Paper and Thin-Layer Chromatography

Core Concepts

In this article, you will first learn about paper chromatography, its procedure, and its applications. Then, you will learn about retention factor and thin-layer chromatography‘s procedure and uses.

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chromatography example

What is Chromatography?

Chromatography is a process that separates components of a mixture. There are two main parts of the chromatography method: the stationary phase and the mobile phase. The stationary phase, usually a porous solid, remains in one place. The mobile phase, commonly a gas or liquid, carries the components of the mixture sample through the stationary phase. The speed at which the components travel will separate them from each other, which is impacted by their solubilities and affinities for the stationary phase. There are many forms of chromatography paper, column, gas, thin-layer that can analyze mixtures in dyes, inks, and more. In this tutorial, we will specifically focus on paper and thin-layer chromatography.

Paper Chromatography

 chromatography  example
Paper chromatography example set up

Paper chromatography is an analytical chemistry technique. It uses migration rate, which deals with chemical interactions, to separate different parts of chemical substances across the paper. The stationary phase in paper chromatography is the chromatography paper, whereas the mobile phase is the liquid solvent that the paper absorbs. The migration rate of each component will depend on the polarities of the paper and liquid, as well as the components’ solubilities in the solvent. These concepts go hand in hand. Recall that “like dissolves like,” so substances with similar polarities will have a higher chance of dissolution.

Commonly, chromatography paper is made of cellulose, which is a polar polymer. The solvent may be polar (like water), somewhat polar (like isopropyl alcohol), or nonpolar (like vegetable oil). For this example, let’s say our solvent is nonpolar, and we are using polar chromatography paper.

Procedure for Paper Chromatography

First, the experimenter spots a small quantity of a sample, such as ink, onto a sheet. The sheet is then suspended in a container with the solvent; the solvent level must be below the spot(s) of ink. Moreover, we must cover the container with a lid so the solvent does not evaporate as it travels up the paper. Due to capillary action, the sample is able to be separated into its different components. As the solvent travels up the paper, the components of the ink will separate and you may see the different colors that make up the ink.

Additionally, we can see which component(s) of the ink are polar and nonpolar. The more nonpolar compounds will travel farther with the nonpolar solvent because they are more soluble in the solvent (“like dissolves like”). The insoluble components (the polar components, in this case) will stay closer to the original location of the spotted ink; its migration rate is low. Compounds will travel faster if they are more nonpolar (in this case), which allows for further analysis. Finally, the experimenter takes the chromatography paper out of the container to dry so they may see the bands on the paper. The pattern of spots left is “chromatogram.”

Stationary and Mobile Phase

The terms “stationary phase” and “mobile phase” come back during the experiment. The mobile phase, or the liquid, is also a phase of time when the molecules are moving with the solvent. The stationary phase, or the paper, is the phase of time when the molecules bind to the paper. Components that are more soluble in the solvent will spend more time in the mobile phase, and components that are more insoluble will spend more time in the stationary phase. 

Retention Factor (Rf value)

Retention factor allows us to identify a certain ink or a specific component. The experimenter may compare the Rf value to a data book that takes into account the solvent and type of paper used; this helps identify the substance. This value compares the distance traveled by a pure sample or a component of a substance to the distance traveled by the solvent.

    \begin{gather*} {R_{f} = \frac{\text{distance traveled by the sample}}{\text{distance traveled by the solvent}}} \end{gather*}

Now, let’s apply the Rf value to a practice problem in which we will attempt to identify which pure dye of two possible samples matches the unknown.

The diagrams below shows the setup of the paper chromatography experiment. The baseline is the origin of the dyes.

paper chromatography development example retention factor
Chromatography Experiment
paper chromatography chromatograms example retention factor

Although the solvent traveled less on the unknown’s paper, we can use the Rf on each dye’s path to identify which known sample matches the unknown. The point to which the solvent traveled is the solvent front. Let’s calculate the Rf for Dye A, Dye B, and the Unknown Dye using the equation from above. Remember that the numerator is the distance traveled by the sample and the denominator is the distance traveled by the solvent (baseline to solvent front).

    \begin{gather*} {R_{f_{A}} = \frac{\text{5 cm}}{\text{20 cm}}} {\text{= 0.25}} \end{gather*}

    \begin{gather*} {R_{f_{B}} = \frac{\text{10 cm}}{\text{20 cm}}} {\text{= 0.50}} \end{gather*}

Then for the unknown dye:

    \begin{gather*} {R_{f_{unknown}} = \frac{\text{7 cm}}{\text{14 cm}}} {\text{= 0.50}} \end{gather*}

The Rf of Dye B (0.50) is the same as the Rf of the Unknown Dye, indicating that the Unknown Dye is Dye A. Since both dyes are the same and have the same chemical composition, they will interact the same with the solvent because of their polarities.

Applications of Paper Chromatography

Various contexts may use paper chromatography, as it is a standard process that can apply to complex mixtures like organic compounds, amino acids, steroids, and carbohydrates. These methods may be used to analyze cosmetics, food, fermentation, and pharmaceuticals. Scientists may use it to separate simple, colored pigments or investigate complex crime scenes by detecting chemicals in blood.

Thin Layer Chromatography (TLC)

Thin-layer chromatography (TLC), like paper chromatography, also uses differing polarity to separate components of a sample. 

In TLC, the experimenter spreads a thin layer of silica gel on a glass surface (TLC plates). The silica gel is polar due to the hydroxyl (OH) groups that can perform dipole-dipole interactions and hydrogen bonding. Next, the experimenter spots the sample on the gel on the plate. The experimenter then places the plate into a container with a lid and a shallow level of solvent. This solvent is the mobile phase like in paper chromatography, but the stationary phase is the plate. Again, the spot of the sample must be above the solvent level in the container.  

Since the stationary phase is polar because of the gel, the polar compounds will make strong interactions with the silica gel and therefore move at a slower rate. The nonpolar compounds will move farther with the solvent because they are weakly absorbed by the stationary phase. Thus, if the solvent is less polar than the gel, the compounds closer to the solvent front are more nonpolar than those near the baseline. 

However, in thin-layer chromatography, the components of the sample usually do not show up as visible patterns like in colored paper chromatography. We can use UV light to see some components, and then trace the data with a pencil. Another method is using a staining agent, like potassium permanganate or iodine. These will react and may make the spots visible.

We can then trace the data and use retention factor in thin-layer chromatography. 

Applications of Thin-Layer Chromatography

TLC may be used to monitor a reaction if the reactant and product differ in polarity. First, we can spot a plate with the starting reactant and calculate its Rf. Then, once the reaction has started, we can spot a second plate and see some reactant as well as some product. Finally, we can spot a third plate and hopefully see only the product. The product and reactant will have different migration rates and retention factors. This is due to their differing polarities, so we will know which spot is which by measuring the Rfs.

reaction monitoring with thin layer chromatography
Reaction Monitoring with TLC

TLC may be in biochemical, pharmaceutical, cosmetic, and nutrition contexts, similar to how and where scientists apply paper chromatography. 

Practice Problems

1. The diagram below shows a developed chromatogram. A nonpolar solvent was used, and the chromatography paper is made of polar cellulose. By using the chromatogram, which dye (A, B, C, or D) is the most polar?

paper chromatography practice problem example

2. On a thin-layer chromatography plate, the spotted pure sample moves 3 cm from the baseline. The solvent moves 12 cm from the baseline. What is the retention factor (Rf)?

Practice Problems Solutions – Paper Chromatography

1. A

2. 0.25