ABSTRACT
References ......................................................................................................................................405
and reach the detector is called the retention time (Rt). These techniques are very powerful because they allow one to separate compounds of similar physical and chemical properties within a complex sample mixture that may be structurally very similar or even isomers of each other. It is also fast, easy, and economical.1-10
The mobile phase can be a gas or a liquid, while the stationary phase generally is some type of solid, either immobilized on a support or contained in a column. When the stationary phase is contained in a column, the term “column chromatography” applies. Column chromatography can be further divided into gas chromatography (GC)11,12 and liquid chromatography (LC).1,2 Based on the aforementioned concepts, there are several primary types of chromatographic methods in common use today. These include paper chromatography (PC),13 thin-layer chromatography (TLC),14,15 LC,1,2 GC,11,12 and supercritical §uid chromatography (SFC). While PC and TLC were named on the basis of the stationary phase, the terms GC, LC, and SFC refer to the physical state of the mobile phase. In PC and TLC, the stationary phases are primarily paper (made of cellulose) and silica, respectively, while the mobile phases are composed of a mixture of organic solvents. LC stationary phases are often some form of silica or other synthetic polymer support, and are usually derivatized with various modifying groups to provide a wide range of selectivities. The mobile phases, on the other hand, are mixtures of aqueous and organic solvents. GC stationary phases are similar to LC in that they are bonded phases with an array of selectivities, but the mobile phase is an inert carrier gas such as helium or nitrogen. Coupled with each mode of chromatographic separation is a unique group of detection techniques. For example, paper and thin-layer chromatographic separations are carried out in an open medium instead of a column format. Consequently, the commonly used techniques for detection are colorimetric methods, where the analytes react with a speci c reagent to form a colored product. These colored products, in the form of bands or spots recovered from the stationary phase, can then be detected or quanti ed by densitometry. The detection techniques for liquid and gas chromatographic separations, however, are quite different. Due to the unique nature of these separations, where analytes elute off the column in sequence, an in-line type of detection is most suitable. For LC, commonly used techniques are UV/visible absorbance, §uorescence, and refractive index. Since the analytes are in the gas phase for GC, they are most often detected by using §ame ionization (FID) and thermal conductivity (TC). Mass spectrometry (MS) is widely employed for detection in both GC and LC applications.1-7,11,12
It is well established that the size of the particle used for the stationary phase affects the separation-the smaller the particle, the better the resolution. This is due in part to the fact that smaller particle size gives rise to a larger surface area per unit weight of the solid phase, and consequently more ef cient interaction with the analytes. Because traditional columns use gravity to pull the mobile phase around the particles, smaller particle sizes often result in higher back pressure so there is a practical limit on how small the particle size can be before the column essentially stops §owing. High-performance liquid chromatography (HPLC)1,2 was developed to overcome this limitation, and also to shorten the analysis time and enhance the separation. In an HPLC system, the sample is introduced through the injector and is then pushed through the column by the constant pumping of mobile phase. It has two major advantages. First, it uses a pump to force the mobile phase through the column at high pressure. The column can be made (or usually purchased) using any of the available solid stationary phases. Hence, HPLC is commonly used to shorten the running times of chromatographic methods that are time restricted by gravity or capillary action. Second, because high pressure is used, much smaller particle sizes of solid support can be used in the columns in conjunction with narrower column internal diameters. Together, these two factors allow higher resolution of the compounds passing through the column. The relationship between particle size, ef ciency of separation, and §ow rate is described mathematically by the van Deemter equation:
HETP the height equivalent to a theoretical plate( ) = + + ⋅A u C u
B
where A represents Eddy diffusion B represents longitudinal diffusion C represents mass transfer u represents linear velocity
which takes into account contributions from molecular diffusion, mass transfer effects, and variable path lengths through a packed bed. Resolution is a measure of the column’s ability to separate mixtures into individual components and is a function of the selectivity, ef ciency, and retentive properties of the column.7 Today, HPLC has become the premier analytical technique in the chemical, pharmaceutical, and nutraceutical industries, and is widely used in all phases of discovery, development, and quality control. By using shorter columns (30-100 mm) and smaller particles (3 μm), in contrast to conventional columns that are 100-250 mm long and packed with 5 μm particles, HPLC allows high ef ciency of separation and faster analysis, with separation times as short as a few minutes. The latest innovation in chromatographic technology is the emergence of ultraperformance liquid chromatography (UPLC). The development of reliable packing material with particle sizes less than 2.5 μm resulted in signi cant increases in column ef ciency compared to HPLC, even at higher §ow rates. Special instrumentation is required since back pressure in systems can exceed 20,000 psi. For somewhat lower performance, UPLC columns can be used with traditional HPLC equipment as long as the §ow rate is adjusted to keep the back pressure within the limits of the system. Overall, the speed of analysis, the number of peaks resolved in a gradient separation, and the sensitivity all are improved dramatically with UPLC.16,17
In addition to the different types of chromatographic methods, there are also various mechanisms of separation. For example, analytes can be resolved based on their molecular shape or size. This mode of separation is referred to as size-exclusion or gel-permeation chromatography. Compounds can be separated also on the basis of charge (ion-exchange chromatography), hydrophobicity (reverse phase and hydrophobic interaction chromatography), biological function (af nity chromatography), and metal binding capability (immobilized metal-ion af nity chromatography [IMAC]). In general, they all can be separated into two mechanisms: adsorption (including partition) in which the analyte molecules are adsorbed onto the stationary phase through binding, and non-adsorption (such as size exclusion). Each mode of chromatography has its own type of bonded phases, and is discussed later.
