Column chromatography

by Marc-AndréC on March 21, 2017 - 4:24pm

Marc-André Côté

 

Column Chromatography

Introduction :

Everybody loves when their shampoo or their dryer sheets smell like roses, jasmine, or many other flowers or enjoyable scents. Except less knows how these odors are found or made. Well some of the scents are synthetic, meaning they are created in laboratory, some others are natural, commonly found in nature (flowers for example). When synthetic scents are produced, the technicians mix A and B together in order to get C, our scent. Although, other unwanted products can be created, and we don’t necessarily want these so in this case, it is a good idea to perform a column chromatography separation. Similarly for the synthetic way, using a flower to obtain a specific aroma requires a separation because it can contain many other compounds for the same flower.

In this process description, column chromatography will be explained, according to Organic Chemistry and Chromatography’s theory. Column chromatography is one of the most used techniques for purifying and separating compounds among many laboratories. Using this technique leads to separate. For instance, one toxic compound from another safe one. How to install the column, to prepare different eluents, the silica gel and the chosen compounds, to elute the solution called analyte through the column, to collect fractions and gather the similar ones together and to evaporate the solvent will be covered.

Material/Components Needed:

To achieve a chromatographic separation, one will need either a chromatography column or a burette, beakers, test tubes, funnels and vacuum tubes. Those will be needed to prepare the column and the compound. One will also have to bring chemical compounds to dissolve the mixture and to help it make its way down the column called ‘’to elute’’. The liquid helping our compound to elute is called the ‘’eluent’’. Finally, the technician will need a rotary evaporator, which is an instrument that evaporates organic liquids using a vacuum and a hot bath. If the compounds eluted are colorless, the technician will need an ultraviolet/Visible lamp. With its help, the uncolored compounds will be glowing. An analytical balance and some thin layer chromatography sheets will also be needed. Thin layer chromatography will be explained in due time.

Step 1: Preparing the Eluents, Mixture and Silica Gel

First of all, a well-prepared technician has researched some properties of the mixture to be separated. The most important of all for the separation is the polarity of the molecules separated. Consider polarity as magnets and apolarity as hairs. Also, be aware that a single molecule can have both hairs and magnets properties. The silica gel is considered a magnet and most compounds are hairs and magnets.

 

Another thing to know is that every molecules are different from one another, in a way that their hairs and magnets’ strengths are different. By changing the strengths of these properties of the eluent during the process, one can achieve a successful separation. Here is a description of the principle.

(Figure 1)

 

           

Relatively to figure 1, compound A has magnets and hairs, so it has a tendency to stay with the silica gel. On the other hand, compound B is only composed of hairs, so it has a preference to stay with the eluent (hairs also). This is why compound B will be more likely to exit the column first.

Once the research has been done, the technician can chose his eluents. Afterwards, the eluents are prepared. There can be as many eluents as needed. The mixture is dissolved in the first one to be used (generally the less polar). The technician also dissolves the silica gel, and a kind of goo is created. These solutions can be left in beakers until the column is up.

To install the column, one needs a laboratory stand along with 2 pairs of clamps. Put the column straight up with the clamps. If the column is not as straight as possible, the final product can be less pure, so it is a really important step. Once the column is clamped up straight, the technician has to put a glass wool inside the column at the very end (before the tap). Now, the goo of silica gel can be poured slowly inside the column, using a funnel. On top of the gel comes a thin layer of sand. The technician has to ensure that the gel is always damp, otherwise he will have to start over. Thereafter, he will need to gently hit the sides of the column using vacuum tubes, to make sure the gel is even. Before the elution can start, the technician has to low down the level of eluent down, about 0.5cm above the layer of sand. The column is now ready to be used.

 

Step 2: The Elution

Now that the column is ready, the technician can proceed to the elution. He simply needs to add the dissolved analytes gently on top of the sand, as to not disturb the straightness of the gel. Once, this is done, he adds as much eluent as required, in order to fill up the whole column. Then the tap is opened so the elution can begin. IF the compounds are colored, he will see the separation live and when the first color comes down to the tap, the technician can start harvesting the drops into test tubes. Refer to figure 2 as an example of a separation.

(Figure 2)

 

Step 3: Pricking the Test Tubes and Gathering the Same Compounds into One Single ‘’Balloon’’:

When the elution is done, the technician will have to prick each test tube in then on a thin layer chromatography sheet. This sheet will tell him what test tubes contain the same compounds, using a capillary tube. See figure 3.

(Figure 3)

Step 4 : Evaporating the Solvent:

Following the image above, the technician would take the three different test tubes, as they contain three different compounds, and put them each in a balloon. He would then put each balloon on a rotary evaporator, to make sure no solvent is present in the purified compound. Afterwards, the technician has his three compounds well-separated. Further tests can be accomplished to certify the purity of the separated compounds.

Conclusion:

Do not forget to be a good laboratory technician, and to always research everything that has to be known about your compounds to be separated. Do not fear the colorless ones, as they can be seen using an ultraviolet/visible lamp. Furthermore, take your time to ensure your column is straight up enough, as it can lead to starting over the preparation of the column. If the technician follows the steps correctly, he will have a 100% purif

Comments

Good article. The procedures were well explained and were easily understandable for non-scientific readers. However, the conclusion seems to be not completed. Beside that great work.

About the author

I'm currently finishing my collegial diploma in analytical chemistry at Cegep de Jonquière. I applied to Univerité de Sherbrooke, and I was accepted so I will continue studying chemistry for a certain time.