Getting Started in HPLC

Section 1A. What is Chromatography?

The figure on the right is an illustration showing the first experiment to be called "chromatography" almost a century ago. A glass tube (the first "column") was filled with powdered chalk (the "packing"), a few drops of an extract of plant leaves (the "sample") was placed on top of the bed of powdered chalk, and then the organic solvent petroleum ether (the "mobile phase") was allowed to percolate down through the bed. Lo and behold, the different plant pigments were carried down through the column at different speeds, with the result that the pigments separated into brightly colored bands, as shown at the right. At the end of the experiment, the powdered chalk was extruded from the tube, the colored bands were sliced out, and the pigments were extracted for further analysis. The name "chromatography" (from the Greek words for "color" and "writing") was coined because of the striking pattern of brightly colored pigment bands against the white background of the powdered chalk. The name has persisted to this day even though the overwhelming majority of compounds analyzed are colorless to the human eye.

Even though this looks quite different from what we do in HPLC, the basic operating principles are identical. The separation is carried out in a system that has two phases: one stationary (the column packing) and the other moving (the mobile phase or solvent). Compounds that have a low affinity for the column packing and/or a high solubility in the mobile phase (like the red pigment shown here) are washed through the column quickly. Compounds that stick tightly to the column packing and/or have a low solubility in the mobile phase solvent (like the green pigment shown here) move more slowly. Developing an HPLC separation requires finding a combination of column packing and mobile phase that provides just the right balance of affinity and solubility to make the compounds of interest move at different speeds.

Watch what happens to the sample pigments as the solvent (light blue) flows down the column.

Separation is complicated by the fact that bands not only separate but broaden as they are swept through the column. Band broadening is bad because excessively broad bands may still overlap even though the band centers are, on average, separated (compare the two figures shown at the right. The center-to-center separation between the blue and red bands is the same in both figures). Band broadening can be minimized by careful selection of the stationary phase particle size and close attention to the way in which the column is packed.

The center-to-center separation between the red and the blue zones is the same in both separations. In the lower figure, the bands overlap because they are too broad.

The practice of chromatography consists of controlling these two aspects of the separation process:
  • differential migration (moving different compounds at different speeds)
  • band broadening (keeping the bands as narrow as possible during the separation).



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Last revised: April 06, 2001.