Chromatography works just as well for organic compounds as it does for proteins. With that in mind what we cover here also applies to organic chemistry compounds so we will cover this material in both contexts.

The Basics

Chromatography is all about affinity and is used to separate compounds on the basis of their different chemical properties. While different chromatography columns operate on different types of affinity they all work on the same basic principles.

A column is typically filled with a porous solid called the stationary phase so-called because it doesn’t move. Then the protein sample or other compound is added to the column and compounds with affinity to the stationary phase will bind to it. For example, polar molecules will stick to a polar stationary phase and negative molecules to a positive stationary phase.

From there a solvent is added to the column called the mobile phase so-called because it moves. Anything that isn’t bound to the column will be washed away or in fancy terms eluted. As time progresses and more of the mobile phase is added to the column even bound molecules will start to wash away. Those with the weakest affinity for the column are the first to go while those with the strongest affinity require the most solvent or the longest time to get washed out.

Putting the Chroma in Chromatography

How do we know if something is stuck or washed out though? Colored or fluorescent dyes are attached to different compounds. For example, size exclusion chromatography sorts compounds by size so the larger the compound the more strongly it will fluoresce since has more space for a fluorescent dye to attach. This is what puts the chroma in chromatography by using some sort of observable colored or fluorescent compound we can see or at least measure the progress of our separation.

Separation by Polarity

One of the most common separation techniques relies on differences in polarity to separate different molecules. For example, we might hope to separate hexanol from hexanoic acid or a protein-rich in polar residues from one that is rich in nonpolar residues.

Thin-Layer Chromatography

In thin-layer chromatography, the stationary phase consists of either a piece of paper or a bit of silica placed between two glass slides. In either case, the stationary phase is polar and the more polar molecule will adhere more strongly. Then a weakly polar mobile phase is allowed to flow from one side of the paper or plate to the other.

Since our initial compounds are dyed we can watch how they move as the solvent progress up the stationary phase. For example, hexanol is less polar than hexanoic acid so to won’t adhere to the stationary phase as strongly. Therefore it would move further up the paper or silica and the same would hold true for a nonpolar versus polar protein.

If we want to quantify the relative polarity of our molecules we can do so using the retardation factor (Rf). The Rf compares how far our compound moves to how far the solvent moved throughout the period of the separation or the solvent front. This is why the solvent is stopped before it reaches the full length of the stationary phase.

R_f=\frac{Spot\; Distance}{Solvent\; Distance}\]

Normal Phase Column Chromatography

Normal phase column chromatography operates on the exact same principle as thin-layer chromatography. Except instead of conducting our experiment between two pieces of glass or on a piece of paper it is done in a column. Again more polar molecules will stick to the stationary phase while more nonpolar molecules are washed away. Here the only difference is that the different fractions can be collected into beakers and used for further analysis.

Reverse Phase Chromatography

Reverse-phase chromatography can be conducted in a column or a thin-layer the only difference here is that the polarity of the two phases is switched. Under reverse-phase circumstances the stationary phase is nonpolar and the mobile phase is polar. Therefore, nonpolar molecules will end up stuck to the stationary phase while polar ones will elute.

Separation by Charge

Another way to separate molecules is on the basis of their charge. Here columns contain a stationary phase with an opposing charge in order to attract the oppositely charged molecule. Typically a salt solution such as 1M NaCl is run over the column washing away any unbound compounds. As more and more of the eluent is added bound molecules will begin to wash away as their and the column’s charges are neutralized by the addition of ions within the eluent.

Anion Exchange Column

Anion exchange columns bind anionic molecules so proteins with net negative charges will get stuck to the column while neutral or positively charged molecules are easily washed away. In order for an anion exchange column to bind to positively charged molecules the stationary phase has to be positively charged since opposite attract.

Cation Exchange Column

A cation exchange column on the other hand binds positively charge molecules by using a negatively charged stationary phase. As with the other column types the greater the net positive charge on a molecule the greater the affinity that molecule will have for the column. The greater the affinity the more eluent needed to wash that molecule away.

It is pretty easy to mix up anion and cation columns because they end up binding one type of charge but have the opposite charge as their stationary phase. Try and remember that the name of each tells you what type of molecule they bind. So a cation exchange column binds cations while an anion exchange column binds anions.

Separation by Affinity Tag

Another way of separating molecules is by affixing tags to them and designing columns that bind with high affinity to those tags. Molecules with the tag will get stuck while molecules lacking the tag are washed away. Although fairly trivial knowledge look out for any mention of histidine or nickel as they are commonly used when designing affinity-based column chromatography.

Separation by Size

Molecules can also be separated by size by using size exclusion or gel filtration chromatography. Although this type of chromatography isn’t technically based on affinity we can still use our affinity framework to explain how they work. Under this framework, the stationary phase is composed of beads that have a high affinity for small molecules. So small molecules get stuck in the column while larger ones are washed through quickly. This occurs because the beads have microscopic pores that only allow tiny molecules to enter.

So while bigger molecules are playing Plinko outside of the beads the small molecules are taking the slow and scenic route through them.

High-Performance Liquid Chromatography (HPLC)

Additionally molecules can be purified or separated using high performance liquid chromatography (HPLC). HPLC works like any other column except it is completely computer controlled and results in more precise and accurate separations. Typically polarity based HPLC is carried out however a wide variety of stationary and mobile phases can be used depending on what needs to be separated.

Gas Chromatography

Moving on from the liquid phase is gas chromatography or GC for short. GC analyzes volatile compounds by first vaporizing them. From there an inert gas such as helium carries the gas through a crushed stationary phase. Here separation occurs due to two factors molecular weight and polarity.

Since the stationary phase is usually polar more polar molecules will adhere as they pass through and slow down. As in normal-phase column chromatography these molecules will take a longer time to reach the detector on a GC and will end up being one of the last traces. In contrast, nonpolar molecules will fly through the stationary phase and straight to the detector.

Additionally, the molecular weight of each gaseous molecule determines its speed with heavier molecules moving more slowly. Therefore polarity being equal smaller molecules will be detected first and larger molecules second.