Getting Started in HPLC

Section 1B. The Chromatography "Family Tree"

   
Chromatography techniques can be characterized in a variety of ways. Many of the possible categories are used only infrequently, so we will deal only with the most commonly used varieties.


 
The first broad distinction among chromatographic techniques is based on the mobile phase:
  • Gas Chromatography (GC) uses a moving gas.
  • Liquid Chromatography (LC) uses a moving liquid.


Liquid Chromatography, in turn, is most commonly categorized on the basis of the physical arrangement of the stationary phase:
  • A sheet of paper, through which the mobile phase moves by capillarity (Paper Chromatography). Paper Chromatography today is more often used as a teaching tool than as an analytical tool.
  • A thin layer of powdered material bound to a glass or plastic support, through which the mobile phase moves by capillarity (Thin Layer Chromatography, or TLC). TLC is used occasionally as a rapid technique for qualitative analysis.
  • A bed of powdered material packed in a glass or plastic column, through which the mobile phase is pulled by gravity ("Classical" or "Open Column" Chromatography). This is used primarily as a preparative technique.
  • A bed of powdered material packed in a small, disposable, plastic cartridge, through which mobile phase is pulled by vacuum or pushed with a small syringe (Solid Phase Extraction, or SPE). This is used primarily as a sample cleanup technique prior to GC or HPLC
  • A bed of powdered material packed in a steel or high-strength plastic column through which the mobile phase is pushed by a constant-flow pump. ("High Performance Liquid Chromatography" or HPLC).


HPLC is subdivided on the basis of separation chemistry. All of these techniques can be done using the same instrumentation.
  • Reversed-phase chromatography uses a non-polar (hydrophobic) stationary phase and a polar (usually including some water) mobile phase. This is the most common type of HPLC separation in use today.
  • Normal-phase chromatography uses a polar (hydrophilic) stationary phase and a non-polar (usually with no water) mobile phase. This was the type of separation to which the term "chromatography" was first applied (remember, the stationary phase was powdered chalk -- calcium carbonate, a very polar material -- and the mobile phase was petroleum ether -- mainly hexane, a very non-polar hydrocarbon solvent). This technique is still used, but is less common than reversed-phase.
  • Ion-exchange chromatography uses a stationary phase support which has been derivatized so as to permanently bind charged groups to the surface. The mobile phase is typically an aqueous buffer. This technique is used primarily for the analysis of ions such as strong acids or bases or for separation of large molecules such as nucleic acids, proteins, or large peptides.
  • Ion-pair chromatography is a "hybrid" technique in which charged groups are temporarily bound to the surface of a "reversed-phase" type of column packing. This technique is often used for the analysis of small, weak-acid or weak-base compounds.
  • Size-exclusion chromatography uses a stationary phase of controlled pore-size distribution. Separation is based on the extent to which analyte molecules can penetrate the network of pores.


Another way to subdivide HPLC is based on whether the mobile phase composition is held constant during the course of the separation ("isocratic" HPLC) or is changed during the course of the separation ("gradient" HPLC).


 
   
 

 


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Last revised: March 30, 2001.