There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, three modes can be specified.
In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packings) and the separation is based on repeated adsorption-desorption steps.
In ion-exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.
In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Mainly for historical reasons, this technique is also called gel filtration or gel permeation chromatography although, today, the stationary phase is not restricted to a "gel".
Concerning the first type, two modes are defined depending on the relative polarity of the two phases: normal and reversed-phase chromatography.
|In normal phase chromatography, the stationary bed is strongly polar in nature (e.g., silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.|
|Reversed-phase chromatography is the inverse of this. The stationary bed is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained.|
Above mentioned types cover almost 90% of all chromatographic applications. Eluent polarity plays the highest role in all types of HPLC. There are two elution types: isocratic and gradient. In the first type constant eluent composition is pumped through the column during the whole analysis. In the second type, eluent composition (and strength) is steadily changed during the run.
Overlay of the four components trace analysis chromatograms. (A) is the isocratic elution, (B) is the gradient elution, shadow line is the gradient profile from 30% acetonitrile in water to 65% acetonitrile.
HPLC as compared with the classical technique is characterized by:
|small diameter (2-5 mm), reusable stainless steel columns;|
|column packings with very small (3, 5 and 10 mm) particles and the continual development of new substances to be used as stationary phases;|
|relatively high inlet pressures and controlled flow of the mobile phase;|
|precise sample introduction without the need for large samples;|
|special continuous flow detectors capable of handling small flow rates and detecting very small amounts;|
|automated standardized instruments;|
|rapid analysis; and|
Initially, pressure was selected as the principal criterion of modern liquid
chromatography and thus the name was "high pressure liquid chromatography" or
HPLC. This was, however, an unfortunate term because it seems to indicate that the
improved performance is primarily due to the high pressure. This is, however, not true. In
fact high performance is the result of many factors: very small particles of narrow distribution range and
uniform pore size and distribution, high pressure column slurry packing techniques,
accurate low volume sample injectors, sensitive low
volume detectors and of course, good pumping
systems. Naturally, pressure is needed to permit a given flow rate of the mobile phase;
otherwise, pressure is a negative factor not contributing to the improvement in
separation. Recognizing this, most experienced chromatographers today, refer to the
technique as high performance liquid chromatography still permitting the use of the