ICAM Separations
What is the equation for Resolution?
2(tRB - tRA)/ (WA + WB) or (tRB - tRA)/ 1/2(WA + WB)
Why is void time equal to the time the solutes spend in the mobile phase?
-mobile phases pass through the entire column -same flow rate -not in stationary phase and mobile phase at the same time -everything is in the mobile phase for the same time
WHat does a Rs= 1.5 indicate?
0% overlap good for quantitative measurements
What does a Rs= 1 indicate?
4% overlap good for qualitative measurements
What is the baseline width (Wb)?
= 4 sigma
What does A=?
A= 2lambda dp
What is the equation for B/u longitudinal diffusion?
B/u = 2(packing quality)Dm/u Dm=diffusion coefficient, u= linear velocity
What is the equation expression for the partition constant (K)?
C a,s/ C a,m
What should we do to get the smallest plate height?
Choose a packed column with small particle size or an open tubular column with a small diameter choose a column with a thin film of stationary phase always operate at or near optimum velocity
What is the equation for mass transfer in mobile phase (Cmu)?
Cmu= (fk(dc)^2u)/Dm
What is the equation for Mass transfer in stationary phase (Csu)?
Csu= (fk(df)^2u)/Ds
What width do we use for asymmetric peaks?
FWHM--full with at half max
What are two solutions for changing conditions during separation so that they are optimal for each pair of analytes as they elute?
Gas chromatography--temperature programming liquid chromatography--solvent programming (gradient elution)
H overall is equal to what?
H = A +B/u + Csu + Cmu
How can we improve R?
Increase N--physical aspects (column and particle size) increase alpha and increase k--chemical aspects (polarity of phases
What is the equation for Resolution (R)?
It can be computed by dividing the distance between two peaks (measured in time) by the average widths of the peaks
What is the most likely direction that this molecule will travel when the mobile phase is flowing?
It will go down with the mobile phase (go with the flow)
For asymmetric peaks, what does N = ?
N= 5.54( tr/W1/2)^2 know how to derive
Combination of what N= ?
N= L/H = 16( tr/Wb )^2
Is there an equilibrium to the partition between mobile and stationary phases?
Not a true equilibrium as there is flow to the system Instead there is a steady state that exists relating to the distribution of solute molecules between the two phases
What is the Purnell equation?
Rs = (sqrt(N)/4) ((alpha-1)/alpha) (kB/(1+kB)) = (efficiency)(selectivity)(retention factor)
What does baseline resolution mean?
Peak A finishes before Peak B starts--peak A comes back to the baselijne before peak B starts
What type of properties are used to separate chemicals?
Physical (usually)
Mass transfer in stationary phase broading is dependent on what?
Retention factor (k) diffusion coefficient (Ds) ** film thickness (dF) --film on outside of column -- the thicker the film, the more ways the analyte can stick ** linear velocity (u) **we can control
What can describe the broadening of the peaks when analytes spend more time on a column?
The broadening can be described based on plate number (N) and the plate height (H)
What is the retention factor (k)?
The ratio of the time a solute spends in the stationary phase to the time it spends in the mobile phase k = ts/t0
What happens to the separation and broadness of peaks when analytes spend a longer time on the column?
The separation becomes greater and the zones become broader
How can you tell if the solute is polar or not?
The solute with the longer retention time is more polar, because it stays longer in the stationary phase with the polar silica
What is plate height?
The variance per unit length sigma^2/L
How do analytes have different retention times if both spend the same time in the mobile phase?
They spend different times in the stationary phase
what does sigma equal to?
Wb/4
What is N?
a quantitative measure of the column efficiency--plate number = L/H H=plate height
What is the definition of resolution?
a quantitative measure of the quality of a separation between two peaks
what is the relationship between retention time, retention factor and peak characteristics?
all are directly proportional to one another if you increase the retention time, the retention factor increases and the peak broadness increases as the peaks get shorter and fatter
What value of Resolution (R) represents a baseline resolution?
an R of 1.5
How do analytes distribute between the different phases?
based on solubility and attraction
What do we do to improve resolution if baseline resolution is not achieved?
change the solvent system so separation is achieved
What is the most common separation method?
chromatography
What is selectivity based on
differences in interaction between analytes and the stationary phase at a given temperature main contributor is the chemical nature of the stationary phase
How does the analyte interact with the different phases?
if high affinity for stationary phase, elutes slower if low affinity for stationary phase, elutes faster
What effect would a large number of different solute paths have on band broadening?
increase the band width
What could affect time, not elution order?
length of column, solvent flow rate
Does a longer or shorter path make peaks shorter and wider?
longer paths --more dilute so spreads out the peak
the void time is also equal to the time the solutes spend in which phase?
mobile
What affects the elution order of solvents?
mobile phase composition, solute composition, stationary phase composition, more polar solvent??
What are the two immiscible phases that must have different chemical and physical properties for chromatography?
mobile phase--moves down the column stationary phase--fixed inside the column
what is mass transfer in mobile phase (Cmu)?
molecules in the mobile phase travel at different velocities, depending on their distance form the wall or particle surface more difference in friction=spreads out peak
what is the mass transfer in stationary phase (Csu)?
molecules of the same type can spend different times dissolved in or adsorbed on the stationary phase more interaction=spreads out peak (shorter and wider)
Are conditions that are optimal for an early eluting substance the same for a later eluting pair?
no--not the same
In an ____ column, all molecules travel the same distance, while in a _____ column, molecules can take paths of different length around the particles
open tube; packed
Longitudinal diffusion (B/u) broadening is dependent on what?
packing quality diffusion coefficient (Dm) linear velocity (u)--we can control
What is multiple paths (A) broadening dependent on?
packing quality (lambda) particle size (dp)--we can control this
The analyte traveling down the column results in a little bit of friction when analyte is closer to the sides. This creates what type of graph?
parabolic relationship
What effect will diffusion of solute molecules have on the broadness of peaks?
peaks will become more broad
What is Longitudinal diffusion (B/u)?
random molecular motion causes diffusion to occur from center of zone outward, I.e. from [high]--> [low]
Mass transfer in mobile phase (Cmu) broadening is dependent on what?
retention factor (k) diffusion coefficient (Dm) **Diameter or column (dc) **Linear velocity (u) ** we can control
How can you determine the time the solute spends in only the stationary phase (ts)?
retention time - mobile phase ts = tR - t0
What factors affect resolution?
retention time affects resolution - if retention times are closer together, the resolution gets worse - the fatter your peaks are, the harder it is to separate them
The compound that elutes faster is going to have a ____ retention time in comparison to the other compounds
shorter
Will band broadening be more or less pronounced from diffusion at slow or fast flow rates?
slow
More diffusion does what to the peaks?
spreads them out and makes them shorter and wider
what is the main contributor to selectivity?
the chemical nature of the stationary phase
Band broadening due to diffusion depends on what?
the flow rate
what is the selectivity factor (alpha)
the ratio of retention factors for two solutes, and is always greater than 1
What is the partition (equilibrium distribution) coefficient (k)?
the ratio of the concentration of the solute in the stationary phase to the concentration of solute in the mobile phase
What is void time?
the time it takes an unretained solute with no interaction to the stationary phase to elute
What peak corresponds to a signal near t=0?
the time it takes to come out of the column no retention void time
what is retention time?
the time needed until a component elutes from the column
True or false: the solute molecules are constantly moving between the mobile and stationary phases?
true--this is a dynamic system
Are the number of paths the same no matter the speed?
yes
is the void time the same for all solutes in the sample?
yes