03_peptide-separation

potentially low abundance of interesting peptides -> sensitivity needed

=> 1.66 * 10^(-24) moles

peptides mixture in solution -> adsorption in mobile phase -> dis-/association along columns in stationary phase

all about physicochemical properties and kinetics

magic triangle

1. increase analyte’s strength -> stationary phase interactions

2. decrease analyte’s strength -> mobile phase interactions

3. decrease mobile phase’s strength -> stationary phase interactions

-> adjusted by elution strength of mobile phase

• (theoretical) Plate height

• efficiency

• selectivity

• resolution

• peak capacity

parameter of chromatography theory

• only theoretical plates

• property of chromatographic column

• different “stages” of condensation reached aka analyte at different plate heights

-> Super Mario jumps up tower - game

parameter of chromatography theory

• number of “plates” as a way of measuring column efficiency

• Height equivalent to a theoretical plate HETP = L/N

• way of describing how “sharp” the chromatographic peaks are

  • auc the same between sharper and rawer ones (proportional to quantity)

  • higher signal intensity (intensity proportional to concentration)

  • better signal-to-noise-ratio

  • H = sigma^2 * L / t(R)^2

parameter of chromatography theory

• describes analyte retention by the column

  -> the larger the difference btw. signals, the better the chromatography

  • overlapping peaks impair calculation of auc

  • alpha = t(R1) / t(R2)

parameter of chromatography theory

• describes overall quality of separation

  • difference in retention time for destinction of two peaks relevant

• function of efficency and selectivity

  -> the narrower the peak (efficiency) and the larger the difference in retention times (selectivity), the better the separation (resolution)

  • R = [ t(R1) - t(R2) ] / 2 * (sigma1 + sigma2)

parameter of chromatography theory

• maximum theoretical number of successfully separated analytes with given column and set of analyte parameters

  -> depending on chromatographic resolution and gradient time

  • peak capacity = net gradient time / peak width (4 * sigma)

-> long, efficient columns and long gradient times = highest peak capacities

• Eddy dispersion

• Longitudinal diffusion

• Restricted mass transfer

=> Van Deemter Diagram

a method to remove peak broadening

• cause of broadening: analytes taking different random paths (with different distances) through the stationary phase

-> smaller particles and better packing to reduce distance and travel time

a method to remove peak broadening

cause of broadening:

travelling of analytes in all dimensions due to Brownian motion (the more active - e. g. caused by heat - the faster)

lower concentration at the edges of the peak than at center

-> higher flow rate

a method to remove peak broadening

mass transfer btw. stationary and mobile phase

restrictes high flow rates due to transfer

-> reduce flow rate

quantitative description of plate heights as a function of mobile phase velocity

• offline - LC

• online LC

in on-line chromatography

most frequenctly used type

stationary phase with C18 material (18 carbon-chain) which bind particles

mobile phase with a gradient of organic solvent

-> slowly particles are going to unbind from C18 according to hydrophobicity

=> hydrophilic ones first, most hydrophobic last

—> different times when the particles are released and washed away by the flow/stream

==> controlled by choice of stationary and mobile phase

on-line chromatiography

most important form of liquid chromatography

direct hydrophobic interactions btw. non-polar peptide sc and non-polar stationary phase

use of electrostatic interactions btw. polar sc of aa in peptide and an amphiphilic ion pairing agent to mediate interaction with non-polar stationary phase

peptide mixture generated by protease digestion of entire proteom overwhelmingly complex -> single dimension often not enough

=> gaining analytical depth

—> orthogonality

1. dimension: off-line chromatography -> collection of fractions

2. dimension: on-line chromatography -> sepeare analyse of each fraction by LC-MS

—> separation not fully orthognal but with random distributions

• high pH reversed phase (acetonitrile elution)

  • SCX (strong cation exchange)

• anion exchange chromatography (salt elution)

• cation exchange chromatography (salt elution)

• mixed materials (salt and/or acetonitrile elution)