Normal phase chromatography involves the use of a column packed with a polar stationary phase (like silica) and a mobile phase of hexane plus another more polar organic solvent such as dichloromethane. The most polar sample components are the most strongly retained, so peaks elute in order of increasing polarity.
Columns for normal phase HPLC. By far the most commonly used column is underivatised silica. It is inexpensive, porous, robust and has a very high surface area. If a rather less retentive column is required for normal phase analysis, the options are cyano (CN), amino (NH2), or DIOL. Silica can be in the form of spherical or irregular particles. Spherical are easier to pack and give lower back pressure and higher efficiency. But the surface area is only about 150sq m/g, compared with about 450 for irregular silica. Hence the loading capacity of irregular silica is much greater. Useful for preparative HPLC!
Eluents for Normal Phase. The base eluent is usually hexane (sometimes heptane), plus another organic solvent such as ethyl acetate, dichloromethane, chloroform, acetone or diethyl ether. Normal phase eluents are generally more expensive than for reversed phase, more flammable, and more UV absorbing. Be especially careful to check the UV cut-off of solvents when working below 250nm with a UV detector.
When to use Normal Phase. Not very often! Most separations will work at least as well using reversed phase. But for some samples which are real;ly non-polar and hence would be hard to remove from a C18 column, normal phase is a better bet. Often these samples are much more easily dissolved in non-polar organic solvents. Normal phase is quite selective between molecules which are quite similar, but with different functional groups.
Silica columns differ from each other quite a bit. So it is not always easy to transfer a method from one column to another. Cyano columns are usually very well behaved, with few if any vices. Be careful when buying amino columns. Sometimes they come equilibrated in methanol:water, which can be a problem i you want to work in heptane!
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