Diol bonded phase, HPLC

In recent years several normal-phase HPLC methods have been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.5). The best of these methods have been able to achieve baseline separation of all four tocopherols and all four tocotrienols, as shown in Figures 11.2 and 11.3. Kamal-Eldin et al. (2000) reported the optimal baseline separation of all eight common tocols using a Diol-bonded phase column and an isocratic mobile phase of hexane/methyl tert-butyl ether (MTBE), 96 4, v/v (Figure 11.2). Similar separations were reported by Moreau et al. (2007) using the same type of column and mobile phase. Schwartz et al. (2008) reported that, with a normal-phase silica column, plastochromanol-8 in rapeseed oil eluted between y-tocopherol and 5-tocopherol. 

In normal phase HPLC, the cyano and amino columns, in conjunction with the diol bonded phase columns, are now often used in preference to unmodified silica or alumina. Whilst silica can demonstrate remarkable selectivity for the separation of closely related isomers (e.g. E/Z isomers are commonly separated on silica), some of the operational demands of silica limit its usefulness. The difficulty of manufacturing reproducible microcrystalline silica and the possible effect of trace amounts of water on... 

Modified-C02 mobile phases excel at stereochemical separations, more often than not outperforming traditional HPLC mobile phases. For the separation of diastereomers, silica, diol-bonded silica, graphitic carbon, and chiral stationary phases have all been successfully employed. For enantiomer separations, the derivatized polysaccharide, silica-based Chiralcel and Chiralpak chiral stationary phases (CSPs) have been most used, with many applications, particularly in pharmaceutical analysis, readily found in the recent literature (reviewed in Refs. 1 and 2). To a lesser extent, applications employing Pirkle brush-type, cyclodextrin and antibiotic CSPs have also been described. In addi-... 

Packed column SFC stationary phases are very similar or identical to those used for HPLC. With neat CO2 mobile phases, polymer or polymer-coated silica stationary phases have typically been used. With modified-C02 mobile phases, bonded-phase silica columns are typically used. For structural separations, diol, amino, or cyano stationary phases are most often used. For stereochemical separations, derivatized polysaccharide-bonded silica columns are most often the stationary phases of choice. A powerful feature of modified-C02 pSFC is the ability to serially connect different stationary phases to obtain enhanced or mul-... 

There are many applications of normal-phase chromatography to separate all eight vitamers using silica or bonded silica phases such as diol or amino (Table 1.5 Figure 1.4). The separation of 3- and y-tocopherols and tocotrienols is the most difficult task, but it can be achieved with normal-phase HPLC. In addition to its... 

The type, if any, of HPLC method used. These were separated into normal phase, reverse phase (C-18, C-8, C-4), other bonded phases (e.g., DIOL, CN, NH2), and instances in which some combination of the above were used. 

One of the first separations of statistical copolymers using gradient HPLC was carried out by Teramachi et al. [34]. Mixtures of poly(styrene-co-methyl acrylate)s were separated by composition on silica columns through a carbon tetrachloride/methyl acetate gradient (see Fig. 11). When increasing the content of methyl acetate in the eluent, retention increased with increasing methyl acrylate content in the copolymer. This behavior fitted the normal-phase chromatographic system used. Similar separations could be achieved on other columns as well, such as polar bonded-phase columns (diol, nitrile, amino columns) [1]. 

Near-critical pSFC applications can be described as those where the mobile phase is solvent-modified CO2, pressurized only enough to maintain a single phase, with temperatures near (typically less than) the critical temperature. Many commercially available HPLC bonded silica phases have been used with modified-C02 mobile phases to achieve normal-phase separations, the choice of stationary phase being dictated by sample polarity. The modifiers added to CO2 acceptably overcome the unwanted analyte-sUica interactions observed with neat CO2 mobile phases. For structural separation of polar compounds such as pharmaceuticals [typically weak acids or bases of molecular weight (MW) < 1000], polar phases such as diol-, amino-, and cyano-bonded silica (or bare sihca) are used. Numerous apphcations for pharmaceutical, natural product, environmental and other compound classes have been reported in the recent hteratiu e (reviewed in Refs. ). For structural separation of higher-molecular-weight, less polar compounds, octyl- or octadecyl silane (ODS)-bonded phases are used... 

A new HPLC technique has been developed for the rapid analysis of sesquiterpene phytoalexins such as capsi-diol, rishitin, luminin, and phytuberol. This method employs a cyanopropyl-bonded-phase column with an iso-cratic mixture of hexane and isopropanol. Flame ionization and UV detection were used for the analysis of capsidiol, rishitin, lubimin, phytuberol, and debneyol. Although both detectors proved to be useful, the signal response with the flame ionization detector was proportional to the mass of each of the phytoalexins, while the signal with the UV detector was proportional to the number of carbon-carbon bonds in each of the compounds. 

Analytical screens are performed with both reverse-phase RP-HPLC and SFC isolation techniques. Analytical SFC should be screened first unless instrumentation availability or project background specifics dictate otherwise. Screening achiral column bonded phases varying in polarity and functionality against different mobile-phase solvent choices is effective for identifying analytical methods for the purpose of impurity isolation. There are currently many unique achiral SFC bonded phase column choices commercially available (2-ethyl pyridine, diethyl amino, dinitrophenyl, pyridine urea, diol, cyano, etc.). SFC column choice provides the most impact in manipulation of relative selectivity for individual... 

An alternative to liquid-liquid extraction is solid-phase extraction (SPE). With SPE a liquid sample is introduced into the top of a plastic syringe shape column containing a small amount (often 100- 500 mg) of a selective adsorbent (Figure 8.1). The adsorbents are of the same types as used for HPLC, typically silica, or bonded silica such as Cl 8, C8, C5, C2, cyano, phenyl, diol and ion-exchange materials. The properties of the adsorbents are similar to HPLC columns and so the same principles apply for retention and desorption of analytes. 

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