Reversed-phase HPLC detectors

Several HPLC systems have been used for the final analysis, including ion-exchange HPLC, reversed-phase HPLC, and ion pair reversed-phase HPLC. Detectors used in all HPLC systems are mainly fluorescence ones, although UV detectors have been sometimes used. 

Castro and Canselier [114] similarly used reverse phase HPLC with methanol-water containing a low concentration of nitric acid as eluent. Quantification was made possible by using a moving-wire flame ionization detector. 

Trathnigg, B., Kollroser, M.J. (1997). Liquid chromatography of polyethers using universal detectors V. Quantitative aspects in the analysis of low-molecular mass poly(ethylene glycols) and their derivatives by reversed-phase HPLC with an evaporative light scattering detector. J. Chromatogr. A 768, 223-238. 

Determination of organolead metabolites of tetraalkyllead in urine can be carried out after solid-phase enrichment and end analysis using reversed-phase HPLC with chemical reaction detector and by LC-MS (thermospray127). The chemical derivation consists of conversion to the dialky Head form, as shown in reaction 1, followed by complex formation with 4-(2-pyridylazo)resorcinol (11) and spectrophotometic measurement at 515 nm128. 

River sediment Alkyltin Reverse phase hplc with ICP mass spectrometric detector 3-I6 pg [83] ... 

Fig. 4.3.7. Reversed-phase HPLC (A + B) and normal-phase HPLC (C + D) chromatograms of wastewater (A + C) and sludge (B + D) taken from an industrial wastewater treatment plant. Detector fluorescence detector. Chromatograms used for calculation of the results shown in Table 4.3.1. 

Fang LL, Wan M, Pennacchio M, Pan JM. Evaluation of evaporative light-scattering detector for combinatorial library quantitation by reversed phase HPLC. Journal of Combinatorial Chemistry 2, 254-257, 2000. 

HPLC and Isolation of Mutagenic Fractions. Analytical and semipreparative reverse-phase HPLC separations were performed by using a water-to-acetonitrile linear gradient (J2). Separations were carried out on a Hewlett Packard Model 10084 B equipped with an automatic sampling device, a solvent programmer, a variable absorbance detector, and an automatically steered fraction collector. The instrument was fitted with a 3.9-mm X 30-cm prepacked analytical column of 10-/zm silica particles bonded with octadecylsilane (Bondapack-Cis) for analytical scale. For semipreparative scale separations, the HPLC was fitted with a 7.8-mm X 30-cm prepacked column packed with 10-/xm silica particles bonded with octadecylsilane. Samples for HPLC were injected at volumes of 20 /xL (flow rate 1 mL/min) and 80 /zL (flow rate 4 mL/min), and the absorption was measured at 254 nm. Fractions... 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

Fig. 42 Reversed-phase HPLC profiles of natural (top) and rearranged (bottom) butterfat triacylglycerols as obtained with the light-scattering detector. HPLC conditions Hewlett-Packard Model 1050 liquid chromatograph equipped with a Supelcosil LC-18 column (25 cm X 0.46-cm ID) coupled to a Varex ELSD II light-scattering detector. Solvent linear gradient of 10-90% propanol in acetonitrile at 25°C over a period of 90 min (1 ml/min) recording stopped at 70 min. Peak identification by carbon and double-bond numbers of triacylglycerols.

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. 

Reversed-phase HPLC has been used to analyze the oxidation products of triacylglycerols in edible oils. The detection is often based on monitoring the conjugated dienes with an ultraviolet detector (234-235 nm). However, the UV detector provides no information about oxidation products without a conjugated diene structure, e.g., products of oleic acid. Information about these compounds is important when oils with a high oleic acid content are studied. The most common universal detector types—refractive index and flame ionization detectors—are not sensitive enough to detect small amounts of oxidation products. 

Fig. 44 Reverse-phase HPLC analysis of purified regioselective product triacylglycerols. Sample size 0.5-1.0 mg 5 fi C-18 column (0.49 X 50 cm) 120-min solvent gradient acetonitrile/methylene chloride (70 30 to 40 60, v/v) flow rate, 0.8 ml/min flame ionization detector. Column cleaned with methylene chloride after analysis. L, linoleic Ln, linolenic O, oleic P, palmitic S, stearic.

Fig. 45 Reversed-phase HPLC of autoxidized trilinolenin (peroxide value = 236.4 meq/kg). Nova-Pak C18 cartridge column (Waters, Milford, MA) (3.9 X 150 mm, 60 A, 4 yam), mobile phase acetonitrile/ dichloromethane/methanol (80 10 10). Ultraviolet (UV) detector (235 nm) and evaporative light-scattering detector (ELSD). Primary oxidation products, double peak at 3.6 min secondary oxidation products elute before primary oxidation products.

All of the fat-soluble vitamins, including provitamin carotenoids, exhibit some form of electrochemical activity. Both amperometry and coulometry have been applied to electrochemical detection. In amperometric detectors, only a small proportion (usually <20%) of the electroactive solute is reduced or oxidized at the surface of a glassy carbon or similar nonporous electrode in coulometric detectors, the solute is completely reduced or oxidized within the pores of a graphite electrode. The operation of an electrochemical detector requires a semiaqueous or alcoholic mobile phase to support the electrolyte needed to conduct a current. This restricts its use to reverse-phase HPLC (but not NARP) unless the electrolyte is added postcolumn. Electrochemical detection is incompatible with NARP chromatography, because the mobile phase is insufficiently polar to dissolve the electrolyte. A stringent requirement for electrochemical detection is that the solvent delivery system be virtually pulse-free. 

Link et al. have used a two-dimensional chromatographic separation approach to characterize yeast ribosome complex proteins (Link, 1999). This technique employs a cation exchange column (SCX) for the first separation and, subsequently, two parallel reversed-phase HPLC columns (Figure 15.4), and thus works extremely rapidly and efficiently. While the first column loads, the second elutes using an acetonitrile gradient The flow from the column is directed to parallel online MS detectors as well as to offline fraction collection with UV detectors. 

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