Reversed-phase HPLC organic solvents

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

Mobile phase as sample solvent. If possible, always use mobile phase as the sample solvent. This ensures the composition (e.g., percent organic, pH) of sample solution matches that of mobile phase and reduces the chance of any problem due to incompatibility of sample solvent and mobile phase. Alternatively, always use sample solvent weaker than that of the mobile phase to ensure that the chromatography is not deteriorated. For example, in reversed-phase HPLC, use less organic solvent in the sample solvent than in the mobile phase. 

HPLC Separations. SAMPLE PREPARATION FOR INJECTION. Isolated residue organics were dissolved in water/acetonitrile solvent mixtures for reverse-phase HPLC separations as follows sample was dissolved in a minimum volume of acetonitrile and diluted with water until... 

F. Rabel and K. Palmer, Am. Lab., August 1992, p. 65.] Between uses, reversed-phase columns can be stored in methanol or in water-organic solvent mixtures that do not contain salts. Normal-phase columns should be stored in 2-propanol or hexane. See also R. E. Majors, The Cleaning and Regeneration of Reversed-Phase HPLC Columns, LCGC 2003,21, 19. 

Compared to refined vegetable oils, the compositions of crude vegetable oils and oil and fat products are more complicated. These samples contain proteins, carbohydrates, and minerals that interfere with HPLC separation and reduce the lifetime of the HPLC column. These compounds need to be largely eliminated from the extract before HPLC analysis. Saponification and heating are used to weaken sample matrices to allow the solvent to fully access all tocopherols and tocotrienols of the sample. Liquid/liquid extraction is used to remove these polar compounds from the organic solvent layer that contains tocopherols and tocotrienols. The normal-phase HPLC method is usually used for crude vegetable oils and vegetable oil products reversed-phase HPLC can be used for animal fat products. 

The most common analytical technique for the analysis of FFAs and their breakdown products has been chromatography. HPLC has been used for the analysis of FFAs (Christie, 1997 Lues et ah, 1998 Zeppa et ah, 2001). Analysis of short-chain fatty acids (C2-C4) may be relatively simple (Zeppa et ah, 2001). However, the analysis of long-chain fatty acids (>C6) may require derivatization. They are extracted using solvents, converted to bromophenacyl esters, and analyzed by reverse-phase HPLC. GC (with sample preparation and derivatization) has been the method of choice for analysis of fatty acids. An ideal but difficult procedure is to extract FFAs from the aqueous phase and organic phase and combine them (IDF, 1991). The challenge is to overcome the effects of partitioning and extraction of compounds that interfere with the analysis. ISO and IDF have detailed some of the extraction methods for lipids and liposoluble compounds in milk products (ISO, 2001b). Several other methods, which are mainly different in the extraction and derivatization steps, were reviewed by Collins et ah (2004). 

Although the separation of amino acids and small peptides by reversed-phase HPLC is becoming an accepted procedure, the application of this technique to the purification of proteins still requires careful evaluation. The biological activities of many proteins are sensitive to denaturation by extremes in pH, by contact with organic solvents or high salt concentrations, by adsorption into glass or hydrophobic moieties, or at an air-water interface (2/). 

Many protein isolations also involve the use of organic solvents at one or more purification steps, and thus one can often use this solvent in HPLC to maximize the recovery of protein samples. For example, apolipoproteins can be solubilized in isopropanol without loss of activity. For this reason Hancock and Sparrow 20) made extensive use of this solvent in the separation of C-apolipoproteins by reversed-phase HPLC. Glasel (//) found that neurophysins had a high solubility in aqueous methanol and used this solvent mixture in HPLC studies. 

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