Reversed-phase chromatography peak identification

A. flos-aquae strains they obtained an excellent linearity in their calibration curve in the range, 20-100 ng (on-column) (Harada et al., 1989). Wong and Hindin studied the applicability of LC-UV for the isolation, identification, and quantification of AN. Both normal phase and reversed phase chromatography were examined, but both phases showed poor chromatograms with broad peaks with extensive band tailing (Wong and Hindin, 1982). 

Figure 11.12 GC analysis of (a) urine sample spiked with opiates 3 p.g/ml) and (b) blank urine sample. Peak identification is as follows 1, dihydrocodeine 2, codeine 3, ethylmor-phine 4, moipliine 5, heroin. Reprinted from Journal of Chromatography, A 771, T. Hyotylainen et al., Determination of morphine and its analogues in urine by on-line coupled reversed-phase liquied cliromatography-gas clrromatography with on-line derivatization, pp. 360-365, copyright 1997, with permission from Elsevier Science.

In 1979, it was stated that poiybrominated aromatic ethers have received little attention (ref. 1). That statement is still applicable. Analyses to characterize this class of commercial flame retardants have been performed using UV (refs. 1-2), GC (refs. 1-6), and GC-MS (refs. 1-4). The bromine content of observed peaks was measured by GC-MS, but no identification could be made. The composition of poiybrominated (PB) diphenyl ether (DPE) was predicted from the expected relationship with polyhalogenated biphenyl, a class which has received extensive attention. NMR (refs. 3-6) was successfully used to identify relatively pure material which had six, or fewer, bromine atoms per molecule. A high performance liquid chromatography (HPLC) method described (ref. 1) was not as successful as GC. A reversed phase (RP) HPLC method was mentioned, but no further work was published. 

Figure 4.29 An example of the use of ternary solvents to control mobile phase strength and selectivity in reversed-phase liquid chromatography. A, methanol-water (50 50) B, tetrahydrofuran-water (32 68) C, methanol-tetrahydrofuran-water (35 10 55). Peak identification 1 - benzyl alcohol 2 phenol 3 3-phenylpropanol 4 2,4-dimethylphenol 5 benzene and 6 -diethylphthalate. (Reproduced with permission from ref. 522. Copyright Elsevier Scientific Publishing Co.)...

Ruiz-Sala et al. (129) described a reversed-phase HPLC method with a light-scattering detector for the analysis of TGs in milk fat. The identification of TGs was carried out by a combination of HPLC and gas-liquid chromatography (GLC), and was based on the equivalent carbon numbers and retention times of different standard TGs. Finally, quantitation of peak areas from HPLC chromatograms was carried out after applying a deconvolution program to the parts of chromatograms with poor resolution. 

Figure 5.7. Separation of deoxyribonucleosides and their 5 - monophosphate esters by multidimensional liquid chromatography on a strong cation-exchange column (column one) and a reversed-phase column. The unseparated nucleosides, peak A, on the ion-exchange column were switched to the reversed-phase coluiim for separation. Peak identification A = nucleosides, B = d-CMP, C = d-AMP, D = d-GMP, E = d-CYD, F = d-URD, G = THD, and H = d-ADO. (From ref. [84] Preston Publications, Inc.).

Mass spectrometry is certainly required for correct identification of an unknown peak (Larson, 1982). Less complicated ways may, however, be employed to test the homogeneity of a peak (1) Elution with different gradient systems and at different pHs can also be used. (2) Quantification should be similar with different derivatives, e.g., those formed with OPA and FMOC chloride (Ejnarsson et al., 1983), and with different principles of chromatography, i.e. reversed-phase and ion-exchange (3) An attractive method was recently described by Joseph and Davies (1983), with fluorimetric and electrochemical detection in series, confirmation of the identities of the OPA derivatives was obtained in a single run. 

Figure 5 Reversed phase HPLC (linear gradient of 10-90% isopropanol in acetonitrile at 25°C) of natural (top) and rearranged (bottom) butterfat triacylglycerols as obtained with light-scattering detector. Peak identification by carbon and double bond number. (Reproduced with permission from Marai L, Kuksis A, and Myher JJ (1994) Reversed-phase liquid chromatography-mass spectrometry of the uncommon triacylglycerol structures generated by randomization of butteroil. Journal of Chromatography A 672 87-99.)...

In our work at Leiden University, apart from HPLC-photodiode array (PDA) detection, HPLC-electrospray mass spectrometry was used as characterization and identification tools. A semipurified taxine extract obtained with acid/base extraction of T. baccata needles was analyzed in reversed phase nine taxines, one taxinine, and six taxanes were found present in the sample. Furthermore, 10-deacetylbaccatin 111 (paclitax-el s main precursor) and other taxanes were also found in the extract. Identification of the peaks was made with online liquid chromatography/mass spectrometry (LC/MS) and off-line nuclear magnetic resonance (NMR) following fraction collection at the end of the HPLC. Retention and spectral data of the identified peaks were used as tool to screen for taxines and taxi-nines in plant and cell culture extracts. Several... 

The number of detectors that are sensitive and selective enough to be applied online with LC is limited because the solvents used are not compatible, as in the case of immunochemical detection after reversed- or normal-phase LC. The technology of coupling is still under development and not yet available in a large number of laboratories not specialized in techniques such as LC-MS. Therefore, LC separations are frequently followed by offline detection. Confirmatory analysis of suspected liquid chromatographic peaks can be made possible by coupling liquid chromatography with mass spectrometry. Atmospheric-pressure chemical ionization LC-MS has been employed for the identification of six steroid hormones in bovine tissues (448). 

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