カラム ・ クロマトグラフィ
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Overview
ソース: 博士ジミー フランコ – メリマック大学講座
カラムクロマトグラフィーは、化合物を浄化するための最も有用な技術のひとつです。充填したカラム固定相と移動相列を通過するこのテクニックを利用します。この手法は、facilely 分離する分子を許可する化合物の極性の違いを利用します。1カラム ・ クロマトグラフィ用の 2 つの最も一般的な固定相がシリカゲル (SiO2) とアルミナ (Al2O3)、最もよく使用される移動相溶媒をされています。2移動相の選択 solvent(s) は、精製されている分子の極性に依存しています。通常より極性化合物が固定相での分子の通過を容易にするためより多くの極性溶媒を必要です。浄化プロセスが完了したら、溶媒は分離材料を行うロータリーエバポレーターを使用して収集した分数から削除できます。
Principles
Procedure
Results
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.
Applications and Summary
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
References
- Mayo, D. W.; Pike, R. M.; Forbes, D. C., Microscale organic laboratory : with multistep and multiscale syntheses. 5th ed.; J. Wiley & Sons: Hoboken, NJ; p xxi, 681 p (2011).
- Armarego, W. L. F.; Chai, C. L. L., Purification of laboratory chemicals. 5th ed.; Butterworth-Heinemann: Amsterdam; Boston; p xv, 609 p (2003).
- Silverman, R. B.; Holladay, M. W., The organic chemistry of drug design and drug action. Third edition / ed.; Elsevier/AP, Academic Press, is an imprint of Elsevier: Amsterdam ; Boston; p xviii, 517 pages (2014).
- Mortensen, D. S.; Perrin-Ninkovic, S. M.; Shevlin, G.; Elsner, J.; Zhao, J.; Whitefield, B. et. al. Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115. Journal of Medicinal Chemistry (2015).
- Davies, D. R.; Johnson, T. M., Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography. Journal of Chemical Education,84 (2), 318 (2007).
- Taber, D. F.; Hoerrner, R. S., Column chromatography: Isolation of caffeine. Journal of Chemical Education, 68 (1), 73 (1991).
Transcript
Column chromatography is a versatile purification method used to separate compounds in a solution. A solution mixture is carried by a solvent through a column containing an adsorbent solid, called the stationary phase. The combined solvent and sample mixture is called the mobile phase.
Molecules in the mobile phase travel through the column at different rates based on their chemical properties and their affinity for the stationary phase. Thus, each compound exits the column at a different time. Once the compounds have been separated and purified they can be further processed or are ready for distribution. This video will introduce the basics of column chromatography, then demonstrate the technique with the purification of organic compounds.
In column chromatography, molecules reversibly adsorb to the stationary phase as they flow through the column, thereby slowing their progress. Compounds that interact weakly with the stationary phase are faster to exit the column, or elute. Compounds that interact strongly with the stationary phase are slower to elute. The stationary phase is an adsorbent powder or gel such as silica gel or alumina. Silica gel and alumina are highly polar so they interact strongly with polar compounds and solvents, and weakly with nonpolar molecules. The stationary phase is loaded into the column as a slurry with the solvent and is then packed by flowing solvent through the stationary phase. When properly packed, the stationary phase is homogeneous from top to bottom and contains no air bubbles or dry patches, as uneven flow caused by these irregularities interferes with the separation of compounds. The solvent, or eluent, is typically an organic solvent supplied from a reservoir. In general, nonpolar solvents only elute nonpolar compounds, whereas polar solvents elute both polar and nonpolar compounds. If a mixture contains compounds of significantly different polarities, a series of increasingly polar solvents may be used to elute all of the compounds of interest. The mobile phase flow rate is usually controlled by a stopcock at the bottom of the column. Pauses in flow are kept to a minimum to avoid diffusion of the compounds. The mobile phase leaving the column, called the eluate, is collected in fractions to preserve the separation of compounds. Now that you understand the principles of column chromatography, let’s go through a procedure for the purification of a mixture of organic compounds.
To begin the procedure, obtain the equipment as noted in the text. Weigh a round-bottomed flask for each compound to be isolated and record the mass. Next, weigh the sample and dissolve it in the minimum volume of solvent needed. The appropriate solvent should be predetermined using thin layer chromatography. The Rf value should be between 0.2–0.3. Then, determine the amount of silica gel required for the stationary phase based on the dry weight of the sample and the difference in migration distance of the compounds of interest based on the TLC pre-screening. Pour the appropriate amount of silica gel into an Erlenmeyer flask. Add the solvent to the silica gel until the slurry is translucent and moves freely when the flask is swirled. Next, select a column large enough that the silica gel will fill it halfway. If the column does not have a glass frit, place glass wool into the column and firmly press it to the bottom with a long rod. Cover the glass wool with about 2 cm of sand to prevent silica from passing through the glass wool. In a fume hood clamp the column to a ring stand, allowing sufficient space below to accommodate the test tubes.
Place a funnel into the column and ensure that the stopcock is closed. Pour the slurry into the column, gently tapping the sides as the slurry settles to exclude air bubbles. Rinse the funnel, flask, and walls of the column with solvent to transfer all of the gel into the column.
Place an Erlenmeyer flask under the column. Open the stopcock and allow the solvent to drain into the flask until the solvent level is just above the silica gel, and then close the stopcock. Pour about 2 cm of sand onto the gel. Gently rinse down any sand stuck to the sides of the column with solvent. Drain the solvent as needed so the sand is mostly dry, but the silica remains completely covered.
To start the separation, add the sample to the column without disturbing the sand. Use small portions of solvent to rinse down any sample adhering to the column walls and to rinse out the sample container. Carefully drain the solvent until the level is just above the silica. Then, with a pipette, gently add 4–5 mL of solvent without disturbing the sand layer. Place a funnel into the column and slowly fill with solvent. Remove the flask and replace with a labeled test tube. With the first test tube in place, open the stopcock and collect the eluate until the test tube is nearly full.
Continue collecting fractions until all desired compounds have been eluted, proceeding sequentially through the labeled test tubes. When finished, close the stopcock.
For each compound isolated, combine the pure fractions in a pre-weighed round-bottomed flask. Remove the solvent from the flask on a rotary evaporator and then weigh the round-bottomed flask containing the dry compound. For more information, see this collection’s video on rotary evaporation.
This sample contained a mixture of tetraphenylporphyrin, or TPP, and fluorenone. The dark reddish-purple TPP was eluted first, followed by the yellow fluorenone. The purity of each isolated compound was confirmed by NMR spectroscopy.
Column chromatography is used in purification and analysis in a variety of scientific fields.
High performance liquid chromatography, or HPLC, is a form of column chromatography that provides excellent separation between compounds and can incorporate specialized detectors such as a radiation detector for radiolabeled molecules. Using HPLC, a radiolabeled phospholipid can easily be isolated from a mixture of many others even if it makes up a small percent of the mixture. This information can help elucidate the production, regulation, and functions of many important biomolecules.
Flash chromatography is a variant of column chromatography in which the mobile phase moves through the column under air or gas pressure rather than by gravity flow alone.
This creates a faster flow rate, minimizing diffusion for better separation. The desired compound is collected in a few pure, concentrated fractions, as shown with thin layer chromatography, resulting in excellent post-purification yield and purity.
The usual column apparatus is not appropriate for separating small volumes, but some mixtures are not compatible with specialized techniques such as HPLC. Small-scale purification is performed with glass pipette columns, with a pipette bulb used for small-scale flash chromatography. This is particularly useful when preparing a sample for specialized purification techniques or as a final step following large-scale purification.
You’ve just watched JoVE’s introduction to column chromatography. You should now be familiar with the principles of column chromatography, a procedure for silica gel column chromatography, and some applications of the technique.
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