Reversed-phase columns commercially available

The use of TFA as a mobile-phase additive in LC-MS can be problematical when using electrospray ionization. In negative ion detection, the high concentration of TFA anion can suppress analyte ionization. In positive ion detection, TFA forms such strong ion pairs with peptides that ejection of peptide pseudo-molecular ions into the gas phase is suppressed. This problem can be alleviated by postcolumn addition of a weaker, less volatile acid such as propionic acid.14 This TFA fix allows TFA to be used with electrospray sources interfaced with quadrupole MS systems. A more convenient solution to the TFA problem in LC-MS is to simply replace TFA with acetic or formic acid. Several reversed-phase columns are commercially available that have sufficient phase coverage and reduced levels of active silanols such that they provide satisfactory peptide peak shapes using the weaker organic acid additives.15... 

Some of the characteristic column volumes outlined in the previous argument were determined by Alhedal et al (11) in the examination of a commercially available reverse phase column packing, Zorbax Cs. These authors examined the exclusion properties of the interstitial volume of the column by measuring the retention volume of a number of salts of different molecular volume.The substances used, in ascending order of ion volume, were as follows,... 

The widespread application of HPLC methodology to chlorophyll analysis demonstrates its flexibility, effectiveness, and reliability. Methods described in this unit are based on the work of Schwartz et al. (1981). This original method allows for resolution of twelve chlorophyll derivatives in 30 min. While minor modifications were made to this method to further simplify the analysis, the final resolution and sensitivity were not compromised. Based on a commercially available reversed-phase column and an aqueous mobile phase, the method can be easily altered for specific separations. This is demonstrated in the Alternate Protocol, where the Basic Protocol was adjusted for the analysis of polar and Cu2+- and Zn2+-containing chlorophyll derivatives. The method for polar derivatives is based on the separation of Can-jura and Schwartz (1991), while the method for... 

The lipophilicity of compounds can be measured with any commercially available HPLC equipment most commonly UV absorption is used as detection system for the chromatography. The retention factor (retention time) of a compound is the analytical parameter needed to measure lipophilicity. To measure the retention factor, the retention time of the compound under investigation has to be determined reproducibly together with the dead time. The dead time is determined using the retention time of a not retained substance on reversed phase columns like buffer salts. 

Separations in RP-HPLC can be highly dependent upon the column utilized. Corran43 and Johns44 list many commercially available reversed-phase columns suitable for protein analysis. 

Cellobiohydrolase has also been bonded to a silica gel matrix and used as an effective stationary phase. It is available commercially as CHIRAL-CBH. The bonded material also acts as a reversed phase column retaining substances largely by dispersive (hydrophobic) interactions. This... 

The application of HPLC to studies of BaP metabolism was an important advance (414). Using reverse-phase colunms, the major metabolites of BaP formed in vitro can readily be separated, as shown in Fig. 7. Systems for separation of the minor phenolic metabolites and for the triol and tetraol metabolites of BaP have also been developed (416, 506). Metabolites are quantified by ultraviolet detection or by scintillation counting of the column eluant. The latter technique is used for mixtures with low levels of metabolites and is more successful when unmetabolized BaP is first removed by silicic acid chromatography. Addition of vitamin E to incubation mixtures prior to analysis helps retard air oxidation (365). High-performance reverse-phase columns are commercially available and have relatively long lifetimes when used for this type of work. 

One of the most important analytical techniques one should incorporate into any LC—MS method is the use of stable isotope dilution. There is no other better internal standard then the heavy labeled version of the compound of interest. The use of either [ C] and/or [ N] labels are preferred as deuterium ([ H]) incorporation alters the retention time of the internal standard. On reversed-phase columns, a [ H]-labeled analog will elute earlier than the protium form and on normal phase columns the analog will elute later. This elution order is due to slight differences in their interactions with the stationary phase. Stable isotope dilution methods provide the assurance that the internal standard experiences the same environment during sample preparation as well as during ionization in the source. This method allows for accurate and precise quantification of the analyte of interest. This technique is becoming increasingly popular however, many labeled internal standards, especially those of novel biomarkers, are not commercially available and must be synthesized. 

Reversed-phase LC is becoming increasingly popular for the separation of amino acids. Reversed-phase columns are readily available commercially and exhibit higher efficiencies than most commercially available ion-exchange columns. They are also compatible with aqueous samples, since water is generally a major component of the mobile phase. Therefore, it is not necessary to employ additional sample preparation steps in order to produce a sample in a nonaqueous environment. 

Reversed-phase HPLC on Cl 8 columns has been the preferred mode for separating carotenoids for quantitative analysis. The popularity of the Cl8 colunm derives from its weak hydrophobic interactions with the analytes (which should make it less destructive than the polar forces in normal-phase OCC), compatibility with most carotenoid solvents and with the polarity range of carotenoids, and wide commercial availability. Many different Cl8 reversed-phase materials are available from different manufacturers, varying in the degree of carbon loading, end capping, and the nature of the bonded phase (i.e., monomeric or polymeric). 

High-performance liquid chromatography (HPLC) This method was applied to identify and quantify characteristic substances in commercially available oakmoss products. This technique provides a powerful complement to the established TLC method. The bonded reverse phase columns are used here, and all the aromatic lichen products are suitable for analysis with this method. Samples are dissolved in methanol and injected in to the appropriate portion column, through which an... 

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