FLASH CHROMATOGRAPHY: AREA & APPLICATIONS
A wide range of columns offer maximum flexibility for every situation. Depending on the nature and the quantity of the sample offers a series of column types which vary in form, size and performance.
Plastic+Glas-coated Glass Columns are available for larger sample amounts and higher pressure applications on a high safety level. The columns are designed for sample amounts from1 – 100 g and pressures up to 50 bar during preparative separations. Easy fixation on a support rod by using the corresponding pivoting clamp.
Plunger Column C-695
Robust, chemically resistant and biocompatible plunger columns are designed for optimum operational performance and safety. Volume changes in soft gel can be equalized and dead volume will be avoided. 1 – 100 g and pressures up to 50 bar during preparative separations. Easy fixation on a support rod by using the corresponding pivoting clamp. An integrated cooling jacket allows separations under constant conditions at a high quality level. Column Length 460 mm.
Precolumn are minimizing dead volumes and enhance the life time of the main column by trapping contaminants. The small Precolumn, fits to Glass Columns of inner diameter of ID 15, 26, 36 and 49 mm. The large Precolumn, fits to Glass Columns of ID 70 and 100 mm inner diameter.
Filling Sets for Glass Columns
Dry Filling Set
The Dry Filling Set is employed for filling glass columns with silica gel using compressed gas. Silica gel in the size range of 25 – 200 μm can be packed with this method.
Slurry Filling Set
The Slurry Filling Set is used for wet filling and conditioning of glass columns with silica gel particles smaller than 25 μm.
For simple separations a column, pump and pump controller may be enough. For a greater level of automation with precision, performance and ease of use the Fraction Collector can be incorporated into most setups.
Fraction Collector C-660
The intelligent, height-adjustable Fraction Collector with with dialogue language options for preparative chromatography. The C-660 collects the separated substances according to time, volume or peak.
Detectors and Recorders/Software
For most applications one of the robust UV/Vis detectors would be sufficient for the systems detection needs. Both detectors are delivered in combination with a preparative flow cell. In the absence of adequate UV/Vis absorption, likely for sugars or polymers, a Differential Refractometer (RI Detector) in combination with a UV/Vis detector is the preferred setup.
UV Monitor C-630
Filter Photometer with four standards built in filters at 200 nm, 220 nm, 254 nm and 280 nm. Delivered with built in Deuterium Lamp and a preparative flow cell.
UV Photometer C-635
Spectral Photometer with a wavelength range between 190 nm and 740 nm. Delivered with built in Deuterium Lamp and a preparative flow cell.
Refractive Index detector mostly used in combination with a UV/Vis detector for the analysis of low UV/Vis absorbing substances.Delivered with a preparative cell. For a maximal flow rate of 100 ml/min..
Procedure for Microscale Flash Column Chromatography
Packing the Column
· Obtain a glass column and make sure that it has either a glass frit or a plug of cotton wool directly above the stopcock to prevent the silica gel from escaping from the column through the stopcock.
· Next, put a ~1/2 inch layer of clean sand above the plug of glass wool. Use only as much as is necessary to obtain a flat surface, with the same diameter as that of the body of the column. Make sure the surface is flat. Add dry silica gel adsorbent, 230-400 mesh usually the jar is labelled "for flash chromatography." One way to fill the column is to invert it into the jar of silica gel and scoop it out & then tamps 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 we use to fill the column, we must tamp it down on the bench top to pack the silica gel. We can also use a pipette 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 pipette. 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 3-pronged clamp.
Solvating the Silica Gel Column:
· Next, tap gently and evenly the sides of the column with a piece of rubber tubing to settle the silica gel.
· Pour a good amount of elution solvent onto the silica gel. Use pressurized gas to force the solvent through the silica. As we force through a few hundred millilitres, we should see the top part of the silica become more homogeneous. This is because we are forcing out air that was entrapped in the silica gel.
· Continue to flush solvent through the silica gel until the entire silica plug becomes homogeneous in appearance. We may have to recycle the solvent coming through the column onto the top of the column several times before all the silica gel is solvated.
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.
· 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.
Load the sample onto the silica gel column:
Two different methods are used to load the column: the wet method and the dry method: wet and dry. Below are illustrations of both methods of loading.
Wet Loading Method
In the wet method, the sample to be purified (or separated into components) 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 to load the sample onto the column is more polar than the eluting solvents. In this case, if we use the wet method of column loading, it is critical that we only use a few drops of solvent to load the sample. If we use too much solvent, the loading solvent will interfere with the elution and hence the purification or separation of the mixture. In such cases, the dry method of column loading is recommended.
Dry Loading Method:
First dissolve the sample to be analysed 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 we are ready to begin the elution process.
Elute the column:
· Add a good part of our elution solvent to the column.
· Apply pressure to force the solvent through the column by pressing on the top of the Pasteur pipette with a pipette 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 pressure should be the minimum necessary to keep a steady stream coming out of the column. Figure below shows the colored compound as it moves through the column after successive applications of the pipette bulb process.
· The collection beaker is changed as soon as the colored compound begins to elute. The process is complicated if the compound is not colored. In such experiments, equal sized fractions are collected sequentially and carefully labelled for later analysis.
Analyze the fractions:
· If the fractions are colored, we 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.
Cleaning the Column:
· Flush all the remaining solvent out of the column using pressurized gas. Flowing air through the column for ~2 hours will give dry, free flowing silica gel.
· Pour out the contents of the column into the silica waste container.In most cases, washing the column with water and acetone is sufficient.
· If necessary, a small amount of liquid soap can be used.When all liquid solvent has been removed from the reservoir, remove the last remnants of solvent by applying a vacuum (from aspirator) to the bottom of the column. Try to avoid scratching the columns with abrasive brushes or soaps. 
NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.
SUBMIT YOUR ARTICLE/PROJECT AT email@example.com
FIND OUT MORE ARTICLES AT OUR DATABASE