Chromatography Flashcards
What is chromatography?
A separation method in which the components to be separated are distributed between two phases, the stationary phase and mobile phase.
What is the general process of chromatography?
Sample is dissolved in a mobile phase. Mobile phase is then forced through an immobile, immiscible stationary phase. Components of each phase have different solubilities. A component very soluble in stationary phase will take longer to travel through it. Separation is due to different solubilities.
With the plate model theory, greater separation occurs when..
*There is a greater number of theoretical plates (N)
*A smaller plate height (H or HETP)
What is a limitation of the plate theory model?
It neglects the concept of solute diffusion and flow paths.
Difference between the plate model theory and rate theory of chromatography.
Plate model assumes equilibrium is infinitely fast whereas rate theory takes into account the time taken for solute to equilibrate between the two phases.
According to the rate theory, what is the band shape of the chromatogram affected by?
*rate of elution (time of separation)
*the different paths available to solute molecules as they travel between particles of stationary phase
What causes broadening of the solute band of the chromatogram?
*Solute molecules travelling through different paths through the stationary phase (A term/eddy diffusion) due to different path lengths.
*analyte diffusing from centre to edges (B term/longitudinal diffusion)
How do you reduce the affects of resistance to mass transfer (C term) in chromatography?
*use a less viscous phase
*keep flow as low as possible- keeping B term (longitudinal diffusion) into consideration (b term is flow dependent)
*use thin coatings of the stationary phase on a solid support
What is the rate theory equation and what does each term mean?
H= A+B/u+C/u
H= height of plate
A=eddy diffusion
B=longitudinal diffusion
C=resistance to mass transfer
u= average velocity of mobile phase
What is the equation used to calculate the number of theoretical plates using the plate model theory and what does each term mean?
N= 5.54(tR/W1/2)^2 or N= 16(tR/Wb)^2
tR=retention time
W1/2= width of peak at half height
Wb=peak width at base
N= number of theoretical plates
What is HPLC?
High performance liquid chromatography (or high pressure liquid chromatography)
*separates, identifies and quantifies any compound that can be DISSOLVED IN A LIQUID.
*the use of high pressure to push a mobile phase solution through column of stationary phase allowing separation of COMPLEX mixtures
How is HPLC carried out?
*small volume of liquid sample injected into a tube (column) packed with tiny particles (3-5uq diameter) = stationary phase
*individual components are pushed through column with a liquid mobile phase using high pressure delivered by a pump.
*separation due to; column packing involving various chemical/physical interactions between molecules and packing particles
*separated particles detected at exit of column by a detector that measured their amount. Detector outputs a ‘liquid chromatogram’
What are the 5 types of HPLC?
*Normal phase- separation of polar analytes by partitioning onto polar bonded stationary phase
*Reversed phase- separation of non polar analytes by partitioning onto non polar bonded stationary phase
*Adsorption-in between first two phases. Separation of moderately polar analytes using ADsorption onto pure stationary phase (silica)
*Ion chromatography- separation of organic and inorganic compounds by partitioning onto ionic stationary phase bonded to solid support
*Size exclusion chromatography-separation of large particles based on path they take through maze of tunnels in stationary phase
How does normal phase chromatography work?
*silica stationary phase has silanol (Si-OH) groups on its surface
*polar analytes migrate slowly through phase due to STRONG interactions with silanol groups
*useful to separate non polar compounds and isomers
*DISADVANTAGE- easy contamination of polar silanol groups by sample components
How does reverse phase chromatography work?
*MOST POPULAR METHOD
*non polar analytes react with hydrophobic C18 groups on silicon surface- polar analytes pass through
