Week 9 Flashcards
definition of chromatography, HPLC?
physical method of separation in which the components to be separated are distributed between two phases: stationary and mobile
HPLC: column chromatography -> separates compounds based on polarities and interactions with stationary phase
what’s the most popular type of chromatography?
reversed-phase chromatography: polar mobile phase and non-polar stationary phase -> more polar analytes are less retained
how has the length of the columns been evolving over the years? what about particle size?
it has been decreasing (now normal is 5 to 10 cm)
smaller particles (<2 microns) can be separated now
isocratic mode: retention time, retention factor, separation factor, plate number
retention time = time between sample injection and detection
retention factor k = quantity of analyte in stationary phase divided by quantity of analyte in mobile phase (measure of how long a compound is retained on the stationary phase relative to the mobile phase)
separation factor alpha= describes the separation of 2 species on the column
plate number N= relates to column efficiency, more plates = better for separation
isocratic mode: resolution, how to optimize it, goal?
baseline separation achieved for R>1.5
maximise these three terms in order: k, alpha, N (square root)
goal is to achieve R>1.5 with shortest analysis time
three terms in the Van Deemter equation (isocratic) (u is flow rate)
A: eddy diffusion
B: longitudinal diffusion (B/u)
C: mass transport diffusion (Cu)
in isocratic mode, for small particles, short or long columns preferred?
short columns -> faster analysis (half of length = half of analysis time)
alternative stationary phases for LC
- monolithic columns (silica rod)
- core shell particles -> reduced eddy diffusion, faster mass transport -> smaller peak width
what is the pressure limit of classic chromatography systems?
400 bar
two modes in which UPLC can be operated
high speed (short columns and high flow rates)
high resolution (long columns and normal flow rates)
what is the general elution problem, solution?
when a mixture contains compounds with widely varying properties, a single set of conditions (e.g., solvent composition, temperature) might not be optimal for all compounds.
- Early Elution -> poor separation and resolution.
- Late Elution -> long run times and broad peaks.
Gradient elution! (composition of mobile phase changes over time)
gradient elution: advantages and disadvantages
+:
- suited for complex samples
- better resolution of early and late eluting peaks
- similar peak widths
- better sensitivity for late eluting peaks
- higher peak capacity
-:
- more complex HPLC instrument
- difficult and time consuming
- long run times due to column equilibration
two most used gradient shapes, explain the first one
linear and segmented
linear described by gradient time, initial and final mobile phase compositions, gradient range
explain band speed and k value for isocratic VS gradient elution
isocratic:
- bands move at constant speed
- k is constant for each peak during separation
gradient:
- bands move at increasing speed
- k is decreasing for a peak
- gradient retention factor k* is constant for a given separation
k*: definition, dependence on gradient slope
value of k when a band has migrated halfway through the column
shallow gradient (k changes slowly) = large k*
steep gradient (k changes fast) = small k*
