ORGANIC cHEMISTRY - Iv

Experiment 4

Aim of the Experiment

To separate mixture of organic compound using column chromatography.

Principle

In chemistry, Column chromatography is a technique which is used to separate a single chemical compound from a mixture dissolved in a fluid. It separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allow them to get separated in fractions. This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments. This method is a type of adsorption chromatography technique.

When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates. The components with lower adsorption and affinity to stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase. The components that move fast are removed first whereas the components that move slowly are eluted out last.

The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as:

R_f = the distance travelled by solute/ the distance travelled by the solvent

R_f is the retention factor.

Materials required

glass column equipped with stopper, cotton, solvents, silica/alumina, glass rod, mixture to be separated etc

Procedure

PREPARING A COLUMN

Take a glass column and place a small cotton plug at the part where it narrows into a capillary tube. Make sure the cotton plug is tight enough to hold the solid phase, but not too tight because that will result in a much slower rate of solvent flow. Also have a small rubber stopper or Teflon cap liner ready to control the solvent flow in the column. Separately, make a slurry consisting of the solid support (alumina or silica gel) and the solvent to be used first. Secure the column vertically with a clamp, and place an empty beaker at the bottom to collect the solvent that goes through the column. Carefully pour small amounts of the slurry into the column with a dropper until the height of slurry that settles inside the column is about 1 – 1.5 inches. During this time the solvent should be allowed to flow freely through the column and collected in the beaker at the bottom. However, the solvent level in the column should not be allowed to fall below the top of the slurry. If you see that this is about to happen, interrupt the solvent flow by blocking the bottom of the column and add more solvent or slurry. In other words, the slurry inside the column should never be allowed to dry out. If this happens it may create cracks and unevenness in the solid phase, which will decrease the efficiency of the separation.

RUNNING A CHROMATOGRAPHIC COLUMN

The sample to be separated is loaded in solution in a suitable solvent, preferably as concentrated as possible. Dilute samples tend to separate less efficiently.

1. When preparing the column as described above, also prepare a series of small test tubes for collecting the different fractions that will come out of the column as the separation proceeds. Also have masking tape and a marker to label the tubes, and of course a test tube rack.

2. After preparing the column as described above, allow the solvent to flow until its level comes very close to the top of the solid support, but not below it.

3. Load the sample solution in small amounts, allowing the solvent to flow after each load until it comes very close to the top of the solid support, but not below it. By doing this you never have large amounts of solvent present above the solid support.

4. After the sample is loaded, add the first eluting solvent (usually a low polarity one) in small amounts, just as you did with the sample. Once again, do not allow the solvent level to fall below the top of the solid support. By doing this you allow for complete adsorption of the sample onto the solid support.

5. Now you’re ready to proceed with the separation. Fill the top of the column with eluting solvent, and allow it to flow. If your sample is colored, you will see the sample move and separate into “bands” as the components of the mixture begin to separate.

6. As the solvent level reaches the top of the solid support, add more solvent to keep it from falling below the top of the solid.

7. As the solvent is flowing and you see the bands move down the column, be ready to collect different bands into separate test tubes. These collections are called fractions. If it is not clear where the band starts, play it safe by starting the collection before the band reaches the bottom of the pipette. It’s better to have an excess of solvent in your fraction than to lose material.

8. The less polar components of the mixture will come out first. The more polar components might lag far behind, moving very slowly. Once the less polar fractions come out, you can gradually increase the polarity of the eluting solvent to speed up the movement of the more polar bands. This is done by gradually increasing the percent of a more polar solvent from zero to whatever it takes to effect the separation. For example if your initial solvent was hexane, you can increase the polarity of the solvent by switching to 20% acetone / 80% hexane.

9. Continue this process until you’re satisfied that all the components of the mixture have come out with good separation. Keep in mind that abrupt changes in solvent polarity might cause poor separations.

Result:

Compound A:----------------

Compound B:----------------

Reference Material

Demo videos for column chromatography

www.youtube.com/watch?v=Zke6xGhbbho&t=123s

www.youtube.com/watch?v=yig3QCfBTzc

Questions

1. Principle of column chromatography?

2. How to get better column separation?

3. Difference between column chromatography and HPLC (High Pressure Column Chromatography)?

4. Name different stationary phase commonly used for column chromatography?