Chemically bonded phases hydrophobicity

Miller, N.T. and Shieh, C.H., Preparative hydrophobic interaction chromatography of proteins using ether based chemically bonded phases, J. Liq. Chromatogr., 9, 3269, 1986. 

Reversed-phase PLC precoated plates are based on silica gel matrices with chemical modifications in such a manner that the accessible polar, hydrophilic silanol groups at the silica gel surface are replaced by nonpolar, hydrophobic alkyl chains via silicon-carbon bonds. For preparative purposes, up to now only PLC precoated RP plates with C-18 modification are available. This abbreviation is often also designated as RP-18, meaning that an octadecyl alkyl chain is chemically bonded to the silica gel surface. 

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

Many other types of solid phase adsorbents, including those based on conventional and specialty materials like restricted access media (RAM), can increase analysis speed and improve assay performance. These types of materials, also known as internal reversed-phase packings, are especially useful for assaying target compounds in biological samples such as serum and plasma. They are chemically modified porous silicas that have hydrophilic external surfaces and restricted-access hydrophobic internal surfaces. The ratio of interior to external surface areas is large. Macromolecules such as proteins cannot enter the pores of the RAM (they are excluded from the hydrophobic internal surface) and they elute quickly through the column. However, the smaller analyte molecules that can enter the pores are retained via interactions with the hydrophobic bonded phase within... 

Reversed-phase chromatography is the predominant technique in HPLC, and chemically bonded silica gel supports are made specifically for the nonpolar stationary phase. In the last decade, as many as 60% of the published LLPC techniques refer to RPC. The reasons for this involve the significantly lower cost of the mobile liquid phase and a favorable elution order that is easily predictable based on the hydrophobicity of the eluate. 

Besides the above differentiation, restricted-access media can be further subdivided on the basis of the topochemistry of the bonded phase. Packings with a uniform surface topochemistry show a homogenous ligand coverage, whereas packings with a dual topochemistry show a different chemical modification of the pore internal surface and the particle external surface (114). Restricted-access media of the former type are divided into mixed-mode and mixed-function phases, bonded-micellar phases, biomatrix, binary-layered phases, shielded hydrophobic phases, and polymer-coated mixed-function phases. Restricted-access media of the latter type include the Pinkerton s internal surface reversed-phase, Haginaka s internal surface reversed-phase diol, alkyl-diol silica, Kimata s restricted-access media, dual-zone phase, tris-modified Styrosorb, Svec s restricted-access media, diphil sorbents, Ultrabiosep phases. Bio Trap phases, and semipermeable surface phases. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

Assume that the hydrophobic bond is a major component of the binding interaction of the gas with the antibody binding site. What is the consequence for that type of chemical bond if performing this reaction is carried out in gas phase ... 

Two main theories, the so-called solvophobic and partitioning theories, have been developed to explain the separation mechanism on chemically bonded, non-polar phases, as illustrated in Figure 2.4. In the solvophobic theory the stationary phase is thought to behave more like a solid than a liquid, and retention is considered to be related primarily to hydrophobic interactions between the solutes and the mobile phase14-16 (solvophobic effects). Because of the solvophobic effects, the solute binds to the surface of the stationary phase, thereby reducing the surface area of analyte exposed to the mobile phase. Adsorption increases as the surface tension of the mobile phase increases.17 Hence, solutes are retained more as a result... 

The most common way to create an RP-IPC system is to use a genuine chemically bonded reversed phase column (e.g. C18 see section 3.2.2.1) and to use large pairing ions with a hydrophobic alkyl chain dissolved in the mobile phase. This technique was introduced by Knox and Laird, who named it soap chromatography [380]. Because of the usually long alkyl chains of the pairing ions, the use of Cl 8 phases is to be recommended in order to avoid effects that are related to the critical chain length (see section 3.2.2.1). 

Another AFM-based technique is chemical force microscopy (CFM) (Friedsam et al. 2004 Noy et al. 2003 Ortiz and Hadziioaimou 1999), where the AFM tip is functionalized with specific chemicals of interest, such as proteins or other food biopolymers, and can be used to probe the intermolecular interactions between food components. CFM combines chemical discrimination with the high spatial resolution of AFM by exploiting the forces between chemically derivatized AFM tips and the surface. The key interactions involved in food components include fundamental interactions such as van der Waals force, hydrogen bonding, electrostatic force, and elastic force arising from conformation entropy, and so on. (Dther interactions such as chemical bonding, depletion potential, capillary force, hydration force, hydrophobic/ hydrophobic force and osmotic pressure will also participate to affect the physical properties and phase behaviors of multicomponent food systems. Direct measurements of these inter- and intramolecular forces are of great interest because such forces dominate the behavior of different food systems. 

In common with other application areas of chromatographic separation, a considerable amount of effort has been expended recently on the development of different elution conditions and types of stationary phases for peptide separations in attempts to maximize column selectivities without adversely affecting column efficiences. Peptide retention will invariably be mediated by the participation of electrostatic, hydrogen bonding, and hydrophobic interactions in the distribution phenomenon. The nature of the predominant distribution mechanism will be dependent on the physical and chemical characteristics of the stationary phase as well as the nature of the molecular forces which hold the solute molecules within the mobile and stationary zones. The retention of the solute in all HPLC modes can be described by the equation... 

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