HPLC columns modes

Most HPLC applications are performed with non-polar columns, thus in the reversed-phase mode (RPLC), since it allows simple and versatile conditions. Another advantage is that in general the applied mobile phase is an aqueous buffer. Moreover in RPLC chemical equilibria such as ion suppression, ion-pair formation, metal complexation, and micelle formation can easily be exploited to optimize separation selectivity. This explains the large number of commercially available non-polar HPLC columns. " ... 

In the case of peptide separation by HPLC, separation modes are combined in series. This approach is called tandem LC. For instance, ion exchange associated with RP is used for peptide separation. Multidimensional protein identification technique (MudPIT) involving use of microcapillary columns (SCX cationic column and RP column) linked in series and eluted into MS is preferred for separation of complex peptide mixtures (Figure 5.4). 

In the past 5 years the frequency of reports on the use of HPLC technology for the determination of trace organic compounds in aqueous environmental samples has been steadily increasing. Many innovative approaches to sample cleanup and analyte isolation have been reported. Reversed-phase separation, with its many mobile-phase adaptations, has been and continues to be the most popular HPLC separation mode. The development of fast columns and microbore columns should provide optimal configurations for particular applications. The operating characteristics of microbore columns also make... 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

Fig. 14 Analytical HPLC of the phylloquinone fraction from an extracted sample of brown rice isolated by semipreparative HPLC. Column, Spherisorb C8 (octyl) mobile phase, methanol/50 mM acetate buffer pH 3.0 (97 3) containing 0.1 mM EDTA, dual-electrode coulometric detection (redox mode), porous graphite electrodes, — 1.5 V (generator electrode), +0.05 V (detector electrode). The arrows signify the fraction containing tritiated phylloquinone 2,3-epoxide (internal standard) and phylloquinone (analyte) that is collected for quantitation by radioisotopic dilution. (Courtesy of M. J. Shearer.)...

In method (c), the NOC after HPLC separation was photolyzed by a UV lamp (254 10 nm), and the charged nitrite species was determined amperometrically (79). The denitrosation reaction was found to be dependent on the wavelength of the UV light, lamp intensity, exposure time, and pH of the solution. The effluent from the HPLC column was passed through a capillary PTFE tubing coiled around a 40-W mercury lamp. The electrochemical detector used permitted either single- or dual-mode detection corresponding, respectively, to detection limits of 60 pg and 20 pg for NDMA. The method was applied to the determination of NDMA in beer and of... 

To demonstrate that the proposed methods are suitable for structural elucidation of isomeric metabolites and derivatives in biological matrices, human plasma was spiked with the mixture of DL, 6-OH-DL, 3-OH-DL, /V-OH-DL, and 1-pyridine-/V-oxide-DL. The resulting sample was extracted and analyzed by LC-MS and LC-MS/MS in ESI and APCI modes as described above. HDX was successfully performed online when the extract was injected directly onto the HPLC column without drying and reconstituting the sample in a deuterated solvent. In general, there were no differences between the results obtained for the spiked plasma extract and for the mixture of the standard compounds, which indicates that the LC-MS methods with HDX described here are applicable for the analysis drug-derived material in plasma or other biological matrices. 

The sample preparation of endohedral metallofullerenes was done by Shino-hara and details are described in the review article [16]. The soot containing M C2 (M=Sc and La) was produced in direct-current (300-400 A) spark mode under He flow at 50 torr and collected under totally anaerobic conditions. The target fullerenes were separated and isolated from the various hollow fullerenes (C60-C110) and other metallofullerenes by the two-stage high-performance liquid chromatography (HPLC) method by using two complementary types of HPLC columns. The purity of the metallofullerenes used for structure analysis relative to other fullerenes was always more than 99.9%. 

The chromatograms show linearity in response with the amount of carbaryl injected in both the absorbance and fluorescence modes. Practical agricultural samples when analyzed for carbaryl, however, require a preliminary column cleanup before injection into the HPLC in order to preserve the integrity of the HPLC Column. 

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