3.1 High performance liquid chromatography  (Page 2/4)

Columns

Different separation mechanisms were used based on different property of the stationary phase of the column. The major types include normal phase chromatography, reverse phase chromatography, ion exchange, size exclusion chromatography, and affinity chromatography.

Normal-phase chromatography

In this method the columns are packed with polar, inorganic particles and a nonpolar mobile phase is used to run through the stationary phase ( [link] ). Normal phase chromatography is mainly used for purification of crude samples, separation of very polar samples, or analytical separations by thin layer chromatography. One problem when using this method is that, water is a strong solvent for the normal-phase chromatography, traces of water in the mobile phase can markedly affect sample retention, and after changing the mobile phase, the column equilibration is very slow.

Reverse-phase chromatography

In reverse-phase (RP) chromatography the stationary phase has a hydrophobic character, while the mobile phase has a polar character. This is the reverse of the normal-phase chromatography ( [link] ). The interactions in RP-HPLC are considered to be the hydrophobic forces, and these forces are caused by the energies resulting from the disturbance of the dipolar structure of the solvent. The separation is typically based on the partition of the analyte between the stationary phase and the mobile phase. The solute molecules are in equilibrium between the hydrophobic stationary phase and partially polar mobile phase. The more hydrophobic molecule has a longer retention time while the ionized organic compounds, inorganic ions and polar metal molecules show little or no retention time.

Ion exchange chromatography

The ion exchange mechanism is based on electrostatic interactions between hydrated ions from a sample and oppositely charged functional groups on the stationary phase. Two types of mechanisms are used for the separation: in one mechanism, the elution uses a mobile phase that contains competing ions that would replace the analyte ions and push them off the column; another mechanism is to add a complexing reagent in the mobile phase and to change the sample species from their initial form. This modification on the molecules will lead them to elution. In addition to the exchange of ions, ion-exchange stationary phases are able to retain specific neutral molecules. This process is related to the retention based on the formation of complexes, and specific ions such as transition metals can be retained on a cation-exchange resin and can still accept lone-pair electrons from donor ligands. Thus neutral ligand molecules can be retained on resins treated with the transitional metal ions.

The modern ion exchange is capable of quantitative applications at rather low solute concentrations, and can be used in the analysis of aqueous samples for common inorganic anions (range 10 μg/L to 10 mg/L). Metal cations and inorganic anions are all separated predominantly by ionic interactions with the ion exchange resin. One of the largest industrial users of ion exchange is the food and beverage sector to determine the nitrogen-, sulfur-, and phosphorous- containing species as well as the halide ions. Also, ion exchange can be used to determine the dissolved inorganic and organic ions in natural and treated waters.