Chromatography - Separations and Purifications - MCAT Organic Chemistry Review

MCAT Organic Chemistry Review

Separations and Purifications

12.3 Chromatography

Chromatography is another tool that uses physical and chemical properties to separate and identify compounds from a complex mixture. In all forms of chromatography discussed here, the concept is identical: the more similar a compound is to its surroundings (whether by polarity, charge, or other characteristics), the more it will stick to and move slowly through its surroundings.


Chromatography separates compounds based on how strongly they adhere to the solid, or stationary, phase (or in other words, how easily they come off into the mobile phase).

The process begins by placing the sample onto a solid medium called the stationary phase, or adsorbent. We then run the mobile phase, usually a liquid (or a gas in gas chromatography) through the stationary phase. This will displace (elute) the sample and carry it through the stationary phase. Depending on the characteristics of the substances in the sample and the polarity of the mobile phase, it will adhere to the stationary phase with differing strengths, causing the different substances to migrate at different speeds. This is called partitioning, and it represents an equilibrium between the two phases. Different compounds will have different partitioning coefficients and will elute at different rates. This results in separation within the stationary phase, allowing us to isolate each substance individually.

There are many different media that can be used as the stationary phase, each one exploiting different properties that allow us to separate out our compound. On the MCAT, the property most commonly used is polarity. For instance, thin-layer chromatography (TLC), which we will shortly discuss, uses silica gel, a highly polar substance, as its stationary phase. Cellulose, another polar substance, may also be used. This means that any polar compound will adhere to the gel well and thus move through (elute) slowly. When using column chromatography, size and charge both have a role in how quickly a compound moves through the stationary phase. Chromatography can use even strong interactions, such as antibody–ligand binding.

As mentioned above, chromatography is based on the speed at which compounds move through media. In practice, however, we will measure either how far each substance travels in a given amount of time (such as in TLC) or how long it takes to elute (as in column or gas chromatography).

The types of chromatography that we will discuss include thin-layer chromatography and paper chromatography, column chromatography, gas chromatography (also called gas–liquid chromatography), and high-performance liquid chromatography, or HPLC.


Thin-layer chromatography and paper chromatography are extremely similar techniques, varying only in the medium used for the stationary phase. For thin-layer chromatography, a thin layer of silica gel or alumina adherent to an inert carrier sheet is used. For paper chromatography, as the name suggests, the medium used is paper, which is composed of cellulose.

For these techniques, the sample that we want to separate is placed directly onto the adsorbent itself; this is called spotting because we apply a small, well-defined spot of the sample directly onto the silica or paper plate. The plate is then developed, which involves placing the adsorbent upright in a developing chamber, usually a beaker with a lid or a wide-mouthed jar. At the bottom of this jar is a shallow pool of solvent, called the eluent. The spots of sample must be above the level of the solvent, or else they will dissolve into the pool of solvent rather than running up the plate. When set up correctly, the solvent will creep up the plate by capillary action, carrying the various compounds in the sample with it at varying rates. When the solvent front nears the top of the plate, the plate is removed from the chamber and allowed to dry.

As mentioned before, TLC is often done with silica gel, which is polar and hydrophilic. The mobile phase, on the other hand, is usually an organic solvent of weak to moderate polarity, so it doesn”t bind well to the gel. Because of this, nonpolar compounds dissolve in the organic solvent and move quickly as the solvent moves up the plate, whereas the more polar molecules stick to the gel. Thus, the more nonpolar the sample is, the further up the plate it will move, as shown in Figure 12.5.

Figure 12.5. Thin-Layer Chromatography Samples are placed at the “X” marks. As the nonpolar solvent moves up the plate via capillary action, the samples that are nonpolar move further up the plate along with the solvent, while the samples that are polar do not move as far.

Reverse-phase chromatography is the exact opposite. In this technique, the stationary phase used is nonpolar, so polar molecules move up the plate quickly, while nonpolar molecules stick more tightly to the stationary phase.

The spots of individual compounds are usually white, which makes them difficult or impossible to see on the white paper or TLC plate. To get around this problem, the developed plate can be placed under ultraviolet light, which will show any compounds that are ultraviolet-sensitive. Alternatively, iodine, phosphomolybdic acid, or vanillincan be used to stain the spots, although this will destroy the compounds such that they cannot be recovered.

When TLC is performed, compounds are generally identified using the retardation factor (Rf), which is relatively constant for a particular compound in a given solvent. The Rf is calculated using the equation:

Because its value is relatively constant, the Rf value can be used to identify unknown compounds.

This technique is most frequently performed on a small scale to identify unknown compounds. It can also be used on a larger scale as a means of purification, a technique called preparative TLC. As the large plate develops, the larger spot of sample splits into bands of individual compounds, which can then be scraped off and washed to yield pure compounds.


The principles behind column chromatography are the same as for thin-layer chromatography, although there are some differences. First, column chromatography uses an entire column filled with silica or aluminum beads as an adsorbent, allowing for much greater separation. The setup for this is shown in Figure 12.6. In addition, thin-layer chromatography uses capillary action to move the solvent up the plate, whereas column chromatography uses gravity to move the solvent and compounds down the column. To speed up the process, one can force the solvent through the column using gas pressure, a technique called flash column chromatography. In column chromatography, the solvent polarity can also be changed to help elute the desired compound.

Figure 12.6. Column Chromatography Sample is added to the top of the column, and a solvent is poured over it. The more similar the sample is to the mobile phase, the faster it elutes; the more similar it is to the stationary phase, the more slowly it will elute (if at all).

Eventually, the solvent drips out of the end of the column, and the different fractions that leave the column can be collected over time. Each fraction will contain different compounds. After collection, the solvent can be evaporated, leaving behind the compounds of interest. Column chromatography is particularly useful in biochemistry because it can be used to separate and collect macromolecules such as proteins or nucleic acids. There are several techniques that can be used to isolate specific materials, which are described in the following paragraphs, as well as in Chapter 3 of MCAT Biochemistry Review.

Ion-Exchange Chromatography

In ion-exchange chromatography, the beads in the column are coated with charged substances so that they attract or bind compounds that have an opposite charge. For instance, a positively charged compound will attract and hold a negatively charged backbone of DNA or protein as it passes through the column, either increasing its retention time or retaining it completely. After all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column.

Size-Exclusion Chromatography

In size-exclusion chromatography, the beads used in the column contain tiny pores of varying sizes. These tiny pores allow small compounds to enter the beads, thus slowing them down. Large compounds can”t fit into the pores, so they will move around them and travel through the column faster. It is important to remember that in this type of chromatography, the small compounds are slowed down and retained longer—which may be counterintuitive. The size of the pores may be varied so that molecules with different molecular weights can be fractionated. A common approach in protein purification is to use an ion-exchange column followed by a size-exclusion column.

Affinity Chromatography

In affinity chromatography, a protein of interest is bound by creating a column with high affinity for that protein. This can be accomplished by coating beads with a receptor that binds the protein or a specific antibody to the protein; in either case, the protein is retained in the column. Common stationary phase molecules include nickel, which is used in separation of genetically engineered proteins with histidine tags, antibodies or antigens, and enzyme substrate analogues, which mimic the natural substrate for an enzyme of interest. Once the protein is retained in the column, it can be eluted by washing the column with a free receptor (or target or antibody), which will compete with the bead-bound receptor and ultimately free the protein from the column. Eluents can also be created with a varying pH or salinity level that disrupts the bonds between the ligand and the protein of interest. The only drawback of the elution step is that the recovered substance can be bound to the eluent. If, for example, the eluent was an inhibitor of an enzyme, it could be difficult to remove.


Gas chromatography (GC) is another method that can be used for qualitative separation. GC, also known as vapor-phase chromatography (VPC), is similar to the other types of chromatography and is shown in Figure 12.7. The main conceptual difference is that the eluent is a gas (usually helium or nitrogen) instead of a liquid. The adsorbent is a crushed metal or polymer inside a 30-foot column. This column is coiled and kept inside an oven to control its temperature. The mixture is then injected into the column and vaporized. The gaseous compounds travel through the column at different rates because they adhere to the adsorbent in the column to different degrees and will separate in space by the time they reach the end of the column. The injected compounds must be volatile: low melting-point, sublimable solids or vaporizable liquids. The compounds are registered by a detector, which records them as a peak on a chart. It is common to separate molecules using GC and then to inject the pure molecules into a mass spectrometer for molecular weight determination, which is referred to as GC–mass spectrometry.

Figure 12.7. Gas Chromatography The sample is injected into the column and moves with the gaseous mobile phase through a stationary liquid or solid phase; a computer identifies the sample components.


High-performance liquid chromatography (HPLC) was previously called high-pressure liquid chromatography. As the name suggests, the eluent is a liquid, and it travels through a column of a defined composition. There are a variety of stationary phases that can be chosen depending on the target molecule and the quantity of material that needs to be purified. This is fairly similar to column chromatography because the various compounds in solution will react differently with the adsorbent material. In the past, very high pressures were used, but recent advances allow for much lower pressures—hence the change in name. In HPLC, a small sample is injected into the column, and separation occurs as it flows through. The compounds pass through a detector and are collected as the solvent flows out of the end of the apparatus. The interface is similar to that used for GC because the entire process is computerized, but uses liquid under pressure instead of gas. Because the whole process is under computer control, sophisticated solvent gradients as well as temperature can be applied to the column to help resolve the various compounds in the sample—hence the higher performance of HPLC over regular column chromatography.

MCAT Concept Check 12.3:

Before you move on, assess your understanding of the material with these questions.

1. What properties of molecules do thin-layer chromatography, paper chromatography, and standard column chromatography take advantage of to separate compounds?

2. What are the three specialized types of column chromatography? What does each use in order to separate the sample?

Type of Column Chromatography

Method for Separating Sample

3. In what way is gas chromatography distinct from all of the other techniques we have discussed?

4. What is the major historical distinction between HPLC and column chromatography? What is the major distinction now?

· Historical distinction:

· Current distinction: