Reversed-phase traditional

Mass spectrometry (MS) is increasingly being combined with reverse phase HPLC or CZE in order to add an additional dimension to the data that a traditional detection system would not provide. A two-dimensional EC-CZE system with mass... 

This sample preparation required both extraction and concentration and this was carried out in the traditional manner with the use of an appropriate solvent. The separation was again achieved exploiting the dispersive interactions between the components of the mixture and the strongly dispersive hydrocarbon chains on the reversed phase. 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

The polarity values of binary acetonitrile/water and methanol/water mobile phases used in RPLC were measured and compared with methylene selectivity (acH2) for both traditional siliceous bonded phases and for a polystyrene-divinylbenzene resin reversed-phase material [82], The variation in methylene selectivity for both was found to correlate best with percent organic solvent in methanol/water mixtures, whereas the polarity value provided the best correlation in acetonitrile/water mixtures. The polymeric resin column was found to provide higher methylene selectivity than the siliceous-bonded phase at all concentrations of organic solvent. 

The ELSD is not plagued with the problem of baseline shift and is significantly more sensitive however it presents other disadvantages, such as the strong non-linearity of the detector response and the possibility of interference from non-volatile compounds in the sample matrix [87]. The HPLC method using traditionally reversed-phase solvents and a hybrid column/pre-column has demonstrated the separation of ethoxylated homologues of broadly distributed linear AEOs... 

Guo, P., Z. Li, Z. Hong, S. Liu, and T. Wu. 1991. Determination of berberine hydrochloride in traditional Chinese medicine containing Coptis chinensis Franch. by reversed phase high performance liquid chromatography. J. West China Univ. Med. Sci. 22 90-92. 

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). 

This protocol focuses on the analysis of chlorophyll a and b, and the more nonpolar derivatives, including pheophytins and pyropheophytins. An octadecyl-bonded, reversed-phase stationary phase is used with a methanol/water mixture and ethyl acetate mobile phases in a gradient elution to provide rapid and complete separation of the major chlorophyll derivatives in 25 to 30 min. This is coupled with traditional UV/visible spectrophotometric detection at 654 nm to selectively screen these photosynthetic pigments in food and plant tissues. 

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). 

Lysine is an essential amino acid. Since lysine is a fairly acid-stable amino acid, its analysis as total lysine by the traditional hydrochloric acid hydrolysis is straightforward. Fairly recent examples for the successful determination of total lysine employing either ion-exchange (82) or reversed-phase (101) HPLC are available. 

Each column type has its own place of use. Column variety is what gives HPLC its versatility. It really depends on your compound and application. Approximately 80% of all separations are done on 5-10-jUm reverse phase Ci8 silica columns. Much of this is tradition. Reverse phase columns offer high-resolution separations for a wide variety of compounds and can be run in aqueous mobile phases. Ion exchange separations require salt solutions for separations, and these are not compatible with mass spectrometers. Size separations have lower resolving power and longer run times, but may be the only way to separate proteins solutions that will irreversibly stick to reverse phase columns. Use small pore size separation columns to remove salt from effluent from other chromatography separations. Zirconium and polymeric column are newer and offer possibilities for unique separations. 

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