Chromatography Flashcards
is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium
Chromatography
a solvent that flows through the supporting medium
Mobile phase
a layer or coating on the supporting medium that interacts with the
analytes
Stationary phase
a solid surface on which the stationary phase is bound or coated
Supporting medium
The analytes interacting most strongly with the stationary phase will take longer to pass through the system than those with weaker interactions. What does this mean?
High affinity
GC; type of stationary phase
1. Gas-solid chrom.
2. Gas-liquid chrom.
3. Bonded-phase gas chrom.
- solid, underivatized support
- liquid- coated support
- chemically-derivatized support
LC; type of stationary phase
1. Adsorption chrom.
2. Partition chrom.
3. Ion-exchange chrom.
4. Size exclusion chrom.
5. Affinity chrom.
- solid, underivatized support
- liquid coated/underivatized support
- support containing fixed charges
- porous support
- support w/ immobilized ligand
tr
tm
Wb
Wh
retention time
void time or dead time
baseline width of peak
half height width of peak
The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
system)
The volume of the mobile phase that it takes to elute a peak off of the column.
Retention volume (Vr)
Is directly related to the
strength of the solute’s interaction with the mobile and stationary phases
Retention volume (Vr)
The amount of mobile phase that it takes to
elute a non-retained component.
Void volume (Vm)
Theory of chromatography:
- Typical response obtained by chromatography (i.e., a chromatogram)
- Solute retention
- Efficiency
Retention on a given column pertain to the particulars of that system:
- size of the column
- flow rate of the mobile phase
Column length formula/ average linear velocity
v = L/tr
Capacity factor (k’): more universal measure of retention, determined from tr or Vr
k’ = (tr -tm)/tm
k’ = (Vr - Vm)/Vm
fundamental definition of k’
k’ = moles A (stationary phase)/ moles A (mobile phase)
k’ is directly related to the strength of the interaction between a solute with the stationary
and mobile phases.
represents the amount of solute present in each
phase at equilibrium
Moles A (stationary phase) and moles A (mobile phase)
When k’ is less than or equal to 1.0,
When k’ is > 30,
When k’ is = 2-10,
separation is poor
separation is slow
separation is optimum
equilibrium constant for the distribution of A between the mobile
phase and stationary phase
Kd
Assuming local equilibrium at the center of the chromatographic peak:
k’ = [A]stationary phase Volume stationary phase/ [A] mobile phase Volume mobile phase
k’ = Kd Volume stationary phase/ Volume mobile phase
As KD
increases, interaction of the solute with the stationary phase becomes more
favorable and the solute’s retention (k’) increases
peak separation also represents different changes in free energy
is related experimentally to a solute’s peak width
- an efficient system will produce narrow peaks
- narrow peaks = smaller difference in interactions in order to separate two solutes
Efficiency
is related experimentally to a solute’s peak width
- an efficient system will produce narrow peaks
- narrow peaks = smaller difference in interactions in order to separate two solutes
Efficiency
related theoretically to the various kinetic processes that are involved in
solute retention and transport in the column
- determine the width or standard deviation (sigma) of peaks
Efficiency
Wb
4 sigma
Wh
2.354 sigma
compare efficiencies of a system for solutes that have
different retention times
Number of theoretical plates (N)
The larger the value of N is for a column, the better the column will be able to separate
two compounds.
- the better the ability to resolve solutes that have small differences in retention
- N is independent of solute retention
- N is dependent on the length of the column
compare efficiencies of
columns with different lengths:
Plate height or height equivalent of a theoretical plate (H or HETP)
Plate height or height equivalent of a theoretical plate (H or HETP)
H = L/N
H simply gives the length of the column that corresponds to one theoretical plate
Why Do Bands Spread?
a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion
What will you do if there are overlapping peaks?
change the design or method
Peaks that are moderately overlapped can often be resolved by increasing column efficiency — by increasing the column plate number to sharpen the peaks (reduce peak volumes)
caused by column overload/overpacking
Fronting peak
caused by underpacking or sample is too viscous
Tailing peak
a process that leads to peak (band) broadening due to the presence
of multiple flow paths through a packed column.
Eddy diffusion
As solute molecules travel through the column,
some arrive at the end sooner then others simply
due to the different path traveled around the
support particles in the column that result in
different travel distances
Eddy diffusion
a process of peak broadening caused by the
presence of different flow profile within channels or
between particles of the support in the column.
Mobile phase mass transfer
A solute in the center of the channel
moves more quickly than solute at the
edges, it will tend to reach the end of
the channel first leading to band broadening
Mobile phase mass transfer
The degree of band-broadening due to eddy diffusion and mobile phase
mass transfer depends mainly on:
1) the size of the packing material
2) the diffusion rate of the solute
band-broadening due to differences in the
rate of diffusion of the solute molecules between the
mobile phase outside the pores of the support
(flowing mobile phase) to the mobile phase within
the pores of the support (stagnant mobile phase).
Stagnant mobile phase mass transfer
Since a solute does not travel down
the column when it is in the stagnant
mobile phase, it spends a longer time
in the column than solute that
remains in the flowing mobile phase.
Stagnant mobile phase mass transfer
The degree of band-broadening due to stagnant mobile phase mass
transfer depends on:
1) the size, shape and pore structure of the packing material
2) the diffusion and retention of the solute
3) the flow-rate of the solute through the column
band-broadening due to the movement of solute
between the stagnant phase and the stationary phase.
Stationary phase mass transfer
Since different solute molecules
spend different lengths of time in the
stationary phase, they also spend
different amounts of time on the
column, giving rise to band broadening.
Stationary phase mass transfer
The degree of band-broadening due to stationary phase mass transfer
depends on:
1) the retention and diffusion of the solute
2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and the
stationary phase
band-broadening due to the diffusion of the solute along the
length of the column in the flowing mobile phase
Longitudinal diffusion
The degree of band-broadening due
to longitudinal diffusion depends on:
1) the diffusion of the solute
2) the flow-rate of the solute through
the column
H =A + B/u + Cu
Van Deemter equation
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of
the mobile phase
where H has a minimum value and the point of maximum
column efficiency; is easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
Optimum linear velocity
Measures of Solute Separation:
parameter used to describe how well two solutes are separated by
a chromatographic system:
separation factor
Does not consider the effect of column efficiency or peak widths, only retention.
separation factor
a second measure of how well two
peaks are separated:
Resolution (Rs)
is preferred over separation factor since both
retention (tr) and column efficiency
(Wb) are considered in defining
peak separation.
Resolution (Rs)
represents baseline
resolution, or complete separation
of two neighboring solutes = ideal
case.
Rs is greater than or equal to 1.5
considered adequate for
most separations.
Rs is greater than or equal to 1.0
affinity = strength of adhesion
the property of how well a component of the mixture sticks to the stationary phase
Adsorption
the property of how well a component of the mixture dissolves in the mobile phase
Solubility
Higher the adsorption to the stationary phase, the slower the molecule will move through the column.
Higher the solubility in the mobile phase, the faster the molecule will move through the column.
The nonpolar solvent acts as the mobile phase. Nonpolar solvents interact more with the mobile solvent, travelling quickly along the polar stationary phase, while polar solutes are attracted to the stationary phase and travel more slowly. This property allows for separation based on polarity.
normal phase chromatography, where the stationary phase is polar, polar molecules will spend more time adsorbed on the stationary phase, while less polar ones will be carried more quickly by the non-polar mobile phase.
