP: non-polar (C18)
mechanism: solvophobic
Reverse Phase analytes
organics, proteins/peptides, aromatics, organic acids and bases
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
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
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
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
Ion Suppression
- exchange eluent counter ion e.g. Na+, with H+/OH- by ion exchange membrane
- react with eluent ion to condense water
Suppressed Conductivity Benefits
- reduces baseline
- direct detection
- higher sensitivity
- no climbing baseline with gradient
- bigger peaks
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
Online Eluent Generator
Creates high purity eluent by electrolysis of water
pulls counter ion from membrane
allows for gradient elution
Ion Exchanger groups
cation exchangers: acids (COO-)
anion exchangers: aminos (NR3+)
