4 Packing Methods of C18 HPLC Chromatography Column
Most chromatographers choose the packing of the guard column based on the packing of the analytical column, and normally the same chromatographic packing as the analytical column can be chosen. However, depending on the actual analytical work, it may not be necessary to match the packing of the analytical column exactly. The principle of selecting a protective column is to choose the shortest possible protective column under the precondition of meeting the requirements of separation analysis and to choose the packing material with weak retention of the separated sample as much as possible.
There are many factors that affect the performance of C18 HPLC column, for example, silica gel purity, column size, particle shape, particle size, surface area, pore size, chemical properties including bonding type, carbon coverage, capping, before choosing, learn more about your samples and impurities, their type structure, polarity, acidity and alkalinity, molecular weight size, etc. Today, HAWACH will introduce the following four major filling methods of the C18 HPLC column.
1. Pressure homogenization method loading
The filler is suspended in a suitable homogenizing solution to make a homogenate, and before it settles, it is quickly pressed into the column at a high flow rate with a high-pressure pump, and a uniformly filled column can be prepared. This is a common method of loading analytical and preparative chromatography columns.
2. Density method of packing
In the early development of HPLC, often use the “equal density method” was to fill the column. The skeleton density of porous silica gel is high. Different grades of silica gel due to different manufacturing methods and differences, but more than 2.2g/cm3 above. Therefore, it is necessary to use high-density solvents to prepare a mixture with a density similar to that of the silica gel skeleton. Often used high-density solvent for iodine and brominated alkanes, and then with the appropriate proportion of other solvents, formulated with the appropriate density of the liquid, so that the filler can be “weightless” suspended in the homogenate, in order to comfortably use the high-pressure pump to press it into the empty column, the production of the uniformly filled column.
3. Dry loading method
For the larger particle size of the packing, you can use the dry method of filling, the basic method is similar to the filling of the gas chromatography column. But the point to note is that in the dry filling preparation of the liquid chromatography column, do not overly violent vibration and knocking. Vibration and tapping can cause the packing to produce an overall inhomogeneity in the column due to the inhomogeneity of its own particle size, i.e., the larger packing particles are close to the column wall, while the finer particles tend to be concentrated in the center of the column. This inhomogeneity of particle distribution in the column will lead to the reduction of column efficiency. A better method is to use a small number of multiple methods to add filler to the column, for example, each time you add the equivalent of a 3-5mm column bed of filler, installed a little that is in the experimental table or tabletop gently knocked vertically dozens of times, after adding some filler, repeat the above operation, until the filling is complete.
4. Viscosity method
High viscosity method, that is, the use of high viscosity homogenizing liquid system to prevent the settling of particles. You can use high viscosity liquid such as ethylene glycol, polyethylene glycol, glycerin, and paraffin oil to mix the homogenizing liquid. However, high viscosity leads to increased fluid resistance, which will certainly prolong the column loading time and require the use of higher column loading pressure, which is very inconvenient in practical terms. Therefore, the high-viscosity method has rarely been used.