Liquid chromatography (LC) is a technique used to separate a sample into its individual components. It involves a liquid mobile phase that flows through a solid stationary phase, allowing separation based on the sample’s interactions with both phases.
This process is often employed in analytical and preparative applications, particularly in liquid-solid column chromatography. The versatility of LC arises from the various combinations of mobile and stationary phases that can be used to analyse complex mixtures.
In High-Performance Liquid Chromatography (HPLC), the mobile phase is passed through the column under high pressure, with a detector identifying the components as they elute.
This scientific process of separating mixtures helps us understand their individual components through elution, as the mobile phase carries the sample through the stationary phase. Various techniques use different substances for both the mobile and stationary phases, and there are many ways to pass the mobile phase through the stationary phase.
Chromatography works because the different components of the mixture will separate at different points as it passes through the stationary phase.
This happens because the components have different levels of attraction to both the mobile and stationary phases, due to the polarity of the substances used. For example, the liquid mobile phase is a non-polar solvent (such as water) and the stationary phase is a polar solid (such as silica).
Each component of the mixture has a unique polarity and hence will have a different affiliation with both the mobile phase and the stationary phase and hence different flow rates during elution. In this example, components that are more polar will move further down the column with the polar mobile phase while components that are less polar will travel more slowly, staying at the top of the column with the stationary phase.
The time it takes for each substance to move through the column is measured to indicate what each component could be.
It is possible to reverse the polarity, to have a polar mobile phase and non-polar stationary phase. This is known as reverse phase chromatography.
Step 1: The stationary phase is placed into the column
Step 2: The mobile phase is passed through column filled with the stationary phase. In HPLC, this is done by pressurising the column.
Step 3: A sample of the mixture in question is injected at the top of the column.
Step 4: The compounds elute through the column and separate as it passes through.
Step 5: A detector is used to measure each of the substances as they leave the column.
Once all the components of the sample leave the column, the detector will indicate what each component is. This is usually shown in graph form, known as a chromatogram, with retention time along the X-axis and signal strength along the Y-axis.
As the signal of each component increases, there is a peak in the chromatogram that indicates the presence of a chemical in the sample.
Each component of the mixture can be identified based on the retention times. All known substances have a typical retention time based on standard measures, so the peaks will always plot around the same retention time. For example, if we know chemical A takes around 5 minutes to retain under these conditions, a peak at 5 minutes is identified as chemical A.
The concentration of each component can also be estimated using a chromatogram. The area under the curve in the graph (i.e., the height of the peak) indicates concentration, so a taller, wider curve suggests a higher concentration.
Liquid chromatography is used in a wide range of industries with lots of different applications, such as:
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