Protein Facility

The Protein Facility utilizes three Beckman Instruments System Gold high performance liquid chromatography systems for the separation of peptides and proteins. One system is devoted to analytical scale separations, one is for micro-analytical purposes such as trypsin digest separations and the last system can handle preparative and semi-preparative scale separations. Users are also trained to run the HPLC systems themselves at a reduced charge. Below is a short description of reversed phase chromatography.


Reversed Phase Chromatography
In reverse phase chromatography, the packing is nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids or bonded hydrocarbons (such as C₁₈, C₈, C₄, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

The C₁₈, C₈, and phenyl bonded phases are most often used in the reverse phase mode. It has been estimated that 60-90% of all analytical LC separations are done on bonded phases in the reverse phase mode. Bonded phases made by covalently bonding a molecule onto a solid stationary phase are intended to prepare "liquid coatings" which will be permanent. Silica is a reactive substrate to which various functionalities can be attached or bonded. The functionalities most widely bonded to silica are the alkyl (C₁₈ and C₈), aromatic phenyl, and cyano and amino groups.


General characteristics of reversed phase chromatography

• Broad scope which allows sample types with a wide range of polarities and molecular weights to be separated.
• General rapidity of mobile phase column equilibration during methods development and gradient regeneration.
• General ease of use.
• Applicability to separation of ionic or ionizable compounds by manipulating secondary chemical equilibrium such as ionization control and ion pairing in the aqueous mobile phase.
  • Buffering the mobile phase in the pH range from 2 to 5 with one of the common buffers, the ionization of the weak acids can be suppressed or controlled allowing them to be retained in their neutral form. Similarly weak bases can be retained in their neutral form at pH 7-7.5.
  • For strong acids and bases ionization control cannot be employed because the stability of alkyl bonded phases is diminished below pH 2 and above pH 7.5. Highly hydrophilic weak acids and bases often remain difficult to retain with ionization control. In such cases ion pair reversed phase chromatography can be used. In this method, counterions (species of opposite charge to the solutes) thereby regulate the retention. Typically alkyl amines or tetra alkyl amines are added to ion pair with acids whereas alkyl sulfates, sulfonates, or phosphates are used to ion pair with bases. The technique is an alternative to ion exchange chromatography for analysis of ionic compounds.
• The possibility of special selectivity such as structural or steric are achievable by specific mobile phase additives:
  • Metal ions are capable of binding to organic compounds in a very selective method which is used for ligand exchange chromatography. The selectivity generated in these metal ion phase systems is based in part on differences of the solute (ligand) binding strength to the metal ion. An alternate approach is the addition of various chelating agents (4-dodecyldiethylene-triamine - C₁₂ dien) in combination with a metal ion. The type and strength of the metal chelate complex-solute binding can be greatly varied depending upon the chemical environment surrounding the metal ion as determined by the chelating agent added.

• HPLC Submission Form (PDF)