Reversed-phase gradient

In most situations the eluent composition is chosen to minimize the effects of hydrophobic interaction, but these secondary effects can be used to advantage. By careful selection of a salt and its concentration, specific selectivities for analytes can be achieved without the use of organic solvents. Therefore, many separations usually run by solvent gradient reversed-phase methods can be completed with a purely aqueous isocratic eluent (13,14). 

Mutton, I. M. Use of short columns and high flow rates for rapid gradient reversed-phase chromatography. Chromatographia 1998, 47, 291-298. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

Davis, M.T., Beierle, J., Bures, E.T., McGinley, M.D., Mort, J., Robinson, J.H., Spahr, C.S., Yu, W., Luethy, R., Patterson, S.D. (2001). Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses. J. Chromatogr. B Biomed Sci. Appl. 752, 281-291. 

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],...

Isoflavone derivatives along with nonvolatile components in soy sauce were also analyzed by gradient reversed-phase HPLC. This HPLC profile of soy sauce was further utilized by a pattern recognition program to understand the quality differences of soy sauces (132), and three isoflavone derivatives (esters of tartaric acid with daidzein, genistein, and 8-hydroxygenistein) were found to contribute significantly to the differentiation in fermented soy sauce. Fermented... 

In the Tiller study, adhesive was applied to a glass bottle and cured. Highly purified water was placed over the adhesive, heated at 50 °C for 3 days, and analyzed with gradient reversed-phase HPLC. An LC/ITMS with ESI was used to profile the polyesters in the adhesive extracts with full-scan mass spectra and corresponding product ion spectra triggered by an ion abundance that surpassed a threshold. 

Fig. 9. Step-gradient reversed-phase HPLC separation of nucleosides in urine. Sample 25 pooled ovarian cancer patient urine column -Bondapak C18, 600 x 4 mm buffer, 0.01 M NHtHjPO, (A) pH 5.3, with 2.5 /c methanol, (B) pH 5.1, with 8.0% methanol flow rate 1.0 ml/min temperature 35°C detection 254 nm, 0.02 AUFS (upper trace) 280 nm, 0.01 AUFS (lower trace). Reprinted with permission from Gehrke et at. (G3). Copyright by Elsevier Scientific Publishing Company, Amsterdam.

Fig. 15. The gradient reversed-phase separation of bovine and porcirte insulin with 0.05 M tetramethylammonium phosphate, pH 3, as the mobile phase, and acetonitrile as the organic modifier. The column used was a LiChrosorb RP18 and the flow rate was 1 ml/min. The following peaks were identified 1, hydroxybenzoic acid 2,/7-hydroxymethyl benzoate 3, bovine insulin 4, monodesamido bovine insulin 5, porcine insulin 6, monodesamido porcine insulin 7, proinsulin. Adapted from Fig. 5 of Biemond et al. (1979).

The effect of elevating the flow rates that the calibrated methods used was studied on these new columns. Resolution (/ ) in gradient reversed-phase LC is a function of mean column efficiency (A ), mean selectivity (a) and the effective capacity factor (k. c) experienced by two closely resolved analytes during the elution process [11.12], such that ... 

Estimation of lipophiiicity and pifa by gradient reversed-phase chromatography... 

Leroy, F., Presle, B., Verillon, F. and Verette, E. Fast generic-gradient reversed-phase high-performance liquid chromatography using short narrow-bore columns packed with small nonporous silica particles for the analysis of combinatorial libraries. ]. Chromatogr. Sci. 39 487-490, 2001. 

Mutton, I.M. Use of Short Columns and High Flow Rates for Rapid Gradient Reversed-Phase Chromatography, Chromatographia 47,291-298 (1998). 

Figure 7.5. HPLC analysis of sweetener, flavors, and preservative in a soft drink sample using gradient reversed-phase chromatography and UV detection. Chromatogram courtesy of PerkinElmer, Inc.

Figure 7.34. HPLC analysis of synthetic oligonucleotides using ion-pair gradient reversed-phase chromatography on a column packed with 2.5-pm nonporous hybrid particles with UV detection at 260nm. Chromatogram courtesy of Waters Corporation.

Furr, H.C. Amedee-Manesme, O. Olson, J.A. Gradient reversed-phase high-performance liquid chromatographic separation of naturally occurring retinoids. J.Chromatogr., 1984, 309, 299-307 [gradient rat human pig liver kidney extracted retinyl esters, tretinoin, vitEunin A]... 

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