Chromatography Systems Part I, Chromatography Concepts
Barbara Kanegsberg and Ed Kanegsberg, January 2005
One classic text defines chromatography as the separation of components of
a mixture by difference in partitioning or distribution between two phases
[1]. For example, liquid/liquid chromatography could be used to separate
components of a mixture based on their polarity. If you wanted to identify
a non-polar organic contaminate mixed with a polar material, a simple
way to separate them would be to add the mixture to a container of oil
and water and shake it. On the principle that like dissolves like, the
non-polar material would partition into the oil; the polar material would
move into the water. Once the oil and water phases separated, the water
phase could be removed, fresh water added, and the process of washing
the oil phase repeated until essentially all of the polar material was
removed. The purified non-polar contaminant in the oil phase could then
be analyzed and identified. The technique could be reversed to identify
a polar component.
The concept of chromatography, as it is generally applied today, involves
the partitioning of mixtures between a stationary phase (the sorbent) and
the mobile phase (a liquid or gas). About a century ago, a botanist, Mikhail
Twsett, described the separation of plant pigments on a glass column packed
with a sorbent [2]. As liquid was poured onto the column, the various plant
pigments were “washed” or eluted at different rates and created
bands of color, depending on the balance between their solubility in the
washing solution and their adherence to the sorbent. Twsett coined the term
chromatography which stems from the Greek word for color. However, since
Tswett is a Russian word for color; the originator may have had both a descriptive
and a biographical intent.
Chromatographic techniques can also be performed using paper as the stationary
phase, or using a thin layer of sorbent applied to a glass or plastic
support (thin layer chromatography). For example, the classic clinical
test for
fetal lung maturity, as evidenced by the ratio of surfactants lecithin
and sphingomyelin
in amniotic fluid, is a TLC method. To achieve appropriate separation,
a two-dimensional method is used. A sample of the fluid is applied near
one
corner of the plate. The plate is placed in a container with a small
amount of solvent. As the solvent is wicked up by capillary action
against the
pull of gravity, components of the amniotic fluid, including the surfactants,
separate or are eluted. To get more resolution, the plate is turned 90
degrees,
so that the separated components are fairly near the bottom edge. The
plate is then placed in a second solvent; the bands are further separated
(gravity
is now acting in a different direction) and then visualized.
Early column chromatography was often performed in very large, long glass
columns using such sorbents as powdered aluminum oxide (alumina) or
even sugar. Various solvents and solvent blends (here we are using
solvents
to mean both aqueous and organic solvents) were used to purify mixtures
for
identification, analysis, or perhaps for synthetic or other uses. Colored “bands” of
material would pass down the column (elution) and collected in beakers. Sometimes,
initial identification of a material was accomplished solely by its elution
properties. One of us (B.K.) fondly remembers early experimental work in
protein characterization where peptides were separated using hand-packed
10 foot alumina columns. An improperly packed column would allow channels
of liquid to pass through; the mixture wouldn’t separate, everyone
would curse, climb up ladders, take the column apart, and start again. Sometimes
it wouldn’t work at all. The point of this trip down memory lane is
not to claim that we used to crawl through 10 feet of snow to accomplish
analytical work, but rather to emphasize that an analytical separation system
is only as good as its basic design, only as good as the ap propriateness
of the technique to the material under consideration.
To those of us accustomed to the hidden complexities of a “packaged” chromatography
system, it is instructive to keep in mind that originally, chromatography
was a very visual technique; and it remained so for most of the twentieth
century. This visualization often allowed us to determine the overall quality
of the separation system. Understanding the attributes and quality of the
contaminant separation system remains important in contamination control
and contamination identification protocols.
Reference:
1 J.D. Roberts, M.C. Caserio. “Basic Principles of Organic Chemistry,” W.A,
Benjamin, Inc. (1965) New York.
2 http: //www.free-definition.com/Chromatography.html
Barbara Kanegsberg and Ed Kanegsberg
are independent consultants in critical cleaning, precision cleaning, surface
preparation, and contamination control. They are the editors of “Handbook
for Critical Cleaning,” CRC Press. Contact them at BFK Solutions
LLC., 310-459-3614; info@bfksolutions.com; www.bfksolutions.com.
