Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry Laboratory	Fall 2002
Lecture Notes:: February 21	© R. Paselk 1999

Characterization of Macromolecules:

Size and Shape

A variety of hydynamic properties are widely used to to determine size and shape. We will look at the most common and important. (We will not discuss viscosity or osmometry - you should be familiar with basic osmometry from General Chemistry and Introductory Biology.)

Major Techniques:

• Sedimentation
  • Velocirty
  • Equilibrium
  • Density Gradient
• Gel Electrophoresis (uniform or pore gradient)
  • SDS; Urea, Guanidine HCl (proteins - gives uniformly charged, rod-shaped particles)
  • Non-denaturant (nucleic acids - already uniformly charged and rod shaped)
• Gel Filtration Chromatography
  • Column (low pressure)
  • HPLC (High Pressure or High Performance Liquid Chromatography)

Introduction to Chromatography

We will begin our discussion by looking at chromatography from two perspectives: 1) types of chromatography (the underlying phenomena upon which chromatographic separations are based), and 2) methods of chromatography (e.g. paper, column, etc.).

Types of Chromatography: What are the underlying physical phenomena for each type of chromatography?

• Partition: based on the differential solubility of the solute in two or more different phases.
• Adsorption: based on the adsorption of solute to an adsorbent (surface) in competition with the solvent and other adsorbants.
• Ion exchange: based on anion-cation interactions. Separation occurs due to differences in charge. Can have either cation or anion exchangers. For example:
  • Cation exchanger - sulfonate/Na⁺. Have Resin-SO₃^-Na⁺.
  • Anion exchanger - Diethylaminoethyl group: DEAE Cl^-. Resin-CH₂CH₂N⁺H(CH₂CH₃)₂Cl^-.
• Gel Filtration (aka: Gel Permeation, Molecular Exclusion, Molecular Sieve): Separates molecules by size, but unlike electrophoresis, large molecules migrate fastest, small molecules are retarded. This occurs because large molecules flow around the resin beads, whereas the smaller molecules can enter the pores in the gel, thus following a longer path. In general, then, the smaller the molecule the greater the volume in the gel interior is available to it, and thus the longer its path and delay. As in gel electrophoresis different degrees of polymerization give differing ranges of MW separations.

Can use gel permeation chromatography to determine MW's. As with SDS-PAGE the system must be calibrated with a set of MW standards, although with LC it is more common to run them separately. Plot K vs. Log MW, where K = (V_e-V_o) / V_s, and V_e= elution volume, V_o= void volume, and V_s= volume of the gel, or V_e vs. log MW as shown in the figures.

Methods of Chromatography: Various substrates and equipment can be used in chromatography. We will look at the most common, emphasizing the underlying types of chromatography seen in each.

• Paper: Paper is made up of cellulose fibers, thus we expect hydroxyl groups (polar, hydrogen bonding) on a hydrophobic ether-like backbone.
  • Thus expect adsorption via hydrogen bonding and hydrophobic/van der Waals forces.
  • Also, polar, and in particular hydrogen bonding, solvents will interact relatively strongly with the cellulose and form a static solvent layer leading to partition.

  Note that with any mixed solvent system, such as aqueous alcohol/ether we expect the aqueous component to form a stationary phase on the cellulose substrate, while the most non-polar solvent component will predominate in the mobile phase flowing through the paper. Non-polar substances will thus tend to move slowly if at all on paper chromatography since it will partition into the stationary phase and adsorb onto the paper, while non-polar substances will migrate quickly with the non-polar solvent. Note this means that any separation on paper will be difficult to predict exactly because multiple modes of separation are involved.

Note that paper can also be modified to give ion-exchange media, which would then separate compounds by a combination of adsorption, partition, and ion-exchange.

• TLC (Thin Layer Chromatography): Like paper chromatogrraphy, TLC is a two-dimensional method. However, because of the very fine paticles used to make up the solid phase on TLC plates the chromatograms tend to run faster. In general TLC is a higher resolution system:
  • The smaller particles and thin layers allow a closer approach to equilibrium.
  • The very uniform particle sizes and porosities available with modern technology make for a very uniform solid phase, and greater uniformity aids in achieving in greater resolution via a more uniform solvent flow.
    

  The most common solid phase for TLC is silica gel, a hydrated form of silicon dioxide polymer. Note that the surface of SiO₂ will have many exposed hydroxide groups (-OH and -O^-). Thus, like cellulose, polar solvent will tend to form a stationary layer. We thus expect adsorption due to hydrogen and polar bonding, and partition into polar solvent stationary phases. Because SiO₂ was traditionally the most common substrate in chromatography, a polar stationary phase with a non-polar mobile phase is referred to as normal phase chromatography, whereas a non-polar stationary phase with a polar mobile phase is referred to as reversed phase chromatogrpahy. As with paper, silica substrates can be modified. A variety of other substates are also available to give a very wide range of chromatographic media, including:

  • Ion exchange: ion exchange groups such as DEAE of sulfonate can be attached to silica gel, resin beads, finely ground paper etc. to give ion exchange. In most media adsorption and partition will also participate in the separation process.
  • Reverse phase: TO BE ADDED IN FUTURE
  • Gel permeation:TO BE ADDED IN FUTURE

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Last modified 21 February 2002