Column chromatography

Column Chromatography:

In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.
The mixture to be analyzed by column chromatrography is applied to the top of the column. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as the solvent drips from the bottom of the column.
Column chromatography is separated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, it is called flash chromatography, a "state of the art" method currently used in organic chemistry research laboratories The term "flash chromatography" was coined by Professor W. Clark Still because it can be done in a “flash."

The Adsorbent:

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the organic chemist for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles.
Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400 mesh for flash columns.
Alumina is used more frequently in column chromatography than it is in TLC. Alumina is quite sensitive to the amount of water which is bound to it: the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity I and deactivated with water before use according to specific procedures. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.
Silica gel and alumina are the only column chromatography adsorbents used in the CU organic chemistry teaching labs; please refer to the references for information on other column chromatography adsorbents.

The Solvent:

The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the successful application of column chromatography as a separation technique. TLC is generally used to determine the system for a column chromatography separation. The choice of a solvent for the elution of compounds by column chromatography is covered in the Chromatography Overview section.
Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent is first used to elute a less-polar compound. Once the less-polar compound is off the column, a more-polar solvent is added to the column to elute the more-polar compound.

Interactions of the Compound and the Adsorbent:

Compounds interact with the silica or alumina largely due to polar interactions. These interactions are discussed in the section on TLC.

Analysis of Column Eluants:

If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. More commonly, the compounds to be isolated from column chromatography are colorless. In this case, small fractions of the eluent are collected sequentially in labeled tubes and the composition of each fractions is analyzed by thin layer chromatography. (Other methods of analysis are available; this is the most common method and the one used in the organic chemistry teaching labs.)

Procedures:

Columns for chromatography can be small or big, according to the amount of material which needs to be loaded onto the column. Pictured below are three glass columns, two of which are used in the organic chemistry teaching labs at CU.
The "column" on the far left in the photo is actually a Pasteur pipet. This size of column is suitable for 10-125 mg of material. The middle column is a 10 mL disposable glass pipet. You can load about a gram of material on this size column. The column on the right would be used for many grams of material.
Of the three columns pictured, only the column on the right is actually manufactured as a chromatography column. Note the stopcock at the bottom of the column. This is to control the flow of solvent through the column, important for gravity column chromatography applications.
The middle column is used for gravity column chromatography in a few of the chemistry majors' laboratory courses (chem 3361 and 3381). Note the piece of flexible tubing which has been added to the bottom of the column.To control the flow of solvent, a pinch clamp would be placed on the flexible tubing at the bottom.
The Pasteur pipet column is used for microscale gravity and microscale flash chromatrography procedures; these procedures (usually) do not require a means of control of gravity-induced solvent flow through the column.
Much larger chromatography columns are available than the one on the right. The size employed depends on the amount of material which needs to be separated. Large-scale flash columns look like this column but have a standard taper connection at the top so they can be connected to a source of pressurized air.
In the Organic Chemistry teaching labs at CU, the most frequently used column is the Pasteur pipet. They work well in microscale flash column chromatography procedures because a pipet bulb fits conveniently on top of them to serve as a source of pressurized air (when you press on the bulb!). Microscale procedures are used at CU Boulder whenever feasible to cut down on waste chemical production.
Here is a picture of a packed column of the type on the right

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Procedure for Gravity Column Chromatography:

Gravity columns are not used in any of the non-major organic lab courses at CU Boulder (chem 3321/3341). The majors (chem 3361/3381) do use this type of chromatography. Gravity columns are a lot slower to run than microscale flash columns. They also are more difficult to set up or "pack" with adsorbent.

Procedure for Microscale Flash Column Chromatography:

Microscale flash chromatography is the method used almost solely in the organic chemistry teaching labs because it is both easy and environmentally friendly. The method is only limited by the fact that it can separate only small amounts of sample. It works best for 25 mg amounts, although we have pushed it to separate 125 mg mixtures if the TLC Rf's of the components of the mixture differ by at least 0.20.

Refrances:

1. http://en.wikipedia.org/wiki/Column_chromatography
2. http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html
3. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4TMYG2D
4. http://fa.wikipedia.org/wiki
5. http://orgchem.colorado.edu/hndbksupport/dist/distsetup/vacdist.html
6. http://fa.wikipedia.org/wiki/سانتریÙ