How Does Chromatography Work?

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Chromatography Techniques

Liquid chromatography (LC)

In most laboratories, chromatography is performed using high-performance liquid chromatography (HPLC) or gas chromatography (GC). In HPLC, liquid serves as the mobile phase, and the stationary phase is most often found inside a column (a polymer or stainless steel tube filled with small spherical particles that have specially modified surfaces to selectively interact with molecules in different ways).  [NOTE: The pressures present in most HPLC systems can be very high, due to the resistance of the flow of mobile phase through the densely packed column. Because of this, the “P” in HPLC is often mistakenly referred to as “pressure”.] Because of the wide assortment of specialized stationary phases and the vast number of reagents that can be used, there is almost an infinite number of ways a scientist can customize the setup of a HPLC system to achieve the desired analyte separation. While HPLC separations are often not as efficient as GC separations, one big advantage of using HPLC is that the sample components can be reused and analyzed further if needed.

Gas chromatography (GC)

In GC, the target analyte is vaporized and introduced to a flowing stream of an inert gas, such as nitrogen, which carries the analyte through the column (most often a large coil of very small fused silica tubing). Because of the nature of GC, the separations are often fast and efficient; however, the one disadvantage to GC over HPLC is that the sample can only be used once. There is no way to reuse or analyze the sample further.

Other chromatography techniques

Other popular techniques include low-pressure liquid chromatography (LPLC), fast protein liquid chromatography (FPLC), gel permeation chromatography (GPC), ion chromatography (IC), affinity chromatography, and numerous others. Methods such as ion exchange chromatography (IEC), affinity chromatography (AF), and fast protein liquid chromatography (FPLC) can provide more specific solutions than the workhorse options of HPLC and GC.

What are the Four Basic Chromatography Steps?

While each chromatographic technique varies slightly, generally they all follow these basic four steps:

STEP 1 – A defined amount of analyte is carefully introduced to the constantly running mobile phase stream.

STEP 2 – The mobile phase carries the analyte to—and through—the stationary phase.

STEP 3 – Once the analyte arrives at the stationary phase, the components that comprise the analyte selectively interact with the stationary phase, with some components interacting more and some less, resulting in the desired separation.

STEP 4 – The mobile phase carries the separated analyte components to a special instrument that can detect the presence of the components in the mobile phase and provide a data signal that can be used to quantify the separation.

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