First, how to choose a column
In modern high performance liquid chromatography, the separation effect depends largely on the choice of chromatographic packing. However, the choice of chromatographic packing is very wide. To make a suitable choice, you must have a certain understanding and understanding.
1. Normal phase chromatography
The stationary phase for normal phase chromatography is typically silica gel (Silica), as well as other bonded phase fillers having polar functional groups such as amine groups (NH2, APS) and cyano groups (CN, CPS).
Since the silicon hydroxy group (SiOH) or other groups on the surface of the silica gel are highly polar, the order of separation is based on the polarity of each component in the sample, that is, the component with the strong polarity is first washed out of the chromatogram. column. The phase of the mobile phase used in normal phase chromatography is relatively lower than that of the stationary phase, such as Hexane, Chloroform, Methylene Chloride, and the like.
2, reversed phase chromatography
Reversed phase chromatography packings are often based on silica gel with a bonded phase bonded to a relatively weakly polar functional group. The mobile phase used in reversed-phase chromatography is more polar, usually water, a mixture of buffer and methanol, and nitrile. The order in which the sample exits the column is that the more polar combination is first flushed out, while the less polar component will have a stronger retention on the column.
Commonly used reverse phase packings are C18 (ODS), C8 (MOS), C4 (, C6H5 (Phenyl) and the like.
Second, the polymer filler
Most of the polymer seasonings are polystyrene-divinylbenzene or polymethylpropionate, and the main advantage is that it can be used at a pH of from 1 to 14. Compared with the silica matrix C18 filler, these fillers are more hydrophobic; macroporous polymer fillers are very effective for the separation of samples such as proteins. The disadvantage of current polymer fillers is that they are less efficient than silica matrix fillers.
Third, other inorganic fillers
Other HPLC inorganic filler columns have also been commercialized. Due to its special nature, it is generally limited to special uses. For example, graphitized carbon is also being used as a reversed phase chromatography packing. The separation of the filler is different from the alkylation phase of the silica gel matrix, and the surface of the graphitized carbon is the basis of retention, and no other surface modification is required. The pillar filler is generally more than an alkyl bonded silica gel or a porous polymer filler. The retention capacity is stronger, graphitized carbon can be used to separate certain geometrical conductors, and because the HPLC mobile phase does not dissolve, such columns can be used at any pH and temperature. Alumina can also be used in HPLC. Alumina particles are rigid and can be made into a stable column bed with the advantage of being used in mobile phases up to pH 12. However, due to the strong action of alumina and basic compounds, the application range is limited, so it is not widely used. The new zirconia filler can also be used in HPLC, commercialized polymer-coated porous zirconia microspheres. The column has a pH range of 1 to 14, and the application range is limited, so it is not widely used. The new zirconia filler can also be used in HPLC, a commercial polymer-coated porous zirconia microsphere column. Application PH range 1~14,
How to choose the filler particle size
At present, the commercial particle size is sold from 1um to over 30um. At present, the separation is mainly carried out with 3um, 5um and 10um fillers. The particle size of the filler mainly affects the two parameters of the packed column, namely column efficiency and back pressure. The smaller the particle size, the higher the column efficiency of the packed column; the less than 3um filler application, under the same selective conditions, the efficiency of the column can improve the resolution, but not the only factor. If the stationary phase is chosen correctly, but the resolution is not sufficient, it is useful to select a smaller particle size packing. The number of columns packed with 3um packing is nearly 30% higher than that of the 5um packing under the same conditions; however, 3um The back pressure of the color phase is twice that of 5um. At the same time, improved column efficiency means shorter columns can be selected under the same conditions to reduce analysis time. In addition, low viscosity solvents can be used as the mobile phase or the column temperature can be increased, such as acetonitrile instead of methanol. To reduce the pressure on the column.