1.1 GENERALITIES ABOUT FLASH CHROMATOGRAPHY
Chromatography generally designs techniques for the separation of compounds inside a mixture. In fact, there are different kind of chromatography like, paper, thin layer and column chromatography. Column chromatography, also called Flash Chromatography (or liquid chromatography) is the most useful methods for the purification and the separation of mixture, because you can use it for both analytical and preparative applications. With this technique, you can purify both solid and liquid compounds. The separation mechanism involves the same principles as Thin Layer Chromatography (TLC) but it is not restricted by quantities to be separated. But how does column chromatography work? Almost all types of chromatographic techniques are based on the affinity of the compounds for the mobile and stationary phases, resulting in the emergence of products from the stationary phase at different speeds. Typically flash chromatography consists in loading a crude reaction mixture to the top of a stationary phase, most of the time silica gel, and elute it with a solvent. This will let the different compounds of the mixture interact with the silica. At the end, if you chose the right eluent and stationary phase, you should separately collect pure products.
Before doing the purification
by flash chromatography, you should first determine the experimental conditions
by using thin layer chromatography. With TLC you will know how many products
are in your crude reaction and you will be able to determine the relative
polarity of each one. So let have a brief overview of the thin layer chromatography
technique.
TLC is a very simple and useful chromatographic method. With this technique
you can optimize your experimental conditions without using a lot of your
compounds and it is fast. This is good if you are using the same stationary
phase for the TLC and the flash chromatography. Using both SiliCycle TLC plates
and SiliCycle UltraPure Flash Cartridges (or silica gel) will give you reproducibility
of your experimental conditions. The mechanism of TLC is based on the affinity
of the compound for the stationary phase and the mobile phase. Every product
will not have the same strength with which it binds to the silica gel (or
the stationary phase) so the retention time will be
different for each product. The principle of the TLC is very easy to understand.
You spot your crude reaction mixture at the bottom of the TLC plate and you
elute the plate into a beaker in which you previously put a small quantity
of solvent (called eluent). The spot where you put your sample on the TLC
should not be mixed with the solvent. You let the solvent elute up to around
1 cm from the top of the TLC plate. Flash chromatography is a simple method
for the purification of crude reaction mixtures. What is very useful with
Flash chromatography is that you can determine your experimental conditions
by TLC and after that do your purification. Note that if you ever used automated
or manual flash chromatography you can use the same stationary phase and solvent.
There are very closely related techniques and both work similarly. See our
TLC Range
1.1.1 DETERMINATION OF THE CONDITIONS BY THIN LAYER CHROMATOGRAPHY
Before doing the purification by flash chromatography, you should first determine the experimental conditions by using thin layer chromatography. With TLC you will know how many products are in your crude reaction and you will be able to determine the relative polarity of each one. So let have a brief overview of the thin layer chromatography technique. TLC is a very simple and useful chromatographic method. With this technique you can optimize your experimental conditions without using a lot of your compounds and it is fast. This is good if you are using the same stationary phase for the TLC and the flash chromatography. Using both SiliCycle TLC plates and SiliCycle UltraPure Flash Cartridges (or silica gel) will give you reproducibility of your experimental conditions. The mechanism of TLC is based on the affinity of the compound for the stationary phase and the mobile phase. Every product will not have the same strength with which it binds to the silica gel (or the stationary phase) so the retention time will be different for each product. The principle of the TLC is very easy to understand. You spot your crude reaction mixture at the bottom of the TLC plate and you elute the plate into a beaker in which you previously put a small quantity of solvent (called eluent). The spot where you put your sample on the TLC should not be mixed with the solvent. You let the solvent elute up to around 1 cm from the top of the TLC plate. When the elution is finished, you should be able to reveal the different products on the plate. The easiest way to reveal your product is with an UV lamp. But this is possible only for unsaturated products. So that is why dyes are often used to be able to see where the products on the TLC plate are.
After revelation of the TLC, you can interpret the results. The better way to know if the experimental conditions that you used will be good enough to do the separation is to use the retention factor (Rf) of every product. The Rf refers to the distance that a product eluted compared to the solvent front. The determination of the Rf parameter is based on a mathematical equation that uses the distances traveled by the compounds and the solvent.
The optimal condition to do a flash chromatography is to use a solvent that will provide an Rf parameter of the product to purified of around 0.25 and all the other components as far as possible from the desired product (at least a difference of Rf of 0.2 unit). To reach that goal and have a good separation, sometime you can’t only use one solvent. That is why you should play a little bit with the mobile phase. There is known possible techniques to determine the mobile phases:
Always the same solvent or mixture
* ISOCRATIC CONDITIONS: This is the most known elution technique that consisting in using the same solvent or composition mixture of solvents during all the separation.
Change gradually the polarity of the solvent mixture
* LINEAR GRADIENT CONDITIONS: Consist to change gradually the polarity of the eluent. Most of the time you start with low polarity solvents and you increased it slowly.
Change by step the solvents
* STEPPED GRADIENT CONDITIONS: This is another useful method ; you used different solvents mixtures polarities and you used it as the eluent gradually
Typically there are some solvents that are commonly used in flash chromatography. You can refer to this book for more details: Introduction to Modern Liquid Chromatography by L.R. Snyder and J.J. Kirkland, New York, Wiley (1979). To find the usual solvents. In that table, solvents in orange are the ones more frequently used in flash chromatography.
The optimization step of the experimental conditions is a critical step before doing the purification by flash chromatography. By using the right conditions you should be able to get a good separation.
1.1.2 SELECTION OF THE COLUMN LENGTH AND SILICA WEIGHT
Another important parameter when you are doing purifications with flash chromatography is to use the proper column size. If your column selection is not adequate, you will not have the best conditions for your purification. There are a general rule that give the correlation between the amount of silica gel to use for the quantity of product you want to purify, and also the diameter of the column that you should use. Below is a guidelines to choose the right column.
General rule for Column Silica gel weight
Typically the mass of the sample to purified should be less than 10 % of the mass of the silica gel inside the column.
* i.e.: 4 g of silica gel to purified sample mass < 0.4 g
Selection of the Column length
The length of a column is directly related to the “number of theoretical plates” that you will have during the separation. The longer is the column length, the better will be the separation because the products will take more time to pass thought the silica gel, so a very efficient separation. However, you shouldn’t take too long column because the time need to do the purification will be very long.
Tips : Generally, you can select the column by looking only at the silica weight. If you have purification problem, you can take a longer column or if you are using Flash Prepacked Cartridges, you can do “stacking columns” (Let pass the product thought more than one column).
After revelation of the TLC, you can interpret the results. The better way to know if the experimental conditions that you used will be good enough to do the separation is to use the retention factor (Rf) of every product. The Rf refers to the distance that a product eluted compared to the solvent front. The determination of the Rf parameter is based on a mathematical equation that uses the distances traveled by the compounds and the solvent.
The optimal condition to do a flash chromatography is to use a solvent that will provide an Rf parameter of the product to purified of around 0.25 and all the other components as far as possible from the desired product (at least a difference of Rf of 0.2 unit). To reach that goal and have a good separation, sometime you can’t only use one solvent. That is why you should play a little bit with the mobile phase. There is known possible techniques to determine the mobile phases:
Always the same solvent or mixture
* ISOCRATIC CONDITIONS: This is the most known elution technique that consisting in using the same solvent or composition mixture of solvents during all the separation.
Change gradually the polarity of the solvent mixture
* LINEAR GRADIENT CONDITIONS: Consist to change gradually the polarity of the eluent. Most of the time you start with low polarity solvents and you increased it slowly.
Change by step the solvents
* STEPPED GRADIENT CONDITIONS: This is another useful method ; you used different solvents mixtures polarities and you used it as the eluent gradually
Typically there are some solvents that are commonly used in flash chromatography. You can refer to this book for more details: Introduction to Modern Liquid Chromatography by L.R. Snyder and J.J. Kirkland, New York, Wiley (1979). To find the usual solvents. In that table, solvents in orange are the ones more frequently used in flash chromatography.
he optimization step of the experimental conditions is a critical step before doing the purification by flash chromatography. By using the right conditions you should be able to get a good separation.
1.1.2 SELECTION OF THE COLUMN LENGTH AND SILICA WEIGHT
Another important parameter when you are doing purifications with flash chromatography is to use the proper column size. If your column selection is not adequate, you will not have the best conditions for your purification. There are a general rule that give the correlation between the amount of silica gel to use for the quantity of product you want to purify, and also the diameter of the column that you should use. Below is a guidelines to choose the right column.
General rule for Column Silica gel weight
Typically the mass of the sample to purified should be less than 10 % of the mass of the silica gel inside the column.
* i.e.: 4 g of silica gel to purified sample mass < 0.4 g
Selection of the Column length
The length of a column is directly related to the “number of theoretical plates” that you will have during the separation. The longer is the column length, the better will be the separation because the products will take more time to pass thought the silica gel, so a very efficient separation. However, you shouldn’t take too long column because the time need to do the purification will be very long.
Tips : Generally, you can select the column by looking only at the silica weight. If you have purification problem, you can take a longer column or if you are using Flash Prepacked Cartridges, you can do “stacking columns” (Let pass the product thought more than one column).