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Nucleotides are the basic building blocks of nucleic acids such as DNA and RNA and consist of a base (adenine, guanine, cytosine, thymine or uracil), a sugar (ribose or deoxyribose) and a phosphate group. They play a central role in many biological processes, including energy storage (e.g. ATP) and signalling.
Analysing nucleotides using high-performance liquid chromatography (HPLC) is a common method for separating, identifying and quantifying these molecules. Various HPLC techniques are used, such as anion-exchange or reversed-phase HPLC, which separate nucleotides based on their charge, polarity and hydrophobic properties. HPLC enables precise and efficient analysis of nucleotide mixtures, which is of great importance in biochemistry, molecular biology and diagnostics.
The difference between nucleotides, nucleosides and nucleobases lies in their chemical structure:
Nucleotides = nucleobase + sugar + phosphate
Nucleosides = nucleobase + sugar
Nucleobases = the pure bases.
The differences between nucleotides, oligonucleotides and DNA/RNA lie in their structure and function:
1. adenosine monophosphate (AMP)
2. guanosine monophosphate (GMP)
3. adenosine diphosphate (ADP)
4. guanosine diphosphate (GDP)
5. adenosine triphosphate (ATP)
6. guanosine triphosphate (GTP)
Column: HALO Penta-HILIC 100x2.1mm, 2.7µm (92812-605)
Mobile phase A: 50/50 acetonitrile/0.025M ammonium phosphate (pH 6)
Mobile phase B: 75/25 acetonitrile/0.025M ammonium phosphate (pH 6)
Gradient:
Time / min | %B |
0 | 90 |
8 | 40 |
Flow rate: 0.3 mL/min
Back pressure: 76 bar
Temperature: 50 °C
Detection: UV, 260 nm
Injection volume: 1 µL
Sample solvent: Mobile phase B
1. unknown 2. 5-AMP 3. 5-GMP 4. 5-IMP 5. 5-TMP 6. 3-AMP 7. 2-AMP 8. 5-ADP 9. 3.5-cAMP 10. 3.5-cGMP 11. 5-GDP 12. 5-IDP 13. 5-TDP
Column: Inertsil ODS-3V 150x4.6mm, 5µm
Mobile phase A: 5mM tetrabutylammonium bromide + 20mM KH2PO4 (pH 3.5)
Mobile phase B: 5mM tetrabutylammonium bromide + 20mM KH2PO4 (pH 3.5)/acetonitrile 40/60
Gradient:
Time / min | %A | %B |
0 | 95 | 5 |
5 | 95 | 5 |
35 | 76 | 24 |
Flow rate: 1 mL/min
Temperature: 40 °C
Detection: 40 °C
Injection volume: 5 µL
HPLC - HILIC:
HPLC - Reversed Phase:
HPLC - Reversed Phase:
HPLC - IEX:
HPLC - Reversed Phase:
HPLC - Reversed Phase:
HPLC - IEX:
HPLC - IEX:
HPLC - Reversed Phase:
UHPLC - Reversed Phase:
HPLC - HILIC:
HPLC - Reversed Phase:
HPLC - HILIC:
HPLC - IEX:
HPLC - Ion chromatography:
HPLC - Reversed Phase:
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