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What is Column Chromatography?
In the field of chemistry, column chromatography is an effective method employed to isolate a specific chemical compound from a mixture dissolved in a fluid.
The process of column chromatography operates by separating substances based on their varying rates of adsorption to the adsorbent material as they traverse through the column. This differential adsorption allows for the compounds to be fractionated and separated. This technique finds utility in both small-scale and large-scale purification of materials for subsequent experimental use. It falls under the category of adsorption chromatography methods. The principles used in thin layer chromatography can be applied on a larger scale in column chromatography to separate mixtures effectively.
In thin layer chromatography, a thin layer of silica gel or alumina is employed as the stationary phase, which is coated onto a glass, metal, or plastic plate. On the other hand, column chromatography functions on a larger scale by packing the same adsorbent materials into a vertical glass column.
Column Chromatography Principle
When the mixture to be separated, along with the mobile phase, is introduced at the upper part of the column, the individual components of the mixture move at varying speeds. Components with lower adsorption and affinity towards the stationary phase exhibit faster movement compared to those with higher adsorption and affinity. As a result, the faster-moving components are eluted first, while the slower-moving components are eluted last.
The adsorption of solute molecules onto the column surface is a reversible process. The speed of component movement is quantified as follows:
Rf = the distance travelled by solute/ the distance travelled by the solvent
Rf is the retardation factor.
In the realms of analytical and organic chemistry, elution refers to the process of extracting one substance from another through solvent washing. This technique is commonly employed to cleanse loaded ion-exchange resins and eliminate trapped ions. In chromatography, elution specifically denotes the extraction of an adsorbed substance from a solid adsorbent material using a solvent. The eluent, also known as the mobile phase, is the solvent that traverses through the column. When the polarity of the eluent aligns with the polarity of the molecules in the sample, the molecules detach from the adsorbent and dissolve in the eluent.
The portion of the mobile phase responsible for transporting the components of the sample is referred to as the eluent. The combination of solute and solvent that exits the column is termed the eluate. The eluate encompasses the mobile phase and analytes. It is a substance that separates and carries the constituents of a mixture through the chromatography column. In liquid chromatography, the eluent takes the form of a liquid solvent, while in gas chromatography, it is a carrier gas.
Column Chromatography Procedure Preparation
Before commencing the experiment on Column Chromatography, it is essential to comprehend the distinct phases involved.
Mobile phase – This phase consists of solvents and fulfills the following roles:
- It serves as a mixture of solvents and the sample, which can be introduced into the column.
- It acts as a developing agent, facilitating the separation of components within the sample to form distinct bands.
- It functions as an eluting agent, responsible for removing the separated components from the column.
- Examples of solvents commonly used as mobile phases, selected based on their polarity, include ethanol, acetone, water, acetic acid, pyridine, and others.
Stationary phase – This phase comprises a solid material that should possess effective adsorption properties and meet the following conditions:
- Particle shape and size: The particles should exhibit uniform shape and size within the range of 60-200μ in diameter.
- Particle stability and inertness: The particles must possess high mechanical stability and chemical inertness. They should not react with acids, bases, or any other solvents employed during the experiment.
- Colorless, cost-effective, and readily available: The stationary phase should be devoid of color, reasonably priced, and easily accessible.
- Allowance for unhindered flow of the mobile phase.
- Suitable for the separation of mixtures containing various compounds.
Using the Column
Imagine you wish to isolate a mixture consisting of two colored compounds: one is yellow, and the other is blue. When these compounds are combined, the resulting mixture appears green. To begin the separation process, it is advisable to prepare a concentrated solution of the mixture, preferably using the same solvent employed in the column. Firstly, you would open the valve, allowing the solvent already present in the column to drain until it reaches the same level as the top of the packing material. Subsequently, you would carefully introduce the solution at the top of the column. Once again, the valve is opened to ensure that the colored mixture is completely absorbed into the upper part of the packing material, resulting in a visual appearance similar to the following example:
Following that, you introduce a new supply of solvent onto the upper portion of the column, taking care to minimize any disruption to the packing material. Subsequently, you proceed to open the valve, allowing the solvent to trickle down through the column, collecting it in a beaker or flask positioned at the bottom. To prevent the column from drying out, it is crucial to continually replenish the top of the column with fresh solvent while the solvent flows through. The subsequent sequence of illustrations illustrates the potential changes that may occur over time.
Explaining What is Happening
Assuming you have already familiarized yourself with the explanation of thin layer chromatography, the following information applies.
The relative polarity of the blue and yellow compounds becomes evident, with the blue compound displaying greater polarity, potentially capable of forming hydrogen bonds. This is indicated by the blue compound’s slower movement through the column. It suggests that the blue compound exhibits stronger adsorption to the silica gel or alumina compared to the yellow compound. On the other hand, the less polar yellow compound spends a significant amount of time in the solvent, causing it to pass through the column much more rapidly. The process of passing a compound through a column using a solvent is referred to as elution, while the solvent itself is commonly referred to as the eluent.
What if You Want to Collect the Blue Compound as well?
Given the current rate of progression, the elution of the blue compound will be exceptionally time-consuming! Nonetheless, there are no limitations on modifying the solvent during the elution process. Let us contemplate substituting the existing solvent with a more polar alternative once all the yellow compound has been collected. This modification will engender two consequences, both of which will expedite the movement of the blue compound through the column.
First and foremost, the polar solvent will compete with the blue compound for available surface space on the silica gel or alumina. When solvent molecules momentarily occupy the space on the stationary phase, the blue molecules have fewer opportunities to adhere to the surface. Consequently, the blue molecules are more prone to continue their migration with the solvent.
Secondly, a heightened attraction will manifest between the polar solvent molecules and the polar blue molecules. This interaction will tend to entice any blue molecules that have adhered to the stationary phase back into the solvent solution.
Overall, the utilization of a more polar solvent results in the blue compound spending more time in the solution, thereby facilitating its swifter traversal through the column.
Now, why not employ this alternative solvent right from the outset? The rationale is that if both compounds in the mixture swiftly traverse the column from the beginning, the likelihood of achieving a satisfactory separation diminishes.
What if Everything in Your Mixture is Colorless?
If you were to employ column chromatography for purifying the product obtained from an organic synthesis, it is highly probable that the desired product will be colorless, even if one or more impurities exhibit coloration. Let us consider the worst-case scenario where everything is colorless.
How can you determine when the desired substance has reached the bottom of the column?
Unfortunately, there is no straightforward or effortless method to accomplish this! What you must do is collect the eluted fractions from the bottom of the column in a series of labeled tubes. The volume of each sample will depend on the size of the column – you may collect samples of 1 ml, 5 ml, or an appropriate volume based on the column dimensions.
Subsequently, you can take a small droplet from each solution and employ it to create a thin layer chromatogram. Placing the droplet alongside a droplet from a pure sample of the compound being synthesized, you can repeatedly conduct this procedure. Through this iterative process, you can determine which samples collected at the bottom of the column contain the desired product exclusively and discard those containing impurities.
Column Chromatography Applications
- Column chromatography finds application in the extraction of active components from mixtures such as plant extracts.
- It proves to be highly advantageous for the separation of compound mixtures.
- This technique is utilized for assessing the drug content in drug formulations.
- It serves as a means to eliminate impurities.
- Moreover, it facilitates the isolation of metabolites from biological fluids.
- Kondeti, Ranjith Reddy, Kranti Sri Mulpuri, and Bharathi Meruga. “Advancements in column chromatography: A review.” World Journal of Pharmaceutical Sciences (2014): 1375-1383. https://wjpsonline.com/index.php/wjps/article/view/advancements-column-chromatography-review