Chapters Reverse-phase liquid chromatography

The column methods are much faster and are automated so that a much larger number of samples can be processed per unit time. An example of this technology, described in more detail in Chapter 10 by Lubman and coworkers, is shown in Figure 1.2, where the first dimension is from a chromatofocusing column, which gives separations in pI much like isoelectric focusing, only here the p/ axis is in bands instead of continuous pI increments. The second dimension is by reversed-phase liquid chromatography (RPLC). 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

A quantitative analysis of the structure-retention relationship can be derived by using the relative solubility of solutes in water. One parameter is the partition coefficient, log P, of the analyte measured as the octanol-water partition distribution. In early work, reversed-phase liquid chromatography was used to measure log P values for drug design. Log P values were later used to predict the retention times in reversed-phase liquid chromatography.The calculation of the molecular properties can be performed with the aid of computational chemical calculations. In this chapter, examples of these quantitative structure-retention relationships are described. 

This work uncovered the fact that a substitution of the amino acid alanine for valine at position 126 in the /3-chain of hemoglobin occurred in a hemato-logically normal adult of Lebanese extraction. The variation /3-globin was initially observed and subsequently purified by reverse-phase liquid chromatography (see Fig. 2-5). Also LC was used to isolate the variant tryptic peptide of /3-T13 that had alanine replacing the valine at amino-acid residue position 126. This is shown in Figure 2-6. (Peptide mapping is discussed later in this chapter.)... 

In reversed phase liquid chromatography (RPLC) silylated silicas are preferred. The surface of these silicas is covered with chemically bonded non-polar groups such as alkyl chains or polymeric layers (Chapter 3.2.3). Silica modified with medium polar groups such as cyano, diol or amino might be used in NP as well as RP mode. Alternatively, cross-linked polymers such as hydrophobic styrene divinyl benzene-copolymers can be used (Chapter 3.2.4). Polymer packings show stability in a pH range 2-14 while silica based packings show limited stability for pH > 7. 

The focus is on concepts in reversed-phase liquid chromatography (RPLC), though the same concepts are usually applicable to other modes of HPLC. International Union of Pure and Applied Chemistry (IUPAC)10 nomenclature is used. The term sample component is often used interchangeably with analyte and solute in the context of this book. As mentioned in Chapter 1, the most common stationary phase is a hydrophobic C18-bonded phase on a silica support used with a mixed organic and aqueous mobile phase. The terms packing and sorbent often refer to the bonded phase whereas solid support refers to the unbonded silica material. 

These are all acid labile (see illustrated mechanism). Most common solid phase resins are also shown, all of which are labile to acidic release conditions too (the rink amide leaves a C-terminal amide in place). During global deprotection and resin removal, scavengers such as phenol, cresol or thioanisole are also included to capture reactive cationic species post deprotection. The desired product oligo-/p°lypeptide is then separated initially by precipitation by means of an agent such as methoxy-tert-butyl ether (MTBE) and purified finally by reversed phase liquid chromatography (see later in Chapter 2). 

The application of high performance liquid chromatography (HPLC) to the analysis of amino acids forms the subject matter of another chapter HPLC is one of the more recent developments in ammo acid determinations, and the use of precolumn fluorogenic denvatization in combination with reversed-phase liquid chromatography has simplified and improved the separation of amino acids. This system combines simplicity of sample preparation with high sensitivity and speed of analysis. Not surprisingly, HPLC is the system of choice for amino acid analysis by an ever-mcreasmg number of laboratories. 

In the preceding chapter the different products/materials, in which alpha acids have to be determined, were mentioned. Undoubtedly hops, hop pellets and hop extracts are the most important. Analysis of alpha acids in wort and beer may also be worthwhile. Of all the possible methods to quantify alpha acids in these products, conductometry and reversed phase liquid chromatography are, at the present time, the only practical possibilities. In this Chapter these methods will be discussed in more detail. 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

The selection of the counter-ion and its concentration are important for the separation of ionic compounds in reversed-phase and ion-exchange liquid chromatography. The addition of hydrophobic ions is an especially powerful method and several surfactants can be used as hydrophobic counter-ions. The theoretical column efficiency of ion-pair liquid chromatography is much better than that of an ion-exchange column, and the regeneration of a column is much faster. Thus, if we can control ion-pair liquid chromatography, we can solve a separation problem. (The important background sources in this area are listed at the end of the chapter.)... 

Holland and Jorgenson reported separating amines using anion exchange and reversed-phase columns in 1995 and since then, there have been numerous reports of combining two LC columns (2D-LC) to achieve efficient sample separation. In addition to the few references mentioned in this section, see Chapter 4 in this handbook on two-dimensional comprehensive liquid chromatography. 

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