Ion-exchange chromatography, HPLC

Several methods have been used to separate the lanthanides chemically solvent extraction, ion exchange chromatography, HPLC using Q-hydroxyisobutyric acid and, in limited cases, selective reduction of a particular metal cation.40-43 The use of di(2-ethylhexyl)orthophosphoric acid (HDEHP) for the separation of various rare-earth elements via solvent extraction has also been reported.44 16 This separation method is based on the strong tendency of Ln3+ ions to form complexes with various anions (i.e., Cl- or N03 ) and their wide range of affinities for com-plexation to dialkyl orthophosphoric acid. When the HDEHP is attached to a solid phase resin, the lanthanides can be selected with various concentrations of acid in order of size, with the smallest ion being the most highly retained. 

Characterization was performed via amino acid analysis after 6 N HCl hydrolysis, nucleoside analysis after enzyme digestion, and mass spectrometry. The homogeneity of the conjugates was analyzed by ion-exchange chromatography. HPLC conditions were as follows column. Mono Q HR 5/5 (Pharmacia) flow rate 0.5 mL/min solvent A 10 mM NaOH, 0.3 M NaCl, pH 12.5 solvent B, 10 mM NaOH, 0.9 M NaCl, pH 12.5 a 50-min linear gradient from 10% B to 60% B. 

The free oil can be determined by an ion exchange HPLC technique. A solution of the sample in ethyl alcohol is analysed by high-performance ion exchange chromatography using a specially prepared ion exchange resin stationary phase, ethanol mobile phase, and differential refractive index detection. 

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 formed 5 -protected 2, 3 -(9,(9-cyclic boranophosphate 26 was finally deprotected by acid treatment and isolated in 70% overall yield by ion-exchange chromatography (Scheme 10) [24], The two pure P-diasteromers of 27 were separated by HPLC. 

Ion-exchange chromatography (both cation and anion) can also be undertaken in HPLC format. Though not as extensively employed as reverse-phase or size-exclusion systems, ion-exchange-based systems are of use in analysing for impurities unrelated to the product, as well as detecting and quantifying deamidated forms. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

Column chromatographic techniques were originally used as preparative tools but over the years major advances have taken place. HPLC is now a highly developed technique and a wide range of stationary phases are available. These enable partition, adsorption, gel permeation, affinity and ion-exchange chromatography to be performed. 

To apply a screening approach to proactive method development, analyses of selectivity samples under a variety of mobile phase conditions are conducted on different HPLC columns. HPLC columns should be as orthogonaT as possible and variations in solvent composition should be designed to maximize the probability of selectivity differences. Alternate separation techniques, such as ion exchange chromatography (IC), supercritical fluid chromatography (SFC), or capillary electrophoresis (CE) may also be used to obtain orthogonality. 

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