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How Separation Works in Column Chromatography Methods

How Separation Works in Column Chromatography Methods

Separation in column chromatography relies on differences. Molecules vary in size, charge, polarity, and solubility. We leverage these differences to distribute molecules between a stationary phase and a mobile phase. But because molecules are so different, it’s not possible to have a single method that works for all.

In my previous article I discussed the basic process of running a chromatography column. And now, with all the different methods available, I think it’s important for you to know some of your options and how they work.

The methods shown below take advantage of several molecular properties to dictate the mode of separation.

cc methods figure 1

Pave the Way

Hydrophobic interaction chromatography separates molecules based on their hydrophobicity. The stationary matrix has hydrophobic groups that interact with the hydrophobic regions of molecules, but they have to find each other first.

Remember the “mix oil and water” experiment? Of course you do! Well, they don’t mix, right? That’s because oil is hydrophobic—it’s not attracted to water. The same happens with a hydrophobic column and water. Because there are no interactions between them, a network of hydrogen bonds among water molecules from the eluent forms instead. This network surrounds hydrophobic groups in the column, preventing your analyte from binding to the matrix.

To enable binding, break the water shield by treating the column with a chaotropic salt solution. This introduces ions that interact with water and thus, exposes the hydrophobic groups. If you use a high-salt solution your molecule will bind strongly to the matrix. At this point you wash out the junk. After, elute your molecule with a low-salt solution as it promotes the reformation of the water shield.

Like Attracts Like

Normal/reversed-phase chromatography separates molecules by polarity. The stationary phase contains either highly polar or highly non-polar functional groups that interact with molecules according to their polarity level.

If we go back to the concept of oil and water not mixing, we can also say that because water is highly polar and oil is highly non-polar, they don’t attract each other but instead are attracted to their own molecules. In other words, it shows how polar attracts polar and non-polar attracts non-polar.

In normal-phase chromatography, the stationary phase is more polar than the mobile phase. So as polar molecules are retained in the column, your elution of molecules will go from non-polar to polar. For reversed-phase chromatography things are, well, the reverse. You use a non-polar stationary phase that retains non-polar compounds and so, you elute first the polar molecules.

Opposites Attract, Too

Ion-exchange chromatography separates molecules based on their ionic interactions. The stationary phase is a resin that supports ionic functional groups and retains molecules of the opposite charge.

So if you want to purify your negatively charged protein, you’ll pass it through a positively charged resin. The molecules that don’t bind, or bind weakly, flush out first while your protein stays in the column. This method is better known as anion exchange chromatography. Its opposite, cation exchange chromatography, uses anionic resins to retain cationic molecules.

One way to elute your molecule is to increase the salt concentration of the mobile phase – note that it’s the opposite of hydrophobic interaction columns. Here, the ions compete with your molecule for binding with the matrix, displacing it to the elution stream. The other approach is to change the pH of your buffer to alter the net charges and thus, the ionic binding.

Hold On Tight

Affinity chromatography separates molecules based on their binding interactions with a specific small molecule that is covalently attached to a stationary matrix.

Think of chromatography by affinity as a lock-and-key mechanism – the small molecule on the matrix is the lock and your molecule is the key. Your crude sample is a set of keys, but your analyte is the only key with the complementary shape (interaction) that matches the lock on the affinity medium. Having your molecule fixed on the matrix allows you to flush out the unwanted stuff. And when you’re ready, break the interaction to collect your analyte.

To apply this method you must use a molecule with well-defined lock-and-key binding properties. Examples of specific binding are those found between an enzyme and substrate, antigen and antibody, and receptor and ligand.

Size Matters

Size-exclusion chromatography separates molecules by their size. This method, also known as gel permeation chromatography, is unlike those described above because it exploits a physical characteristic instead of chemical interactions.

The stationary phase is a resin of porous beads that traps small molecules but not large ones. So compounds with high molecular weights and large diameters are excluded because they’re too big to fit through the holes of the beads.

Suppose you load a polymer sample onto the column. During the run, your polymer freely moves around and between the beads while the small impurities constantly enter and exit beads. It’s like comparing someone driving on back roads with someone taking the highway. So the travel is longer for small particles, but your large analyte moves with the elution stream, exiting the column first.

Have you used other column chromatography methods? Please add to the list by commenting below!

Further Reading

  1. Skoog DA, Holler FJ, Nieman, TA. Principles of Instrumental Analysis: Chapter 28. 5th Brooks Cole; 1997.
  2. Hydrophobic Interaction and Reversed Phase Chromatography: Principles and Methods. GE Healthcare Bio-Sciences Corp. Handbook 11-0004-21.
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