Metal interaction chromatography stationary phase

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed phase (RPC) [4], and affinity chromatography (AC) [5]. Besides analytical applications, other possible utilization of micropellicular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular... 

Metal interaction chromatography is an HPLC technique that can separate many biopolymers because of their differential ability to form complexes with metal ions [1]. It employs a stationary phase with an appropriate metal immobilized via chelating functions bound to the surface. Retention and separation of the sample components occur largely by their interaction with the chelated metal. Although immobilized metal affinity chromatography (IMAC) is a common name for the technique, it is called metal interaction chromatography (MIC) in this book to conform to the nomenclature used for the other interactive chromatographic methods for biopolymer separation by HPLC. 

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Affinity Chromatography was initially defined as a method based on specific and reversible molecular interactions between biologically active substances. However, the method has extended to non-biological stationary phases, such as metal-chelate complexes. This technique is used for separation and purification of proteins and other biologic materials, such as viruses and cells.47,48 A survey of various stationary phases and affinity interactions is given in figure 8.2. 

Size exclusion chromatography (SEC, also known as gel permeation chromatography) is a method of separating compounds of different molecular masses and sizes. Because steric interactions between analytes and the stationary phase are relatively weak, unstable forms of metals can be separated from more stable complexes and from adducts stabilized by ionic interactions. Unfortunately, the process of sorption and ionic interactions between the investigated substances and the stationary phase can decrease metal recovery by as much as 50 % these interactions are also responsible for the instability of retention times [146]. The separation can be performed both in the aqueous environment and in the presence of organic solvents. Because the technique is not selective, it is utilized primarily as the first stage of multidimensional chromatography [147]. 

The use of chiral stationary phases (CSP) in liquid chromatography continues to grow at an impressive rate. These CSPs contain natural materials such as cellulose and starch as well as totally synthetic materials, utilizing enantioselective and retentive mechanisms ranging from inclusion complexation to Ti-electron interactions. The major structural features found in chiral stationary phases include cellulose, starch, cyclodextrins, synthetic polymers, proteins, crown ethers, metal complexes, and aromatic w-electron systems. 

Recently, PHEMA microspheres have been more and more extensively used for IMAC. Separon-IDA-Fe(III) or Cu(II) was prepared. It was found that Cu(II) interacted preferentially with histidine and tryptophan residues, while Fe(III) preferred phosphate residues, as demonstrated by the separation of lysozyme, ribonuclease A, myoglobin, and transferrin on the Cu(II) column and ovalbumin on the Ee(III) column, respectively.The PHEMA-Congo Red-Cu(II) and PHEMA-Cibacron Blue E3GA-Zn(II) microspheres were applied for adsorption of BSA. Without incorporating the metal ions, the dyed sorbents were already good stationary phases for affinity chromatography. As shown in Fig. 2, the addi-... 

Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surfooe of the stationary phase, which is strongest when nonpolar solvents are used as mobile phase. Previously, it was also called adsorption chromatography. However, the technique has expanded from the exclusive application of metal oxide adsorbents such as silica and alumina as stationary phases to the use of polar bonded phases. Thus the name adsorption chromatography has become too narrow. 

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