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Flash Chromatography

Flash chromatography is a quick and efficient method for separating and purifying compounds in a mixture. Here's a step-by-step guide on how to run flash chromatography:

Materials Needed

- Flash chromatography column
- Solvent system (e.g., hexane, ethyl acetate)
- Sample mixture to be separated
- UV lamp or detector (if applicable)
- Collection tubes or flasks

Preparation

1. **Column Selection:** Choose a column with the appropriate size and stationary phase (e.g., silica gel) for your sample.
2. **Packing the Column:** Slurry pack the column with the stationary phase (e.g., silica gel) in a suitable solvent. Ensure that the column is packed evenly without air bubbles.
3. **Equilibration:** Pass a few column volumes of the eluent (solvent or solvent mixture) through the column to equilibrate the stationary phase.

Choosing a Stationary Phase

In flash column chromatography the most commonly used stationary phases are silica gel (SiO2) and alumina (Al2O3). Other stationary phases are available for specific uses (Table 1).
Figure 2 shows the general structure of normal phase, reversed phase and specialized silica. Normal phase silica has terminal silanol groups which give it its polar adsorption properties. In reversed phase silica, the silanol groups are replaced by apolar groups such as C-18 carbon chains, which makes it well suited for purification of highly polar compounds. The carbon chains can also be capped at their ends to produce specialized silica.

Table 1. Stationary phase selection guide
Sample Characteristics Stationary Phase Conditions
Low to medium polarity

Normal phase silica
Neutral alumina

 Normal Phase
High polarity

Reversed phase silica
Cyano functionalized silica

 Reverse Phase
Basic

Basic alumina
Amine functionalized silica
Normal phase silica + Et3N
Neutral alumina + Et3N

 Normal Phase
Acidic

Acidic alumina
Normal phase silica + AcOH
Neutral alumina + AcOH

 Normal Phase
Acid sensitive

Neutral alumina
Cyano functionalized silica
Florisil

Normal Phase
Normal Phase
Reverse Phase
Charged

Reversed Phase Silica
Cyano functionalized silica

 Reversed Phase
Figure 2. Different stationary phases

While adding Et3N or AcOH to the eluant can prevent streaking of basic and acidic compounds on normal phase silica, it also implies having to remove it from the purified product usually by extraction or high vacuum evaporation. Using a specialized silica with an acidic or basic moiety bound to it eliminates this problem. Alumina can also be used as a stationary phase.

 

Packing the Column

1. Preparation: Place a small piece of glass wool or a frit at the bottom of the column to prevent the stationary phase from escaping.
2. Adding Sand:  Add a small layer of sand on top of the glass wool to create a flat surface for the stationary phase.
3. Slurry Packing: Prepare a slurry by mixing the stationary phase (e.g., silica gel) with an appropriate solvent. Pour the slurry into the column, ensuring that it settles evenly. Tap the column gently to remove air bubbles and achieve a uniform packing.  Determine the silica gel to compound ratio. Easy separations require ratios between 30-50:1 (by weight), while harder separations call for ratios of up to 120:1.
4. Top Layer of Sand: Once the column is packed with the stationary phase, add another layer of sand on top. This layer helps to distribute the solvent and sample evenly and prevents disturbance of the silica gel during sample loading.

By following these steps, you can ensure that your flash chromatography column is properly packed for efficient separation.

Prepare and load your sample

There are three different ways to load a product onto a column: neat, in solution or dry.

  • Neat is rarely used because it can overload the column and lead to cracking.
  • Dry loading is seldom used because of the risks associated with it (acidity and volatility of silica gel).
  • Loading of a solution of the product is by far the easiest way and will be described here.

Ideally the crude product should be dissolved into the least polar solvent possible such as hexane or pentane. Unfortunately, a lot of products will be insoluble in those solvents. In this case, the easiest is to dissolve the crude mixture into dichloromethane. Dichloromethane will dissolve most organic products while still being relatively non polar. The product should also be dissolved in the minimum amount of solvent in order to start with the thinnest band possible. If too much solvent is added it is best to concentrate it back down and start again.
Once the crude mixture is in solution, open the stopcock and using a long glass pipette carefully add the solution to your column by running it along the wall of the column as far down as you can. Once all the solution has been added, let it absorb onto the silica gel. Then rinse your crude material flask with a small volume of eluant and carefully add it to your column, rinsing the walls of the column. Let it absorb and repeat two more times. The idea is to get all the crude material absorbed onto the silica gel using the smallest amount of solvent possible so as to not dilute it too much.
After the three rinses, fill your column with eluant. At first using a pipette until you have a few centimeters of solvent then straight from the Erlenmeyer. Always run the solvent down the walls of the column to avoid disturbing your silica gel.

Running the column

With your column full of solvent you can start applying pressure. It is usually safe to elute the first column volume into an Erlenmeyer flask as your product should not come out until the third column volume (Rf=0.3 => CV=3.3). Once you've eluted a column volume, switch to your test tubes, filling each up to about 80-90%. Always make sure there is solvent above the silica. The column should never run dry. Once all your initial eluant is used up gradually increase the polarity until you get to twice the polarity of your TLC solvent. Increasing the polarity too rapidly will result in cracking of the silica and should therefore be avoided.
While you are running the column, you should be checking your fractions by TLC. This may be a little overwhelming at first, but eventually it will become easier. If it is too hard, you can stop the pressure while spotting, but constant changing of the pressure can also lead to cracks in the silica and should be avoided. When spotting TLCs, spotting every other tube usually gives you a good enough idea of the separation all while saving time and TLC plates. Once all the products of interest have eluted out of the column, you can safely flush and dry your column.

 

 

Fraction Collection

1. **Collect Fractions:** Collect the eluate in separate tubes or flasks. Label each fraction clearly.
2. **Analysis:** Analyze the fractions using techniques such as thin-layer chromatography (TLC) to identify the compounds present.

Cleaning and Storage

1. **Cleaning the Column:** After the separation is complete, wash the column with a suitable solvent to remove any residual compounds.
2. **Storage:** If the column is reusable, store it in an appropriate solvent to prevent drying out.

Safety Considerations

- Always wear appropriate personal protective equipment (PPE) such as gloves and safety glasses.
- Work in a well-ventilated area, especially when using volatile solvents.
- Dispose of solvents and waste materials according to your institution's safety guidelines.

By following these steps, you can effectively separate and purify compounds using flash chromatography. Adjustments may be necessary based on the specific requirements of your sample and the complexity of the mixture.