Chromatography: purifying your understanding

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Maybe you were studying for the MCAT or just in your college chemistry lab when it dawned on you: why are there so many different types of chromatography? What do they have in common and what are their differences? 

In all its forms, chromatography is an extremely useful tool for separation and purification of mixtures of different chemical substances. Separation allows us to further analyze components, and some everyday applications of this include drug testing from blood samples, food and beverage testing for ingredients that could make consumers sick, and even vaccine preparation! Therefore, chromatography is nicely paired with analytical chemistry, where methods such as mass spectrometry and NMR are used to determine the identity of an unknown substance. 

So, what do they all have in common? There is always a mobile phase (liquid or gas) and a stationary phase over which a sample is run. The key to separation lies in the difference in polarity of the mobile and stationary phases. Usually, it is the stationary phase that is more polar and the mobile is less or nonpolar. A sample consisting of a more polar substance (we’ll call substance A) and a less polar substance (substance B) will be separated out because A will prefer to be dissolved in the polar stationary phase while B will prefer to be dissolved in the less polar mobile phase. Remember: like dissolves like

The main forms of chromatography and their principles:

Paper Chromatography


  • Stationary phase: paper with water molecules**
  • Mobile phase: liquid solvent
  • What is measured: Retention factor
  • Takeaway: Greater the Rf, the less polar the compound

Paper is made of cellulose, a sugar and, therefore, a polar molecule. Furthermore, cellulose molecules will attract water vapor molecules, making the stationary phase relatively polar. Therefore, most of the time we think of an effective mobile phase as being a nonpolar solvent, such as hexane. Retention factor, or Rf, is calculated as the distance traveled by a compound over the distance traveled by the solvent. In our example here, nonpolar compounds will travel further with the nonpolar liquid solvent, giving them greater Rf values. Polar compounds will be attracted to the paper and will be less likely to move far with the mobile phase; they will have lower Rf values.  

Thin Layer Chromatography


  • Stationary phase: glass with silica gel, aluminum, or cellulose
  • Mobile phase: liquid solvent
  • What is measured: Retention factor
  • Takeaway: Greater the Rf, the less polar the compound

The experimental setup of TLC can be thought of as similar to paper, but with a different stationary phase.  Here, a surface such as glass or plastic is coated with a polar substance, the most common being silica gel, aluminum oxide, and cellulose. Rf is measured the same way here, with the same trend as above.

Gas/Liquid Chromatography


  • Stationary phase: Polar liquid coating the column
  • Mobile phase: Inert gas
  • What is measured: Retention time
  • Takeaway: Greater the retention time, the MORE polar

Here, the stationary phase is actually a liquid that lines the column through which a sample is run. The mobile phase is an inert gas, such as hydrogen, nitrogen, or helium, which carries the sample without reacting with it. A detector at the end of the column will pick up when each compound passes, generating a series of retention times. A greater retention time means the compound spent more time being attracted to the polar liquid coating the column, rather than traveling straight through with the inert gas. Therefore, greater retention time corresponds with more polar compounds. 

Column Chromatography


  • Stationary phase: Solid silica gel or alumina
  • Mobile phase: liquid solvent
  • What is measured: Order of elution
  • Takeaway: First eluents are the least polar

In column chromatography, a polar solid (silica and aluminum are used here as in TLC) is packed into a column and the sample is loaded on top. Then the liquid phase is poured on top and allowed to run downward. At the bottom, a stopcock is opened to allow the liquid to run through. The solvent will carry the least polar components of the sample the fastest, as they will have the least attraction to the silica. We collect the sample in fractions, with the first fractions being solvent alone, the next being solvent carrying the nonpolar components, and finally the fractions containing the more polar compounds. 

A Final Note

This has been a brief rundown of the most important types of chromatography that separate compounds based on polarity. Size-exclusion and ion exchange chromatography are two different forms that should be explored individually, as they separate based on size and charge, respectively. The secret to answering chromatography questions lies in a keen understanding of relative polarities of compounds, so, as always, be sure to brush up on your organic chemistry!