A thorough understanding of the chromatographic mobile phase and a stationary phase is integrally important in order to sufficiently comprehend the basics of chromatography. Chromatography is an analytical separation, identification and purification method which consists of two foundational units: the stationary phase and the mobile phase.
We have already discussed the importance and diversity present in chromatographic stationary phases in our previous article. This article on the other hand is particularly focused on elaborating the chromatographic mobile phases. Keep reading further to know all there is to know about a mobile phase in chromatography.
What is mobile phase
The mobile phase refers to the solvent or the combination of different solvents that moves past the stationary phase in a chromatographic separation. It carries the analyte components with it while flowing over the stationary phase, allowing them a chance to interact with the stationary phase molecules and to get separated. The analyte components separate on the basis of their relative distribution between the mobile phase and the stationary phase.
A stronger interaction with the mobile phase as opposed to the stationary phase means faster flow of analyte molecules and weaker retention onto the stationary phase. The mobile phase is preferably a liquid in most of the chromatographic techniques except in gas chromatography. The mobile phase in case of a gas chromatographic separation is, as its name suggests, a gas.
Let us delve further into the topic to discuss about the mobile phase compositions with particular reference to different types of chromatography.
Mobile phase in 2-dimensional/planar chromatography
Planar chromatography includes two main types i.e., paper chromatography and thin-layer chromatography (TLC) respectively. Organic solvents are usually used for performing these two types of chromatography. These organic solvents are selected on the basis of the solubility of the analyte components. Both paper and thin layer chromatography are examples of normal-phase chromatography so the mobile phase will be comparatively less polar than the stationary phase in this case.
Alcohols such as ethanol and methanol or non-polar solvents such as hexane are commonly employed as mobile phases. The mobile phase travels up the chromatographic plane (paper or TLC plate) through capillary action.
Relatively less polar components from the analyte mixture flow with the mobile phase to a farther end. Contrarily, more polar analyte components will stay retained onto the stationary phase for long. Solute spots will appear at distinct positions with differing Rf values so that the different solutes can be identified as separate entities.
Mobile phase in 3-dimensional/column chromatography
Column chromatography refers to any chromatographic operation in which the stationary phase is packed into a column. Both liquid chromatography and gas chromatography fall under the column chromatography category.
Mobile phase in liquid chromatography
A series of organic solvents are often employed to prepare mobile phase compositions for liquid chromatography. The polarity of the organic solvents can be exploited as per requirement to perform normal phase or reverse phase liquid chromatographic processes. The most widely used mobile phase solvents are as shown below, arranged in an ascending order of polarity.
All the organic solvents are used as their aqueous solutions. The composition of mobile phase could either be fixed for column chromatographic process or it may vary gradually as the separation proceeds. The process of using a fixed mobile phase composition throughout is called isocratic elution. On the other hand, performing column chromatography by gradually varying the mobile phase composition is known as gradient elution.
The following step-by-step guide will give you a clear idea about the role of varying a mobile phase composition in reverse phase liquid chromatography.
Step I: A suitable mobile phase composition is prepared by mixing a definite ratio of the chosen organic solvents say acetonitrile, ethanol and cyclohexane.
Step II: A column packed with methylated silica is used and the analyte mixture introduced from the top of the column.
Step III: The analyte components are allowed some time to interact with the stationary phase.
Step IV: The mobile phase is then introduced and allowed to flow through the column. Analyte components interact with the mobile phase and stationary phase on the basis of their relative polarities.
Step V: Less polar components get strongly adsorbed onto the stationary phase while more polar components pass through the column quickly with the mobile phase.
Step VI: After collecting the highly polar components, the mobile phase composition is then varied, decreasing the proportion of the polar ethanol and increasing the amount of less polar cyclohexane in it.
Step VII: The least polar components strongly adhered to the stationary phase will eventually move into the mobile phase and will elute out of the column.
Result: Decreasing the polarity of the mobile phase helped overcome the strong forces of attraction between the solute molecules and the stationary phase.
The analyte molecules may interact with the stationary phase via Vander Walls forces of attraction or π-π interaction. In contrast to that, the solute molecules get solubilized in the mobile phase by electrostatic forces of attraction or hydrogen bonding etc. Like attracts like.
Mobile phase in gas chromatography
A gaseous mobile phase is used to perform gas chromatography. The mobile phase is this case acts more as a carrier gas. It sweeps the solute molecules with it rather than interacting with those molecules chemically as witnessed in the instance of liquid mobile phases. The choice of carrier gas depends on the required separation efficiency and speed of analysis. Some of the commonly employed gaseous mobile phases are as given in the table below with their associated pros and cons.
|Small molecular weight
High flow rate
|Lowest molecular weight
Fastest flow rate
Explosive in nature
|Relatively high molecular weight
Lower flow rate
|Inert, noble gas
|Higher molecular weight and density
Lowest rate of flow
According to the information provided in the table above, one can decipher that hydrogen and helium are the most popular mobile phases that are used in gas chromatography. We should always keep in mind however that helium is always preferred over hydrogen as a mobile phase, considering the explosive nature of the later.
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2. Danielson, N. D. (2003). Liquid Chromatography. Encyclopedia of Physical Science and Technology (Third Edition). R. A. Meyers. New York, Academic Press: 673-700.
3. M.Younas (2017). Organic Spectroscopy and Chromatography.