Representative reverse phase

Table 3 Representative Reverse-Phase HPLC Methods Cited in the Literature for a- and /3-Acid Analysis...

The labelhng yield obtained for I-DOTATATE was greater than 95% according to the Sep-Pak elution profile and the chromatographic behaviour of the reaction mixture using the ITLC-SG-saline system (Rj of 0.0-0.3 for -°1-DOTATATE, of 0.8-1.0 for "). Electrophoresis of non-purified DOTATATE showed that the radiolabelled peptide did not migrate from the point of application. Representative reversed phase HPLC radiometric and UV profiles are shown in Fig. 16.1. Two radiochemical species were found with retention times (R ) of 23 and 25 min. The R of the unlabelled peptide was found to be 20 min, which allowed the collection of fractions containing the radiolabelled peptide free from cold peptide. 

FIGURE 10.9 (a) Representative reversed-phase UPLC-Q-TOF MS TICs of a urine sample analyzed in positive and negative ion modes (b) PLS-DA scores plot (OSC filtered) of healthy, insulin-sensitive subjects (A) and prediabetic, insulin-resistant individuals ( ) (c) corresponding PLS-DA loading plot. The variables are labeled with m/z, and m/z 194.34 is labeled by an arrow and (d) differences in the TIC peak height of m/z between insulin-sensitive subjects (A) and insulin-resistant individuals ( ). p < 0.05 vs. insulin-resistant individuals analyzed by the Wilcoxon rank sum test. (Reprinted from Chen, J. et al., Anal. Chem., 80, 1280, 2008. With permission.)... 

TABLE I I Representative Reversed Phase LC Column Screening Conditions... 

The stationary phase constitutes about 12% of the column volume, which is equivalent to only about 17% of the mobile phase content of the column. The values given in Table 2 are probably representative of most reverse phase columns but will differ significantly with extremes of pore size and pore volume. 

Figure 12.12 Coupled SEC-RPLC separation of Plioflex rubber stock (a) SEC (b) RPLC ti ace of fraction 1, Wingstay 100 (Eive-peak pattern is representative of diarylphenylenedi-amine isomers) (c) RPLC ti ace of fraction 2, mixed disulfide and MBTS (2,2 -thiobis (ben-zothiazole)). Obtained under the same conditions as given for Eigure 12.11. Reprinted from Journal of Chromatography, 149, E. L. Johnson et al, Coupled column chromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

Figure 13.9 Coupled-column RPLC-UV (215 nm) analysis of 100 p.1 of an extract of a spiked soil sample (fenpropimoiph, 0.052 mg Kg ). LC conditions C-1, 5 p.m Hypersil SAS (60 m X 4.6 mm i.d.) C-2, 5 p.m Hypersil ODS (150 m X 4.6 mm i.d.) M-1, acetonitrile-0.5 % ammonia in water (50 50, v/v) M-2, acetonitrile-0.5 % ammonia in water (90 10, v/v) flow-rate, 1 ml min clean-up volume, 5.9 ml transfer volume, 0.45 ml. The dashed line represents the cliromatogram obtained when using the two columns connected in series without column switcliing. Reprinted from Journal of Chromatography A, 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in envir onmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

The separation was carried out on a column 3 cm long, 4.6 mm in diameter and packed with a Cl8 reversed phase on particles 3 p in diameter. It is seen that the separation appears to be good and all the peaks represent individual solutes. However, by plotting the adsorption... 

Virtually all interactive mechanisms that control retention in chromatography are, in fact, mixed interactions as shown by the previous application examples. It has already been suggested that reverse phases can exhibit almost exclusively dispersive interactions with solutes. However, as they are almost always employed with aqueous solvent mixtures then, polar and dispersive interactions will still be operative in the mobile phase. Consequently, the examples given here will be taken where the mixed interactions are either unique or represent a separation of special interest. 

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

Solvent strength determines the value, but not the selectivity. The mobile phase can be established by using the polarity index P proposed by Snyder. The highest values of P represent the strongest solute adsorbed in conventional TLC but represent the weakest for the separation in reversed phases. Sometimes aqueous polar mixtures cannot totally wet the chemically bonded layer. For this reason, checking... 

Lipophilicity represents the affinity of a molecule or a moiety for a lipophilic environment. It is commonly measured by its distribution behavior in a biphasic system, either liquid-liquid (e.g. partition coefficient in 1-octanol-water) or solid-liquid (retention on reversed-phase high-performance liquid chromatography or thin-layer chromatography system). 

Residues of alachlor and acetochlor are determined by similar methods involving extraction, hydrolysis to the common aniline moieties, and separation and quantitation by reversed-phase FIPLC with electrochemical detection. The analytical method for acetochlor is included as a representative method for residue determination of alachlor and acetochlor in plant and animal commodities. Propachlor and butachlor residues, both parent and metabolite, are determined by similar analytical methods involving extraction, hydrolysis to common aniline moieties, and separation and quantitation by capillary GC. The analytical method for propachlor is included as a representative method. The details of the analytical methods for acetochlor and propachlor are presented in Sections 4 and 5, respectively. Confirmation of the residue in a crop or... 

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

Miniaturized columns have provided a decisive advantage in speed. Uracil, phenol, and benzyl alcohol were separated in 20 seconds by CEC in an 18 mm column with a propyl reversed phase.29 A19 cm electrophoretic channel was etched into a glass wafer, filled with a y-cyclodextrin buffer, and used to resolve chiral amino acids from a meteorite in 4 minutes.30 A 6 cm channel equipped with a syringe pump to automate sample derivatization was used to separate amino acids modified with fluorescein isothiocyanate.31 Nanovials have been used to perform tryptic digests on the 15 nL scale for subsequent separation on capillary Electrophoresis.32 A microcolumn has also been used to generate fractions representing time-points of digestion from a 40 pL sample.33 A disposable nanoelectrospray emitter has been... 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

Fig. 2.55. Gradient reversed-phase HPLC analysis of flavonoids in white onions (a) and celery (b). ODS column of 150 X 3.9mm i.d particle size 5pm. Mobile phase 20min gradient of 15-35 per cent acetonitrile in water adjusted to pH 2.5 with TFA. Fowrate lml/min. Upper and lower traces represent samples before and after hydrolysis, respectively. Detection wavelength 365 nm. IS = internal standard Qc = quercetin Ap = apigenin Lt = luteolin. Reprinted with permission from A. Crozier et al. [159],...

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