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What is Chromatography? - Definition, Types & Uses

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  • 0:01 A Mixture of Colors
  • 1:24 Basics of Chromatography
  • 5:05 Types of Chromatography
  • 8:18 Lesson Summary
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Lesson Transcript
Instructor: Gail Marsella
Chromatography is a versatile method of separating many different kinds of chemical mixtures. In this lesson, learn the different types and uses of the technique.

A Mixture of Colors

In the early 1900s, a Russian botanist named Mikhail Tswett became interested in the individual chemical compounds in plants. He noted that mixing ground-up plant material extracts with different solvents produced different colored solutions. One of his experiments involved pouring a plant extract through a glass tube packed with powdered calcium carbonate. As the liquid passed through the solid powder, bands of color appeared; these were the individual compounds, separated from each other by the interaction of the solid (which remained fixed in the tube) and the liquid extract (which flowed through the tube and out the other end). Tswett had invented chromatography, a word he derived from the Greek words for color (chroma) and writing (graphe).

Figure 1: A liquid moves through a finely divided solid, and a mixture placed at the top gradually separates into its component parts as it moves along.
chromatography column

Since then, chromatography has become a cornerstone of separation science, that branch of chemistry devoted to separating compounds from mixtures. There are two main categories of chromatography: preparative and analytical.

Analytical work (which may be used in an environmental lab to look for pollutants) uses small sample sizes, and the objective is to separate compounds in order to identify them. Preparative work (which may be used in the pharmaceutical industry) uses large quantities of samples and collects the output in bulk, and the point of the chromatography here is to remove impurities from a commercial product.

Basics of Chromatography

In any chromatographic technique, a stationary phase usually a solid, thick liquid, or bonded coating that stays fixed in one place, and a mobile phase or eluent (usually a liquid or gas) that moves through it or across it.

A sample to be separated, when placed on the stationary phase, will gradually move along in the same direction as the mobile phase. If a sample compound (or analyte) has no interaction with the stationary phase, it will run right through and come out of the system (elute) at the same rate as the mobile phase. On the other hand, if an analyte has no interaction with the mobile phase, it will stick directly to the stationary phase and never elute. Neither of these are good outcomes.

In a well-designed chromatography process, the chemist will choose stationary and mobile phases that will both have at least some interaction with the analytes. Any individual sample molecule will interact first with one phase and then the other, back and forth repeatedly, but the fraction of each analyte overall in each phase will remain constant. This distribution ratio among the selected phases must differ for each analyte in order for them to separate. (Compounds will not separate chromatographically if they have the same distribution ratio on a particular system.)

Figure 2: Distribution ratios. The analytes continually cross back and forth over the interface between the stationary and mobile phases, but the green check marks will move faster because on the average they spend more time in the mobile phase, which in the diagram is moving to the right.
distribution ratio

Analytes with greater attraction to the stationary phase will still flow through the system because they spend part of their time moving in the mobile phase, but they will tend to lag behind those analytes that have a greater interaction with and, thus, spend more time in the mobile phase. Gradually, as they progress through the system, the analytes separate from each other (usually in order of their distribution ratio), and can be captured or at least detected in relatively pure form as they elute.

Separation is only the first part of chromatography. In Tswett's original experiment, he could see where the various compounds were by color. Most chemicals are colorless, however, so there must be a detector to notice when compounds elute.

Figure 3: a flame ionization detector or FID (usually used with systems that have an inert gas as the mobile phase.) A: analyte enters the FID as it elutes from the chromatography system. B, C, and D: analyte heated and mixed with hydrogen and oxygen. E and F: analyte burns in a flame and the ions are pushed forward. G: collector plates detect ions. H: signal transmitted to electronics. I: exhaust port for flame products.
flame ionization detector

While there are various types of detectors, their output is usually a chart with peaks corresponding to the various compounds (a chromatogram). The amount of time any given analyte stays on the column is its retention time and the area under its peak is proportional to its concentration, so the chemist can figure out how much of one analyte is present relative to the other compounds.

Figure 4: A chromatogram of a perfume sample, showing multiple fragrance components. The X-axis shows the retention time of each peak in minutes, and the Y-axis shows the response of the detector. Notice that some of the peaks overlap, which indicates an incomplete separation.
chromatography output peaks

One thing chromatography alone cannot do is identify the molecular structure of the sample compounds; it can separate them from each other and detect them, but it does not tell what they are. Other kinds of analytical work would be needed for identification.

Types of Chromatography

There are hundreds of stationary phase, mobile phase, and detector combinations in wide use throughout the chemical, medical, and biotechnology industries, but we can identify five major types.

Thin Layer Chromatography, or TLC, uses a flat plate coated with a stationary phase instead of a packed column. One edge of the plate is dipped into the mobile phase, which moves up through the solid material, carrying and separating the analytes. Colorless analytes can be sprayed with a chemical that colors them, so the chemist can see the result. TLC is usually preliminary to more in-depth analysis; it is, however, fast and requires very little equipment.

Gas Chromatographyor GC in which the mobile phase is an inert gas, usually helium, which simply carries along any molecules in the gaseous state. A GC system has an oven for heating the column, and so works best for samples that evaporate readily and do not decompose at high temperature. The stationary phase is a finely divided solid (often coated) packed into a coiled column. The analytes interact either directly with the solid surface (adsorption chromatography) or with the surface coating (partition chromatography). A popular variant on the packed column is the capillary column, a very long, coiled, open, narrow-bore tube with a coating on the inside walls. Capillary columns are particularly efficient at separating analytes with similar chemical structures.

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