Nucleotides

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.

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Technical Data

Difference between Nucleotides, Nucleosides and Nucleobases

The difference between nucleotides, nucleosides and nucleobases lies in their chemical structure:

 

  • Nucleotides: They consist of three components - a nucleobase (adenine, guanine, cytosine, thymine or uracil), a sugar (ribose in RNA, deoxyribose in DNA) and one or more phosphate groups. Nucleotides are the building blocks of DNA and RNA and also play a role in energy metabolism (e.g. ATP).
  • Nucleosides: Nucleosides consist of a nucleobase and a sugar (ribose or deoxyribose), but without the phosphate group. They are therefore a precursor of nucleotides and are formed by the absence of the phosphate group.
  • Nucleobases: Nucleobases are the nitrogenous bases contained in nucleotides and nucleosides. There are five main bases: Adenine, guanine, cytosine, thymine (only in DNA) and uracil (only in RNA).

 

Nucleotides = nucleobase + sugar + phosphate

Nucleosides = nucleobase + sugar

Nucleobases = the pure bases.


Difference between Nucleotides, Oligonucleotides and DNA/RNA

The differences between nucleotides, oligonucleotides and DNA/RNA lie in their structure and function:

 

  • Nucleotides: Nucleotides are the basic building blocks of nucleic acids. They consist of a nucleobase (adenine, guanine, cytosine, thymine or uracil), a sugar (ribose or deoxyribose) and a phosphate group. They serve as building blocks for larger molecules such as DNA and RNA and also play an important role in other biological processes such as energy transfer (ATP).
  • Oligonucleotides: Oligonucleotides are short chains of nucleotides, typically between 2 and 20 nucleotides long. They are often used in research as probes, primers or for genetic engineering applications, as they contain specific sequences that can bind to DNA or RNA segments.
  • DNA/RNA: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are long polymers of nucleotides. DNA consists of deoxyribonucleotides and stores genetic information, while RNA consists of ribonucleotides and is involved in protein synthesis and gene regulation in various forms. DNA is usually double-stranded, while RNA is usually single-stranded.

Applications

HILIC separation of adenosine phosphates with AMT HALO Penta-HILIC

Peak identities

1. adenosine monophosphate (AMP)

2. guanosine monophosphate (GMP)

3. adenosine diphosphate (ADP)

4. guanosine diphosphate (GDP)

5. adenosine triphosphate (ATP)

6. guanosine triphosphate (GTP)

Test conditions

Column: HALO Penta-HILIC 100x2.1mm, 2.7µm (92812-605)

Reversed phase analysis of nucleotides with GL Sciences Inertsil ODS-3V

Peak identities

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

Test conditions

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

Downloads

Manufacturer overview


AMT

HPLC - HILIC:

  • HALO Penta-HILIC, 2.7µm

ChromaNik

HPLC - Reversed Phase:

  • Sunniest RP-AQUA, 3µm
  • SunShell RP-AQUA, 2.6µm







Sepax

HPLC - Reversed Phase:

  • HP-C18, 5µm

 

HPLC - IEX:

  • Proteomix SAX, 5µm



Thermo

HPLC - Reversed Phase:

  • Accucore aQ, 2.6µm

 

HPLC - HILIC:

  • Accucore Amide-HILIC, 2.6µm

 

HPLC - IEX:

  • DNAPac PA100

 

HPLC - Ion chromatography:

  • IonPac AS11-HC, 4µm

Tosoh

HPLC - Reversed Phase:

  • TSKgel ODS-120T, 5µm

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