Module 6: Chromatography / Electrophoretic Methods

the goal of chromatography and electrophoresis

to separate the target analyte from potential inteferants

the advantage of modern chromatography over simple separations

w/ modern chromatography, we are able to separate things with similar K_D values

retention time (t_r)

time substance stays in stationary and mobile phase combined; highest peak

void time (t_m )

time in mobile phase

retention factor (k’)

ratio solute spends in stationary phase compared to time spent in mobile phase

(t_r - t_m)/ t_m

the ____ the retention factor, the broader the peak, and the longer the elution time

larger

partition chromatography

relies on differences in polarity and equilibrium partitioning between the liquid SP and MP

ion-exchange chromatography

separates based on charge of solute, and whether that charge is more compatible with the charge of the mobile and stationary phase

molecular exclusion chromatography

separates based on size of solutes

electrophoretic separations

no SP; separates according to the charge of the applied magnetic field

2 ways to improve resolution

• increase separation between peaks

  • increase interactions between SP

  • increase the column’s selectivity for one solute so it selects for it

• make the peaks more narrow by reducing the width

column selectivity (\alpha)

ratio of retention factors for 2 different solutes; must be over 1

column efficiency

measures band broadening after the solute is added

• previously thought to be dependent on the number of theoretical plates

gas chromatography

the mobile phase is an inert gas that is carried through a column (SP)

2 types of mobile phase with gas chromatography

packed column and capillary column

elution order in GC’s SP is determined by ___

boiling point

3 ways to inject a sample in GC

• split injection- highest [ ] of analyte

• splitless injection

• on-column injection- lowest [ ] of analyte

5 detectors for GC

1. Thermal Conductivity Detectors

2. Flame Ionization Detector

3. Electron Capture Detector

4. Nitrogen-Phosphorous Detector

5. Mass Spectroscopy

thermal conductivity detector

GC detector that measures the heat conductivity levels of different gasses

• needs a high [ ] of analyte

• universal bcuz the carrier gas (He) is a better conductor than every other gas possible; so it is able to provide a difference for everything

flame ionization detector

GC detector that measures the current thats a result of the release of electrons brought on by combustion

electron capture detector

GC detector that measures the electric current given off when a solute is ionized

• only responds to structures with electronegative atoms

high performance liquid chromatography

uses pressure to push solutes through closed columns with fine particles that lead to high-resolution separation

the main difference between the 2 main modes of HPLC

in reverse phase chromatography, the SP is non-polar, and in normal phase chromatography, the SP is polar

partitioning

elution method that implies that the solute is fully in SP

adsorption

elution method that implies that the solute has surface contacti with the SP, but is not fully embedded