Re versed-phase chromatography

HPLC detection [578,601], gradient elution [602], RPLC [603], HPLC columns [549] and derivatisa-tion reactions [604]. The Journal of Chromatography A 656 (1993) deals extensively with re versed-phase chromatography. 

Fig. 16. Separation of cytochrome c (1), myoglobin (2), and chicken egg albumin (3) by re-versed-phase chromatography on a monolithic poly(styrene-co-divinylbenzene) column at flow rates of a 5 ml/min b 25 ml/min. (Reprinted with permission from [53]. Copyright 1996 American Chemical Society). Conditions column 50 mmx8 mm i.d., mobile phase linear gradient from 20 to 60% acetonitrile in water... 

If the size of the literature is a reliable indicator, the analysis of compo-uents fotmd In nvironmfntnl samples has not been developed t the same extent as clinical applications of re versed-phase chromatography. More attention has been paid to the analysis of volatile species by gas phase chromatography. This is due in part to the difficulty in identifying large molecular weight complex molecules which are present in water at trace levels. However, determination of a variety of analytes in water, soil, or other matrices has been reported and the wider use of RPC in the evaluation of water quality especially can be expected. The apolar phases used in RPC may be a boon in the determination of dilute analytes. Frei (4M) has discussed how relatively unpolar compounds dissolved in water can be concentrated at the top of a reversed-phase column and then eluted as a narrow band with an appropriate solvent. This technique can be used for the analysis of environmental samples in which the analyte of interest is in exceedingly low concentration. 

Figure 4. Fractionation of low molar mass birch lignosulfonates (fraction B in Figure 1) by re versed-phase chromatography.

A. C. Haneke, J. Crawford, and A. Aszolos, Quantitation of daunorubicin, doxorubicin, and their aglycones by ion-pair re-versed-phase chromatography, J. Pharm. Sci., 70 1112 (1981). 

HPLC Separation of Selenoamino Acids Selenium proteins and amino acids have a wide variety of ionic characteristics that are pH dependent [149, 151, 152] and can be separated by anion, cation, and re versed-phase chromatography [94, 97, 98, 133, 135, 152-157]. Maher and his coworkers [30, 32, 37] used both anion and cation HPLC to confirm the identity of Se species (Figs 20.1 and 20.3). It should be noted that selenocysteine is released from proteins complexes with cysteine and chromatographs differently than the commercially available selenocystine [158]. Although the major selenoamino acid in mullet has been tentatively identified as selenocysteine (Fig. 20.3), further confirmation is required. 

RE VERSED-PHASE CHROMATOGRAPHY OF NUCLEIC ACID FRAGMENTS... 

Because the capacity factor k is proportional to 1/D in normal-phase chromatography and to Z) in re-versed-phase chromatography, it follows that k in IPC is inversely proportional to the pairing ion concentration in the normal-phase situation but directly proportional in the reversed-phase case. 

Much effort has been made to detect steroids in biological fluids. Even simple TLC methods have been used for qualitative analysis [38], One method that been used for quantification involves an immunoassay, but several problems exist with that method, most notably cross-reactions and interference with other substances [39], On the other hand, a number of chromatographic methods have been developed to overcome these problems. The majority of analytical methods involved GC, which has good detection limits, but requires previous derivatization [40] of the steroids to accomplish volatilization. Many methods have also been reported using HPLC with UV detection or LC-MS [40, 41], Previously used stationary phases for LC was e.g., Sephadex LH-20, Celite and Lipidex, but they could not be operated with high pressure [42], These columns were therefore slow to run and the separation of steroids was very time-consuming [43], Nowadays applications mainly use HPLC as a separation method with both normal-phase and re-versed-phase chromatography. 

Bannier, A. Brazier, J.L. Ribon, B. Quincy, C. Determination of ketoprofen in biological fluids by re-versed-phase chromatography. J.Pharm.Sci., 1980, 69, 763-765... 

CA Hoeger, R Galyean, RA McClintock, JE Rivier. Practical aspects of preparative re versed-phase chromatography of synthetic peptides. CT Mant, RE Hodges, eds. High-Performance Liquid Chromatography of Peptides and Proteins Separation, Analysis, and Conformation. Boca Raton, FL CRC Press, pp 753-764. 

Schenk et al. reported the quantification of cytokines in cell extracts using micro-HPLC packed microcolumns coupled with immunochemical detection. Various types of chromatographies were associated with the immunochemical detection system in order to perform the separation of cytokines. These included cation-exchange, size exclusion, and re-versed-phase chromatography, with the latter being the most successful. 

In a previous paper [ref. 44], hydrophobic interactions of polyvinyl alcohol gel packings with small molecules, such as bases of nucleic acids, fatty acids, peptides and so on, have been reported. Therefore, it should be noted that some biological molecules could be separated by the combination mode of SEC and re-versed-phase chromatography on this type of columns. 

An example of the detection of some catacholamines and related compounds (39) using the electrochemical detector is shown in Figure 23. The chromatogram shows eighteen compounds important in central nervous system physiology. The mixture was injected into a buffered solution and separated by re versed-phase chromatography. 

Figure 3 Separation of tocopherols and tocotrienols by (a) nomial-phase and (b) re-versed-phase chromatography. Chromatographic conditions (a) Column, Zorbax SIL, 250 X 4.6 mm mobile phase, hexane isopropanol (99 1, v/v), (b) Column, Zorbax ODS, 250 X 4.6 mm mobile phase, acetonitrile methanol dichloromethane (60 35 5, v/v) detection, UV, 295 nm. Peak identification 1, a-tocopherol 2, a-tocotrienol 3, -tocopherol 4, y-tocopherol 5, y-tocotrienol 6, 5-tocopherol 7, 5-tocotiienol. (From Ref. 91 -)... 

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