Lab Procedures, Tips & Safety

What is Chromatography?

Core Concepts

In this article, you will learn the how chromatography separates compounds. In addition, You will learn about several common chromatographic methods seen in real world lab applications!

Topics Covered in Other Articles

What is Chromatography?

Chromatography is a technique used to separate the different chemical compounds of a mixed sample. It uses two phases: a nonmoving “stationary” phase and a mobile phase which moves through the stationary phase, carrying along the chemical compounds.

Take a look at this setup below. The paper acts as the stationary phase, and a solvent is used as the mobile phase. Specifically, this is an example of paper chromatography.

When the solvent travels up the paper, it carries along with it the chemicals present in your original sample. Furthermore, each compound will travel at a different speed based on its chemical properties, separating the compounds from each other. This is the basic principle behind all chromatographic methods.

Types of Chromatography

There are various chromatographic techniques used to isolate and analyze chemical mixtures. With attention to this, notable methods make-up the remainder of this article.

Gas Chromatography (GC)

In gas chromatography, the liquid or gas sample moves through a specialized column by the help of a carrier gas in an instrument called a gas chromatograph, pictured below. The instrument detects which chemicals exit the column over time.

Due to the vaporization methodology, this technique is used to analyze the components of a sample, not for isolation. Specifically, it can help answer questions like “Is my sample pure?” or “how much benzene is in my solution?”

Thin Layer Chromatography (TLC)

In TLC, the stationary phase is commonly silica is fixed to a glass plate. The mobile phase is a mixture of solvents. TLC is less time and resource intensive; therefore, it is usually implemented to quickly check sample contents or reaction progress. Because of the similarities between thin layer and column chromatography, a sample can be analyzed by TLC to estimate how it will behave in column chromatography. Additionally, TLC plates can be stained, or subjected to UV light to help visualize its contents.

Liquid Chromatography (LC)

Overall, liquid chromatography encompasses a wide range of techniques. Let’s look at some of the types of LC:

Column Chromatography

Column Chromatography typically uses a glass column filled with silica gel (stationary phase), and a mixture of organic solvents serves as the mobile phase. Depending on the mixture of solvents, your sample can elute off, or exit, the glass column faster or slower. Changing the solvent proportions can also allow you to achieve more or less separation between the compounds in your sample. This is a great isolation technique since the components of the sample are preserved through the process, and the process can be scaled up or down.

Column Chromatography

TLC Under UV light

Size Exclusion

Size exclusion is similar to liquid chromatography, but uses a special solid phase media that causes different sized particles to take longer or shorter paths through the column. Larger particles, like proteins, elute first, then smaller ones.

Size Exclusion Column: Small molecules/proteins (thin line) take a longer path than large molecules/proteins (thick line).

Ion Exchange

Ion exchange chromatography is a way of separating compounds based on their relative charges. A column is filled with a charged solid phase. When sample is run through the column, your compound of interest will have an affinity for the charged resin and not be flushed through. The desired compound will only elute off of the column when the buffer solution is changed, and therefore the charge of your compound of interest changes.

Affinity

Affinity like ion exchange chromatography is a technique used to isolate a desired compound from a mixture. It differs from ion exchange in that the compound of interest binds to a solid-state attached ligand instead of being attracted to the solid state by ionic interactions.

High-performance Liquid Chromatography (HPLC)

High-performance, or high-pressure, liquid chromatography is a form of liquid chromatography that operates at higher pressures. It also is typically more automated.

Wrap-Up

TypeHow it worksAdditional Information
PaperSolvent moves up paper.
GasSample moves through a long/thin column by a carrier gas.Used in analytical chemistry, good for small samples.
ColumnSolid silica is packed into a column and organic solvent run through.
Thin LayerSample is carried by organic solvent through a thin layer of silica bound to glass.Can be used to quickly get a snapshot of what is in your mixture.
HPLCAutomated liquid chromatography under high pressure.
Size ExclusionSample is separated by size using a special solid phase. Larger species elute first.
Ion ExchangeChemical of interest stays with the oppositely charged solid phase, a buffer is used to change the chemical’s charge and allow it to elute.
AffinityCompound of interest has a specific interaction with the chosen solid phase.

Further Reading

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