Chromatography is a proven method used to separate complex samples into their constituents, and it is undisputedly the most important procedure for isolating and purifying chemicals. It is classified into two types based on the physical state of the mobile phase used – liquid chromatography (LC) and gas chromatography (GC).
Chromatography includes a series of procedures that have in common the separation of components of a mixture by a series of equilibrium operations that result in the separation of the entities as a result of their partitioning between two distinct phases – one stationary with a large surface and other moving which is in contact with the first.
VIDEO Liquid chromatography
LC is one of the most popular separation techniques used in labs for the separation of a sample mixture on the basis of the interactions between the individual molecules in the sample with the aforementioned stationary and mobile phases. It can be carried out either in a column or on a sheet with a liquid mobile phase and solid support as the stationary phase.
The mobile phase travels down the stationary phase bringing along the components of the sample separated during chromatography. In LC, the interaction between sample molecules and the chromatography medium may be based on several factors such as size, charge, affinity binding, or hydrophobicity.
An advanced form of the LC technique that uses high pressure to force sample through the column is called high performance liquid chromatography or high pressure liquid chromatography (HPLC). It is currently the most extensively used method of quantitative analysis in pharmaceutical analysis laboratories and in the pharmaceutical industry as a whole.
GC is another widely used chromatography technique. Here the mobile phase is usually an inert gas such as helium or argon (also known as the carrier gas), while the technique itself is performed in a capillary or packed column made up of inert materials. Even though packed columns are cheaper and user-friendly, capillary columns provide greater resolution and are relatively expensive.
The stationary phase is either a granular solid (
i.e. gas-solid chromatography), or a granular solid coated with a thin film of nonvolatile liquid ( i.e. gas-liquid chromatography). A majority of analytical gas chromatographs employ capillary columns, where the stationary phase directly coats the walls of a tube with a small diameter.
GC is usually used to separate vaporizable or volatile compounds and test their purity. It is also used to quantify the different components in a mixture. Although volatility of a sample is a prerequisite for GC analysis, the modification of the functional group of a certain molecule by a process known as derivatization enables the analysis of compounds that otherwise could not be easily monitored by GC.
In GC, the separation of components of a mixture depends on the length and temperature of the column, as well as on the flow rate of the carrier gas. These conditions must be optimized for a particular analysis.
In both GC and LC, detection of the individual components in the sample can be carried out by several methods. The most common and sensitive detection method is mass spectrometry, which identifies compounds based on the atomic sample organization of the molecules and their charge state.
Differences between of LC and GC
The key differences between liquid and gas chromatography are tabulated below.
Mobile phase is a liquid
Mobile phase is a gas
Separation is based on interaction of solute with the chromatography medium
Separation is primarily based on the boiling points of solute molecules
Can be performed in a sheet or a column
Can be carried out only in a column
Can be used to separate any soluble compound,
e.g. amino acids, proteins, drugs, nucleic acids, lipids, antioxidants, carbohydrates, and natural and artificial polymers
Can be applied in the separation of volatile compounds and gaseous mixtures
Usually carried out at room temperature so heat-sensitive compounds can be safely analyzed using the technique
Performed at higher temperatures so thermally labile substances might get denatured
Solute retention here is based on the interaction of solutes with the mobile and stationary phases so it is easy to optimize results
Separation is based on the boiling points of the solute molecules so it is not very flexible in terms of optimizing separation
This is a relatively slower technique
The analysis is faster and usually measured in minutes, although it can take as little as a couple of seconds
Usually gives a greater peak or broader band resulting in lower resolution
Provides comparatively better resolution
Uses polar solvents like water or methanol
Uses any solvent that vaporizes
Reviewed by Afsaneh Khetrapal, BSc (Hons)
Additional Review by Dr Tomislav Meštrović, MD, PhD References
http://arlok.com/articles/High-Performance%20Liquid%20Chromatography.pdf Blumberg LM. Theory of Gas Chromatography. In: Poole CF, editor. Gas Chromatography, First Edition. Elsevier, 2012; pp. 19-78.
Laird CK. Chemical Analysis: Gas Analysis. In: Walt Boyes, editor. Instrumentation Reference Book. Butterworth-Heinemann, 2009; pp. 327-340.