Anion exchange, nucleoside separation

Fig. 3-93. Separation of nucleosides using anion exchange chromatography. — Separator columns 2 IonPac AS4A eluent see Fig. 3-92 flow rate 1.5 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations 10 ppm cytidine (1), 5 ppm adenosine (2), 10 ppm thymidine (3) and uridine (4), 13 ppm 2 -deoxyguanosine (5), 15 ppm guanosine (6), and inos-ine (7).

Figure 3.180 Separation of nucleosides using anion-exchange chromatography. Separator columns 2 lonPac AS4A-SC eluent 3 mmol/L Na2C03 -h 1 mmol/L NaOH flow rate 1.5 mL/...

From the known, differential complexing between boronic acids and polyhydroxy compounds, it follows that carbohydrate mixtures may be separated by column-chromatographic methods that exploit the differences. Nucleoside and nucleotide boronates have been separated on columns of anion-exchange resins,90 and sugars and alditols have been shown to be differentially retained on such resins in the sulfonated phenylboronic acid form,64 but perhaps the best uses of column chromatography in this connection have incorporated the resolving powers of insoluble polymers to which boronic acid groups have been covalently bonded. Such insoluble forms of boronates have been synthesized either by substitution of polysaccharide derivatives, or by polymerization of suitable arylboronic acids. 

Singhal and Cohn (S26) used exclusion chromatography with anion-exchange columns for the separation of nucleosides and bases. Brown et al. (B30) optimized this technique and obtained a separation of the naturally occurring free nucleosides and bases in cell extracts. Although these separations were useful, the analysis time of 2 h and relatively low efficiencies of the columns limited their usefulness in routine analyses. 

The assay is carried out both at 1 jaM and 1 mM substrate concentrations [162]. The enzyme preparation is incubated with tritium-labelled cyclic nucleotide in the presence of Mg, the reaction is stopped by brief heating in a water bath, the 5 -nucleotide formed is converted to the nucleoside by snake venom 5 -nucleotidase, the nucleoside is separated from the remaining cyclic nucleotide by anion exchange chromatography (Dowex 2), and the radioactivity of both compounds is counted. 

Several functional groups have been used to obtain cellulose anion exchangers [aminoethyl (AE), diethyl-aminoethyl (DEAE)], or cation exchangers [car-boxymethyl (CM), phosphate (P)] for thin-layer chromatography. PEI cellulose is not a chemically modified cellulose, but a complex of cellulose with polyethyleneimine. These cellulose exchangers are particularly useful for the separation of proteins, aminoacids, enzymes, nucleobases, nucleosides, nucleotides, and nucleic acids. 

The quantitative separation of phosphorus acids and esters by ion-exchange chromatography has been reviewed. The separation of nucleoside polyphosphates by anion-exchange chromatography and the affinity chromatography of enzymes on immobilized adenosine monophosphate have also been described. 

The ionizable phosphate group present in organic phosphorus species provides the basis for separation by anion exchange chromatography. This approach has been applied to the separation of phosphate, inositol phosphates, organic condensed phosphates, nucleic acids, nucleoside phosphonates, phospholipids and other organic phosphorus species. 

In the past HPLC separations of nucleosides have been carried out in a variety of modes, including anion-exchange (Floridi et al., 1977) and cation-exchange (Breter et al., 1977) however, since the introduction of stable packings reversed phase has become the preferred method. Typical chromatographic conditions for the separation of nucleosides include the use of dilute phosphate buffers with organic modifiers such as methanol or acetonitrile on ODS stationary phases. The effects of variations in these parameters is described below. 

In 1949, Cohn applied ion-exchange chromatography to the separation of nucleic acid derivatives and demonstrated elegant separations of nucleotides on anion-exchange resins such as Dowex-1 (which has quaternary ammonium charged groups on a polystyrene matrix). It was shown at that time that both RNA and DNA could be hydrolyzed by phosphodiesterases to yield nucleoside 5 -phosphates and it was therefore evident that polynucleotides could be regarded as assemblies of nucleoside 5 -phosphates. At this point, the adenosine phosphates were the only free nucleotides known to occur in tissues, apart from the coenzymes. Because the free adenosine phosphates were 5 -esters, a precursor relationship between these compounds and the pol mucleotides seemed likely. 

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). 

PGA extracts were analyzed by HPLG using described methods (6). Purine nucleosides and bases were separated with a reversed-phase column. Purine nucleotides were separated by anion-exchange HPLG. Simultaneous UV monitoring and radioactivity detection were performed with an on-line radioactivity flow detector. 

Figure 8.137 Separation of nucleoside di- and triphosphates together with NAD and NADP by capillary anion-exchange chromatography coupled with high-resolution mass spectrometry. Separator column lonPac AS19 column dimensions 250 mm x 0.4 mm i.d. eluent KOH (EG) gradient 8-40 mmol/L linearly from 0 to 15 min, then to 80 mmol/L in 10 min, to...

Diethylaminoethyl-cellulose (DE AE) is the functional group incorporated into the paper. It is an anion exchanger which is generally used to separate proteins and enzymes and similar materials but which is also used for nucleic acids, nucleotides, deoxynucleotides, and nucleosides. Separation on DEAE-cellulose is not as sharp as on PEI-cellulose but there is considerable data on the separation of nucleic acids on these layers. 

ECTEOLA is named for the epichlorohydrin and triethanolamine groups which are combined with cellulose. DEAE-cellulose and ECTEOLA-cellulose layers have about the same ability to resolve nucleic acid derivatives. ECTEOLA is especially useful for nucleic acids, nucleotides and nucleosides as an anion exchanger. Its strength also lies in its rapid separation of pyrimidines from purines. 

The hydrophobic properties of a series of 5-aIl l-, 5-alkenyl- and 5-alI yl-substituted deoxyuridines were defined from their retention behaivour on silica and reversed-phase columns. Zeatin and its riboside were determined in plant tissues by solid-phase extraction and cation-exchange h.p.l.c. Sugar and nucleoside H-phosphonates have been separated by anion-exchange h.p.l.c., and by flow injection analysis in which the components are hydrolysed, oxidised and the orthophosphate so generated is detected colorimetrically. ... 

Column Chromatography.- Gel-filtration has been used for determining the levels of glucose, sucrose, raffinose, stachyose, and verbascose in developing lupin seeds.Nucleobases, nucleosides, and nucleotides have been separated by ion chromatography (on a low-capacity anion-exchange resin) with dual conductimetric and u,v. 

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