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Column Chromatography 101: A Clear and Simple Explanation

Column Chromatography 101

Table of Contents

What is column chromatography

Column chromatography is a widely used laboratory technique in chemistry and biochemistry for separating and purifying mixtures of chemical compounds based on their different properties. It operates on the principle of differential partitioning, which means that compounds in a mixture are separated based on how they interact with two phases: a stationary phase and a mobile phase. Here’s how column chromatography works:

  1. Column Setup: In column chromatography, a vertical glass or plastic column is packed with a stationary phase material. Common stationary phase materials include silica gel, alumina, or other substances with finely divided particles. The stationary phase is typically held in place by a glass frit or a layer of sand at the bottom of the column.

  2. Sample Loading: The mixture to be separated is dissolved in a suitable solvent and then carefully introduced at the top of the column. This mixture forms a concentrated band at the beginning of the column, known as the sample plug.

  3. Elution: A mobile phase, often a liquid solvent or a mixture of solvents, is introduced into the column and allowed to flow downward by gravity or under pressure. As the mobile phase travels through the column, it interacts with the stationary phase and the sample plug.

  4. Separation: Different compounds in the sample have varying affinities for the stationary and mobile phases. Compounds that have a stronger affinity for the stationary phase will move more slowly through the column because they spend more time interacting with the stationary phase particles. Conversely, compounds that have a stronger affinity for the mobile phase will move more quickly through the column.

  5. Fraction Collection: As the compounds in the sample move through the column, they separate into distinct bands or zones based on their affinities. Fractions of the eluent are collected in test tubes or other containers as they exit the column. Each fraction contains a mixture of compounds that were traveling at a similar rate through the column at that particular time.

  6. Analysis: The collected fractions are then analyzed to determine the composition of each fraction. This analysis can involve techniques like spectroscopy, chromatography, or other analytical methods to identify and quantify the separated compounds.

Column chromatography is a versatile and essential technique used in various fields of chemistry, including organic chemistry, biochemistry, and analytical chemistry. It is employed for tasks such as isolating and purifying chemical compounds, monitoring chemical reactions, and separating complex mixtures into their individual components. The effectiveness of the separation depends on factors like the choice of stationary phase, the mobile phase, and the nature of the compounds being separated.

How does column chromatography work

Column chromatography is a widely used technique in chemistry to separate and purify mixtures of compounds based on their different properties, such as size, polarity, and solubility. It works on the principle of differential partitioning of substances between a stationary phase and a mobile phase. Here’s how it works:

  1. Setup: Column chromatography typically involves a glass or plastic column filled with a solid stationary phase and a mobile phase (usually a liquid solvent). The stationary phase is often a fine powder or a gel-like material, such as silica gel or alumina, packed into the column.

  2. Sample Application: The mixture to be separated, containing different compounds, is dissolved in a small volume of a suitable solvent and carefully applied to the top of the column. The sample is loaded onto the column using a pipette, and it forms a small spot or band on top of the stationary phase.

  3. Elution: The separation process begins when the mobile phase (solvent) is introduced into the column. The solvent flows down through the column by gravity or using a pump. As the solvent travels down the column, it interacts with the sample mixture.

  4. Separation: Different compounds in the sample interact differently with the stationary and mobile phases. Compounds that have a higher affinity for the stationary phase will move more slowly through the column, while those with a higher affinity for the mobile phase will move faster.

  5. Fraction Collection: As the compounds move through the column, they separate into distinct bands. These bands correspond to different compounds in the mixture. Fractions are collected from the bottom of the column, typically using test tubes or other collection vessels. Each fraction contains a mixture of compounds that have similar affinities for the stationary and mobile phases.

  6. Analysis: After all the fractions have been collected, they can be analyzed using various techniques, such as spectroscopy or chromatography, to identify and quantify the separated compounds. Researchers can then select the fractions containing the pure compounds of interest.

Key factors that influence the separation in column chromatography include the choice of stationary phase, the type and composition of the mobile phase, the flow rate of the mobile phase, and the nature of the compounds being separated.

Column chromatography is a versatile and powerful technique commonly used in research laboratories for tasks like isolating natural products, purifying synthesized compounds, and analyzing complex mixtures. Its effectiveness relies on the differential interactions between the compounds and the stationary and mobile phases within the column.

Purpose of column chromatography

The primary purpose of column chromatography is to separate and purify mixtures of chemical compounds based on their differing physical or chemical properties. This technique is widely used in various fields of chemistry and biochemistry for several specific purposes:

  1. Compound Isolation: Column chromatography allows researchers to isolate specific compounds from complex mixtures. By exploiting differences in properties like size, polarity, or charge, it’s possible to separate and recover individual substances of interest.

  2. Purification: It’s common for chemical compounds to contain impurities after synthesis or extraction. Column chromatography can be used to purify these compounds, removing unwanted substances and yielding a higher-purity product.

  3. Analysis: Column chromatography is also used for analytical purposes, such as determining the composition of a mixture or assessing the purity of a compound. By analyzing the fractions collected during the separation process, researchers can gain valuable insights into the nature of the sample.

  4. Fractionation: In some cases, the goal is not to isolate a single compound but to fractionate a mixture into several components. Column chromatography allows for the separation of a mixture into distinct fractions, each containing compounds with similar properties.

  5. Reaction Monitoring: Researchers often use column chromatography to monitor chemical reactions. They can analyze samples taken at different time points during a reaction to track the progress of the reaction and identify reaction intermediates or byproducts.

  6. Sample Cleanup: In analytical chemistry and biochemistry, column chromatography is used to clean up samples before further analysis. This removes interfering substances, making the sample suitable for subsequent experiments or measurements.

  7. Sample Concentration: By concentrating a dilute sample onto a column and then eluting the compound of interest, column chromatography can also serve as a means of sample concentration.

  8. Preparative Scale: On a larger scale, preparative column chromatography is employed to produce substantial quantities of pure compounds for industrial or research purposes. This is often necessary for pharmaceutical production, natural product extraction, or other applications requiring significant quantities of a single substance.

The choice of stationary phase, mobile phase, and operating conditions in column chromatography can be tailored to the specific characteristics of the compounds being separated, making it a versatile and essential technique in chemical analysis and purification. It plays a crucial role in various scientific disciplines, including organic chemistry, biochemistry, pharmaceuticals, and environmental chemistry.

Size Exclusion Chromatography Columns ?

Size exclusion chromatography (SEC), also known as gel filtration chromatography or gel permeation chromatography, is a chromatographic technique used for separating and purifying molecules based on their size or molecular weight. SEC columns are a fundamental component of this technique. Here’s how size exclusion chromatography columns work:

  1. Column Setup: In size exclusion chromatography, a column is packed with a stationary phase made of porous beads. These beads can be made of materials like agarose or cross-linked polymers. The beads have pores of a specific size distribution, which allows molecules to enter and exit these pores based on their size.

  2. Sample Injection: A sample containing a mixture of molecules, such as proteins, nucleic acids, or polymers, is injected into the top of the SEC column. The sample solution is typically dissolved in a buffer or mobile phase that does not interact with the stationary phase.

  3. Separation by Size: As the sample flows through the column, molecules within the sample encounter the porous beads in the stationary phase. Larger molecules cannot enter the pores and therefore flow through the column more quickly, taking the path of least resistance. In contrast, smaller molecules can enter the pores and become temporarily trapped or slowed down. Consequently, larger molecules elute (exit) from the column earlier than smaller ones.

  4. Elution: The molecules are separated based on their size and shape as they travel through the column. Smaller molecules are temporarily excluded from the pores and spend more time traveling through the interstitial spaces between the beads. This results in their slower elution from the column. Conversely, larger molecules are less affected by the porous beads and exit the column more rapidly.

  5. Fraction Collection: Fractions are collected from the bottom of the column over time. Each fraction contains a group of molecules with similar sizes. Researchers can analyze these fractions to determine the distribution of molecular sizes in the original sample.

  6. Analysis: The collected fractions can be further analyzed using techniques such as spectrophotometry, gel electrophoresis, or other analytical methods to quantify and characterize the separated molecules.

Size exclusion chromatography is especially valuable for biomolecular research, as it allows for the separation and purification of proteins, nucleic acids, and other biomolecules without subjecting them to harsh chemical conditions or high temperatures. It is a non-destructive and gentle separation technique that provides valuable information about the size distribution of the molecules in a sample.

How to improve column chromatography separation

Improving the separation efficiency of column chromatography can be crucial for obtaining better results, especially when dealing with complex mixtures or challenging compounds. Here are several strategies to enhance the separation in column chromatography:

  1. Choose the Right Stationary Phase:

    • Select a stationary phase material that best suits your separation needs. Different materials, such as silica gel, alumina, or polymer-based resins, have varying properties and selectivities.
    • Consider using a stationary phase with a smaller particle size for higher resolution. Smaller particles provide more surface area and better separation but may require higher pressure.
  2. Optimize Mobile Phase Composition:

    • Adjust the composition of the mobile phase (solvent or solvent mixture) to enhance separation. Changes in solvent polarity, pH, or additives can influence compound elution.
    • Conduct a solvent screening or gradient elution to find the most suitable conditions for your specific mixture.
  3. Control Flow Rate:

    • Regulate the flow rate of the mobile phase through the column. Slower flow rates can improve separation by allowing more interaction time between compounds and the stationary phase.
    • Avoid excessively high flow rates that can cause band broadening and reduced resolution.
  4. Sample Loading:

    • Carefully load your sample to ensure that you do not overload the column. Overloading can lead to poor separation and band broadening.
    • Consider pre-concentrating or diluting your sample to optimize loading conditions.
  5. Column Packing:

    • Pack the column carefully and uniformly to prevent channeling and uneven flow. Use appropriate techniques, such as slurry packing or dry packing, depending on the stationary phase material.
    • Ensure that the column is properly packed without air bubbles or gaps.
  6. Column Length and Diameter:

    • Adjust the column length and diameter based on the scale of your separation. Longer columns generally provide better resolution, while wider columns can handle larger sample volumes.
  7. Monitor and Collect Fractions Carefully:

    • Continuously monitor the eluent as it exits the column, either by UV detection or other suitable methods, to detect compound peaks.
    • Collect fractions at the appropriate time to capture the compounds of interest while minimizing overlap between peaks.
  8. Reiterate if Necessary:

    • If your initial separation is not satisfactory, consider running the column chromatography again using the collected fractions as samples. This can further improve purity and separation.
  9. Use Specialized Techniques:

    • Consider employing specialized column chromatography techniques, such as high-performance liquid chromatography (HPLC) or preparative thin-layer chromatography (TLC), for specific separation challenges.
  10. Practice and Experience:

    • Developing expertise in column chromatography often requires practice and experience. Familiarity with your specific compounds and the chromatographic system can lead to better results over time.

Remember that optimization may involve a combination of these strategies, and it may take some experimentation to achieve the desired separation quality. Additionally, consulting with experienced colleagues or chromatography experts can be valuable in troubleshooting and refining your separation techniques.

How to choose solvent system for column chromatography ?

Selecting the appropriate solvent system for column chromatography is a critical step in achieving successful separations. The choice of solvent system depends on the nature of your sample, the stationary phase, and the compounds you want to separate. Here’s a step-by-step guide to help you choose the right solvent system for your column chromatography:

  1. Know Your Sample:

    • Understand the chemical properties of your sample, including its polarity, solubility, and stability.
    • Identify the key compounds you want to separate and their chemical characteristics, such as polarity, acidity, or basicity.
  2. Select the Stationary Phase:

    • Choose the type of stationary phase (e.g., silica gel, alumina, polymer-based resin) based on its compatibility with your sample and the specific separation goals.
    • Consider the particle size and pore size of the stationary phase material, as these parameters can influence separation efficiency.
  3. Determine Compound Polarity:

    • Assess the polarity of the compounds in your sample. Polarity is a fundamental factor that influences separation.
    • If your sample contains both polar and nonpolar compounds, you may need a gradient elution approach or a mixed solvent system.
  4. Review Solubility:

    • Ensure that your sample is soluble in the chosen solvents or solvent mixture. Incompatibility can lead to poor sample loading and separation.
  5. Consider pH:

    • The pH of your mobile phase can impact the ionization of acidic or basic compounds in your sample. Adjust the pH if necessary to optimize separation.
  6. Choose Initial Solvent System:

    • Start with a solvent system that you believe will provide a good starting point based on your knowledge of the sample’s properties. Common starting solvent systems include hexane/ethyl acetate, methanol/water, or dichloromethane/methanol.
  7. Perform Solvent Screening:

    • If you are unsure about the best solvent system, conduct a solvent screening experiment. This involves running small-scale column chromatography experiments using different solvent systems to assess separation efficiency.
    • Gradually change the solvent composition or polarity until you achieve satisfactory separation.
  8. Use Gradient Elution:

    • For complex mixtures or samples with a broad range of polarities, consider using gradient elution. This involves starting with a less polar solvent and gradually increasing the polarity over time.
    • Gradient elution can help resolve compounds with a wide range of polarities more effectively.
  9. Optimize Flow Rate:

    • Adjust the flow rate of your mobile phase to control the separation. Slower flow rates may be required for more complex separations.
  10. Monitor and Adjust:

    • Continuously monitor the progress of your separation using appropriate detection methods (e.g., UV-Vis, TLC).
    • If you encounter issues like co-elution or poor resolution, consider modifying the solvent system or gradient conditions accordingly.
  11. Document Your Findings:

    • Keep detailed records of your chromatography runs, including the solvent systems used, flow rates, and any modifications made. This documentation can be valuable for future reference.
  12. Seek Expert Advice:

    • If you are dealing with particularly challenging separations or if you are new to column chromatography, consider seeking guidance from experienced colleagues or chromatography experts.

Choosing the right solvent system for column chromatography may require some experimentation, but careful consideration of the sample’s properties and systematic optimization can lead to successful separations.

Difference between TLC and column chromatography

Both TLC (Thin Layer Chromatography) and column chromatography are techniques used to separate mixtures based on the different interactions of components with a stationary and mobile phase. However, they differ in scale, application, and complexity:


  • TLC – Analytical technique, works with very small samples (micrograms to milligrams).
  • Column Chromatography – Preparative technique, used to purify larger quantities ( milligrams to grams or even kilograms).


  • TLC – Primarily used for qualitative analysis (identifying components in a mixture) and monitoring progress during column chromatography.
  • Column Chromatography – Primarily used for purification of desired compounds from a mixture.


  • TLC – Relatively simple setup, fast, and inexpensive.
  • Column Chromatography – More complex setup, requires more time and solvent, and can be prone to errors.

Here’s an analogy:

Imagine you have a box of mixed legos.

  • TLC – Like quickly sorting legos by color on a flat tray. It gives you a general idea of what colors are present but doesn’t give you individual pieces.
  • Column Chromatography – Like meticulously separating all the legos piece by piece using a complex system of compartments. It takes more time and effort but allows you to isolate specific lego pieces.

So, as these information, you can know TLC is a quick and easy way to get a preliminary picture of a mixture, while column chromatography is the workhorse for purifying desired components.

Column Chromatography vs Paper Chromatography

Paper chromatography and column chromatography are both chromatographic techniques used for separating mixtures of compounds, but they differ in several key aspects, including their principles, applications, and the physical setup. Here are the main differences between paper chromatography and column chromatography:

  1. Principle of Separation:

    • Paper Chromatography: In paper chromatography, separation occurs based on differences in the distribution of sample compounds between a stationary phase (the paper) and a mobile phase (the solvent). Compounds move up the paper through capillary action, and separation is achieved as the sample components travel at different rates.

    • Column Chromatography: Column chromatography relies on the differential partitioning of compounds between a stationary phase (typically a solid support inside a column) and a mobile phase (liquid solvent). Compounds are separated as they pass through the column due to differences in their affinities for the stationary and mobile phases.

  2. Physical Setup:

    • Paper Chromatography: Paper chromatography is typically performed on a sheet of absorbent paper (e.g., filter paper or chromatography paper). The sample is spotted at the base of the paper, which is then immersed in a solvent or mobile phase. As the solvent rises up the paper, it carries the sample compounds with it.

    • Column Chromatography: Column chromatography involves a vertical glass or plastic column packed with a stationary phase material (e.g., silica gel or alumina). The sample is loaded onto the top of the column, and the mobile phase is passed through the column, causing separation as compounds interact with the stationary phase.

  3. Applications:

    • Paper Chromatography: Paper chromatography is commonly used for qualitative analysis and separation of smaller quantities of compounds. It is suitable for tasks such as separating pigments in ink or plant extracts, identifying unknown substances, or checking the purity of a sample.

    • Column Chromatography: Column chromatography is used for both qualitative and quantitative analysis and is better suited for larger-scale separations. It is widely used in research and industry for isolating, purifying, and analyzing a wide range of compounds, including natural products, pharmaceuticals, and biomolecules.

  4. Resolution and Separation Efficiency:

    • Paper Chromatography: Paper chromatography typically provides lower resolution and separation efficiency compared to column chromatography. It is more suitable for simple separations and qualitative analysis.

    • Column Chromatography: Column chromatography offers higher resolution and better separation efficiency due to its larger column size, controlled flow rates, and the ability to optimize parameters like the stationary phase and mobile phase composition.

  5. Scale of Operation:

    • Paper Chromatography: Paper chromatography is often used for small-scale separations, making it suitable for educational and analytical purposes.

    • Column Chromatography: Column chromatography can be performed at various scales, from analytical (milligrams) to preparative (grams to kilograms), making it versatile for a wide range of applications.

In summary, paper chromatography and column chromatography are chromatographic techniques with different principles, applications, and setups. Paper chromatography is simple and best suited for qualitative analysis and smaller-scale separations, while column chromatography offers greater flexibility, resolution, and scalability for both qualitative and quantitative separations, especially in research and industrial settings.

Column Chromatography vs Paper Chromatography

What is the role of empty chromatography columns ?

Empty chromatography columns, sometimes referred to as “blank columns” or “void columns,” do not contain any stationary phase material, such as silica gel or alumina. Instead, they are typically used in chromatography for specific purposes or as a part of the chromatographic process. Here are some key roles and uses of empty chromatography columns:

  1. Sample Loading and Equilibration: Empty columns are often used to load and equilibrate the column with the mobile phase before the actual chromatographic separation begins. This helps remove any air bubbles, prewets the column with the solvent, and ensures a consistent flow of the mobile phase when the stationary phase is introduced.

  2. System Priming: Empty columns can be employed to prime the entire chromatography system. This is done by connecting an empty column to the system and pumping the mobile phase through it to remove any air or contaminants from the tubing, detectors, and other components.

  3. Guard Columns: In some chromatography systems, an empty column may be used as a “guard column” positioned in front of the analytical column. The purpose of the guard column is to protect the more expensive and delicate analytical column from contamination and to extend its lifespan. The guard column can be replaced or cleaned more frequently.

  4. Column Maintenance: Empty columns can be used for column maintenance and cleaning procedures. For example, flushing the column with a suitable solvent can help remove impurities, sample residues, or any adsorbed materials from the stationary phase.

  5. Column Storage: When not in use, empty columns can be used for storing analytical or preparative columns. This helps protect the stationary phase from dust, moisture, and contamination.

  6. Calibration and System Checks: Empty columns may be used as part of system calibration and performance checks. By passing known standards or test solutions through an empty column, operators can verify the system’s performance and the accuracy of detectors.

  7. Mobile Phase Compatibility Testing: Empty columns can be used to test the compatibility of different mobile phases with the column hardware and to check for leaks or other issues before conducting actual chromatographic separations.

  8. Teaching and Training: In educational settings, empty columns may be used to demonstrate chromatographic principles and procedures to students before they work with actual stationary phases and samples.

In summary, empty chromatography columns play various roles in the preparation, maintenance, and operation of chromatography systems. They are particularly useful for ensuring that the chromatography system is properly primed, equilibrated, and ready for successful separation. Additionally, they can serve protective and diagnostic functions within the chromatography process.

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