Ion-paired reverse phase liquid chromatography for the detection, separation and quantification of nucleotides If you are working in the field of molecular biology, there is hardly a day that goes by without the use of nucleotides. But beyond the use of the four well known deoxynucleotides in PCR, there are several other uses of nucleotides. In the field of enzymology, nucleotides are used as substrates of various enzymes. For example, kinases and phosphatases use nucleotides as substrates while phosphotransferases transfer phosphate group from one nucleotide substrate to another. If you want to study the kinetics of such enzymes, you need to quantify either the substrate or the product. Detection of nucleotides can be done in several ways. …show more content…
The only addition is the use of an ion-pairing agent. It is a compound that has a hydrophobic group which sticks to the stationary phase and a charged group which hangs out of the stationary phase, making it charged. So now the hydrophobic column has become polar. Ion-pairing agents are either acidic like tetrabutylammonium hydrogen sulfate or basic like sodium dodecyl sulfate. Acidic ion-pairing agents are positively charged and will bind anionic molecules while basic ion-pairing agents are negatively charged and will bind cationic molecules. The choice of ion-pairing agent will depend upon your experiment but there are a lot of options available out …show more content…
The two most important factors that can affect your separation are the type of organic solvent used and its concentration in the mobile phase. Two most widely used organic solvents are methanol and acetonitrile. Methanol is more polar than acetonitrile and hence is less powerful in separating compounds with stronger affinity to the stationary phase. However, this is not the only factor considered while choosing your organic solvent. Sometimes methanol just works better than acetonitrile in separating out a specific set of nucleotides. It would be wiser to try out both organic solvents and see which one works best for you. Another important factor is pH. Nucleotide separation is usually carried out at pH 6-8. At very low pH, nucleotides are not negatively charged and hence will not bind to the stationary phase. At very high pH, the ion-pairing agent will be neutral and hence not bind to the nucleotides. The concentration of ion-pairing agent should also be optimal in order to allow a higher binding capacity. Using a higher than optimal concentration will increase the affinity of the nucleotides for the stationary phase and make elution a difficult