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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

GL Sciences

HPLC - Reversed Phase:

  • Inertsil ODS-2, 5µm
  • Inertsil ODS-3V, 5µm
  • InertSustain NH2, 5µm
  • Inertsil NH2, 5µm
  • InertSustain C30, 5µm
  • Inertsil ODS-4, 5µm

 

HPLC - Mixed-Mode:

  • InertSustain AX-C18, 3µm

 

UHPLC - HILIC:

  • InertSustain Amide, 1.9µm

 

HPLC - IEX:

  • Inertsil AX, 5µm

Hamilton

HPLC - Reversed Phase:

  • PRP-1, 5µm

Helix

HPLC - Mixed-Mode:

  • Amaze HD
  • Amaze HA, 3µm
  • Coresep 100, 2.7µm
  • Amaze TCH, 3µm
  • Coresep SB, 5µm

Imtakt

HPLC - Mixed-Mode:

  • Intrada Organic Acid

 

HPLC - Reversed Phase:

  • Unison UK-Amino

 

UHPLC - Reversed Phase:

  • Dacapo DX-C18, 2.5µm

Macherey-Nagel

HPLC - IEX:

  • Nucleogel SAX, 8µm

 

HPLC - Reversed Phase:

  • Nucleodur PolarTec, 3µm
  • Nucleosil C18, 5µm

 

HPLC - HILIC:

  • Nucleodur HILIC, 5µm
  • Nucleoshell HILIC, 2.7µm

Osaka Soda

HPLC - IEX:

  • Capcell PAK NH2, 5µm

 

HPLC - Reversed Phase:

  • Capcell PAK ADME, 5µm
  • Capcell CORE AQ, 2.7µm

Sepax

HPLC - Reversed Phase:

  • HP-C18, 5µm

 

HPLC - IEX:

  • Proteomix SAX, 5µm

Shimadzu

HPLC - IEX:

  • Shim-pack WAX-1, 3µm

 

HPLC - Reversed Phase:

  • Shim-pack GIST C18-AQ, 3µm
  • Shim-pack Scepter C18, 3µm

 

UHPLC - Reversed Phase:

  • Shim-pack XR-ODSIII, 2.2µm

 

HPLC - HILIC:

  • Shim-pack Mix-HILIC, 5µm

Shodex

HPLC - Mixed-Mode:

  • RSpak NN-814
  • Asahipak GS-320 HQ

 

HPLC - Reversed Phase:

  • RSpak DE-613
  • RSpak DE-413

 

HPLC - IEX:

  • IEC DEAE-825

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

Waters

UHPLC - HILIC:

  • Atlantis Premier BEH Z-HILIC, 2.5µm
  • ACQUITY BEH Amide, 1.7µm

 

UHPLC - Reversed Phase:

  • ACQUITY HSS T3, 1.8µm
  • ACQUITY Premier HSS T3, 1.8µm
  • XTerra MS C18, 2.5µm

 

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