Lecture 3: Efficiency and quantification P1 (finished) Flashcards
narrow peaks help you what?
separate compounds
Retention time is found where on the peak?
at the top
what another word for retention time?
hold- up time.
To diffuse the width of the peak you can measure form where?
base line or half way up the peak
Do nretained solute molecules travel down the column at the same rate as the carrier gas?
yes
quality of the separation is expressed mathematically by measuring what?
width
relative positions
peaks in the chromatogram
what are the fundamental parameters?
- retention factor (k),
- selectivity factor () ,
- column efficiency (N).
- resolution (R).
what is retention factor the measure of?
measure of how an analyte is
partitioned between stationary and mobile phase
whats the retention time equation?
k’= time analyse spends in sp/ time analyse spend sin mp
k ideally should be no more than what?
10
what is retention factor?
The retention factor is the ratio between the time a solute spends in the stationary and mobile phases.
Parameter influencing retention factor is?
Stationary phase
retention factor k slide
do it
in selectivity factor alpha is?
always more than 1
what is alpha defined as?
ratio of the two retention factors with the
larger one always being the numerator.
whats the Selectivity factor ( alpha) equation?
alpha=k’2/k’1 = t’2/t’1
what’s the t’2 and t’1 relationship int he Selectivity factor equation?
Where t’2 > t’1, so alpha > 1
what does k’ indicate?
k’ indicates where the peaks elute relative to Tm
what does the selectivity factor alpha show?
shows where the peaks elute relative to each other.
alpha represents what?
selectivity
whats k’1?
Retention factor of first peak
whats k’2?
Retention factor of second peak
what is selectivity a measure of?
Selectivity is a measure of the time or distance between two peaks.
what happens to the peaks if alpha = 1?
the two peaks have the same retention time and co-elute.
what is selectivity defined as?
Selectivity is defined as the ratio in retention factors.
what parameters influence retention factor?
- Stationary phase
- Mobile phase
- Temperature
what does Band broadening do?
reduces the efficiency of the separation being carried out
what type of band should be appleid to the column?
Important to ensure that a narrow band of mixture is applied to the column
injection volumes should be kept to a ?
minimum
is diffusion faster in liquids or gases?
10^4 times slower in liquids
in what type of chromatography is band broadening a bigger problem?
GC
what should happen at Pre and post column?
keep dead spaces and tube lengths to a minimum
what is the standard measure of column efficiency?
is…the number of theoretical plates (N) generated by the column.
what is N?
number of theoretical plates (N) generated by the column.
whats the equation for N?
N= 16 tr^2 / W^2
in simple terms what does the N equation measure?
peak narrowness
what doe stem theoretical plate model suppose?
supposes that the chromatographic column contains a large number of separate layers, called theoretical plates.
what happen sin these plates according to the theoretical plate model?
Separate equilibrations of the sample between the SP and MP occur
How analyte moves down the column?
by transfer of equilibrated mobile phase from one plate to the next.
do plates actually exist?
do not
really exist
Theoretical Plate Model helps us do what?
- understand the processes at work in
the column. - measuring column efficiency
N is?
column efficiency ( and plate number)
Are columns with high plate numbers less or more efficient ?
more efficient
A column with a high N will lead to what type of peak?
narrower peak at a given RT than lower N
what parameters influence column efficiency?
- Column length (increasing colum length increases efficiency)
- Particle size (decreasing particle size increases efficiency)
what snaother way to measure column efficiency?
height equivalent to a
theoretical plate
whats H units?
mm
H =?
L/N
whats L in the equation?
length of column (mm)
The shorter each theoretical plate, the…..
more plates are “contained” in any length of column.
what does smaller plate height mean?
smaller plate high means narrower band width means narrower peak means better separation
what is R?
resolution
The measure of good chromatography is whether or not the peaks are what?
adequately separated
what does resolution describe?
Resolution describes the ability of a column to separate the peaks of interest.
what does resolution take into consideration?
efficiency (N), selectivity (a) and retention (k).
whats the minimum value for measurable separation and adequate quantitation?
one
A value of __is required to discern a valley between two equal-height peaks.
0.6
Values of 1.7 or greater generally are desirable for what?
rugged methods.
A value of what is considered a baseline separation and ensures the most accurate quantitative result?
1.6
whats the R equation?
R=square root N/a times (a-1/a) (k’2/1+k’av)
whats K’av?
k’av is the average retention factor for the two peaks.
the valley between two peaks should do what?
return to the baseline.
whats the minimum value for R?
1.2
what ha stew hgihest influence eon resolution?
Selectivity
Small changes in selectivity lead to big changes in ?
resolution
Efficiency describes what?
the separation power of the column.
Retention has a significant influence at?
small k-values.
How can R be improved?
Increasing k’ , increasing N, changing alpha
what the issue with increasing k’ to improve resolution?
the peaks become broader resulting in a loss in sensitivity.
How does increasing N improve R?
improves R, but only by a square root relationship.
whats the best way to improve R?
Changing is by far the most powerful way to improve resolution
why is alpha the best way to improve R?
small change can have a dramatic effect
Resolution is a function of column efficiency of which the standard measure is what?
theoretical plates (N)/metre
how is N related to the length of the column?
N = L/HETP
what is HETP ?
the height for a single theoretical plate.
If all other parameters remain fixed, what will a 2 fold increase in column length do?
will lead to a two fold increase in N.
when a 2 fold increase in column length is done whats the negative outcome?
doubles the analysis time because the solute bands will take twice as long to migrate through the column.
