Vidéo: Column Chromatography: Principle, Separation of Compounds from a Mixture

Column Chromatography

Vue d'ensemble

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.

Procédure

1. Silica Gel Slurry

Pour the silica gel into an Erlenmeyer flask. The weight of the packing material should be roughly 50x that of the sample being separated. If the compounds being separated have very similar R_f values, then it may require using a larger amount of silica per sample, which is the case in this example.
1. Place 10 g of silica in the Erlenmeyer flask, since 50 mg of sample (45 mg of fluorenone and 5 mg of tetraphenylporphyrin) are being isolated.

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Résultats

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

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Applications et Résumé

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

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).

1. Silica Gel Slurry

Pour the silica gel into an Erlenmeyer flask. The weight of the packing material should be roughly 50x that of the sample being separated. If the compounds being separated have very similar R_f values, then it may require using a larger amount of silica per sample, which is the case in this example.
1. Place 10 g of silica in the Erlenmeyer flask, since 50 mg of sample (45 mg of fluorenone and 5 mg of tetraphenylporphyrin) are being isolated.
Add the solvent system (hexane/dichloromethane, 70%: 30%) to the Erlenmeyer flask containing the silica gel. Add enough solvent to ensure that all of the silica gel is well solvated. The silica will not dissolve, but the mixture will be visually noticeable when solvated. Once the solvent has been added swirl the Erlenmeyer flask to ensure that all of the silica is well solvated.

2. Preparation of the Column

Select the appropriately sized column. Typically the column should be filled about half way with silica gel slurry. The larger the sample being purified, the larger the column required.
Plug the bottom of the column with a piece of glass wool. Using a long rod, make sure the wool is firmly lodged in the bottom of the column just above the stopcock.
Once the wool is firmly in place, apply a thin layer of sand over the glass wool.
Note: If the column is equipped with a glass frit above the stopcock, this step should be omitted.
Clamp the column in the vertical position to a ring stand.
Using a funnel, gently pour the prepared slurry of silica gel into the column. You may need to add additional solvent to transfer the slurry from the Erlenmeyer flask to the column. Using a pipette, wash down any silica gel that sticks to the sides of the column.
1. As the silica gel is settling in the column, gently tap the sides of the column to ensure that the silica gel packs tightly and excludes any air bubbles.
Open the stopcock and allow solvent to drain into a clean Erlenmeyer flask until just before the silica gel and the solvent front meet. The silica gel should never go dry until the procedure is complete.
Place a thin layer of sand on top of the silica gel (Figure 1). Using a pipette, wash down any sand that may have stuck to the sides of the column.
Drain any additional solvent until the sand is dry, but not down to the silica gel layer.

Figure 1. The proper setup for a column chromatography experiment prior to the addition of the sample.

3. Adding the Sample to the Column

Dissolve the sample in the smallest amount of solvent possible (using the same solvent that was used to make the silica gel slurry).
Using a pipette, gently add the sample to the top of the column.
Once the sample has been applied to the top of the column, open the stopcock and allow the solvent to drain through the sand layer but not the silica gel layer. Use a very small amount of solvent to wash down any sample that may have clung to the sides of the column. Drain this additional solvent through the sand layer as well.

4. Eluting the Sample through the Column

Using a pipette, very gently add 4–5 mL of solvent in such a manner that does not disturb the sand layer.
Place a funnel at the top of the column and very slowly and gently fill the remainder of the column with solvent.
Open the stopcock and allow the solvent to drain through the column.
Begin collecting the mobile phase as it drains from the column into test tubes.
1. Test tubes should be placed in a test tube rack in a sequential manner.
Add additional solvent to the top of the column as needed until all the desired compounds have eluted from the column.

5. Recovering the Constituents

If the compounds are colored, then they can be visually identified. However, if the compounds are colorless, they will have to be identified using ulta-visible (UV) light (if the compounds contain conjugation) or with the appropriate stain. The purity of the compounds can be verified using thin layer chromatography.
Identify the test tubes that contain the desired compound(s).
Merge all of the fractions that contain the desired isolated compound(s) into a pre-weighed round-bottom (RB) flask. Do this for each compound being isolated.
Evaporate the solvent by placing the RB flask on the rotary evaporator.
Once all of the solvent has been removed, weigh the RB with the dried product and subtract the initial weight of the RB to obtain a yield.

Passer à...

0:00

Overview

1:01

Principles of Column Chromatography

3:06

Column and Sample Preparation

5:23

Separation of Compounds

6:24

Results

7:10

Applications

8:58

Summary

Vidéos de cette collection:

article

Now Playing

Column Chromatography

Organic Chemistry

357.9K Vues

article

Introduction à la catalyse

Organic Chemistry

34.0K Vues

article

Montage d'un chauffage à reflux

Organic Chemistry

165.5K Vues

article

Réaliser des réactions en dessous de la température ambiante

Organic Chemistry

70.2K Vues

article

Transfert de solvants via une rampe à vide (ligne Schlenk)

Organic Chemistry

41.4K Vues

article

Dégazage des liquides par la technique "cycle geler-pomper-dégeler"

Organic Chemistry

55.8K Vues

article

Préparation de réactifs anhydres et équipement

Organic Chemistry

79.0K Vues

article

Purification des composés par recristallisation

Organic Chemistry

703.0K Vues

article

Séparation des mélanges par précipitation

Organic Chemistry

156.9K Vues

article

Extraction solide-liquide

Organic Chemistry

236.2K Vues

article

Utilisation d'un évaporateur rotatif (ou Rotovap) pour éliminer un solvant

Organic Chemistry

211.8K Vues

article

Distillation fractionnée

Organic Chemistry

331.8K Vues

article

Préparation de cristaux pour analyse par diffraction des rayons X

Organic Chemistry

32.2K Vues

article

Performing 1D Thin Layer Chromatography

Organic Chemistry

287.6K Vues

article

Spectroscopie à résonance magnétique nucléaire (RMN)

Organic Chemistry

246.3K Vues

Copyright © 2025 MyJoVE Corporation. Tous droits réservés.