Reversed-phase chromatography nucleosides

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... 

Figure 7.32. HPLC analysis of bases and nucleosides using gradient reversed-phase chromatography and UV detection at 254nm. Chromatogram courtesy of PerkinElmer, Inc.

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. ... 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Singhal, R.P., Bajaj, R.K., Buess, G.M., Smoll, D.B., and Vakharia, V.N. (1980) Reversed-phase boro-nate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs. Anal. Biochem. 109, 1-11. 

In addition to classical reverse phase separation of peptides on octadecyl derivatized silica monoliths, sugars and peptides as well as proteins and nucleosides have been analyzed on a 20-cm-long silica-based poly(acrylic acid) column (ID. 200 pm), employing HILIC and weak cation-exchange chromatography, respectively [194]. Furthermore, HILIC fractionation of polysaccharides delivered remarkable and promising results [84,194]. 

Hartwick, R.A., Assenza, S.P. and Brown, P.R., 1979. Identification and quantification of nucleosides, bases and other UV absorbing compounds in serum using reversed phase high performance liquid chromatography. I. Chromatographic methodology. In A. Ziatkis (Editor), Advances in Chromatography. Lausanne Symp., pp. 725—736. 

Figure 5.7. Separation of deoxyribonucleosides and their 5 - monophosphate esters by multidimensional liquid chromatography on a strong cation-exchange column (column one) and a reversed-phase column. The unseparated nucleosides, peak A, on the ion-exchange column were switched to the reversed-phase coluiim for separation. Peak identification A = nucleosides, B = d-CMP, C = d-AMP, D = d-GMP, E = d-CYD, F = d-URD, G = THD, and H = d-ADO. (From ref. [84] Preston Publications, Inc.).

Relatively few papers have reported the estimation of nucleosides in animal cells using HPLC. However, the metabohc state of cells can be determined in this way and this allows the progress of certain diseases to be monitored (Nissinen, 1980). A recent determination of nucleosides in cell extracts was carried out after initial acid precipitation, neutrahsation and centrifugation followed by chromatography on a reversed phase Cjg column using an aqueous sodium acetate-acetonitrile mobile phase (Harmenberg, 1983). 

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... 

In a second procedure, poly(ADP-ribose) was first separated from the bulk of the nucleic acids and proteins by dihydroxyboryl-Sepharose affinity chromatography 147,190). The isolated polymer was treated with snake venom phosphodiesterase and bacterial alkaline phosphatase to yield the nucleoside 2, l"-ribosyladenosine from internal residues. This product was then treated with chloroacetaldehyde to produce the fluorescent derivative, l,iSr -ethenoribosyladenosine, which was then separated from other derivatized residues by reversed-phase high performance liquid chromatography picomole amounts were quantified by fluorescence detection. This procedure facilitates the accurate determination of minute quantities of endogenous poly(ADP-ribose) (102, 190). Niedergang et al. (147) have also utilized a fluorimetric assay for determination of the enzymatic digestion products of the polymer, ADP-ribose, or iso-ADP-ribose. 

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). 

General Reviews. — Pre- and post-column fluorescence deiivatization procedures used in the h.p.l.c. analysis of carbohydrates and nucleosides have been reviewed, and a section on the reversed-phase h.p.l.c. analysis of glucosyl ester and glucoside derivatives has been included in a review on the chromatography of gibberellins. ... 

R. P. Singhal, R. K. Bajaj, C. M. Buess, D. B. Smoll and V. N. Vakha-ria, Reversed-phase boronate chromatography for the separation of 0-methylribose nucleosides and aminoacyl-tRNAs, Anal. Biochem., 1980,109(1), 1-11. 

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. ... 

Veldkamp, A.I. Sparidans, R.W. Hoetelmans, R.M.W. Beijnen, J.H. Quantitative determination of abacavir (1592U89), a novel nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1999, 736, 123-128. 

Faux, J. Venisse, N. Le Motil, G. Dupuis, A. Bouquet, S. Simultaneous determination of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography after solid-phase extraction, Chromatographia 2003, 58, 421-426. [amprenavir indinavir lopinavir nelflnavir ritonavir saquinavir efavirenz nevirapine prazepsun]... 

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