Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry Laboratory	Fall 2008
Lecture Notes:: Lab	© R. Paselk 1999-2008

Characterization of Molecules

Introduction to Chromatography

The most widely used method of analytical separation is chromatography. Chromatography can also be used as a qualitative method to determine what is present. With modern instrumentatio it is also often used quantitatively by the addition of specific deteion methods. So what is chromatography as a phenomena? Although there are a variety of methods, all involve a mobile phase in which a sample is disolved, and a stationary phase (immiscible in practice) which is fixed in place. For an effective separation to take place the two phases must be chosen so that the analyte distributes itself between these two compartments. Depending on the relative residence times in the phases, the analyte may be held up or flow freely.

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) a classification of the 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. The strength of binding is described by the adsorption isotherm, the relationship between concentration of adsorbant and fraction bound at constant temperature. (This has similar formulation to the binding curves we've seen for protein systems such as myoglobin and largely shares the mathematical description for acid-base titration.)
• 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^-.
• Affinity: based on specific biological interactions, so has incredible possibilities for specific isolation from complex mixtures.. In practice a ligand is bound to a stationary phase and molecules which bind the ligand will be retarded or held back. For example, an antigen might be bound which would result in the isolation of the specific antibodies binding it. In a similar fashion can use an enzyme inhibitor as ligand.
• 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: We can classify chromatography in two quite distinct ways:

1. by the physical means in which phases are brought into contact, and
2. by the type of mobile phase used and the process of separation (which of the types of chromatography discussed above are used).

First we can note that there are two fundamental geometries:

1. Planar chromatography - the stationary phase is held on a flat plate or in the interstices of a fibrous sheet. Note that this from of chromatography will generally only be practical for a liquid mobile phase.
2. Column chromatography - the stationary phase is held in a "narrow" tubethrough which the mobile phase flows.

Second and more fundamentally we can classify by mobile phase and process. We will look at the most common substrates and equipment used in chromatography, noting the types of chromatography seen in each.

• Paper chromatography: 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: A reverse phase system is one where the solvent system is polar, and the stationary phase is non-polar. Such stationay phases are generally synthetic, with the most common being silica gel particles coated with covalently linked organosilanes. The most widely used reversed phase substrate is "C-18" or "C₁₈", an octadecylsilane coated silica gel. In effect the C₁₈ groups provide an oil monolayer in which non-polar substances can dissolve. Thus reverse phase chromatography tends to be almost a partition chromatography, with some charecteristics of adsorption thrown in. Other common reverse phase systems use C₈ (octyl) and C₁ (methyl) silanes as coatings. Both the C₁₈ and C₈ systems are genreally "blocked" with C₁ as well. That is, after complete reaction with the appropriate silane, mehtyl silane is reacted to cover any exposed Si-OH groups which were unreactive to the original silane due to steric hindracne etc.
  • Gel permeation: As above, gel permeation separates molecules by size (though it can also be modified to add additional separtion modes). See below under liquid chromtography for additional practical considerations.
• Gas Chromatography (GC). Three common types:
  • Gas Liquid Chromatography (GLC) -
  • Gas bonded-phase Chromatography (GC) -
  • Gas solid Chromatography (GC) -
• Liquid chromatography (LC). Five common types:
  • partition (liquid-liquid)
  • liquid-bonded phase
  • adsorption (liquid-solid)
  • ion-exchange
  • Gel filtration/size exclusion/etc. - As above, gel permeation separates molecules by size (though it can also be modified to add additional separtion modes). Gel chromatography has a number of advantages in separating molecules (and in determining MW):
    • The behavior of most substsances in gels is independent of temperature, pH, ionic strength and buffer composition. Thus separations (and MW determinations) can be carried out under conditions where biomolecules are stable, and even in complex mixtures as long as a specific assay is available. For example, one could determine the MW of an enzyme in an unpurified homogenate, then analyse the eluante fractions for activity.
    • Elution volume is related in a simple way, as seen above, to MW.
    • There is virtually no adsorption to the gel matrix, so very labile substances can be separated without denaturation.

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Last modified 25 September 2008