Liquid-bonded phase partition chromatography

In liquid-bonded-phase partition chromatography, the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds. 

Gradient elution works well with bonded-phase partition chromatography and adsorption chromatography but is difficult to use with liquid-liquid chromatography. 

The structure of silica gel tends to change with time and this creates problems of irreproducibility in the separations. To remedy this situation and reduce the gel s polarity, the reactivity of silanol groups can be used to covalently bind organic molecules. Bonded stationary phases behave like liquids. However, the separation mechanism now depends on the partition coefficient instead of adsorption (Fig. 3.9). Bonded phases, whose polarity can be easily adjusted, constitute the basis of reversed phase partition chromatography, which is used in the majority of analyses by HPLC. 

LIQUID CHROMATOGRAPHY BY BONDED PHASE - PARTITION - ADSORPTION ION EXCHANGE c Developments Since 1975... 

Liquid bonded-phase chromatography Partition chromatography that makes use of a stationary phase that is chemically bonded to the column packing. 

C. Cellulose Cellulose is a polymerized polysaccharide characterized by the cellobiose unit. The presence of free OH groups in cellulose permits hydrogen bonding with low-molecular-weight liquids such as alcohols or water. Cellulose is useful for the separation of hydrophilic substances primarily by the mechanism of normal-phase partition chromatography. For a discussion of separation mechanisms and the cellulose-water complex , see Ref. 176. 

Other modes of LC operation include liquid-liquid partition chromatography (LLC) and bonded phase chromatography. In the former, a stationary liquid phase which is immiscible with the mobile phase is coated on a porous support, with separation based on partition equilibrium differences of components between the two liquid phases. This mode offers an alternative to ion exchange in the fractionation of polar, water soluble substances. While quite useful, the danger exists in LLC that the stationary phase can be stripped from the column, if proper precautions are not taken. Hence, it is typical to pre-equil-ibrate carefully the mobile and stationary phases and to use a forecolimn, heavily loaded with stationary phase 9). 

It may be difficult to imagine a liquid mobile phase used with a liquid stationary phase. What experimental setup allows one liquid to move through another liquid (immiscible in the first) and how can one expect partitioning of the mixture components to occur The stationary phase actually consists of a thin liquid film chemically bonded to the surface of finely divided solid particles, as shown in Figure 11.8. It is often referred to as bonded phase chromatography (BPC). Such a stationary phase cannot be removed from the solid substrate by heat, reaction, or dissolving in the mobile phase. 

FIGURE 11.8 An illustration of partition chromatography. A thin liquid film chemically bonded to the surface of finely divided solid particles is the stationary phase. 

In summary, partition chromatography is a type of chromatography in which the stationary phase is a liquid chemically bonded to the surface of a solid substrate, while the mobile phase is either a liquid or gas. The mixture components dissolve in and out of the mobile and stationary phases as the mobile phase moves through the stationary phase, and separation occurs as a result. Examples of mobile and stationary phases will be discussed in Chapters 12 and 13. 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

The great versatility of HPLC lies in the fact that the stability of the chemically bonded stationary phases used in partition chromatography allows the use of a wide range of liquids as a mobile phase without the stationary phase being lost or destroyed. This means that there is less need for a large number of different stationary phases as is the case in gas chromatography. The mobile phase must be available in a pure form and usually requires degassing before use. The choice of mobile phase (Table 3.6) is influenced by several factors. 

Done, J. N. J. Chromatog. 125 (1976) 43. Sample loading and efficiency in adsorption, partition and bonded-phase high-speed liquid chromatography. 

Partition chromatography. A liquid stationary phase is bonded to a solid surface, which is typically the inside of the silica (Si02) chromatography column in gas chromatography. Solute equilibrates between the stationary liquid and the mobile phase, which is a flowing gas in gas chromatography. 

In gas chromatography,1 2 gaseous analyte is transported through the column by a gaseous mobile phase, called the carrier gas. In gas-liquid partition chromatography, the stationary phase is a nonvolatile liquid bonded to the inside of the column or to a fine solid support... 

Bare silica can be used as the stationary phase for adsorption chromatography. Most commonly, liquid-liquid partition chromatography is conducted with a bonded stationary phase covalently attached to the silica surface by reactions such as... 

I Liquid-liquid partition chromatography, where the sample components are partitioned between a moving liquid phase and a stationary liquid phase deposited on an inert solid. The two solvent phases must be immiscible. The stationary phase may he a large molecule chemically bonded lo the surface of a solid (bonded liquid phase) lo prevent loss by solubility in the moving phase. This method can also be subdivided into normal-phase systems, in which Ihe moving phase is less polar than the stationary phase, and reverse-phase systems, in which it is more polar. 

In MLC, the mobile phase consists of surfactants at concentrations above their critical micelle concentration (CMC) in an aqueous solvent with an alkyl-bonded phase (52). Retention behavior in MLC is controlled by solute partitioning from the bulk solvent into micelles and into stationary phase as well as on direct transfer from the micelles in the mobile phase into the stationary phase. Eluent strength in MLC is inversely related to micelle concentration. A linear relationship exists between the inverse of retention factor and micelle concentration. Similar to what is observed in RPLC, a linear relationship exists between retention in MLC and , the volume fraction of the organic modifier. Modeling retention in MLC is much more complicated than in RPLC. The number of parameters is important. Micelles are obviously a new domain in both liquid chromatography and electrophoresis. Readers interested in the topic will appreciate Ref. 53, a special volume on it. 

LC Tan, PW Carr. Revisionist look at solvophobic driving forces in reversed-phase liquid chromatography. II. Partitioning vs. adsorption mechanism in monomeric alkyl bonded phase supports. J Chromat A 775 1-12, 1997. 

In general, there is a wide variety of chromatographic modes (types) that can be employed for the HPLC determination of food components, but only a few have been used for the determination of NOC. These include partition/adsorption on silica gel, liquid-liquid partition on polar-bonded phase (e.g., cyano, amino) or nonpolar hydrophobic-bonded phase (e.g., reversed-phase), and anion-exchange chromatography. Macrae (61) discussed the theories behind the various modes of chromatography. 

J. J. Kirkland, High speed liquid-partition chromatography with chemically bonded organic stationary phases, J. Chromatogr. Sci. 9 206 (1971). 

If retardation is caused by adsorption on granular solids or other fixed surfaces, the technique is called adsorption chromatography, as noted above. If the solid surfaces merely act as a scaffold to hold an absorbing liquid (which may be of a chosen polarity) in place—perhaps within the pores of solid particles—we have partition chromatography. Many cases lie between these extremes this occurs whenever the solid acts as a support for liquid but retains some adsorptive activity [7]. A special case exists with chemically-bonded phases (CBPs), which usually consist of a one-molecule thick layer of hydrocarbon (often C18) chemically bonded to the solid surface. (These nonpolar phases are frequently used for nonpolar solutes in RPLC.) Partitioning into such a thin layer is affected by the nearby surface, especially since the configuration and motion of each attached molecule is restricted by its fixed anchor to the surface [8]. 

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