Lab Procedures, Tips & Safety

Lab Procedure: Thin Layer Chromatography

Core Concepts 

In this tutorial, you will learn what is chromatography. You will also learn about TLC analysis, thin layer chromatography, and the steps required to perform one.

Topics Covered in Other Articles 

Vocabulary: 

  • TLC plate: inert glass or plastic slide coated in silica gel; receives drops of sample for TLC analysis 
    • Stationary phase: silica gel; does not travel up the TLC plate 
    • Mobile phase: liquid solvent or solvent mixture; travels up TLC plate, carrying drops of sample at varying speeds 
    • Hydroxyl groups: polar functional groups in silica gel; participate in dipole-dipole interactions with other polar substances 
  • Development chamber: isolated beaker filled with solvent in which TLC plate is placed 
  • Rf value: ratio of the distance moved by solute and distance moved by solvent; quantifies the distance travelled by the tested solution on a scale of 0 to 1

What is Chromatography

The definition of chromatography, is a procedure where you separate a mixture by passing it through a medium where the components of the mixture move at different rates. The mixture is usually first dissolved in a gas or liquid. Types of chromatography include thin layer chromatography (TLC) and column chromatography. Types of column chromatography include gas chromatography, liquid chromatography, Ion exchange chromatography, size exclusion chromatography, and chiral chromatography.

Introduction to Thin Layer Chromatography

Scientists use the analytical technique of Thin Layer Chromatography, known as TLC, to separate compounds. The TLC process involves spotting a TLC plate with a compound in order to isolate and identify its constituents and check reaction progress. 

Thin layer chromatography in the chemistry lab

TLC plates are coated in silica gel, a solid substance containing silicon dioxide with alternating silicon and oxygen atoms. At the surface of the silica gel lie polar hydroxyl groups with the chemical formula: -OH. The components of a given compound interact with these polar compounds in different ways according to the varying polarities of their functional groups. These interactions directly impact the speed at which they migrate up a TLC plate once it is placed in a solvent system, as shown above. This facilitates the separation of different “dots” of compound components. TLC analyses allow scientists to obtain well defined, separated spots that denote the different components of a mixture. 

Researchers may employ TLC as a tool to monitor the progress of a reaction, to purify small amounts of compounds, or even to select effective solvent systems for other chromatographic procedures. College students use TLC when using the scientific method. Because of its simplicity, cost effectiveness, and efficiency, TLC functions as one of the most widely used analytical analyses in the chemistry world. 

Solvent Systems 

Choosing an appropriate solvent system, or mobile phase, is viewed as the most important parameter of an efficient, effective TLC process. To select a good solvent system, scientists consider their solution’s solubility in the system as well as its different affinities with the mobile and stationary phases on the plate. 

Typically, non-polar solvents are employed in TLC analyses. These allow non-polar compounds to move further up the plate, keeping most polar compounds closer to the starting line. Examples of non-polar solvents include hexane and pentane. Polar solvents prompt polar components to move further up the plate. They keep most nonpolar compounds closer to the starting line. Examples of polar solvent systems include ethyl acetate, methanol, and dichloromethane. 

The most effective solvent systems will move all components off of the starting line, resulting in Rf values within the range of 0.2-0.8. 

Mobile and Stationary Phases 

The functional groups of a given solution dictate its behavior on the TLC plate. As discussed, the mobile phase, or solvent system, carries the solution up the plate, or stationary phase. But the speed at which the solution travels depends predominantly on its relative polarity

A substance containing polar functional groups, or a strong polar net dipole, interacts “significantly” with the stationary phase. This means that the substance is strongly absorbed to the stationary phase. It should move slowly up the TLC plate, “sticking” to the silica gel. 

On the other hand, a substance with nonpolar functional groups will exhibit the opposite  behavior. These nonpolar functional groups have a weak polar net dipole, so they do not interact significantly with the stationary phase. This type of substance should move up the TLC plate at a speed similar to that of the solvent system; the substance should not stick to the silica gel and thus moves faster and further. 

How To Perform a TLC Analysis 

The following steps describe the methods involved in a Thin-Layer Chromatography analysis:

Setting Up:

  1. Draw a line near the bottom of your TLC plate to mark the starting point for the substances that will travel up the plate. Draw this line with a pencil – a pen’s ink will run and smudge the plate.
  2. Insert a capillary tube into the solution being analyzed. Spot the bottom of the TLC plate, at the line, with the tube contents. Be sure to press firmly during this step to ensure a complete transfer of your substance from the tube to the plate.
  3. Add your chosen solvent system to a beaker, called the development chamber. Then, insert the spotted TLC plate; lean the plate against the side of the beaker, and make sure that the spotted solutions sit above the solvent. If the solutions touch the solvent before the TLC begins, your samples will likely wash away.

Observing the TLC Process:

  1. Cover the beaker so the solvent doesn’t evaporate. Allow the TLC plate to develop; the solvent will move up the plate, carrying the different substances with it.
  2. The solvent, or mobile phase, should rise up the TLC plate, or stationary phase, via capillary action. As the components of your solution travel up the plate, they will interact with the silica hydroxyl groups in various ways, depending on their respective functional groups. 
  3.  Once the solvent reaches the top of the plate, mark this boundary with a second line. Then allow the TLC plate to dry.

Collecting and Quantifying Your Results:

  1. To examine the developed plate, use a UV light source to illuminate the locations of each dot. Trace everything you see–this includes splotches, dots, lines, and streaks. You may also utilize a staining agent, such as iodine, to complete this step. Staining agents react with particular functional groups, rendering their spots more visible; submerging your TLC plate in a staining agent, then placing it on a hot plate to dry, will accomplish this. 
  2. Assign a retention factor, or Rf value, to every individual dot on the plate. This amount represents the distance travelled by your substance as a fraction of the distance traveled by the solvent system. To calculate Rf values, first measure the distance between the two lines that you drew to quantify the distance the solvent traveled. Next, measure the distance between the starting line and the dot. Divide the latter by the former; repeat this process for each dot on the plate. Your measurements should be in centimeters (cm), and because you divide one by the other, your final Rf value will not require units.

Rf Value = (distance traveled by solution) / (distance traveled by solvent front)

These Rf values allow you to compare the solubility of different dots in the solvent system on the same scale. Two dots with the same Rf values likely contain identical molecules. Conversely, two dots with different Rf values probably consist of dissimilar molecules. Smaller Rf values indicate less soluble, more polar solutions; larger Rf values denote highly soluble, less polar solutions.

Applications of TLC to Today’s World 

Chemists may use TLC to monitor reactions. For example, they can determine whether a reactant remains after a specific reaction by calculating Rf values of the reactant and product. If they detect multiple Rf values, the reaction has not fully finished. If the reaction reached completion, they should only find one Rf value–that of the product. 

TLC can also inform the selection of an effective solvent system for column chromatography, a larger-scale physical separation technique. Scientists strive to use solvent systems that maximize the distance between two components on their column chromatography plate. This system may contain a mixture of two or more solvents, such as hexanes, ether, or ethyl acetate, among others. TLC allows scientists to develop a ratio in which to mix such components. For instance, if two components being studied end up close together on the TLC plate, scientists could conclude that the solvent system used was not particularly effective. They would draw the same conclusion if the two components separate a bit but do not travel far from the starting line. As they test different solvent system combinations through trial and error, they can continuously alter the ratios of these systems. 

Most current applications for thin layer chromatography analysis appear in the clinical, pharmaceutical, and food testing industries. Doctors rely on TLC to isolate and compare different compounds from blood, urine, and other body fluids. This process facilitates the identification of abusive drugs in patients’ systems. 

In the pharmaceutical industry, TLC fuels quality assurance investigations. Scientists employ it to identify harmful substances and quantitatively determine drug purity. 

Lastly, TLC helps researchers determine storage stability of different foods and food products. They can detect healthy levels of sweeteners, preservatives, and other additives using this analytical technique. 

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