Column Chromatography is another common and useful separation technique in organic
chemistry. This separation method involves the same principles as TLC, but can be applied to separate larger quantities than TLC. Column chromatography can be used on both a large and small scale. The applications of this technique are wide reaching and cross many disciplines including biology, biochemistry, microbiology and medicine. Many common antibiotics are purified by column chromatography.
To understand to uses of this separation technique, we can use the last experiment as an
example. In the TLC experiment, we separated and analyzed the different components that makeup over-the-counter painkillers. The technique of TLC was useful in determining the type and number of ingredients in the mixture, but it was not helpful for collecting the separated components. We could only separate and visualize the spots. If we needed to collect the separated materials, column chromatography could be used. We could load 100 mg of a crushed Anacin tablet on a column made up of a silica stationary phase and separate the aspirin from the caffeine and collect each of these compounds in separate beakers. Column chromatography allows us to separate and collect the compounds individually. In this experiment, Column Chromatography
(abbreviated CC) will be used to separate the starting material from the product in the oxidation of fluorene to flourenone and TLC will be used to monitor the effectiveness of this separation.
Choosing a Stationary Phase
As with TLC, alumina and silica are the two most popular stationary phases in column
chromatography. For these common phases, the partitioning works in an analogous manner. The more polar sample will be retained on the stationary phase longer. Thus the least polar compound will elute from the column first, followed by each compound in order of increasing polarity.
Although the interactions between the mobile and stationary phase are based on the same
principles for CC and TLC, be careful when predicting the order of elution. Since the direction of the solvent flow in TLC moves up and in CC the solvent flows down, it appears that the order is “upside-down”. In TLC the more polar molecules will have lower Rf values, but in CC they will be retained longer on the column.Remember this when considering the polarities of the stationary phase as well as the polarity of the compounds being separated when predicting the order of elution.
Stationary phases for CC can come in a variety of sizes, activities, acidic and basic variations for both alumina and silica. The types of stationary phase chosen are determined experimentally, or often based on results from a previous TLC experiment. The type of adsorbent, the size of the column, the polarity of the mobile phase as well as the rate of elution all affect the separation. These conditions can be manipulated to get the best separation for your mixture.
Solvent systems for use as mobile phases in CC can be determined from previous TLC
experiments, the literature, or experimentally. Normally, a separation will begin by using
nonpolar or low polarity solvent, allowing the compounds to adsorb to the stationary phase, then SLOWLY switching the polarity of the solvent to desorb the compounds and allow them to travel with the mobile phase. The polarity of the solvents should be changed gradually. On a macroscale, the mixing of two solvents can create heat and crack the column leading to a poor separation.
Some typical solvent combinations are ligroin-dichloromethane, hexane-ethyl acetate and hexane-toluene. Often an experimentally determined ratio of these solvents can sufficiently separate most compounds. Solvents such as methanol and water are normally not used because they can destroy the integrity of the stationary phase by dissolving some of the silica gel.
Columns can be as thin as a pencil to a diameter of several feet in industrial processes. They can separate milligram to kilogram quantities of materials. In this experiment, we will be separating a mixture of approximately 50 mg, so a small column can be used. Figure 8.1 shows the typical set-up we will be using during this experiment. It is essential to have several clean tared Erlenmeyer flasks, reaction tubes, beakers, test tubes or vials available to collect the solvent and compounds as they elute. Once you have the general set-up prepared, you can move on to packing the stationary phase in the column.
Packing the Column
There are several acceptable methods when packing a column. These include dry packing
(there are two versions of dry packing discussed here) and the slurry method. The slurry method normally achieves the best packing results, but there are several occasions when the dry packing method works just as well if not better.
Dry packing is the method of choice for a microscale column. Begin by filling the column with a nonpolar solvent. Slowly add the powdered alumina or silica while gently tapping the side of the column with a pencil. The solid should “float” to the bottom of the column. Try to pack the column as evenly as possible; cracks, air bubbles, and channels will lead to a poor separation.
For the second dry pack method, the stationary phase is deposited in the column before the solvent. In this case fill the column to the intended height with the stationary phase and then slowly add the nonpolar solvent. The solvent should be added slowly as to avoid uneven channeling. This method is typically used with alumina only, since silica gel expands and does not pack well with this dry method.
The slurry method is often used for macroscale separations. Combine the solid stationary
phase with a small amount of nonpolar solvent in a beaker. Thoroughly mix the two until a
consistent paste is formed, but is still capable of flowing. Pour this homogeneous mixture into the column as carefully as possible using a spatula to scrape out the solid as you pour the liquid. The slurry method normally gives the best column packing, but is also a more difficult technique to master. Whether the dry or slurry method is chosen, the most important aspect of packing the column is creating an evenly distributed and packed stationary phase. As mentioned, cracks, air bubbles and channeling will lead to a poor separation.
Once the column is loaded, open the stopcock and allow the solvent level to drop to the top of the packing, but do not allow the solvent layer to go below this point. Allowing this solvent level to go below the stationary phase, (known as letting the column to “run dry,”) should always be avoided. Since it allows air bubbles and channel formation to occur leading to a poor separation.
Adding the Sample
Once the packing is complete, the sample can be loaded directly to the top of the column.
Normally, a minimum amount of a polar solvent, 5-10 drops, is used to dissolve the mixture. The solution is then carefully added to the top of the column using a pipet without disrupting the flat top surface of the column. A thin horizontal band of sample is best for an optimal separation. After the sample is loaded, a small layer of white sand is added to the top of the column. This will help to keep the top of the column level when adding solvent eluent. Once the mixture is added and the protective layer of sand is in place, continuously add the solvent eluent while collecting small fractions at the bottom of the column. Using a pipet to add the first bit of solvent on top of the packing, sample, and sand will minimize disturbance of the column and diluting the sample. Collecting small fractions (1-3 mL) is important to the success of your column separation.
Fractions that are too small can always be pooled together; however, if the collected fractions are too large, you may get more than one compound in any particular fraction. If this occurs, the only way to complete the separation is to redo the chromatography. Since column chromatography is time consuming, collecting large fractions is discouraged.
Monitoring the Column
If the mixture to be separated contains colored compounds, then monitoring the column is very simple. The colored bands will move down the column along with the solvent and as they approach the end of the column, collect the colors in individual containers. Use the color as your guide. However, most organic molecules are colorless. In this case, the reaction must be monitored by TLC. Spot each fraction on a TLC plate. Four or five fractions can be spotted on a single TLC plate. Develop the plate and use the observed spot or spots to determine which compound is in each of the collected fractions. Spotting some of the starting material or the product (if available) on the TLC plate as a standard will help in the identification.
Isolating the Separated Compounds
Once you believe all the materials have been removed from the column, the colors of the
materials or TLC results should indicate which fractions contain the compound(s) you are
interested in isolating. Combine the like or same fractions and evaporate the solvent. The pure separated compound will be left behind. Recrystallization may be used to further purify a solid product. However, on a milligram scale, there is usually not enough material to do this.
Column chromatography is often very time consuming. Allowing the solvent to elute through the column one drop at a time takes patience. One method to speed up the process is to use Flash Chromatography. This method uses a pressure of about 10 psi of air or nitrogen to force the mobile phase through the column. Because the rate of the mobile phase is increased, in general, this method gives a poorer separation. However, by using a finer grade of alumina or silica, flash chromatography can be used to increase the speed without lowering the quality of the separation.
HPLC (High Performance Liquid Chromatography) is a variation on the traditional liquid
chromatographic methods. High pressure pumps are used to force solvent through a tightly packed column connected to a variety of different types of very sensitive detectors. Modern HPLC is used extensively in biochemistry to separate cellular components such as proteins, lipids, and nucleic acids. Mixtures of these types require aqueous mobile phases such as methanol-water or acetonitrile-water and these liquids do not work well on normal silica or alumina stationary phases. Instead of these polar phases, very nonpolar ones, called “reverse-phase” packing are used. These are manufactured by bonding lots of hydrocarbon molecules to the surfaces of a silica gel particles so that the silica gel is like a very nonpolar “grease ball.” In this situation, the order of elution will be exactly opposite the behavior on an alumina or silica column. On a reverse-phase column, the more nonpolar materials will adhere to the stationary phase (or like material) longer
and the polar compounds will elute first.
Many compounds can be separated using typical chromatographic stationary phases and
solvents. These separations depend on the difference in polarity of the molecules to be separated. However, how would you separate two compounds with the identical polarity, such as enantiomers? This separation technique is of great importance in the pharmaceutical industry where the FDA controls the amounts of impurities, including enantiomers, in prescribed drugs. For example, Thalidomide, a drug administered in the 60’s has two enantiomers. This drug was used as a sedative and an anti-depressant, but was found to cause abnormalities in the fetuses of pregnant
women. Although this drug was pulled from the market due to the resulting birth defects, there is recent literature that suggests that only one of the enantiomers caused the defects. If the enantiomers could be completely separated, Thalidomide might be used as an FDA approved drug and be helpful to people today.
Chiral stationary phases can be used to separate enantiomers. By giving the stationary phase a “handedness,” one enantiomer will be specifically retained on the column. These columns are very expensive and specific to the particular type of separation, but have led to great achievements in separation science.
There are many, many different types of chromatographic methods including gel electrophoresis and size exclusion chromatography that have not been discussed here. Hopefully, with the basic chromatographic background provided, you can apply this knowledge to the many different types of chromatography used in many different professions today.