Reproducibility Reverse-phase liquid chromatography

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.)...

Figure 1 Electrochemical detection of catechol, acetaminophen, and 4-methyl catechol, demonstrating the selectivity of differential pulse detection vs. constant potential detection. (A) Catechol, (B) acetaminophen, and (C) 4-methylcatechol were separated by reversed phase liquid chromatography and detected by amperometry on a carbon fiber electrode. In the upper trace, a constant potential of +0.6 V was used. In the lower trace, a base potential of +425 mV and a pulse amplitude of +50 mV were used. An Ag/AgCl reference electrode was employed. Note that acetaminophen responds much more strongly than catechol or 4-methylcatechol under the differential pulse conditions, allowing highly selective detection. (Reproduced with permission from St. Claire, III, R. L. and Jorgenson, J. W., J. Chromatogr. Sci. 23, 186, 1985. Preston Publications, A Division of Preston Industries, Inc.)...

Kele, M. and Guiochon, G., Repeatability and Reproducibility of Retention Data and Band Profiles on Reversed-phase Liquid Chromatography Columns. 1. Experimental Protocol./. Chromatogr. A, 830 41-54, 1999. 

Reverse phase liquid chromatography is very versatile, fast and highly reproducible. Aqueous solutions are normally used and the modifiers used are very cheap and highly pure. Separation is predictable based on the polarity, pH profile, solubility and other physicochemical characteristics of the solute molecules. Analysis time is rather short and reequilibration is generally fast. Multiple components with minor differences in polarity can be separated by appropriate choice of gradient profiles. 

Figure 2 Contour plot of the comprehensive normal-phase x reversed-phase liquid chromatography analyses of carotenoids present in red orange juice with peaks and compound classes indicated. Reproduced from P. Dugo V. Skerikova T. Kumm A. Trozzi P. Jandera L. Mondello, Anal. Chem. 2006, 78, 7743-7750.

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.)...

The OPA reagent was first reported in 1971 by Roth as a postcolimm fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 

Monosaccharides are released by acid hydrolysis then labeled with the flurophore to allow sensitive detection 2-aminobenzoic acid (2-AA) for the neutral monosachharides, or l,2-diamino-4,5-methylenedioxybenzene. 2HC1 (DMB) for the sialic acids. Analysis by reversed-phase liquid chromatography provides reproducible quantitative results. 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

Reversed-phase high-performance liquid chromatography is an analytical technique of tremendous importance for the separation and quantitative analysis of compounds with widely varying chemical properties. Due to the versatility, sensitivity, and reproducibility of the technique, high-performance liquid chromatographic separations have become routine in biochemical and biomedical research, especially in investigations involving nucleic acid constituents. 

The D4 content of the samples was determined by reverse-phase high-pressure liquid chromatography (HPLC) with a Varian 5500 liquid chromatograph. A DuPont Zorbax ODS (Cis) column was used with a Wilmad infrared detector set at 12.45 xm to monitor the Si-CHa vibration. The mobile phase was an 83 17 mixture of acetonitrile and acetone at a flow rate of 0.8 mL/min. A Rheodyne injector valve operating on compressed air was used with a 10-p,L sample loop for reproducible injection volumes. Ethyl acetate was used to dissolve the samples for analysis. 

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