Chromatography is a method of separating substances completely. There are several types of chromatography, of which column chromatography is the most frequently used in laboratories that deal with organic compounds.

A type of adsorption chromatography is column chromatography. It uses an adsorbent called silica gel to separate organic compounds that are mixed in with each other.

Column chromatography is an experimental operation that is performed daily in laboratories that deal with organic compounds, such as those that require organic synthesis and structural determination of compounds.

So what are the principles of separating organic compounds by column chromatography? What are the methods and procedures of column chromatography? These are explained in an easy-to-understand manner.

Normal-Phase Chromatography for Complete Separation of Organic Compounds

Adsorption chromatography is a method of separating chemical substances by using the properties of adsorbing substances to separate them. There are several types of adsorption chromatography, one of which is column chromatography. Column chromatography is also referred to as normal-phase chromatography.

Although they are not exactly the same, you can generally think of adsorption chromatography and normal-phase chromatography as the same thing. The following equipment is column chromatography.

In column chromatography, the stationary phase of the column (a small cylinder) is filled with a substance. Absorbed by the stationary phase, the compound is gradually moved forward.

Velocity of Movement Through the Stationary Phase (Silica Gel) Varies with the Material

Silica gel is used as the stationary phase in column chromatography. Sometimes alumina is used in column chromatography, but silica gel, not alumina, is the most common choice. There is no need to consider the use of alumina.

Silica gel is a solid and is used as a stationary phase in column chromatography. The structural formula of silica gel is as follows

As you can see from this structural formula, silica gel is extremely polar. There are a lot of oxygen atoms with high electronegativity around the silicon.

Silica gel has these properties. When a compound passes through a column containing silica gel, it has a different velocity. These differences in speed are used to separate compounds.

HPLC (High Performance Liquid Chromatography) Is Reverse Phase Chromatography

Note that among the also well-known chromatographs, HPLC (high-performance liquid chromatography) and column chromatography are different.

Column chromatography utilizes highly polar materials such as silica gel and alumina as the stationary phase. Thus, column chromatography, which uses highly polar materials as the stationary phase, is normal-phase chromatography.

On the other hand, reversed-phase chromatography uses less polar materials (highly hydrophobic materials) as the stationary phase. The nature of the substances used for the stationary phase (column) is different.

  • Normal-phase chromatography: uses highly polar materials as the stationary phase
  • Reversed-phase chromatography: uses highly hydrophobic materials as the stationary phase

In HPLC (high performance liquid chromatography), reversed-phase chromatography is the main method. Think of it as different in nature from column chromatography.

Highly Polar Compounds Interact with the Stationary Phase as an Adsorbent

So why do different compounds move through silica gel at different rates when using column chromatography? This is because they interact with the silica gel. That is, the compounds are adsorbed onto the silica gel.

Water and oil do not mix. This is because water has a high polarity and oil has a high fat solubility. Substances with high polarity are soluble in water. On the other hand, even compounds that are highly fat-soluble and insoluble in water are soluble in oil. Substances with the same properties have a high affinity for each other.

Think about it this way, substances with high polarity tend to interact with silica gel, which is also highly polar. In other words, they will be adsorbed by the silica gel.Even if the solvent is flowing, the compound will gradually move forward while being adsorbed by the silica gel and leaching into the organic solvent of the mobile phase.

In contrast, what about less polar compounds (highly hydrophobic compounds)? They do not interact with the highly polar silica gel and will not be adsorbed by it. As a result, they will move forward quickly when the solvent is flushed.

This is the reason why the speed of organic compounds varies when the organic solvent is poured into a tube of silica gel.

Velocity (Degree of Elution) Varies Depending on the Polarity of the Organic Solvent in the Mobile Phase

Also, in column chromatography, you have to decide on a mobile phase. The stationary phase is a choice of silica gel. However, you have to decide which type of solvent to run.

The most common organic solvents used in column chromatography are ethyl acetate and hexane.

As you can see from the structural formula, ethyl acetate has a high polarity. On the other hand, hexane has a lower polarity. In column chromatography, ethyl acetate and hexane are generally mixed together. The mobile phase (organic solvent) of column chromatography used in this process is called the developing solvent.

Because of the high polarity of ethyl acetate, the substance passes quickly through the silica gel as the ratio of ethyl acetate in the developing solvent is increased. Even though the compounds are adsorbed on silica gel, they can easily elute into ethyl acetate, which has a high polarity, and move quickly through the column chromatography.

On the other hand, what if the ratio of hexane is increased? Because hexane is highly hydrophobic, it is difficult for a compound to dissolve in an organic solvent with a high hexane ratio. As a result, the higher the percentage of hexane, the slower the material will move through the column.

When creating a developing solvent, the ratio of ethyl acetate and hexane must be determined based on an understanding of their properties and characteristics.

Determine the Mobile Phase (Developing Solvent) from the Rf Value of Thin-Layer Chromatography

So, how do we decide on a developing solvent? Before performing column chromatography, there is one experiment that must be done. That is thin layer chromatography (TLC). Column chromatography without thin layer chromatography is not a common practice in chemistry.

TLC is a thin glass plate with silica gel on it. When you do thin layer chromatography, you spot the solution you want to measure. You then create a developing solvent and immerse the TLC in the developing solvent so that the compounds can be separated.

The Rf-value can be measured for each compound. The speed of a compound in silica gel is called Rf value. The Rf value is shown below.

The Rf value depends on the developing solvent. We adjust the ratio of ethyl acetate to hexane so that the target compound has an Rf value of about 0.3.

The higher the Rf value, the faster the compound moves through the silica gel, and there is a risk of separation of the compound along with other impurities in the solution. On the other hand, if the Rf value is too low, the target compound will not come out of the column even though the developing solvent is flowing for a long time.

So, TLC is performed to find the developing solvent with Rf value around 0.3. Then, the developing solvent is flushed into the column.

-The Developing Solvent Can Be Dichloromethane and Methanol.

The most common method of column chromatography is to use an organic solvent mixed with ethyl acetate and hexane. In some cases, however, other expansion solvents may be used.

In addition to ethyl acetate and hexane, a mixture of dichloromethane and methanol is also used as a developing solvent. Sometimes the dichloromethane/methanol combination is better for separating compounds with very high polarity.

Put the Silica Gel and Run the Developing Solvent Through It

We have explained the principles and characteristics of column chromatography. So, when you actually do column chromatography, how does it work?

First, you have to put a silica gel in the column. Then, the compound is placed on top of the silica gel. Once you have completed the preparations up to this point, you just need to flow the developing solvent. As the mobile phase flows, the compound will gradually move forward as well.

Naturally, the mobile phase, the organic solvent, comes out of the end of the column. The organic solvent is collected in a test tube. Once the solution containing the target compound is collected, the separation of the compound is complete.

Now let’s consider the compounds estriol, estradiol and estrone as an example.

Suppose you have a solution of these compounds mixed together. When this solution is separated by column chromatography, what speed difference would you consider the compounds to produce?

The polarity is estriol > estradiol > estrone, in that order. If we focus on the hydroxy group (-OH), the nature of the substance is easy to predict.

In column chromatography, the higher the fat-soluble compound, the less affected it is by silica gel. In other words, we can expect the substances to flow out in the following order

Column chromatography is an experimental operation that focuses on the polarity of a substance and separates organic compounds. Once the principles are understood, it is possible to examine at what speed compounds flow at different speeds.

If the Compound Is Not Separated, Change the Length of the Column

However, the compounds may not be separated in actual chemical experiments. The ability to separate compounds is an advantage, but in some cases, the disadvantage of column chromatography is that you have to consider the experimental method.

A common example of this is the close Rf values between compounds, making it difficult to separate them, as shown below.

In this case, the most obvious thing to do is to increase the amount of silica gel you put into the column. The longer the stationary phase (silica gel), the easier the material will separate.

For example, in a 100-meter run, a second of difference is a big difference. On the other hand, if you’re running a full marathon, a time difference of 10 minutes or more is common for a full marathon of 42 km. The longer the distance, the bigger the difference will be.

Lowering the Rf Value Will Prevent Failure

You can use a lower Rf value. Even if it’s the same column length, the lower the Rf value, the easier it is to separate the compounds.

For example, if you’re running 100 meters and there are no obstacles, the difference in time is minimal. On the other hand, what if there is a pond, a large block or a maze within 100m? The total time will be completely different for each person.

Similar to this principle, the lower the Rf value, the higher the separation ability of the compounds. Of course, the movement speed of compounds is very slow, so even if you run the solvent for a long time, there is a disadvantage that the compounds do not show up. However, it makes it easier to separate compounds.

If you can’t separate compounds by column chromatography, you should apply both a longer stationary phase and a lower Rf value. Otherwise, you will fail to separate the compounds.

Understand the Principles and Learn How and What to Do with the Experiment

Column chromatography is a widely used experimental technique in laboratories that handle organic compounds because of its ability to separate compounds. Therefore, it is important to understand the principle, characteristics, advantages and disadvantages before conducting experiments.

Use silica gel as a stationary phase, and understand that silica gel has a high polarity. Then the higher the polarity of the compound, the more it will be adsorbed on the silica gel and the slower the rate will be.

You also have to determine the mobile phase. We use organic solvents to create the developing solvent. First, the ratio of ethyl acetate and hexane is determined. The higher the ratio of ethyl acetate, the more easily the compound dissolves in the solvent and the faster the rate. In other words, the Rf value will be higher.

After learning these things, we decide on a developing solvent, measure the Rf value, and perform column chromatography. Once you understand these experimental procedures, you will be able to separate only the target compound even when many compounds are mixed together.