Reverse-phase HPLC parallel

In principle, the analytical results obtained by the GPC spin column/HPLC ESI-MS methodology described in this chapter should be similar to the results obtained using the tandem chromatographic method of GPC/reversed-phase HPLC ESI-MS described in Chapter 3. There are practical advantages for each method. Since each of the chromatographic and mass spectral steps are done serially for the GPC spin column/HPLC ESI-MS methodology, each of the steps can be performed and optimized individually. In the event of mass spectrometer failure, the production of spin column eluate samples can proceed and samples can be stored for future analysis. In contrast, the parallel methodology of tandem GPC/ reversed-phase HPLC ESI-MS requires the simultaneous optimization of multi-... 

Link et al. have used a two-dimensional chromatographic separation approach to characterize yeast ribosome complex proteins (Link, 1999). This technique employs a cation exchange column (SCX) for the first separation and, subsequently, two parallel reversed-phase HPLC columns (Figure 15.4), and thus works extremely rapidly and efficiently. While the first column loads, the second elutes using an acetonitrile gradient The flow from the column is directed to parallel online MS detectors as well as to offline fraction collection with UV detectors. 

Flowing FS-MMLLE with on-line hyphenation to FtPLC has also been investigated. Sandahl et al. were the first to interface FS-MMLLE with reversed-phase HPLC for the on-line extraction of methyl-thiophanate in natural water, obtaining an LOD of 0.5 pg L-1.89 Also, a parallel FS-SLM and FS-MMLLE design was coupled on-line to reverse-phase HPLC for the extraction of methyl-thiophanate (by MMLLE) and its metabolites (by SLM) in natural water.90 In addition, on-line coupling of FS-MMLLE and normal-phase HPLC has been successfully applied in the determination of vinclozolin (Ee =118 and LOD = 1 pg I. ) in surface water91 and of in-sample ion-paired cationic surfactants (Ee > 250 and LOD = 0.7-5 ug L-1) in river water and wastewater samples.92... 

As is common for many biological systems, the hydrophobic effect plays an important role in both models. The effect contributes a favorable energetic state to the close association of nonpolar (hydrophobic) surfaces in aqueous environments. In the absence of such an association, the nonpolar surfaces must be exposed to an aqueous environment and considerable energy is required to form a solvent cavity for the molecules. There may well be a useful parallel between these models and the mechanism of separation of proteins by reversed-phase HPLC. 

Although this variation in behavior was initially found for only a small set of peropyrene isomers, it has since been seen for many other PAHs of various structural types by using both normal- and reverse-phase HPLC (7, 9). The parallel trends in both the chromatographic and spectral behaviors were also seen. 

In Table 2 appear complexes of transition metals with acetylenic compounds. A study was made of the structure and interconversion of acetylene complexes with binuclear transition metals, where acetylene lies parallel or perpendicular to the metal-metal bond" . Carbonyl alkyne complexes with binuclear iron give good separations in reverse-phase HPLC 4... 

As mentioned above, ESI instruments were coupled to liquid chromatography. The biggest impact in ESI MS has been the adaptation to reduced flow capabilities in the 10-500 nl/min range. Wilm and Mann [115] and Emmett and Caprioli [116] developed such improvements in parallel. The combination of nano-flow LC with micro-capillary reversed phase HPLC and nano-ES has i) dramatically improved the sensitivity of ESI-MS/MS and ii) enabled the automation of protein identification by using an auto sampler for loading of samples onto the LC [117]. 

In a parallel study Hullin, Kim and Salem (1989) have described the reversed-phase separation of individual molecular species of PE and PS (rat brain and human red blood cells) as the trinitrobenzenesulphonic acid (TNP) derivatives. This allowed excellent resolution of the diacyl and alken-ylacyl species of PE. The identity of the resolved species was confirmed by TS/MS. The TNP derivatives fragmented in a manner similar to other PLs, with DGs and MGs providing the base peaks. Under reversed-phase HPLC conditions, the retention times of the TPN derivatives depended both on the polar head-group and on the fatty acid composition of the PL. The TNP derivatives of PS eluted at a lower methanol concentration than the corresponding species of PE. The brain PS was resolved into 11 components, the identities of which were confirmed by LC/TS/MS (Kim and Salem, 1987). 

Retinal is not always well resolved from retinol in reversed-phase HPLC systems resolution is affected both by the stationary phase and by the mobile phase composition. Curley et al. found that methanol water was superior to acetonitrile water for resolution of retinol from retinal on an end-capped C18 column, whereas acetonitrile water was superior for separating retinal from retinol on a non-end-capped column (130) this difference presumably is due to hydrogen bonding between the analytes and the mobile phase or stationary phase. Plots of log (k ) versus carbon number are linear and parallel for reversed-phase separation of alcohol versus aldehyde/ketone analogs of apo-retinoids and apo-carot-enoids using methanol water mobile phases, but nonparallel for acetonitrile water mobile phases (H.C. Furr, unpublished observations). Thus, separation of retinol and retinal is more dependent on the specific column and mobile phase than are most retinoid separations. 

Fluorous reverse phase silica gel (FRPSG) has been used in the purification of synthetic DNA fragments.In solid phase DNA synthesis, truncated sequences are often separated from the desired product after deprotection using HPLC or electrophoresis. In order to perform, parallel syntheses and separations of nucleotides the trityl-on purification procedure was developed, in which a lipophilic support material is used to separate the desired and undesired product, followed by deprotection. If the protecting group is labelled with a fluorous group, fiuorous-fiuorous interactions between the FRPSG and the protected nucleotide can be used to aid separation of the aqueous mixture. 

The retention model developed by Eon and Guiochon [7,8] to describe the adsorption effects at both gas-liquid and liquid-solid interfaces, which was later modified by Mdckel et al. [6] to account for the retention at chemically bonded reversed-phase materials in HPLC, is not applicable to ion chromatography. But if the dependence of the capacity factors of various inorganic anions on the column temperature is studied, certain parallels with HPLC are observed. The linear dependences shown in Fig. 3-2 are obtained for the ions bromide and nitrate when the In k values are plotted versus the reciprocal temperature (van t Hoff plot). However, in the case of fluoride, chloride, nitrite, orthophosphate, and sulfate, the k values were found to be constant within experimental error limits in the temperature range investigated. Upon linear regression of the values in Table 3-1, the following relations are derived for bromide and nitrate ... 

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