Partition<unk>  HILIC Partition Technique Useful Range Partition<unk>  was one of the first kinds of<unk>  that chemists developed. The partition coefficient principle has been applied in paper<unk> , thin layer<unk> , gas phase and<unk> –liquid separation applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition<unk>  uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper<unk> ; or takes advantage of some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules partition between a<unk>  stationary phase and the eluent. Just as in Hydrophilic Interaction<unk>  (HILIC; a sub-technique within<unk> , this method separates analytes based on differences in their polarity. HILIC most often uses a bonded polar stationary phase and a mobile phase made primarily of acetonitrile with water as the strong component. Partition<unk>  has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. HILIC bonded phases have the advantage of separating acidic, basic and neutral solutes in a single<unk> c run. The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups (e.g., hydroxyl-) and groups capable of hydrogen bonding inducing more retention. Coulombic (electrostatic) interactions can also increase retention. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times. Normal–phase<unk>  Normal–phase<unk>  was one of the first kinds of<unk>  that chemists developed. Also known as normal-phase<unk>  (NP-HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g., Chloroform), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers. The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. This behavior is somewhat peculiar to normal phase<unk>  because it is governed almost exclusively by an adsorptive mechanism (i.e., analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed-phase<unk>  below). Adsorption<unk>  is still widely used for structural isomer separations in both column and thin-layer<unk>  formats on activated (dried) silica or alumina supports. Partition- and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase<unk>  because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or alumina<unk> c media. This layer changes with any changes in the composition of the mobile phase (e.g., moisture level) causing drifting retention times. Recently, partition<unk>  has become popular again with the development of Hilic bonded phases which demonstrate improved reproducibility, and due to a better understanding of the range of usefulness of the technique. Displacement<unk>  The basic principle of displacement<unk>  is: A molecule with a<unk>  affinity for the<unk>  matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution<unk> . In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the<unk>  matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode<unk> , especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement<unk>  has advantages over elution<unk>  in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly<unk> er concentration. Reversed-phase<unk>  (RPC) A chromatogram of complex mixture (perfume water) obtained by reversed phase<unk>  Reversed phase<unk>  (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release. RP-HPLC operates on the principle of hydrophobic interactions, which originates from the<unk>  symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase. This solvophobic effect is dominated by the force of water for "cavity-reduction" around the analyte and the C18-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3 J/cm², methanol: 2.2 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis. Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C–H, C–C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. On the other hand, analytes with<unk> er polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO− or -NH3+ in their structure) are less retained as they are better integrated into water. Such interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention. Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C–C bonds elute later than those with a C=C or C–C triple bond, as the double or triple bond is shorter than a single C–C bond. Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 J/cm² per Mol for NaCl, 2.5 J/cm² per Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition
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