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 like gas, thin-layer and paper chromatography seen in real world lab applications!

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What is Chromatography?

Chromatography is a technique used to separate the different chemical compounds of a mixed sample. Imagine that you have a jar filled with a bunch of different colored candies, like red, blue, and green. If you wanted to know which candies were in the jar and how many of each color there were, you could use chromatography to help you figure it out. 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.

Chromatography is a really useful tool for scientists because it helps them understand what is in a mixture and how much of each component there is. It is used in many different fields, including chemistry, biology, and environmental science, to analyze all sorts of mixtures, like inks, dyes, drugs, and even food.

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)

Gas chromatography (GC) is a laboratory technique used to separate and analyze the components of a mixture. It is based on the fact that different compounds will travel at different rates when carried by a gas, such as helium or nitrogen.

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.

To perform gas chromatography, a small amount of the mixture to be analyzed is injected into the gas chromatograph, which consists of a gas source, a column packed with a solid or liquid stationary phase, and a detector. The gas carries the mixture through the column, and the different components of the mixture will interact with the stationary phase to varying degrees. As a result, each component of the mixture will travel at a different rate through the column, with more polar compounds (which have a stronger interaction with the stationary phase) moving more slowly than nonpolar compounds (which have a weaker interaction with the stationary phase).

As the components of the mixture pass through the column and emerge from the other end, they are detected by the detector, which generates a signal proportional to the amount of each component present. The detector’s output is plotted as a function of time, resulting in a graph known as a chromatogram. The chromatogram allows the analyst to identify and quantify the various components of the mixture based on the time it takes them to emerge from the column.

Gas chromatography is a highly sensitive and selective technique that is widely used in a variety of fields, including chemistry, biology, and environmental science. It is particularly useful for analyzing volatile compounds, such as those found in fuels and industrial chemicals.

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)

Thin layer chromatography (TLC) is a laboratory technique used to separate and identify the components of a mixture. It is similar to paper chromatography, but it uses a thin layer of a solid adsorbent material, such as silica gel or alumina, coated onto a glass plate or plastic sheet as the stationary phase. The mobile phase is a mixture of solvents.

To perform TLC, a small amount of the mixture to be analyzed is applied to a spot near the bottom of the TLC plate. The plate is then placed in a container with a small amount of solvent, which will travel up the plate by capillary action. As the solvent moves up the plate, it will dissolve and carry the components of the mixture with it.

Each compound in the mixture will travel at a different rate based on its solubility in the solvent and its affinity for the stationary phase. More polar compounds, which are more soluble in the solvent, will tend to move faster. Nonpolar compounds, which are less soluble in the solvent, will tend to move more slowly.

After the solvent has traveled a certain distance up the plate, the plate is removed from the container and allowed to dry. The various compounds in the mixture will have formed distinct bands on the plate, with each band corresponding to a different compound. The distance that each compound traveled can be measured and used to identify the compound.

TLC is a relatively simple and inexpensive technique that is widely used in the analysis of a variety of mixtures, including drugs, plant extracts, and industrial chemicals. It is particularly useful for separating and identifying complex mixtures, and for identifying the purity of compounds.

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.

Paper Chromatography

aper chromatography is a laboratory technique used to separate and identify the components of a mixture. It is based on the fact that different compounds will travel at different rates when absorbed by a porous material, such as paper.

To perform paper chromatography, a small amount of the mixture to be analyzed is applied to a spot near the bottom of a piece of chromatography paper. The paper is then placed in a container with a small amount of solvent, such as water or alcohol. As the solvent moves up the paper by capillary action, it will also dissolve and carry the components of the mixture with it.

Each compound in the mixture will travel at a different rate based on its solubility in the solvent and its affinity for the paper. More polar compounds, which are more soluble in the solvent, will tend to move faster. Nonpolar compounds, which are less soluble in the solvent, will tend to move more slowly.

After the solvent has traveled a certain distance up the paper, the paper is removed from the container and allowed to dry. The various compounds in the mixture will have formed distinct bands on the paper, with each band corresponding to a different compound. The distance that each compound traveled can be measured and used to identify the compound.

Paper chromatography is a relatively simple and inexpensive technique that is widely used in the analysis of a variety of mixtures, including inks, dyes, and pigments. It is also commonly used in the field of biochemistry to identify and quantify the components of complex mixtures such as proteins and enzymes.

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

Column Chromatography

tlc - thin later 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.

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