Other Separation Modes

Besides the four major HPLC separation modes, several others often encountered in HPLC or related techniques are noted below. 

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Although the overwhelming majority of theoretical papers in liquid chromatography are dealing with the various aspects of RP-HPLC separation, theoretical advances have also been achieved in some other separation modes. Thus, a theoretical study on the relation between the kinetic and equilibrium quantities in size-exclusion chromatography has been published, hi adsorption chromatography the probability of adsorbing an analyte molecule in the mobile phase exactly r-times is described by... 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

Drugs possessing a steroid structure are particularly easy to separate by CEC (see also the section on Steroids). Euerby et al. [203] published the separation of tipredane and five related compounds. A conventional capillary packed with 3 mm Spherisorb ODS-1 can be used for this purpose using acetonitrile-Tris pH 7.8 buffer (8 2) (50 mmol/1). Under these conditions it is also possible to separate the C-17 diastereoi-somer of the active compound without the addition of a chiral modifier (b-cyclodextrin is needed to achieve a comparable result in other separation modes) (Fig. 10.25). The elution order of individual compounds was exactly the same as with reversed phase chromatography, and it was concluded that with unionized species HPLC methods should be directly transferable to the CEC mode. 

MI-SPE has been applied in either on-line or off-line mode for several analytes. In the first case, MIP is packed as an HPLC precolumn. Column switching and pulsed elution modes are employed in on-line MI-SPE. One advantage of this design is the automation capability and the direct coupling to HPLC or other separation modes. Despite these advantages, the off-line mode, which has been successfully applied to bio and environmental and food analysis, is the most commonly practiced method. 

Chen and Pentoney have reported a competitive immunoassay method for digoxin in serum with sensitivity in the low KT11 M range (68). The high-resolution power of CE makes the simultaneous analysis of two, or more than two, species feasible, whereas this may not be the case in most other separation modes (e.g., HPLC). Chen and Evangelista also reported a simultaneous competitive immunoassay of morphine and phencyclidine in urine (69). The immunoassay could be performed routinely and reproducibly in less than 5 min with detection limits of 4 nM for phencyclidine and 40 nM for morphine. 

Solvents commonly used in normal phase chromatography are aliphatic hydrocarbons, such as hexane and heptane, halogenated hydrocarbons (e.g., chloroform and dichloromethane), and oxygenated solvents such as diethyl ether, ethyl acetate, and butyl acetate. More polar mobile phase additives such as isopropanol, acetone, and methanol are frequently used see Table 2). The technique is particularly suited to analytes that are very hydrophobic, e.g., fat-soluble vitamins such as tocopherols (6J and other hydrocarbon-rich metabolites that exhibit poor solubility in the water-miscible solvents employed in other separation modes. In addition, since the geometry of the polar adsorbent surface is fixed, the technique is useful for the separation of positional isomers the proximity of functional groups to the adsorbent surface, and hence the strength of interaction, may well differ between isomers. 

In RPLC, the solute continuously partitions between the stationary phase and the mobile phase. The nature of the partitioning between the two phases is very similar to partitioning between two immiscible liquids. For example, the process is noncompetitive and the sorption isotherms are hnear. As a result, peaks are usually symmetrical, and the separations are very reproducible. RPLC is by far the most popular hquid chromatographic technique currently in use. Other separation modes are usually considered only after RPLC fails to deliver desirable results. 

Nevertheless, the utility of SEC in copolymer analysis is not impaired, if one applies SEC keeping the limitations described above in mind. The rapidity, simplicity, and capability of obtaining much data on copolymers by SEC overcome those drawbacks. In this chapter, the methods of determining molecular mass averages and MMD, as well as composition and chemical heterogeneity, are described. Other separation modes of HPLC used with the size-exclusion mode, such as the adsorption mode, are also explained. 

Other separation modes using LC are available, such as adsorption chromatography or chiral chromatography. However, these separation techniques are not frequently used with detection... 

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