Reverse phase method development molecular weight

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

We will now proceed to discuss method development strategies. First, we will contemplate the choice of isocratic or gradient methods. Then we will develop an efficient method development strategy. The focus of the method development is the analysis of low-molecular-weight ionizable compounds by reversed-phase HPLC. 

Trace enrichment is a valuable tool to use to simplify any laborious sample preparation technique. It is basically automation of the well-known SPE method of sample preparation. As such, it saves the analyst time, money, and solvent, and all but eliminates sources of analytical error. In addition to the traditional bonded reversed phase and ion exchange packings, newer packings have been developed with different surface and inner pore bonded phases. These RAMs have polar outer surfaces that repel biopolymers and non-polar or ionic inner surfaces that collect drugs and other smaller-molecular-weight components. 

The use of the reverse phase evaporation method permits inclusion of 50 and more percent of the substance to be encapsulated from the water phase into the liposomes. Besides, a variety of methods have been developed to obtain lyophilized liposomal preparations possessing good storage stability. The in vitro release rate of different compounds from liposomes, including proteins of moderate molecular weight, is usually under 1% per hour, assuming that the incubation temperature sufficiently differs from the phase transition temperature of a given phospholipid, since the maximal permeability of liposomes is usually observed at temperatures close to the phase transition temperature of the liposomal phospholipid. In vivo, this parameter can vary within wide limits from minutes to hours and depends on the liposome membrane composition, cholesterol content, and disposition within the body. 

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