Reversed-phase chromatography nucleotides

These sorbents may be used either for selective fixation of biological molecules, which must be isolated and purified, or for selective retention of contaminants. Selective fixation of biopolymers may be easily attained by regulation of eluent polarity on the basis of reversed-phase chromatography methods. Effective isolation of different nucleic acids (RNA, DNA-plasmid) was carried out [115, 116]. Adsorption of nucleosides, nucleotides, tRN A and DNA was investigated. It was shown that nucleosides and nucleotides were reversibly adsorbed on... 

Since amino acids and nucleotides are all polar and hydrophilic, they will be eluted quickly by the column. The mobile phase (see below) is also selected on the basis of polarity, with a medium- to high-polarity solvent required. The opposite of reverse phase chromatography is normal phase, where the column packing is medium to high polarity and the mobile phase is nonpolar. This technology is generally not applied to the analysis of polar molecules such as amino acids or nucleotides. Some peptides are more hydrophobic, making this method potentially more useful for peptides than for amino acids or nucleotides. 

A reversed-phase chromatography technique for the preparative separation of polar compounds has been demonstrated with the separation of a simple nucleoside mixture. A group separation of the 2 -deoxyribonucleoside, nucleotide and nucleobase constituents of normal and modified nucleic acids was achieved by gel permeation chromatography, and further separation was achieved on a hydrophilic acrylate polymer operating in the partition mode. Mononucleotides are selectively bound at low pH to Fe(ni) immobilized on agarose gel due to their free phosphate ester group, and can be recovered with a neutral eluant. Nucleosides and molecules with phosphate diester groups are not retained. ... 

Jin, C.H., Polyakova, Y., and Kynng, H.R. Effect of concentration of ionic hqnids on resolution of nucleotides in reversed-phase hquid chromatography. Bull. Kor. Chem. Soc. 2007 28, 601-606. 

Patthy, M., Balia, T. and Aranyi, P. (1990) High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates separation of myo-inositol phosphates, some common nucleotides and sugar phosphates. Journal of Chromatography A 523, 201-21 5. 

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. 

A comparison of ion-exchange, reversed phase and reversed phase ion-pair chromatography in the separation of nucleotides from bacterial cells concluded that the latter method was preferable for analytical separations (Fig. 11.1.10). The resolution obtained allowed... 

Fig. 11.1.10. Separation of nucleotides by reversed phase ion-pair chromatography. Chromatographic conditions column, Ultrasphere 5 (im ODS (250 x 4.6 mm) mobile phase, Buffer A (5 mM tetrabutylammonium phosphate, 4% acetonitrile and 50 mM potassium dihydrogen phosphate, pH 6.0), Buffer B (acetonitrile), a gradient from 0-40% buffer B was used over 60 min flow rate, 1.5 ml/min detection, UV at 254 nm. Reproduced from Payne and Ames (1982), with permission.

Optimal mobile phase conditions for the separation of nucleotides using reversed phase ion-pair chromatography have been found to be a phosphate buffer which contains tetrabutylammonium phosphate (TBAP) as the counter-ion (Darwish and Prichard, 1981). Phosphate concentrations between 50 mM and 120 mM gave the best resolution, whilst the addition of TBAP at concentrations between 0.5 mM and... 

M.W. Taylor, H.V. Hershey, R.A. Levine, K. Coy and S. Olivelle. Improved method of resolving nucleotides by reversed-phase high-performance liquid chromatography. J. Chrom. 219 133-139 (1981). 

Deoxyribonucleic acid and ribonucleic acid, as well as their nucleotides, nucleosides, and base constituents play an important role in many vital biochemical processes of medical interest. To better understand these processes, fundamental investigations into the structure, occurrence, search for modifications, and biochemical impact of structural variation are required. Thus, reliable high-resolution analytical methods for the separation and identification of the nucleic acid constituents (often at extremely low concentration levels) had to be developed. Chromatography (including reversed-phase hquid chromatography), ion exchange chromatography, dHPLC, and electrophoresis... 

Lim CK and Peters TJ (1989) Isocratic reversed-phase high-performance liquid chromatography of ribonucleotides, deoxynucleotides, cyclic nucleotides and deoxycy-clic nucleotides. Journal of Chromatography 461 259-266. 

Purine compounds were quantitated by high-performance liquid chromatography (HPLC). These procedures permited the simultaneous measurement of concentration and radioactivity of a purine compound separated by either reversed-phase (nucleosides and base) or anion-exchange (nucleotides) gradient HPLC. These HPLC procedures have been described in detail (3). 

High Pressure Liquid Chromatography.—Reverse phase h.p.l.c. using octadecyl bonded groups has been developed for the rapid and efficient separation of nucleosides, nucleotides, and protected oligonucleotides on both analytical and preparative scales. ... 

Monosodium glutamate (MSG) and the 5 -nucleotides are generally recognized as the primary food components that provide the umami sensation [105,106]. MSG is readily measured by ion chromatography or reverse phase HPLC [103]. The 5 -nucleotides are most commonly determined by HPLC as well [107,108] but other methods have found use, e.g., derivative spectrophotometry [108]. 

Schwenn J D, Jender H G 1980 Reversed-phase high-performance liquid chromatography of adenine nucleotides application to the kinetics of an adenosine 3 -phosphate 5 -sulfatophosphate sulfotransferase from plants. J Chromatogr 193 285-290... 

Although the FI A technique does not include a separation procedure, the simultaneous determination of 5 -mononucleotides essentially requires a separation step before detection of these compounds. Thus, high-performance LC (HPLC) techniques were employed for the quantitation of 5 -mononucleotides. The 5 -mononucleotides were quantified not to determine flavor enrichment but as components of nutritional or clinical importance, especially in infant formulas [16]. Three main modes of LC are applied for nucleotide analysis ion-exchange chromatography, reversed-phase LC (RP-LC), and ion-pair RP-LC [16]. 

Seifar, R. M., C. Ras, J. C. van Dam, W. M. van Gulik, J. J. Heijnen, and W. A. van Winden. 2009. Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry. Anal. Biochem. 388 213-219. 

---