Source: Laboratory of Dr. Jimmy Franco - Merrimack College
Column chromatography is one of the most useful techniques for purifying compounds. This technique utilizes a stationary phase, which is packed in a column, and a mobile phase that passes through the column. This technique exploits the differences in polarity between compounds, allowing the molecules to be facilely separated.1 The two most common stationary phases for column chromatography are silica gel (SiO2) and alumina (Al2O3), with the most commonly used mobile phases being organic solvents.2 The solvent(s) chosen for the mobile phase are dependent on the polarity of the molecules being purified. Typically more polar compounds require more polar solvents in order to facilitate the passage of the molecules through the stationary phase. Once the purification process has been completed the solvent can be removed from the collected fractions using a rotary evaporator to yield the isolated material.
The sample mixture is placed on the top of the column and absorbed onto the top of the stationary phase. Subsequently, the mobile phase is applied to the column and used to elute the mixture through the stationary phase. Column chromatography exploits a molecule's polarity to separate the compounds. The difference in polarity leads to variances in the rate at which the molecules travel through the column, which effectively separates the compounds from one another. The mobile phase is collected in small fractions in test tubes as it elutes off the column, thus allowing for the isolation and purification of the compounds. Lastly, the solvent is removed using a rotary evaporator to yield the isolated compound(s).
Column chromatography's versatility and convenience has made it one of the most widely used techniques for purifying compounds. Unlike recrystallization (another commonly used purification technique) compounds purified with column chromatography do not have to be solid. Column chromatography is also capable of isolating a number of compounds from a mixture. Another advantage of column chromatograph is that very little needs to be known about the compound's physical properties in order to use this purification method, making this technique very valuable when synthesizing or isolating novel compounds, in which little is known about the compound(s).
Solvent
The rate at which a compound traverses through the column is highly dependent on the mobile phase being utilized. Typically, the more polar the solvent the faster the compounds will pass through the column. Polar solvents have a greater affinity for the solid phase, limiting the interactions between the compound(s) and the solid phase, allowing the compounds to elute more rapidly. Caution must be taken to ensure that the solvent system chosen for the column chromatography has the appropriate polarity to create separation between the compounds in the mixture. The solvent choice is crucial to successful separation using column chromatography. To identify an optimal solvent system, a series of thin layer chromatography (TLC) experiments should be conducted prior to performing the column chromatography experiment. In some case it may be necessary to use a binary solvent system.
Selecting a Solvent System
1. Silica Gel Slurry
2. Preparation of the Column
Figure 1. The proper setup for a column chromatography experiment prior to the addition of the sample.
3. Adding the Sample to the Column
4. Eluting the Sample through the Column
5. Recovering the Constituents
The sample containing a mixture of tetraphenylporphyrin (TPP, 5 mg) and fluorenone (45 mg) has been successfully separated and each compound has been isolated. The TPP eluted first off the column as a dark purple-reddish band and the fluorenone subsequently eluted off the column as a yellow band (Figure 2). The eluted fractions were collected in test tubes and identified by their distinctive colors (Figure 3). The fractions containing the isolated compounds were merged into separate RBs and the solvent was removed using a rotary evaporator to afford highly pure TPP and fluorenone. The purity of the chromatographed compounds was validated by nuclear magnetic resonance (NMR) spectroscopy. Compounds can additionally be verified by melting point, but only if the melting point for the desired compound(s) has been previously determined.
Figure 2. As the compounds traverse through the stationary phase they begin to separate. In this experiment the TPP (dark purple-reddish band) travels through the column slightly faster than the fluorenone (yellow band).
Figure 3. As the compounds elute off the column they are collected in test tubes. The compounds being separated in this experiment are colored, so they can be visually identified.
Summary
Column chromatography is a convenient and versatile method for purifying compounds. This method separates compounds based on polarity. By exploiting differences in the polarity of molecules, column chromatography can facilely separate compounds by the rate at which the compounds traverse through the stationary phase of the column. One of the benefits of column chromatography (especially when compared to recrystallization) is that very little about the compounds needs be known prior to the purification process. The other advantage to using column chromatography is that it can be used to purify both solids and oils, while recrystallization can only be used to purify solids. This technique can also be used to isolate a number of compounds from a mixture.
Applications
Column chromatography is one of the most convenient and widely used methods for purifying compounds. Often, synthetic reactions will produce multiple products and column chromatography can be used to isolate each of the compounds for further examination. Column chromatography is extremely valuable when synthesizing or isolating novel compounds, as very little needs to be known about a compound and its' physical properties prior to the purification process.
The pharmaceutical industry routinely uses column chromatography to purify compounds as part of its early stage drug development process.3 Often in these preliminary stages researchers will construct libraries of compounds around a lead compound, then subsequently use column chromatography to purify the newly synthesized compounds.4 The extensive use and versatility of this purification technique has prompted educators to incorporate the technique into the undergraduate curriculum.5,6
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