Reverse phase chromatography silica based columns

The setnp for polymer HPLC is qnite similar to SEC systems with a few modifications. Liqnid Adsorption Chromatography (LAC) requires the adsorption and desorption on a stationary phase. Therefore, in most cases isocratic separation is not sufficient. Gradients with respect to pH valne, ionic strength, eluent composition, or temperature are applied. The most common approach is to use eluent composition gradients. In contrast to SEC where polymeric phases dominate, silica-based column packings are the most important stationary phase. Both normal phase and reversed phase separations have been described. A summary of different applications in copolymer separation is offered by Pasch [27]. Detectors used in gradient LAC are mainly UV/DAD detectors and ELSD. 

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]. 

The factors that control separation and dispersion are quite different. The relative separation of two solutes is solely dependent on the nature and magnitude of the Interactions between each solute and the two phases. Thus, the relative movement of each solute band would appear to be Independent of column dimensions or particle geometry and be determined only by the choice of the stationary phase and the mobile phase. However, there is a caveat to this statement. It assumes that any exclusion properties of the stationary phase are not included in the term particle geometry. The pore size of the packing material can control retention directly and exclusively, as in exclusion chromatography or, indirectly, by controlling the access of the solute to the stationary phase in normal and reverse phase chromatography. As all stationary phases based on silica gel exhibit some exclusion properties, the ideal situation where the selective retention of two solutes is solely controlled by phase interactions is rarely met in practice. If the molecular size of the solutes differ, then the exclusion properties of the silica gel will always play some part in solute retention. 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

Reversed-phase chromatography is the most popular mode of analytical liquid chromatography for phenolic compounds. In most cases, the reported systems for the separation of phenolics and their glycosides in foods are carried out on reversed-phase chromatography on silica-based Cl8 bonded-phase columns. Occasionally, silica columns bonded with C8 were applied in the analysis of phenolic acid standards and coumarins (7), and C6 columns for the analysis of ferulic acid in wheat straw (8). 

Most cases of isocratic HPLC reported were carried out on reversed-phase chromatography on silica-based ODS (C,8 bonded phase) columns (1) for citrus juices. For mobile phases utilizing C,8 columns, isocratic solvents of 80-82% aqueous acetic acid with acetonitrile were commonly used. A ternary mobile-phase system of water-acetonitrile-glacial acetic acid... 

Silica monoliths are a new class of bonded-phase column in which the inside of the column is completely filled with silica foam that has had a bonded phase attached to it. It should run like a high-efficiency reverse-phase column, but with much lower back-pressure. Realize that they are a new type of column and you may have to adjust your chromatography to get them to run in the same way as your other silica-based columns. Look at it as an adventure into the future. 

Kirkland JJ.Truszkowski FA, Ricker RD Atypical silica-based column packings for high-performance liquid chromatography. /. Chromatogr. A (2002) 965 25-34. Kirkland JJ Development of some stationary phases for reversed-phase high-performance liquid chromatography. J. Chromatogr. A (2004) 1060 9-21. 

H. A. Claessens, M. A. van Straten, and J. J. Kirkland, Effect of buffers on silica based column stability in reversed phase high performance liquid chromatography, J. Chromatogr. A 728 (1996), 259-270. 

S.6.4.2 Reversed-phase ion-pair liquid chromatography. Reversed-phase ion-pair chromatography is an alternative approach for controlling the retention of ionic compounds. This approach is particularly useful for the separation of amines on silica-based columns and it has had a profound effect on the analysis of this class of compounds. In particular, the combination of reversed-phase ion-pair liquid chromatography and electrochemical detection revolutionized the analysis of neurotransmitters in the brain (Tomlinson et al., 1978 and refs, therein). 

Figure 7.23. HPLC analysis of a protein mixture using reversed-phase chromatography using a silica-based column and UV detection at 215nm. Reprinted with permission from reference 35.

The stationary phases used in reversed-phase chromatography, when it was first introduced, comprised of a non-polar substance (e.g. squalene) coated on to a silica-based support. These are now seldom used. The stability of such systems is low, because the forces holding, say, squalene to even a silylated silica are so weak that the stationary phase is easily washed from the column. A compromise reversed-phase packing material was developed, which had a polymeric hydrocarbon stationary phase on the support, but although quite successful it has now been superseded by a chemically bonded stationary phase of which some examples are discussed below. 

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