How to Perform Column Chromatography

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7-10-2017, 07:00
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Column Chromatography (CC) is a common lab technique used in college-level chemistry courses. The purpose of CC is to separate individual chemical components from an impure mixture containing two or more components. CC is related to Thin-Layer Chromatography (TLC); however, TLC reveals the purity of a mixture (how many individual chemical components it contains), while CC separates the components of a mixture for further use.

Preparation

  1. Read and understand the lab procedure. The goal of column chromatography is to separate and collect the desired chemical components from a mixture. Separation will occur within a column containing a stationary adsorbent and a mobile eluent (solvent). An impure sample is introduced to the column and eluted (or separated) by running eluent through the column. Separation will occur because your impure mixture is made up of different chemical components that will be unevenly adsorbed (attached) to the surface of the adsorbent. The adsorbent is a porous material, commonly alumina or silica gel, and is the medium through which your mixture will separate. Ideally, as the eluent runs through your column it should interact with a single component, thus extracting one component from the mixture while the other(s) remain adsorbed at the top of the column. Several eluents may be used to separate multiple components of your mixture.
  2. Obtain appropriate equipment and chemical species. Gather the equipment and chemical species necessary to perform the procedure. Also, familiarize yourself with your resources and surroundings before you begin your work. Materials needed to carry out this process will vary; however, at minimum, this project will require the items listed under Things You'll Need, listed a the end of this article.

Assemble the Column

  1. Assemble the column apparatus. Using a ring stand and an appropriate clamp, position the column so that the tip of the column is several inches above the base of the stand. The space between the base of the stand and tip of the column should be large enough that the collection flask can be easily positioned to collect the column’s output, but small enough so that the liquid output does not splash when it lands in the flask.
  2. Insert a small piece of glass wool, or cotton, in the column just above the stopcock. The purpose of the glass wool is to prevent the solid phase adsorbent from escaping the column.
  3. Fill the column approximately half-full with your least polar solvent, or eluent. It is important to begin with your least polar solvent because this will separate the least polar component of your mixture first. If necessary, when you have successfully eluted the first component you can switch to a second, more polar solvent to elute another component. Several eluents may be used to elute all components of your impure mixture.
  4. Add a 1-cm layer of sand above the glass wool plug. Carefully pour sand into the column until there is a 1-cm layer of sand resting atop the plug. Use additional, small quantities of your least polar solvent to wash down any sand that adheres to the walls of your column. The purpose of the sand is to prevent the fine powder adsorbent from escaping through the glass wool plug and potentially clogging the narrow tip of the column.
  5. Introduce your adsorbent to the column by powder packing. Slowly (see Warnings) add your powdered adsorbent directly into the column (powder packing). Tap or gently swirl the column to remove any adsorbent from the column walls and allow the solid to settle in the column.
    • There should be no air bubbles present within the column––air pockets will cause the elements of your mixture to separate unevenly and will compromise the purity of your results. If your column contains obvious air bubbles, empty your column and prepare another.
  6. Add sand to the column until there is a 1-cm layer of sand above the adsorbent layer. Again, carefully pour sand into the column until there is a 1-cm layer of sand resting atop the adsorbent layer. Use additional, small quantities of your least polar solvent to wash down any sand that adheres to the walls of your column. The purpose of the top layer of sand is to keep the adsorbent in place.
  7. Adjust the solvent level so that it is several millimeters above the top layer of sand. Position a collection flask beneath your column, open the stopcock, and allow the solvent level to recede until it is several millimeters above the top layer of sand. Your column is now ready to use.

Elute Components from Column

  1. Introduce your mixture to the column and flush it past the sand layer using your least polar solvent. Using a pipette, introduce your mixture to the column—do not disturb the protective layer of sand. Open the stopcock and allow the level of your mixture/solvent solution to fall until it is several millimeters above the top layer of sand. Your mixture is now "on the column."
  2. Elute the first components of your mixture. Perform the steps listed below to separate and collect the first component of your mixture using your least polar solvent. Remember, never allow the solvent level to fall below the top layer of sand.
    • Add a small amount of your solvent (2-4 millimeters) to the column.
    • Open the stopcock to allow the solvent level to fall until it is several millimeters above the top layer of sand.
    • Repeat the previous sub-steps until the component reaches the bottom of the column (see Warnings). Never allow the solvent level in the column to fall below the top layer of sand.
    • When the component reaches the bottom of the column, collect it in a clean and clearly labelled collection flask then set it aside for further analysis.
  3. Elute additional components with slightly more polar solvents. One or more components of your mixture will not separate with your least polar solvent and, therefore, will remain adsorbed to the adsorbent at the top of the column. Separate these components using a slightly more polar solvent by changing the solvent used and following the same procedure used to elute the first components of your mixture. To change the solvent:
    • Open the stopcock to allow the level of the less polar solvent to fall until it is just above the top layer of sand.
    • Carefully introduce your new solvent (2-4 millimeters) to the column. Open the stopcock to adjust the new solvent level so that it is several millimeters above the top layer of sand. Again, do not disturb the protective layer of sand.
  4. Analyze the purity of the eluted solutions using Thin-Layer Chromatography. (This step is optional.) Column chromatography is a separation technique, therefore at the conclusion of the procedure you should have several beakers that contain the separate components of your initial mixture. To analyze the purity of your eluted products, it is common to perform Thin-Layer Chromatography, which you can learn more about in the article wikiHow to How to Perform Thin-Layer Chromatography.

Clean Up

  1. Empty the contents of the column into a clean beaker by removing the stopcock and applying air pressure. Point the open end of the column into a beaker. Then remove the stopcock. Apply air pressure gently to the tip to empty the contents of the column into the beaker. If there is anything stuck in the column (i.e. plug of glass wool), wet the column with acetone and reapply air pressure.
  2. Dispose of solid and liquid byproducts in the appropriate waste containers. Dispose of solid materials, including the contents of the column and plug, in the appropriate “solid waste” container. Dispose of liquids in the proper waste containers as well. Never wash solid materials or liquids down the sink or place them in an inappropriately labeled waste container.
  3. Wash all equipment and return it to its proper place.

Tips

  • Never allow the solvent level in the column to fall below the top layer of sand.
  • Choose the appropriate eluents for your impure mixture using Thin Layer Chromatography. Appropriate eluents will separate one component of your mixture at a time.

Warnings

  • Add adsorbent to the column slowly. When the adsorbent combines with the solvent in the column, an exothermic reaction occurs and heat is liberated. Slowly adding the adsorbent to the column prevents the contents of the column from becoming unnecessarily hot and boiling over, in which case you will need to dismantle your column and prepare another.
  • In the case that the components of your mixture are colored, it will be easy to see them separate and move down the column, as well as collect them in separate flasks. However, in the case that the components of your mixture of are colorless, then you will need to take extra steps to monitor the flow of components out of your column. In the scope of this discussion we assume the components are easily differentiated by color and therefore will be easily identified and collected.

Things You'll Need

  • A column with a stopcock (to control flow of liquid)
  • A glass wool, or cotton, plug
  • Sand
  • An adsorbent
  • The necessary eluents (solvents)
  • Collection flasks
  • Pipette
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