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JoVE Lab Manual
Lab: Chemistry

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Column Chromatography

Column Chromatography

Learning Objectives

At the end of this lab, students should know...

What are the two phases in column chromatography?

Column chromatography involves a mobile phase and a stationary phase.

How are mixtures separated in column chromatography?

Compounds separate based on their interactions with the stationary phase.

What compounds elute first?

Compounds interacting weakly with the stationary phase elute first because they interact stronger with the mobile phase and thus travel through the stationary phase faster than those with strong stationary phase interactions.

What physical properties are used to separate mixtures in column chromatography?

The most common properties utilized to separate mixtures are based on size, charge, polarity, and hydrophobicity of the constituent substances.

How do column properties such as the diameter and length affect the separation of mixtures?

The diameter of the column determines the amount of sample that can be loaded on to the column. A small thin layer of sample provides the most efficient separation; therefore, a smaller diameter means less sample can be loaded and vice versa. The length of the column is dependent on the affinity of the substances to the stationary phase of the column. A longer column is required for substances with similar affinity, while a shorter column can be used for substances with very different affinities.

List of Materials

  • 50-mL glass beaker
  • 100-mL glass beaker
  • 5- or 10-mL glass graduated cylinder
  • 50-mL glass graduated cylinder
  • Small powder funnel
  • Glass stirring rod
  • Labeling tape
    5 rolls
  • Labeling pen
  • Ruler
  • Pasteur pipette bulbs
  • Laboratory stand
  • Burette clamp
  • 50-mL glass burette
  • Weighing boats
  • Cotton or glass wool plugs
  • Long-stem Pasteur pipettes
  • Sand (40-100 mesh)
    15 g
  • Silica gel powder for column chromatography (35-70 mesh, 100 grade)
    50 g
  • Ethanol (95%)
    400 mL
  • Green food coloring containing tartrazine and erioglaucine
    5 mL
  • 5-mL glass volumetric flask with cap
  • Long blunt-ended wooden/metal rod
  • 250-mL plastic wash bottle
  • Paraffin film
    1 box
  • Spatulas
  • Lab wipes
    3 boxes
  • Acetone
    250 mL
  • Analytical balance (at least 1)
    Dependent on lab size
  • Deionized water
    Dependent on lab size

Lab Prep

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

  1. Preparation of the Dye Solution

    Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.

    • Before you get started, put on a lab coat, safety glasses, and nitrile gloves, and make sure that the lab has a labeled organic waste container.
    • Prepare 5 mL of green food dye solution in ethanol. First, tare a clean empty 5-mL volumetric flask and slowly add 2.5 g of green food dye to the flask.
    • Add about 1.5 mL of 95% ethanol to the flask, stopper it, and seal it with plastic paraffin film.
    • Hold the stopper firmly in place and invert the flask several times to mix the ethanol and food dye together.
    • Then, add 95% ethanol to the fill line, reseal the flask, and invert it until the solution color appears even throughout.
    • Label the flask with its name and concentration and place it in a central area.
  2. Preparation of the Laboratory
    • Ensure that the lab has labeled waste containers for silica gel and glass.
    • Fill a plastic wash bottle with acetone and set it by the organic waste container. This is for students or teaching assistants, who will clean the columns after the lab.
    • Place a container of silica gel powder, a container of sand, a box of laboratory wipes, several clean spatulas, and enough weighing boats for each student group by a calibrated balance in a well-ventilated area.
    • Place a bottle of 95% ethanol in a nearby hood. Then, make sure that the stopcocks for the columns fit securely but still turn easily.
    • Close the stopcocks after testing them and distribute the columns to the benchtop workspaces.
    • Make sure that each workspace has a lab stand or a scaffold and an appropriate clamp for the column being used.
    • Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
       5    50-mL beakers
       1    100-mL beaker
       1    5- or 10-mL graduated cylinder
       2    50-mL graduated cylinders
       2    Small powder funnels
       1    Glass stirring rod
       1    Roll of laboratory tape
       1    Marking pen
       1    Ruler
       2    Pipette bulbs
    • Lastly, place glass or cotton wool, at least one long, blunt-ended metal or wooden rod, and a box of Pasteur pipettes in the central area with the dye solution.

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