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The Analysis of Macromolecules
[edit]Separation of macromolecules
[edit]- Size exclusion chromatography (SEC)
- Stationary phase is a porous material called Sephadex
- Smaller molecules can penetrate every pore of the stationary phase, so are retained longer
- Larger molecules cannot penetrate any of the pores, so elute faster
- YouTube animation of SEC in action: http://www.youtube.com/watch?v=wjuWb6XpRmQ
- It is possible to calculate the approximate molecular weight of a macromolecule using SEC
- Determine the column void volume, Vo (volume a particle sees if it cannot penetrate any pores) by flowing dextran blue (a huge molecule, m.w. 2 MDa) through the column
- Determine elution volumes Ve of several macromolecules of known molecular weight
- Plot a calibration curve: log(m.w.) vs. Ve/Vo
- Measure Ve of the analyte (a macromolecule of unknown m.w.), read off m.w. from calibration curve
- Stokes radius
Measuring the size of macromolecules
[edit]Movement and distance
[edit]FRET
[edit]Visualisation
[edit]SEM
[edit]- Scanning electron microscopy (SEM)
- Fire high-energy electrons at a sample in a vacuum chamber
- Electron beam rasters across the sample
- Detect backscattered electrons and secondary electrons
- Backscattering is greater for atoms of higher atomic number