Gradient reversed-phase HPLC method

For phenolic acids in bilberry juice, a reversed-phase HPLC method (16) using a linear-gradient elution of (a) 2% aqueous acetic acid and (b) acidified, aqueous acetonitrile on two Cl8 columns was able to separate the 12 phenolic acids and flavonoids (three flavonol glycosides and three flavonols) in ethyl acetate extract. Phenolics in blueberries were extracted, isolated, and... 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

The dansyl derivatives, which have a napthtalene structure, are excellent derivatives for primary amines (Mietz and Karmas, 1978). They are easily formed and detected by a uv (HPLC) detector in amounts as little as 10 ng, therefore the method does not require high sensitivity or fluorometric detectors. Gradient elution improves the separation, allowing for a broad range of derivatives to be separated in a relatively short time (40 min). Use of reverse-phase microparticular (5-10 /u,m) columns can further improve the HPLC separation (Gouygou et al, 1992). Desiderio et al (1987) described the use of a reversed-phase HPLC method for the quantification of putrescine, cadaverine, spermidine, and spermine from brain extracts as the dansyl derivatives. 

The reversed-phase HPLC method is a traditional method for the analysis of peptides and proteins. A good choice for the stationary phase could be a short-alkyl reversed-phase (e.g., C4) with wide pores (30 nm). It has been shown that large pore supports give distorted peaks with small collagens and triple helical peptides, resulting in poor resolution. The formation of broad peaks has been ascribed to conformational instability of the separated solutes and slow cis-trans isomerization of the peptide bonds. The best sorbents of those examined were diphenyl or non-porous Cig reversed-phases standard water-acetonitrile gradients were recommended as mobile phases. 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

The peptides generated by proteolysis are separated using reverse-phase HPLC to minimize mass overlap and ionization suppression caused by ion competition in the electrospray source [40]. The optimized LC gradient parameters efficiently separate peptides while minimizing loss of deuterium through back exchange with solvent. Increased sensitivity can be achieved by using capillary HPLC columns and nanoelectrospray methods [47]. 

Fractionations by all these methods are well-documented by chapters in this book. Our own work has emphasized fractionation by reverse-phase HPLC. By using water-to-acetonitrile elution gradients,... 

Polar chlorophyll derivatives and metalloporphyrin derivatives such as Cu2+ and Zn2+ pheophytins can also be analyzed by C18 reversed-phase HPLC. Appropriate standards must be used see UNITF4.2 for polar chlorophyll derivatives, or see Support Protocol 2 for Cu2+ and Zn2+ pheophytin standards. Gradient solvent conditions and flow rates are given in Tables F4.4.3 and F4.4.4. Otherwise, the separation is performed as described for chlorophylls and nonpolar derivatives (see Basic Protocol). Using this method, separation of polar chlorophyll derivatives can be achieved in 20 to 25 min, and separation of the metalloporphyrin derivatives in 20 to 25 min. Examples of chromatograms obtained for polar derivatives, Zn2+ pheophytins, and Cu2+ pheophytins are shown in Figures F4.4.2, F4.4.3, and F4.4.4, respectively. 

Usually, mobile phases of acetonitrile and acetone have been used in the analysis of TG from milk fat, most often in isocratic elution (114,115) and in gradient eiution, and they provide a resolution of 50 chromatographic peaks (Numela). One of the main difficulties in the analysis of TG is the identification of the chromatographic peaks, because of the small number of mixed TGs in a pure state. Bornaz et al. (115) and Dotson et al. (114) identified butterfat chromatographic peaks from the relationship between the retention time and the theoretical carbon number according to the model proposed by El-Hamdy and Perkins (87). An alternative method is the fractionation of total TG in milk fat by reversed-phase HPLC and analysis of the fatty acids in each fraction (116,117). 

A method for the determination of ethoxyquin in paprika that avoided the previous separation steps from other colored substances was proposed by Vinas (133). Analysis is carried out by reverse-phase HPLC using the gradient elution technique and UV detection at 270 nm. Using fluorimetric detection with excitation at 311 nm and emission at 444 nm, a detection limit of 0.2 jig /ml was reached. The method can be applied to the determination of ethoxyquin in commercial samples in the presence of paprika (Capsicum annuum) carotenoids. 

Bockhardt et al. (78) derived a manual extraction and automated [4-(4-dimethyl-aminophenylazo)benzensulfonyl] derivatization procedure from the method previously proposed by Krause et al. (44). Reversed-phase HPLC was carried out on a Spherisorb ODS-2 column at 50°C with gradient elution and detection at 436 nm, reaching detection limits (DLs) of 0.3-0.8 pmol. 

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