Reverse phase high-power liquid chromatography

Synthetic peptides often lack the conformational stability required for a successful drug therefore determination of peptide stability in serum constitutes a powerful and important screening assay for the elimination of unstable peptides in the pipeline of drug development (see Note 1). Peptide stability in serum can rather easily be determined by reverse phase-high-performance liquid chromatography (RP-HPLC) and mass spectroscopy (MS) from both in vitro and in vivo studies. 

Ogorka, J., Schwinger, G., Bruat, G. and Seidel, V. Online coupled reversed-phase high-performance liquid chromatography-gas chromatography-mass spectrometry. A powerful tool for the identification of unknown impurities in pharmaceutical products. ]. Chromatogr. 626 87-96, 1992. 

CE, with its high resolving power, rapid method development, easy sample preparation, and low operational cost, is reported to be an excellent technique for resolving caseins (including different genetic variants), peptides derived from them, and whey proteins (16-19). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (17, 20). The application of CE to the assessment of proteolysis in milk and different cheese types has acquired an enormous importance in recent years. Reviews on the application of CE to this field can be found in papers by Otte et al. (21) and Redo et al. (22). 

SPPS has been enabled through the development of powerful methods for the analysis and subsequent purification of the complex mixture of products typically produced by SPPS. In particular, the development of reversed phase, high performance liquid chromatography (HPLC) and macromolecular mass spectrometry [22], matrix assisted laser desorption/ionization mass spectrometry (MALDI) [23] and electrospray ionization mass spectrometry (ESI-MS)... 

Folley, L.S., Power, S.D. and Poyton, R.O. (1983) Separation of nucleotides by ion-pair, reversed-phase high-performance liquid chromatography use of Mg(ll) and triethylamine as competing hetaerons in the separation of adenine and guanine derivatives, jouma/ of Chromatography 28i, 1 99-207. 

In the past decade, we have benefited from major improvements in the sensitivity and efficiency of both chromatography and electrophoresis. High-performance liquid chromatography (HPLC) has become firmly ensconced as a powerful method for protein analysis. Ironically, reversed-phase chromatography, which employs denaturing conditions, has found a particularly wide application, at least when only analytical information is sought. This is the case in most routine analytical work required for many biotechnological applications. 

Liquid chromatography (LC) is a separation technique which is used widely in many different areas of analytical chemistry and provides a powerful tool for the separation and quantification of substances in various matrices. Nowadays, the majority of the high-performance liquid chromatography (HPLC) methods are carried out in reversed-phase mode using a non-polar stationary phase and a polar-mobile phase. 

Analysis of DCLs by a combination of high-performance liquid chromatography (HPLC) and MS (LC-MS) has proven very powerful, provided the library members are stable enough to allow chromatographic separation. LC-MS analysis can yield both the quantity and the identity of individual library members. Care should be taken that the exchange between library members is slow on the timescale of the experiment. This is usually the case for hydrazone and disulfide libraries, without requiring special precautions. It has recently been reported that even the composition of DCLs of hydrolytically labile imines can be analyzed directly by HPLC, provided an acidic mobile phase is used. Beau et al. successfully used a common reversed-phase HPLC solvent system based on acetonitrile/water mixtures containing 0.1% trifluoroacetic acid [10]. Imine hydrolysis is slow under these conditions because at low pH the zwitterionic hemi-aminal intermediate that is believed to be important in the hydrolysis process is more difficult to attain (Scheme 2.1). 

There are a variety of HPLC columns and most of them are based on silica particles. The most popular column used today is the reversed-phase Cl8 column that uses a silica support with a bonded organic surface layer. Traditional HPLC analytical columns are usually 4.6 mm x 250 mm with a 5 pm particle size, as used in the USP official method. Modern columns for HPLC-mass spectrometry (LC-MS) applications are more efficient and offer the same or better resolving power in a much smaller package (2.1 mm x 150 mm with a 3.5 pm particle size or smaller). Ultra high-performance liquid chromatography (UHPLC) columns (sub 2pm particle size, 1-2 mm in diameter and 30-100 mm in length) are becoming more and more popular. 

Optical rotation and refractometric detectors were combined to characterize a series of dextrins by gel permeation chromatography and high performance liquid chromatography (HPLC). Gel permeation fractionates according to the hydrodynamic volume when adsorption is avoided/ and for this reason separates isomaltodextrins from linear maltodextrins. Specific rotation power [a] is directly obtained and confirms the chemical structure. Elution of cyclodextrins is also tested and discussed. HPLC reverse phase chromatography separates the anomers as shown by optical rotation and allows good resolution in the range of low DP. 

High-performance liquid chromatography (HPLC) This method was applied to identify and quantify characteristic substances in commercially available oakmoss products. This technique provides a powerful complement to the established TLC method. The bonded reverse phase columns are used here, and all the aromatic lichen products are suitable for analysis with this method. Samples are dissolved in methanol and injected in to the appropriate portion column, through which an... 

For the moment, high-performance liquid chromatography is still the most versatile tool that can be used for the determination and the small-scale isolation of various flavin compounds. In the analytical field, HPLC has already completely superseded paper chromatography and more or less completely superseded thin-layer chromatography. A modem HPLC apparatus, including a fluorescence detector, leaves little to be desired in regard to sensitivity, reliability, and reproducibility. The on-line combination with mass spectrometry which needs expensive equipment and special expertise, is, however, a powerful technique for the separation and unequivocal identification of unknown flavin compounds from different sources. Various approved reversed-phase or ion pair HPLC systems are available... 

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