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.
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
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.
of chromatography consists of controlling these two
aspects of the separation process: