What is stationary phase in chromatography

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A stationary phase and a mobile phase are the foundational units of any chromatographic process. Chromatography is an analytical separation technique which separates components of a complex mixture by their differential distribution between two distinct phases. These two phases include a stationary phase and a mobile phase respectively. In this particular article, we will learn all there is to know about the stationary phase in chromatography.

What is stationary phase

Stationary phase is the phase over which the mobile phase or solvent carrying the analyte components moves past. It is experimentally convenient to keep the stationary phase fixed or unmoved while the mobile phase flows over it in a particular direction.

A broader definition of the concept does not necessarily consider the stationary phase ‘’stationary’’ i.e., fixed in one place. Rather, it states that the direction or rate of migration of the two phases must be different in a chromatographic method. We will however for our convenience and for studying the basics of chromatography will consider a stationary phase as a fixed entity only.

As there are many different types of chromatography so all the different forms of chromatography essentially require different types of stationary phases. Learn more about chromatographic classification into different types here.

Stationary phase in 2-Dimensional Chromatography

Planar chromatography obtains its name from the fact that the stationary phase in this case is held on a plane i.e., lying flat. This type of chromatography has two different kinds including paper chromatography and thin layer chromatography.

Stationary phase in paper chromatography

A fine quality cellulose paper with a specific pore size is used to perform paper chromatography. A Whatman filter paper No.42 with a pore size of 2.5 µm is a good choice for this purpose. An interesting fact about paper chromatography is that in this case the paper itself is not the stationary phase, as is a common misconception. Rather, the water trapped in the layers of cellulose fiber of the paper is the stationary phase in paper chromatography.

The paper is used just as a support to hold the stationary phase molecules in place. Separation of analytical components take place by partitioning of the solute molecules between the aqueous based stationary phase and the molecules of the organic solvent used as a mobile phase. Paper chromatography thus is a form of normal phase liquid-liquid chromatography. Both the stationary and the mobile phases in this case are liquid and the stationary phase is more polar than the mobile phase.

Stationary phase in thin layer chromatography

A rectangular piece of glass plate coated with a thin layer of silica is used to perform thin-layer chromatography (TLC). Silica gel in this case acts as the stationary phase while the glass plate acts as a support in keeping the stationary phase upright. In this case, the silica gel acts as a solid adsorbent while the organic solvents are used as liquid mobile phases. TLC thus falls under the category of solid-liquid chromatography. It is also a normal phase chromatographic method because the stationary phase is more polar than the mobile phase.

TLC glass plates. Image by Sorbtech.com

You may also like to check a video tutorial on TLC.

Stationary phase in 3-Dimensional Chromatography

For more extensive chromatographic separations, the stationary phase is packed into a column. A column is a glass tube, specifically designed for performing chromatographic procedures. It is used for different types of chromatography such as liquid chromatography including high performance liquid chromatography (HPLC), ion exchange chromatography and size exclusion chromatography etc. Gas chromatography on the other hand is either performed using a packed column or an open tubular column. Keep reading to unwind these concepts in further detail.

Stationary phase in liquid column chromatography

Silica gel is the most popular stationary phase that is used to perform a column chromatography. Silica gel has a porous structure which offers a high surface area for adsorption of analyte molecules. The hydroxyl (-OH) functional groups present on its surface allow a diversity of modifications of the silica gel to facilitate a chromatographic process. Usually, a column packing process requires the following steps:

Step I: Silica activation prior to column packing by treating the silica gel with a concentrated acid solution followed by its drying.

Step II: The dried powder is then converted into a slurry by adding a definite quantity of the respective mobile phase solvent.

Step III: The column is packed by first blocking its lower end by placing a cotton plug followed by a thin layer of sand. This prevents the risk of stationary phase bleeding which may otherwise clog the opening valve attached at the bottom of the chromatographic column.

Step IV: The silica slurry is then added from the top end and allowed to settle down.

Step V: All the excess moisture is drained off from the column by opening the valve.

A uniformly packed column without any cracks or bleeds ensures a successful stationary phase packing, ready to carry out the desired chromatographic separation.

Stationary phase in high performance liquid chromatography

The stationary phases used to pack an HPLC column includes silica gel, alumina and different types of synthetic and natural polymers. HPLC is preferably performed in reverse phase. Methylation of silica surface with long hydrocarbon chains generate a reverse phased stationary phase column in high performance liquid chromatography .C-8 (octyl) and C-18 (octadecyl) silica based commercially packed stationary phase columns are highly preferred in HPLC.

The stationary phase material in HPLC can be further categorized on the basis of particle size as shown below.

Stationary phaseParticle sizeComposition
Microporous10-20 µmSilica beads
Pellicular40 µmSilica particles attached on a porous support say a glass surface
Bonded phase3-10 µmChemically bonded silica

Smaller the particle size, greater the surface area thus a faster chromatographic separation expected.

Stationary phase in ion exchange chromatography

Ion exchange chromatography, as its name suggests, is based on the separation of analyte components on the basis of charge. This implies that some charge must be present on the stationary phase surface as well in order to interact with the charged solute particles. The stationary phases in ion exchange chromatography are thus developed as cation or anion exchangers.

A cellulose polymer composed of β-D glucose units is usually employed as the stationary phase support in ion exchange chromatography. Charged species are then covalently bonded to the outwardly extended functional groups of the cellulose polymer to prepare a cation or an anion exchange resin. Cations are positively charged species such as a quaternary amine (-N(CH3)4)+. Covalently attached to the stationary phase support it helps to retain negatively charged anions from the analyte mixture, forming an anion exchange resin.

On the other hand, anions such as sulfonate (-SO3) covalently attached to stationary phase support forms a negatively charged cation exchange resin. It retains and separate the oppositely charged cations from the complex analyte mixture.

Stationary phase in size exclusion chromatography

Size exclusion is a chromatographic technique which facilitates the separation and analysis on the basis of molecular size. The stationary phase support in this case is usually silica gel with a porous morphology. The solute molecules are retained on the stationary phase by penetrating into the internal cavities present in the stationary phase called pores.

Molecules bigger than the pore size, pass through quickly while molecules smaller in size than the stationary phase pores get trapped for long time intervals. A larger quantity of the mobile phase is consequently required to remove these firmly retained analyte molecules from the stationary phase. The difference in retention times and retention volumes ensure an adequate chromatographic separation.

More on size exclusion chromatography here.

Stationary phase in gas chromatography

The chromatographic stationary phases used in gas chromatography (GC) are either packed similarly as discussed for HPLC or open tubular columns are used. The open tubular column is developed by a uniform coating of the stationary phase such as silica or methylated silica on the inner tube wall. This type of stationary phase columns allows an open/unrestricted path to the mobile phase flow.

The open tubular columns are sometimes also known simply as capillary columns in GC. They allow high resolution, shorter analysis time and a greater sensitivity to the chromatographic process. The open tubular columns can be further categorized on the basis of variations in stationary phase coatings as discussed below.

Type of GC ColumnStationary phase coating
Porous layer open tubular (PLOT)A porous layer of solid silica particles coated on the surface
Wall coated open tubular
(WOT)
A liquid stationary phase coated on the inner wall of the column
Surface coated open tubular
(SCOT)
An external solid support coated with liquid stationary phase attached on the inner wall of the column
Fused silica coated open tubular
(FSCOT)
A liquid stationary phase fused with silica coated on a polymer support
POT
WOT
FSCOT & SCOT

Learn more interesting information by reading our article written exclusively on gas chromatography.

Summary

Type of ChromatographyStationary Phase
Paper ChromatographyWater
Thin Layer ChromatographySilica
High Performance Liquid ChromatographySilica (normal phase)
Octadecyl silane (reverse phase)
Ion Exchange ChromatographyPositively or negatively charged species covalently bonded to a polymer
Size Exclusion ChromatographyPorous silica beads
Gas ChromatographySilica (solid stationary phase)
Squalene and polydimethyl siloxane (Liquid stationary phases)

Additional Helpful Sources on Chromatography

  1. Thin Layer Chromatography: Background and Principles
  2. A video tutorial on column packing

References

1.Poole, C. F. (2003). The essence of chromatography, Elsevier.

2. Rotzsche, H. (1991). Stationary Phases in Gas Chromatography

3. Tanaka, N., Y. Tokuda, K. Iwaguchi and M. Araki (1982). “Effect of stationary phase structure on retention and selectivity in reversed-phase liquid chromatography.” Journal of Chromatography A 239: 761-772.

What is stationary phase in chromatography

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