Columns, reversed HPLC separation mechanism

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]. 

In an attempt to rationalize the large variety of buffers used to date in protein HPLC separations, it is necessary to briefly return to the separation mechanism based on the hydrophobic effect. This mechanism related the retention of a sample on a reversed-phase column to the nonpolar surface area of a sample molecule. A possible explanation of the dramatic effect of ion-pairing reagents on the retention of proteins on reversed-phase columns can then be based on the modification of surface polarity of the protein molecule on association with suitable counterions. In the absence of salts dissolved in the mobile phase, the peptide or protein sample probably has some counterions associated with the sample. Alternatively, the basic side chains of the protein may be neutralized by a salt bridge with an acidic residue which is adjacent in the three-dimensional structure. In either case, the contribution of the ammonium group to the surface polarity is relatively small. Figure 13 shows the result of association of the amine cation with a highly polar anion such as phosphate, which has a substantial sphere of hydration. In this case, the nonpolar area of the... 

Combining different separation methods, governed by different separation mechanisms, to multidimensional methods is suitable for multiplying the potential of the individual techniques. Reversed-phase chromatography high-performance columns (RP-HPLC) can be coupled with normal phase TLC [9,10]. 

Ion Chromatography. Ion chromatography (IC) is a mode of HPLC in which ionic analyte species are separated on cationic or anionic sites of the stationary phase. The detection techniques largely fall under three categories electrochemical, spectroscopic, or post-column reactions. In general, IC provides an orthogonal separation mechanism to traditional reversed-phase HPLC (RP-HPLC).54 63 This technique can be exploited to quantify ionic... 

With normal-phase HPLC, oil samples were analyzed as is by simple dilution in n-hexane. A Du Pont Zorbax amino-bonded phase column, 25 cm x 0.46 cm ID, was used, with n-hexane and dichloromethane as solvents. For reversed-phase HPLC, Vydac 201TP5 columns were used (25 cm x 0.46 cm ID for analytical scale and 25 cm x 1 cm ID for preparative scale). Samples for reversed-phase HPLC were fractionated in order to remove the saturated hydrocarbons which can interfere with the separation mechanism. The samples dissolved in n-hexane were passed Baker silica solid-phase extraction cartridges. The PAH fraction was then collected by eluting with a 1 1 mixture of dichloromethane and methanol. Acetonitrile and dichloromethane were used in the HPLC gradient. 

Of these, partition chromatography, especially the reversed-phase (RP) mode, is the most widely used technique for the separation of several classes of compounds. In RP-HPLC (see Table 5.1), the stationary phase is a nonpolar matrix and the mobile phase is a polar solvent (e.g., water mixed with a polar organic modifier such as methanol, isopropanol, or acetonitrile). Mobile phase and stationary phase both play prominent roles in the separation mechanism. A sample is applied onto the head of a column filled with an appropriate stationary phase. The... 

Despite the fact that, in some cases, small differences in AH and A5 can be observed (for various solutes, but on the same column with the same mobile phase), these differences could be found to be essentially insignificant when compared to a change in stationary-phase bonding density using enthalpy-entropy compensation. Enthalpy-entropy compensation is a term used to describe a compensation temperature, which is system independent for a class of similar experimental systems.Melander et have used the enthalpy-entropy compensation method in studies of hydrophobic interactions and separation mechanisms in reversed phase HPLC. Mathematically, enthalpy-entropy compensation can be expressed by the formula 9 ... 

The anthocyanins are most often separated by HPLC on a Cig column with long gradients to achieve the best chromatographic resolution [31-33]. A new approach using a HPLC column that combines both ion-exchange and reversed-phase (RP) separation mechanisms showed significant improvement in chromatographic... 

Mobile-phase ion chromatography, a technique which uses traditional ion chromatography equipment, a nonpolar poly(styrene/divinylbenzene) separation column, a suppressor column, and conductivity detection, is suitable for analysis of cationic surfactants. Dilute perchloric acid in 70 volume percent acetonitrile is used as eluent (13). The separation mechanism is the same as in the reversed-phase HPLC methods discussed above. 

Discussed below are various modes of separations in HPLC. Included here is brief coverage of mobile-phase selection for various modes of chromatography and elementary information on mechanism, choice of solvents and columns, and other practical considerations. It should come as no surprise that reversed-phase HPLC is discussed at greater length in this section because it is the most commonly used technique in HPLC (more detailed discussion is provided in Section 15.8). Clearly,... 

Irrespective of the mechanism of resolution, HPLC CSPs work by providing a chiral environment for analyte stereoisomers to interact with. Resolution relies upon the formation of reversible, transient diastereomers on the CSP that have different free energies of interaction and therefore stability. The stereoisomer forming the most stable diastereomer with the CSP will be the most retained and vice versa. Free energy differences are typically small in such systems but may be large enough to produce useable resolutions provided the column efficiency is sufficient [41]. If column efficiency is insufficient to allow complete separation of the stereoisomers, inaccurate integrations can result in erroneous... 

In certain cases, such as the separation of PAHs obtained from a coal liquefaction process, using reversed-phase HPLC is complicated as sample preparation is elaborate. This is due in large part to the fact that most complex fuel-related materials contain compounds that are not usually soluble in acetonitrile, the solvent of choice in reversed-phase HPLC. Here, NPC, which employs a variety of solvents, offers an alternative to the analysis of such samples. Separation of five well-studied coal liquefaction process stream samples was achieved and 19 isomers were resolved when NPC was used [33]. The method employed a tetrachlorophthalimidopropyl-modified silica column (TCPP) with a charge-transfer mechanism. 

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