Question

Analytical Chemistry Tutorial: Introduction to Chromatography

Answer 1

disclaimer: this is meant for an A-chem course so the approach to chromatography is more focused on theory and instrumentation. this may not be useful for a biology or biochem class.

Answer 2

\({\bf{Terminology:}}\)
- sample is dissolved in the mobile phase and is forced through the immiscible stationary phase
- strength of interactions btwn the molecules in mobile and stationary phases determines retention time, or how long it takes before the compound is eluted or exits the chromatograph
- elution is done by flushing the chromatograph with excess mobile phase
- elute: the portion of the sample in the mobile phase

Answer 3

\({\bf{Classification:}}\) either by the mobile phase/stationary phase (liquid, gas, or supercritical fluid, or by the physical apparatus used to conduct the separation (planar vs column)

\({\bf{Quantifying~Chromatography:}}\)
distribution coefficient = (as)/(am) where a is the activity of the analyte in the stationary phase and am is the activity in the mobile phase

for low concentrations and non-ionic compounds activity approaches concentration and thus they can be used interchangeably

retention time = tr = ts + tm a.k.a the sum of the times it spends in the stationary + mobile phases

avg. linear rate of solute migration = v = L/tr
where L is the length of column
this is also equal to u cross (fraction of solutes in mobile phase compared to total moles of solute)

avg. linear velocity of mobile phase = u = L/rm

volume flow rate = pi * r^2 * u0 * epsilon
where u0 is the initial linear velocity of mobile phase, and epsilon is the portion of the total column "available" to liquid (column porosity)

Answer 4

cont:
the retention factor kA = (KA * Vs)/(Vm)
where KA is the distribution constant for A and V are the volumes in the stationary/mobile phase.

avg. linear rate of solute migration can now be re-written as

v = u cross 1/(1 + as/am) = u cross 1/(1+kA)

letting v = L/tr, u = L/tm, this equation becomes

(L/tr) = (L/tm) cross 1/(1+kA)

this can be re-arranged into ka = (tr-tm)/tm to make this entirely dependent on retention times.

Answer 5

\({\bf{Selectivity~Factor:}}\) or the ability of a chromatography to separate individual components from the sample

alpha = KB/KA
note: capital K designates distribution constant with B being the more readily retained compound

alpha is also equal to kb/ka with lowercase k representing the retention factors

using ka = (tr-tm)/tm we can re-write the selectivity factor in terms of t only

alpha = [(tR)B - tm]/[(tR)A - tm]

Answer 6

Source material is sections 26A-B of Principals of Instrumental Analysis, 6th edition by Skoog, Douglas A., Holler, James F., Crouch, Stanley R.