Column chromatography is an essential technique used for the separation and purification of various chemical compounds. It is a powerful method that is used extensively in the pharmaceutical, biotech, and chemical industries. In this comprehensive guide, we will discuss the principles of column chromatography, its applications, and how to optimize its performance for efficient separation.

Introduction

Column chromatography is a type of liquid chromatography that involves separating and purifying a mixture of compounds using a stationary phase and a mobile phase. The stationary phase is usually a solid support, such as silica gel, while the mobile phase is a liquid solvent, such as hexane or methanol. The mixture of compounds is loaded onto the top of the column, and as the mobile phase passes through the column, the components of the mixture separate and are eluted in order of their affinity to the stationary phase.

Principles of Column Chromatography

The separation process in column chromatography is based on the principles of adsorption, partitioning, and ion exchange. Adsorption occurs when the components of the mixture adhere to the surface of the stationary phase. Partitioning occurs when the components of the mixture partition between the stationary and mobile phases based on their solubility. Ion exchange occurs when charged components of the mixture bind to the stationary phase through electrostatic interactions.

Stationary Phase

The stationary phase is a critical component of column chromatography. It is responsible for the separation and purification of the mixture. The most commonly used stationary phase is silica gel. Silica gel has a high surface area and a wide range of pore sizes, which makes it an excellent adsorbent for a broad range of compounds. Other stationary phases that are commonly used include alumina, cellulose, and ion-exchange resins.

Mobile Phase

The mobile phase is the liquid solvent that flows through the stationary phase and carries the mixture of compounds with it. The choice of the mobile phase depends on the type of compounds being separated and the properties of the stationary phase. Nonpolar solvents, such as hexane and heptane, are typically used for the separation of nonpolar compounds, while polar solvents, such as methanol and water, are used for the separation of polar compounds.

Column Preparation

The first step in column chromatography is to prepare the column. The column is typically a glass or plastic tube with a fritted disk at the bottom to support the stationary phase. The stationary phase is packed into the column by adding a slurry of the stationary phase in the mobile phase. The slurry is allowed to settle, and the excess mobile phase is drained off. The packing process is critical to the performance of the column, and it is essential to ensure that the stationary phase is evenly distributed and properly packed.

Sample Loading

Once the column is packed, the sample is loaded onto the top of the column. The sample should be dissolved in the appropriate solvent and filtered to remove any impurities. The sample is loaded onto the top of the column using a pipette or syringe. It is essential to ensure that the sample is loaded onto the column slowly and evenly to prevent channeling.

Elution

Once the sample is loaded onto the column, the elution process begins. The elution process involves passing the mobile phase through the column at a steady rate. The elution rate should be slow enough to allow for proper separation of the components but fast enough to prevent excessive band broadening. The eluate is collected in fractions, and the fractions are analyzed using various analytical techniques, such as thin-layer chromatography or HPLC.

Optimizing Column Performance

Several factors can affect the performance of a column, including the particle size of the stationary phase, the choice of mobile phase, the flow rate, and the size of the column. To optimize the performance of a column, it is essential to consider these factors carefully.

Particle Size

The particle size of the stationary phase has a significant impact on the performance of the column. Smaller particles provide a higher surface area and result in better separation of the components. However, smaller particles also lead to higher pressure drops and longer separation times. It is essential to choose the appropriate particle size based on the properties of the mixture being separated.

Choice of Mobile Phase

The choice of mobile phase also affects the performance of the column. The mobile phase should be chosen based on the properties of the mixture being separated and the stationary phase. The mobile phase should be selected to optimize the solubility of the compounds in the mixture and their affinity for the stationary phase.

Flow Rate

The flow rate of the mobile phase is critical to the performance of the column. A slow flow rate results in better separation of the components, but it also leads to longer separation times. A fast flow rate results in shorter separation times, but it can lead to poor separation and broadening of the bands. It is essential to find the appropriate flow rate to balance separation and speed.

Column Size

The size of the column also affects the performance of the separation. A longer column results in better separation of the components, but it also leads to longer separation times and higher pressure drops. A shorter column results in faster separation times but can lead to poor separation and broadening of the bands. It is essential to choose the appropriate column size based on the properties of the mixture being separated.

Conclusion

Column chromatography is a powerful technique that is widely used for the separation and purification of chemical compounds. In this comprehensive guide, we discussed the principles of column chromatography, its applications, and how to optimize its performance for efficient separation. By following the steps outlined in this guide and carefully considering the factors that affect the performance of the column, you can optimize your column chromatography and achieve efficient separation of the mixture.

Column Chromatography

In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column. 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 placed inside 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. An example of each of these adsorbents is shown below.

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 of 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 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.

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. Thin-Layer Chromatography (TLC) is generally used to determine the system for a column chromatography separation.

Often a series of increasingly polar solvent systems are used to elute a column. A less-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.

Analysis of Column Eluents

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 fraction is analyzed by TLC. (Other methods of analysis are available; this is the most common method and the one used in the organic chemistry teaching labs.)

Procedures

Column chromatography procedures

Column Chromatography Procedures

Columns for chromatography can be big or small, 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 Pasteur pipet column on the left is used for microscale gravity and microscale flash chromatrography procedures (about 10-125 mg of material); these procedures usually do not require a means of control of gravity-induced solvent flow through the column. 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 the bulb is squeezed). Microscale procedures are used at CU Boulder whenever feasible to cut down on waste chemical production.

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. This column is made from 10 mL disposable glass pipet and can separate about a 1 g of material.

The column on the right is the only one that is actually manufactured as a chromatography column. Note the stopcock at the bottom. This is to control the flow of solvent through the column, important for gravity column chromatography applications. Much larger chromatography columns are available than this. 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.

Procedure for Microscale Flash Column Chromatography

Microscale flash chromatography is the primary method used 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 R_f's of the components of the mixture differ by at least 0.20. In microscale flash chromatography, the column does not need either a pinch clamp or a stopcock at the bottom of the column to control the flow, nor does it need air-pressure connections at the top of the column. Instead, the solvent flows very slowly through the column by gravity until you apply air pressure at the top of the column with an ordinary Pasteur pipet bulb.

	Step 1: Prepare the column Plug a Pasteur pipet with a small amount of cotton; use a wood applicator stick to tamp it down lightly. Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out.
	Add dry silica gel adsorbent, 230-400 mesh. Usually the jar is labeled "for flash chromatography." One way to fill the column is to invert it into the jar of silica gel and scoop it out...
	..then tamp it down before scooping more out.
	Another way to fill the column is to pour the gel into the column using a 10 mL beaker.
	Whichever method you use to fill the column, you must tamp it down on the bench top to pack the silica gel. You can also use a pipet bulb to force air into the column and pack the silica gel.
	When properly packed, the silica gel fills the column to just below the indent on the pipet. This leaves a space of 4-5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand using a small three-pronged clamp.
	Step 2: Pre-elute the column Add hexanes (or other solvent, as specified by the procedure) to the top of the silica gel. The solvent flows slowly down the column; on the column above, it has flowed down to the point marked by the arrow.
	Monitor the solvent level, both as it flows through the silica gel and the level at the top. If you are not in a hurry, you can let the top level drop by gravity, but make sure it does not go below the top of the silica. Again, the arrow marks how far the solvent has flowed down the column.
	You can speed up the process by using a pipet bulb to force the solvent through the silica gel. Place the pipet bulb on top of the column, squeeze the bulb, and then remove the bulb while it is still squeezed. You must be careful not to allow the pipet bulb to expand before you remove it from the column, or you will draw solvent and silica gel into the bulb.
	When the bottom solvent level is at the bottom of the column, the pre-elution process is completed and the column is ready to load.

	If you are not ready to load your sample onto the column, it is okay to leave the column at this point. Just make sure that it does not go dry -- keep the top solvent level above the top of the silica (as shown in the picture to the left) by adding solvent as necessary.
	Step 3: Load the sample onto the silica gel column Two different methods are used to load the column: wet and dry. In the wet method, the sample to be purified is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent. This solution is loaded onto the column. Sometimes the solvent of choice is more polar than the eluting solvents. In this case, if you use the wet method of column loading, it is critical that you only use a few drops of solvent to load the sample. If you use too much solvent, the loading solvent will interfere with the elution and hence the separation of the mixture. In such cases, the dry method of column loading is recommended.
	The column at the left is being loaded by the wet method.
	Once the sample is in the column, fresh eluting solvent is added to the top and you are ready to begin the elution process.
	For the dry method, first dissolve the sample to be analyzed in the minimum amount of solvent and add about 100 mg of silica gel.
	Swirl the mixture until the solvent evaporates and only a dry powder remains.
	Place the dry powder on a folded piece of weighing paper...
	... and transfer it to the top of the prepared column.
	Add fresh eluting solvent to the top. Now you are ready to begin the elution process.
	Step 4: Elute the column Force the solvent through the column by pressing on the top of the Pasteur pipet with a pipet bulb. Only force the solvent to the very top of the silica: do not let the silica go dry. Add fresh solvent as necessary.

	The colored bands will travel down the column as the compound is eluted.
	As soon as the colored compound begins to elute, the collection beaker is changed. The process is complicated if the compound is not colored. In such experiments, equal sized fractions are collected sequentially and carefully labeled for later analysis.
	Step 5 (Optional): Elute the column with the second elution solvent If you are separating a mixture of one or more compounds, at this point you could change the eluting solvent to a more polar system, as previously determined by TLC. Elution would proceed as in step 4. Step 6: Analyze the fractions If the fractions are colored, you can simply combine like-colored fractions, although TLC before combination is usually advisable. If the fractions are not colored, they are analyzed by TLC (usually). Once the composition of each fraction is known, the fractions containing the desired compound(s) are combined.

As soon as the colored compound begins to elute, the collection beaker is changed. The process is complicated if the compound is not colored. In such experiments, equal sized fractions are collected sequentially and carefully labeled for later analysis.

Procedure for Gravity Column Chromatography

Gravity columns are used only in the majors organic lab courses at CU Boulder (chem 3361/3381). Gravity columns are a lot slower to run than microscale flash columns. They also are more difficult to pack with adsorbent. There are two common methods of packing a gravity column: the slurry method and the dry pack method. Both of these procedures were written for the middle columns shown above; you will need to vary the quantities if you use a different column.

In the slurry method of column packing, you mix the adsorbent with the solvent and then pour this slurry into the prepared column. The nature of the slurry is a bit different depending on whether you use silica gel or alumina; some slurries are easier to work with than others. This procedure was written for alumina slurries. The advantage of slurry methods is that they eliminate air bubbles from forming in the column as it packs.

Place a piece of glass wool in the bottom of the column, and gently tamp the glass wool down with a glass rod. Attach the column to a ring stand and make sure that the column is securely fastened in a vertical position. Add a pinch clamp to the bottom of the column and close the clamp. Fill the column about half-way with a non-polar solvent, such as hexanes. Weigh 8 g of alumina into a beaker. Place 15 mL of hexanes in a 125 mL Erlenmeyer flask and slowly add the alumina powder, a little at a time, while swirling. Use a Pasteur pipet to mix the slurry, then quickly pipet the slurry onto the column (you can pour it instead if you prefer). Place an Erlenmeyer flask under the column, open the pinch clamp, and allow the liquid to drain into it. Continue to transfer the slurry to the column until all the alumina is added. Add more hexanes as necessary; the hexanes collected in the Erlenmeyer flask can be re-used to add more alumina to the column. When finished packing, drain the excess solvent until it just reaches the top level of the alumina. Close the pinch clamp. Your column is now packed and ready for use. Sometimes a small amount of sand is added to the top of the column to prevent it from being disturbed when fresh solvent is added.

The dry-pack method is easier, but can lead to bubbles in the column. Obtain an empty column, plug it with a small piece of glass wool, and affix a pinchclamp to the bottom of the column. Clamp the column in a vertical position, close the pinchclamp, and fill the column with solvent. Using a dry funnel, sprinkle 8 g of alumina into the solvent, and allow solvent to drain from the column to prevent overflowing. Let the alumina settle and gently tap the column so that the alumina will pack tightly into the column. Drain the solvent until the solvent level is just even with the surface of the alumina.

Loading and eluting gravity chromatography columns: The sample to be analyzed is dissolved in a very small amount of solvent and added to the top of the column. The pinch clamp is opened and the solvent is allowed to drain just to the top of the column. A small amount of the eluting solvent is added and allowed to drain in until the mixture is a little way into the adsorbent, then the column is filled to the top with eluting solvent. The column is now ready to run -- continue adding solvent at the top and collecting fractions at the bottom until the compounds elute at the bottom. If applicable, change the eluting solvent to a more polar solvent during the eluting process. Never let the solvent level drop below the top of the adsorbent. The process is discontinued when the compound(s) desired is (are) off the column.

References

A. Wikipedia: https://en.wikipedia.org/wiki/High-performance_liquid_chromatography

B. Knauer: https://www.knauer.net/en/search?q=chromatography

C. Kromasil: https://www.kromasil.com/support/faq.php

D. Shimadzu: https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/what_is_hplc.html

E. ChemistryView: https://www.chemistryviews.org/details/education/9464911/What_is_HPLC/