LC-EC determination of nucleotides and nucleic acids: Application to enzyme assays and the analysis of DNA fragments

Malliaros, David P.; Debenedetto, Mark J.; Guy, Pamela M.; Tougas, Terrence P.; Jahngen, Edwin

doi:10.1016/0003-2697(88)90262-x

Cited by 11 publications

(4 citation statements)

References 19 publications

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“…These compounds play an important role in cell biology and biochemistry; therefore, sensitive and accurate detection methods for them are in great demand. High-performance liquid chromatography and capillary electrophoresis are frequently used to detect these analytes in complex mixtures. − UV absorbance detection is the most widely used detection method; however, its lack of selectivity necessitates great care in sample preparation to avoid interferences. An alternative is to derivatize adenine-containing compounds with chloroacetaldehyde and detect by laser-induced fluorescence, but the requirement of a long reaction time required and a UV laser limit the utility of this method.…”

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confidence: 99%

Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase

et al. 2001

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A biotinylated-DNA aptamer (molecular weight 16,600) that binds adenosine and related compounds in solution was immobilized by reaction with streptavidin, which had been covalently attached to porous chromatographic supports. The aptamer medium was packed into fused-silica capillaries (50-150-microm i.d.) to form affinity chromatography columns. Frontal chromatography analysis indicated that the dissociation constants (Kd) of cyclic-AMP, AMP, ATP, ADP, and adenosine were 138 +/- 18, 58 +/- 2, 38 +/- 2, 28 +/- 6 and 3 +/- 1 microM, respectively, for aptamer immobilized on a controlled pore glass support. Similar values were obtained for aptamer immobilized on a polystyrene support except for a slightly higher Kd for adenosine. The Kd for adenosine is similar to the previously reported value of 6 +/- 3 microM for adenosine-aptamer in solution indicating that immobilized aptamers can have affinity similar to that of the solution forms. Columns had 20 nmol of binding sites/100 microL of support media, which is 3.3-fold higher than that previously reported for immobilization of IgG on similar media, indicating that the aptamer can be immobilized with higher density than antibodies. Variation of mobile-phase conditions revealed that ionic strength and Mg2+ level had strong effects on retention of analytes while pH and buffer composition had less of an effect. It was demonstrated that the column could selectively retain and separate cyclic-AMP, NAD+, AMP, ADP, ATP, and adenosine, even in complex mixtures such as tissue extracts.

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Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase

et al. 2001

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“…1, a single irreversible wave was obtained on a carbon fiber disk electrode for adenyl and guanylyl nucleotides. The electrochemical response is due to the purine base; therefore the oxidation potential required for steady state oxidation current (E max ) is identical for all adenosine-containing compounds at +1.40 V. Similarly, all guanosine-containing compounds have the same, albeit slightly lower, E max of +1.25 V. The oxidation peak potential shifts slightly positive with pH decreasing from 7 to 2 as observed before; 24 however, preliminary studies and previous work 6,[8][9][10] suggested that a mobile phase pH of at least 5.0 would be suitable for separation. Therefore, all further electrochemical optimization studies were performed with electrolyte at pH 5.0 and an applied potential of +1.50 V.…”

Section: Electrochemical Detectionmentioning

confidence: 54%

“…In such cases, high performance liquid chromatography (HPLC) may be used; however, this method is usually incapable of detecting all of these compounds at once. [6][7][8][9][10][11][12][13][14][15][16] In addition, with mass detection limits in the 2-5 pmol range, the method is not acceptable for microscale analysis of tissue. While capillary electrophoresis (CE) has excellent mass sensitivity, the concentration detection limit by UV absorbance detection is too poor to detect the lowest level compounds.…”

Section: Introductionmentioning

confidence: 99%

“…Purine bases are electroactive [23][24][25] meaning that EC detection can be accomplished without a requirement of derivatization which greatly simplifies the method. 6 Potential problems with EC detection of purines are the high potential required for oxidation creating high background and fouling of the electrode by the oxidation products.…”

Section: Introductionmentioning

confidence: 99%

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Microscale determination of purines in tissue samples by capillary liquid chromatography with electrochemical detection

Deng

Kauri

Qian

et al. 2003

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A microscale method for purines involved in intracellular signaling and energy metabolism, including ADP, ATP, cyclic-AMP, NADH and GTP, was developed. The analytes were separated on a fused-silica capillary liquid chromatography column (50 microm inner diameter by 25 cm long) packed with 7 microm reversed-phase particles and detected with a carbon fiber cylinder microelectrode at +1.50 V versus Ag/AgCl reference electrode. With an acetonitrile gradient, the separation was carried out within 15 min. With a 100 nl injection volume, the detection limits varied from 0.9 to 8 fmol depending upon the analyte. The low detection limits make the method suitable for analysis of small tissue samples. As a demonstration of the method, islets of Langerhans were analyzed for their adenosine-related messenger content.

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Biopolymer Chromatography

Huber¹

2000

Encyclopedia of Analytical Chemistry

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Biopolymers are biological macromolecules including peptides, proteins, nucleic acids, and polysaccharides whose physical and chemical properties differ considerably from those of small molecules. Biopolymer chromatography is a branch of liquid chromatography which is adapted to the specific requirements of biopolymers in order to obtain rapid and high‐resolution chromatographic separations of these molecules. Because of the low diffusion coefficients of biopolymers, special stationary phase configurations and surface modifications are necessary to permit sufficiently fast mass transfer kinetics between the mobile and stationary phase. Exploiting different molecular properties of the biopolymers, such as size, charge, hydrophilicity, hydrophobicity, the ability to form complexes with metal ions, or biological function, the most popular liquid chromatographic modes for biopolymer separation are size‐exclusion, ion‐exchange, normal‐phase, reversed‐phase, ion‐pair reversed‐phase, metal‐interaction, and affinity chromatography. Because of their large size and the multitude of different functional groups present in biopolymers, more than one mechanism may be responsible for interaction with the stationary phase, resulting in enhanced selectivity due to mixed‐mode interactions. Adsorption at multiple sites results in very steep elution isotherms as a function of the mobile phase solvent strength. Therefore, elution with a gradient of increasing solvent strength is usually applied in biopolymer chromatography. The choice of a suitable chromatographic environment is of special importance ifthe native confirmation of a biopolymer, which is essential for its biological activity, is to be preserved for subsequent experiments. Generally, conditions that come close to the physiological environment are most appropriate. The most important detection techniques for biopolymers are ultraviolet/visible absorbance detection, fluorescence detection, electrochemical detection, mass spectrometry, and low‐angle laser light scattering photometry. Miniaturization and multidimensional separation together with mass spectrometric detection allow the routine separation and identification of femtomole–attomole amounts of hundreds to thousands of different biopolymers contained in complex mixtures of biological origin.

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LC-EC determination of nucleotides and nucleic acids: Application to enzyme assays and the analysis of DNA fragments

Cited by 11 publications

References 19 publications

Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase

Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase

Microscale determination of purines in tissue samples by capillary liquid chromatography with electrochemical detection

Biopolymer Chromatography

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