JoVE Lab Manual
Lab: Chemistry
Lab: Chemistry
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Säulenchromatographie
Column chromatography is a technique that uses a packed column to separate compounds based on their interactions with a stationary phase, which is usually in the form of microscopic beads. The spaces between the beads are filled with solvent. Opening the column allows the solvent to flow through the stationary phase. When a mixture is applied to the top of the packed column followed by more of the solvent, the mixture moves into the mobile phase and flows through the stationary phase.
Each component of the mixture interacts with the stationary phase in a different way. Some components have weak interactions with the stationary phase and so move quickly through the column, while others have strong interactions with the stationary phase and thus move more slowly. This separates the different compounds into bands, which are collected in small fractions. This enables the purification of each compound.
So, what kind of properties can be used to separate mixtures? One of the most commonly used properties is polarity. For this, silica gel, which is a form of silicon dioxide, is often used as the stationary phase. Silica gel interacts with compounds by dipole-dipole interactions and hydrogen bonding through the -OH groups that form on its surface. Thus, polar compounds will interact strongly with the stationary phase, while non-polar compounds will interact weakly.
Other properties that can be exploited for separation include size, charge, and hydrophobicity. A common way to load the stationary phase into the column is as a slurry of the stationary phase and solvent. Then, the stationary phase is packed by flowing more solvent through the column to compact the slurry. It is essential that the stationary phase is packed uniformly without air bubbles, empty channels, or dry patches. These can disrupt flow and cause mixing of bands.
When choosing a column, the diameter is based on the volume of the sample to be separated. The sample should cover the top of the column in a thin, even layer. A thicker sample layer leads to broader bands. The column length depends on how well the compounds separate on the stationary phase. Compounds with similar affinities for the stationary phase require a long column for adequate separation. However, a mixture of compounds with very different affinities for the stationary phase can be separated on a shorter column.
In this lab, you will explore column chromatography by first packing and preparing a silica gel column. Then, you'll use your column to separate the colored components in green food dye.
Säulenchromatographie
Column Chromatography
Multiple techniques exist to purify and separate compounds in an organic chemistry laboratory. One of the most reliable separation techniques for microscale separations, where less than 10 grams of the compound is available for analysis, is column chromatography. This technique separates compounds in a mixture based on their physical properties, such as their solubility, polarity, hydrophobicity, size, and charge.
Chromatography Basics
The main components of any chromatography system are the stationary phase, the mobile phase, and the sample mixture to be analyzed. In column chromatography, the stationary phase is typically microscale beads, which are packed uniformly in a vertical column. A continuous flow of the mobile phase, also known as the solvent, is added to the top of the column, which flows through the stationary phase via gravity or at a controlled flow rate by a pump.
The sample is dissolved in a small amount of the solvent and then added to the top of the column. More solvent is added to force the sample to flow through the stationary phase. The compounds in the mixture partition between the mobile phase and the stationary phase based on their differing properties and form discrete bands.
Compounds with strong interactions with the stationary phase will move slower than compounds with weak interactions with the stationary phase. Thus, compounds with weak interactions will exit the column, or elute, earlier than those with strong interactions. The strength of a compound’s interaction with the stationary phase is defined by the retardation factor (Rf), which is the ratio of the distance traveled by a component to that traveled by the mobile phase. The retardation factor is determined by first performing thin layer chromatography.
A high retardation factor value indicates that the sample interacts strongly with the stationary phase and takes a long time to elute. A low retardation factor value indicates that the sample does not interact with the stationary phase very strongly and elutes more quickly. Since compounds partition into individual bands and elute from the column at different times due to their different Rf values, they can be isolated individually by collecting the solvent from under the column in fractions.
Column Chromatography Considerations
A chromatography column is a vertically oriented glass or plastic tube. The size of the column can range from a few centimeters (such as a Pasteur pipette) to meters (such as with industrial columns). The diameter of the column depends on the amount of sample to be separated, whereas the length of the column depends on the difficulty of separation. The most efficient partitioning into discrete bands requires thin sample layers; therefore, determining the appropriate diameter is a vital step. Loading too much sample volume relative to the column’s diameter will create broader and less defined bands than the same volume of sample in a column with a larger diameter.
When choosing the length of the column, the Rf values of the components must be considered. Components with similar retardation factors may be challenging to separate and require a longer column to resolve the individual bands. Compounds with very different retardation factors may not need a long column, as they should partition easily. When selecting the stationary phase, it is important to choose one that results in different retardation factors for each component.
Preparing the column is the most critical part of running a chromatography column. The mass of the stationary phase packed in the column should be at least 20x that of the sample. If the column does not have a porous disk at the bottom, it should be packed with cotton and a thin layer of sand. This prevents the loss of the stationary phase through the exit of the column.
There are two methods to pack the column: the dry packing method and the slurry method. In the dry method, the stationary phase is transferred into the column in its powder form. The column is then washed several times with the mobile phase/solvent to ensure that the solvent penetrates every part of the silica gel column. This method, if not done correctly, can increase the probability of dry patches, channels, and air bubbles forming in the column. These defects will affect the ability of the column to partition components of a mixture.
The slurry method provides a more uniform packed column that does not contain air bubbles, dry areas, and channels. For this method, the stationary phase is mixed with a solvent to form a consistent slurry. The slurry is then transferred to the column while tapping at the sides to get rid of any air bubbles formed. The stopcock of the column, if there is one, should be open during this step to allow the solvent to drain.
Silica Gel Chromatography
Many different properties are exploited to separate a mixture of compounds, such as size, charge, and hydrophobicity. One property used to separate compounds in organic chemistry is polarity. For this, silica gel and alumina gel are the most commonly used stationary phases. Silica gel is extremely polar and forms strong dipole-dipole interactions with polar compounds. In addition, silica gel can form hydrogen bonds with the analyte due to the presence of -OH groups on its surface.
Components of the mixture that are most polar bind strongly to the silica gel and travel slowly through the column, while non-polar components are more soluble in the mobile phase and travel quickly through the column. Thus, it is essential that the components of the mixture have different polarities. Components that interact strongly with the stationary phase are eluted by flowing a polar mobile phase through the column.
References
- Shuler, M.L., Kargi, K., DeLisa, M. (2017). Bioprocess Engineering, Basic Concepts. Upper Saddle River, NJ: Prentice Hall.
- Harris, D.C. (2015). Quantitative Chemical Analysis. New York, NY: W.H. Freeman and Company.
