Chromatography
by Chris Woodford. Last updated: February 25, 2022.
Most of us have got our papers wet at some time or another, but have you ever noticed
what happens to the ink as the water spreads? It doesn't always
smudge and blur, as you might expect. Sometimes it splits up into
weird colored streaks that creep across the page. When that happens,
you're seeing chromatography in action. In this case it's totally
accidental, but we can also use it by design to split up mixtures and
other substances into their components. Chromatography is actually
one of the most useful analytical techniques chemists have at their
disposal, helpful in everything from identifying biological materials
to finding clues at crime scenes. What is it and how does it work?
Let's take a closer look!
Photo: High-performance liquid chromatography (HPLC) is a very fast and precise kind of
liquid chromatography used in research labs. Here it's being used to analyze an antibiotic for controlling
diseases in wheat. Photo by Jack Dykinga courtesy of US Department of Agriculture/Agricultural Research Service.
What is chromatography?
Chromatography is a pretty accurate description of what happens to ink on wet paper,
because it literally means "color writing" (from the Greek words
chroma and graphe). Really, though, it's a bit of a
misnomer because it often doesn't involve color, paper, ink, or
writing.
Chromatography is actually a way of separating out a mixture
of chemicals, which are in gas or liquid form, by letting them creep
slowly past another substance, which is typically a liquid or solid.
So, with the ink and paper trick for example, we have a liquid (the
ink) dissolved in water or another solvent creeping over the surface
of a solid (the paper).
The essential thing about chromatography is that we have some mixture in one
state of matter (something like a gas or liquid) moving
over the surface of something else in another state of matter (a liquid or solid) that stays where it is. The
moving substance is called the mobile phase and the substance
that stays put is the stationary phase. As the mobile phase
moves, it separates out into its components on the stationary phase.
We can then identify them one by one.
Photo: What makes ink blur on wet paper? What you're seeing here is chromatography in action. Different inks and papers produce very different effects. Try experimenting for yourself!
How does chromatography work?
Think of chromatography as a race and you'll find it's much simpler than it sounds. Waiting on the
starting line, you've got a mixture of chemicals in some unidentified
liquid or gas, just like a load of runners all mixed up and bunched together. When
a race starts, runners soon spread out because they have different
abilities. In exactly the same way, chemicals in something like a
moving liquid mixture spread out because they travel at different
speeds over a stationary solid. The key thing to remember is that chromatography is a surface effect.
As the liquid starts to move past the solid, some of its
molecules (energetic things that are constantly moving about) are
sucked toward the surface of the solid and stick there temporarily before being
pulled back again into the liquid they came from. This exchange of
molecules between the surface of the solid and the liquid is a kind of adhesive or
gluing effect called adsorption (with a d—don't confuse it with
absorption, with a b, where molecules of one substance are permanently
trapped inside the body of another). Now remember that our liquid is actually a
mixture of quite a few different liquids. Each one undergoes
adsorption in a slightly different way and spends more or less time
in either the solid or the liquid phase. One of the liquids might
spend much longer in the solid phase than in the liquid, so it would
travel more slowly over the solid; another one might spend less time
in the solid and more in the liquid, so it would go a bit faster.
Another way of looking at it is to think of the liquid as a mixture
of glue-like liquids, some of which stick more to the solid (and
travel more slowly) than others. This is what causes the different
liquids within our original liquid mixture to spread out on the
solid.
Artwork: How chromatography works: here the mobile phase is a liquid (blue) and the stationary phase is a solid (gray). The green molecule spends most time in the liquid so moves fastest. The yellow molecule spends more time on the surface of the solid, so moves slower. The red molecule spends even more time on the solid surface, so moves slowest.
For chromatography to work effectively, we obviously need the components of the mobile
phase to separate out as much as possible as they move past the
stationary phase. That's why the stationary phase is often something
with a large surface area, such as a sheet of filter paper, a solid
made of finely divided particles, a liquid deposited on the surface
of a solid, or some other highly adsorbent material.
What are the different types of chromatography?
There are many different ways of using chromatography. These are some of the best known:
Paper chromatography
Photo: Simple paper chromatography. Draw some blobs of ink on paper (Crayola washable children's fiber
tips are perfect), roll the paper into a cylinder, and place it in a wine glass with a small amount of water. As the water creeps
up the paper, the colors will separate out into their components. That's chromatography in action!
This is the "spot of ink on paper" experiment you often do in school (also the effect
we described at the start when you get your papers wet). Typically
you put a spot of ink near one edge of some filter paper and then
hang the paper vertically with its lower edge (nearest the spot)
dipped in a solvent such as alcohol or water. Capillary action makes
the solvent travel up the paper, where it meets and dissolves the
ink. The dissolved ink (the mobile phase) slowly travels up the paper
(the stationary phase) and separates out into different components.
Sometimes these are colored; sometimes you have to color them by
adding other substances (called developers or developing fluids) that
help you with identification.
Column chromatography
Instead of paper, the stationary phase is a vertical glass jar (the column)
packed with a highly adsorbent solid, such as crystals of silica or silica gel,
or a solid coated with a liquid. The mobile phase drips (or is pumped at high
pressure) through the column and splits into its components, which are
then removed and analyzed.
There are quite a few variations, including:
- Liquid-column chromatography, where the
mixture being studied is placed at one end of the column and an extra
added substance called an eluant (sometimes spelled eluent) is poured in to help it travel
through.
- Thin-film chromatography is a variation of this technique in
which the "column" is actually a film of glass,
plastic, or metal coated with
a very thin layer of adsorbent material.
- High-performance liquid chromatography (HPLC), where the
mixture is forced through the column at high pressure (roughly 400 times atmospheric pressure).
This is faster, more precise, and more sensitive.
Photo: Column chromatography: You take your column, containing the stationary phase,
and load it with your sample at the top (dark gray). As you add eluant (solvent) to the sample, it splits into its
components (let's say they are colored red, yellow, and blue). These travel at different speeds and
emerge one at a time at the bottom, where you can collect them in different containers.
Gas chromatography
So far we've considered chromatography of liquids traveling past solids, but one
of the most widely used techniques is a type of column chromatography
using gases as the mobile phase. Gas chromatography is a largely
automated type of chemical analysis you can do with a sophisticated
piece of laboratory equipment called, not surprisingly, a gas
chromatograph machine.
First, a tiny sample of the mixture of substances being
studied is placed in a syringe and injected into the
machine. The components of the mixture are heated and instantly
vaporize. Next, we add a carrier (the eluant), which
is simply a neutral gas such as hydrogen or helium, designed to help
the gases in our sample move through the column. In this case, the
column is a thin glass or metal tube usually filled with a liquid
that has a high boiling point (or sometimes a gel or an adsorbent
solid). As the mixture travels through the column, it's adsorbed and
separates out into its components. Each component emerges in turn
from the end of the column and moves past an electronic detector
(sometimes a mass spectrometer),
which identifies it and prints a peak on a chart. The final chart has
a series of peaks that correspond to all the substances in the
mixture. Gas chromatography is sometimes called vapor-phase
chromatography (VPC) or gas-liquid partition chromatography (GLPC).
Photo: Gas chromatography is largely automated, but it still
takes a trained operator to work one of these machines. Here, the sample is about to be injected. Photo by courtesy of
NASA Kennedy Space Center (NASA-KSC).
What is chromatography used for?
“... the pigments separate themselves from top to bottom
in different colored zones.... such a preparation I call... the chromatographic method.”
Mikhail Tswett, 1906
Chromatography was developed in Russia in 1906 by an Italian-born botanist named
Mikhail
Tswett (sometimes spelled Tsvet; 1872–1919), who used it for studying
plant pigments such as chlorophyll. (Like all great scientists, Tswett used and built on techniques
developed by earlier researchers, so there is dispute over whether he can truly be described as the
"inventor" of chromatography.)
During the 20th century, chemists found chromatography was a superb technique for studying and
separating all kinds of complex mixtures. It's now widely used in
forensic science (for identifying samples taken from crime scenes),
in pollution monitoring (for identifying small concentrations of
unknown pollutants in air and water samples), and for studying
complex mixtures in such things as food, perfume, petrochemical, and
pharmaceutical production. One of chromatography's big advantages is
that it works with tiny samples and low concentrations (particularly
helpful when it comes to such things as forensic science and drug or
pollution testing).
Photo: What's your poison? A sample of vehicle exhaust is injected
into a gas chromatography machine so the pollutants it contains can be analyzed.
Photo by Warren Gretz courtesy of US DOE/NREL
(US Department of Energy/National Renewable Energy Laboratory).
