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
How does Ion-Exchange chromatography work?
*exchange of ions with counter ions which are attached to solid silica support
*cationic exchange or anionic exchange
*used to analyse biological components such as amino acids, proteins
How does size- exclusion chromatography work?
*based on analytes molecular size
*large molecules cannot penetrate pores- migrate quickly
*small molecules can penetrate pores- migrate slowly
What parameters affect HPLC results?
*Sample parameters- concentration, volume of injection
*mobile phase- solvent, flow rate/ pressure, pH of solvent
*column parameters- column material, column material particle size, column length/diameter
*detector- wavelength, sensitivity
How should the mobile phase be prepared?
*use highly purified buffer salts and reagents (HPLC grade preferred)- any impurities can lead to chromatographic artefacts or small particles can collect in narrow tubing or column to cause a void,
*degass solvents- small gas bubbles can collect in pump head and detector-void. Effects separation process, baseline drift and peak shape- spikes
*filter solvent- remove particulate matter that can cause wear to pumping system.
How is degassing carried out?
*Vacuum filtration- not always reliable
*Helium Purging- very effective, helium expensive
*ultrasonication- converts ultra high frequency to mechanical vibrations- removes air bubbles.
What factors effect mobile phase performance?
*solvent strength and selectivity
*pH
*flow rate
*column temp
What does solvent strength refer to?
The ability of a solvent to elute solutes from a column
Increased solvent strength=increased elution
In RPC
Increased organic strength=increased elution
Water is weak=increased retention
Strength depends on polarity
In NPC
Water is strong=decreased retention
How do you calculate % ionised of a molecule?
% ionised= 100/ 1+10^(charge(pH-pKa))
Charge; bases=1 acids=-1
How does temperature and flow rate affect HPLC?
Higher temp= reduced viscosity= reduced retention time
Higher flow rate=reduced retention time and analysis time
What two types of pumps are used in HPLC?
*constant pressure pumps
*constant flow pumps (most common) both provide ISOCRATIC compositions
Split into
Single piston reciprocating pump-expels liquid through one way check valve
Double piston reciprocating pump-provides constant almost pump free flow
What is isocratic elution?
When same mobile phase used throughout elution of entire sample
*used for simple separations
* used in quality control applications
What is gradient elution?
Strength of mobile phase increased with time during sample elution
*best for complex samples
*used in method development for unknown mixtures
What are characteristics of the HPLC column?
*stainless steel tube filled with small particle packing
*50-300mm long
*internal diameter= 4/4.6mm
What are characteristics of an ideal HPLC detector?
*high sensitivity
*negligible baseline noise
*convenient, reliable
*inexpensive to purchase and operate
What is derivatization and when is it done?
A chemical reaction of compound to another product to improve its ability to be analysed
*pre column deriv- before separation
*post column deriv- after separation
*deriv compound then injected into column
What is an example of derivatization?
Attaching a chromophore to improve detection sensitivity of a low detection molecule.
Used in fluorescent spectroscopy- allows detection for non-fluorescent molecules to fluorophores.
