Stationary-phase separation capability

No single stationary phase is capable of separating every acid - although capillary-gas chromatography (where the columns have dimensions such as 50 m by 0.25 mm interior diameter) usually separates most components including positional isomers. Generally two different types of column are necessary to resolve all the individual components. 

The selectivity (separation capability) of an HPLC system is dependent upon the combination of mobile and stationary phases. Since ions are being generated directly from the mobile phase by electrospray, its composition, including the identity and concentration of any buffer used, and its flow rate are important considerations. 

The addition of metal ions to the mobile phase frequently yields Improved separations of solutes capable of forming complexes (conversely the addition of ligands to the mobile phase may allow the separation of metal ions based on differences in the distribution constants of the complexes between the mobile phase and, stationary phase) [353-355]. A number of important... 

A number of ketones, pharmaceutical compounds, alcohols and hydroxy acids have also been resolved on this phase [724,765-767]. A chiral polysiloxane phase with tartramide substituents has been used for the separation of enantiomers capable of hydrogen bonding interactions with the stationary phase, such as enantiomers containing carboxylic, hydroxyl and amine functional groups [768]. 

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]. 

Surfactants are separated according to adsorption or partitioning differences with a polar stationary phase in NPLC. This retention of the polar surfactant moiety allows for the separation of the ethylene oxide distribution. Of all the NPLC packings that have been utilized to separate nonionic surfactants, the aminopropyl-bonded stationary phases have been shown to give the best resolution (Jandera et al., 1990). The separation of the octylphenol ethoxylate oligomers on an amino silica column is shown in Fig. 18.4. Similar to the capabilities of CE for ionic surfactants, the ethylene oxide distribution can be quantitatively determined by NPLC if identity and response factors for each oligomer are known. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

The selection of a stationary phase depends largely on trial and error or experience, with consideration given to the polar nature of the mixture, as noted in Table 12.3 or a similar table. The usual procedure is to select a stationary phase, based on such literature information, and attempt the separation under the various conditions of column temperature, length, carrier gas flow rate, etc., to determine the optimum capability for separating the mixture in question. If this optimum resolution is not satisfactory (see Section 12.6), then an alternate selection is apparently required. 

Since the commercial introduction of the P-CAC in 1999, several industrial applications have been shown to be transferable to the system. Moreover, users in the biopharmaceutical and foodstuff industry have seen their productivity increasing dramatically as a result of using the P-CAC technology. Furthermore, a P-CAC has been shown capable of continuously separating stereoisomers when using chiral stationary phases even when there is more than one chiral center in the desired molecule. Below some of the applications are described in more details. Others are proprietary and hence cannot be disclosed. 

Interestingly, in HPLC the stationary phase and the mobile-phase is able to interact with the sample selectively. Besides, such interactions as hydrogen bonding or complexation which are absolutely not possible in the GC-mobile phase may be accomplished with much ease in the HPLC-mobile phase. Furthermore, the spectrum of these selective interactions may also be enhanced by an appropriate chemical modification of the silica surface the stationary phase. Therefore, HPLC is regarded as a more versatile technique than GC and capable of achieving more difficult separations. 

The first consideration when investigating HPLC method development protocols is the chemical structure of the analyte, in particular, the presence of functional groups capable of interacting with the stationary phase and containing or in the vicinity of the stereogenic elements [79]. Since the natural target of macrocyclic antibiotics is the A-acyl-D-alanyl-D-alanine terminus (see Section 2.1), the early choice of suitable substrates for this kind of CSPs was that of amino acids [45]. However, it turned out that the macrocyclic CSPs were very successful not only in amino acids enantioresolution, but also in the separation of a wide variety of different structures. 

CEC is a miniaturized separation technique that combines capabilities of both interactive chromatography and CE. In Chapter 17, the theory of CEC and the factors affecting separation, such as the stationary phase and mobile phase, are discussed. The chapter focuses on the preparation of various types of columns used in CEC and describes the progress made in the development of open-tubular, particle-packed, and monolithic columns. The detection techniques in CEC, such as traditional UV detection and improvements made by coupling with more sensitive detectors like mass spectrometry (MS), are also described. Furthermore, some of the applications of CEC in the analysis of pharmaceuticals and biotechnology products are provided. 

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