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.
Contact:
René Harmsen
T:033-4571705
F:033-4571706
Postbus 496 3800 AL Amersfoort
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)
1.1.3 LOADING SAMPLES IN FLASH CHROMATOGRAPHY
One of the most critical steps when you are doing purifications by flash chromatography is loading the sample on the column. With both automated and manual chromatography, if the sample is not loaded properly on the top of the column, the efficiency of the purification will be affected. With flash chromatography you can purify liquid and solid samples, but the sample loading will be different.
With automated flash chromatography, solid sample are possible to separate also; you just have to do a sample column with the impregnated silica gel and place this column at the beginning of the system to let the solvent pass thought it first. This was a brief overview of the purification technique that most organic chemists usually use. Now, in the next sections, we present our SiliCycle UltraPure Flash Cartridges compatible with the principally used brands; BiotageTM, IscoTM and Jones FlashmasterTM.
1.2 SILICYCLE ULTRAPURE FLASH CARTRIDGE PRODUCT LINE
You can utilize the superior performance of our UltraPure Flash Cartridges Silica Gel in all your separations with our pre-packed flash cartridges. Increase the reproducibility by using the same silica gel for all your flash systems. We offer a full line of cartridges compatible with the most used technologies: BiotageTM, IscoTM and Jones FlashmasterTM systems.
1.2.1 CHARACTERISTICS OF SILICYCLE ULTRAPURE FLASH CARTRIDGES
Using UltraPure Flash Cartridges, you will not only save time and money, you will also generate higher purity samples and reduce the number of false positives in your screening, resulting in higher quality data you will be confident in using. All our Flash Cartridges are packed with sorbents based on our fines free UltraPure Silica gel which is the highest purity silica gel on the market. When you use SiliCycle’s cartridges we guarantee you will have access to:
Wide variety of sorbents
* SiliCycle leads the market of functionalized silica gel by offering innovative products. All silica included in this new exclusive line of silica-based functionalized silica gels can be packed into a Flash Cartridge. With UltraPure Flash Cartridges, you can not only do purification itself, but also :
Catch and release - Metal Scavenging - Coupling reactions And more!
Excellent packing and storage qualities
* Each SiliCycle’s Flash Cartridge is packed with our UltraPure Silica gel. In consequence, the silica inside the cartridge presents the same characteristics than our bulk silica gel: very high purity, acid washed and tight particle size silica gel with no fines. In addition to the quality of our silica gel, we have developped an excellent packing method, that avoids uneven flow paths inside the cartridge (Figure 5). Also, all flash cartridges are air-tight sealed to insure storage stability and protection from moisture.
Very good separation and tighter peaks
* Our manufacturing process eliminates fines and extractable in our UltraPure Silica gel, resulting in a very tight particle size distribution. This ensures more uniform cartridge packing, as well as better resolution and separation
Less time and solvent used for conditioning cartridges
* Our cartridges need to be conditioned not cleaned so you don’t have to spend time and waste solvents cleaning the cartridge before use. SiliCycle’s proprietary technology generates a silica gel with the lowest trace metal content on the market today. Table 5 shows the trace metal analysis for SiliCycle’s gel and two other manufacturers’ flash silica gel.
No silica contamination in your final product
* Not only do we eliminate fines which can pass through the frit and contaminate your product, we also end-cap our functionalized products which makes them insoluble in all solvents including 5% ammonia in methanol. Normally, silica gel contains trace quantities of a variety of different metals, which can affect the separation and the purity of your product. With the UltraPure Flash Cartridge, you will not have contamination by the silica gel.
High recoveries
* End-capping of our functionalized gels removes all residual polar silanol groups so there are no active groups for your compounds to adhere to.
Reproducible Flow Rate
* Our fine free silica gel and state of art packing process guarantee a consistent, reproducible flow rate from one cartridge to the next and from lot-to-lot. Also, tight control of trace metals lot-to-lot also improves your reproducibility.
1.2.2 AVAILABLE SILICYCLE ULTRAPURE FLASH CARTRIDGES
We offer a full line of cartridges compatible with all BiotageTM, IscoTM and Jones FlashmasterTM systems. For your convenience all cartridges can be packed with any of our functionalized silica gels. SiliCycle have developed many functionalized silica gel. Most can be put inside Flash cartridges. In this section of the document (see table 5), we present only a brief overview of the “standard sorbents” that you can use for flash chromatography. The standard irregular silica gel that we are using for functionalized silica gel presents these physical properties:
Physicals characteristics of our standard silica gel matrix
R10030B : UltraPure I Flash Silica
Shape : Irregular Silica Gel
Particle size Distribution : 40-63 µm
Pore size : 60 Å
Specific surface area : 500 m2/g
If you need other silica characteristics for your Flash Cartridges, we can provide you other types of silica gel. Please refer yourself to our catalog or to our link to see the different kind of silica that we can provide.